and QIMR Berghofer Medical Research Institute

Identification of Anti-malarial Drug Candidate Inhibitors using Ultra-high Throughput Screening Methods and Subsequent Mechanism of Action Studies Aimed at the Treatment and Prevention of Plasmodium falciparum.

Timothy Patrick Spicer Student Number: 4297236 Candidature for PhD.

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2019 Faculty of Medicine

1 Abstract

The World Health Organization estimates almost 3.2 billion people remain at risk of malaria with

200 million new cases each year 1. Approximately 600,000 people die each year from malaria,

most as a result of infection with the protozoan parasite Plasmodium falciparum. Almost 75% of

those that die are under the age of five 2,3. While malaria is a preventable and treatable mosquito

borne illness and antimalarial drugs do exist, they are becoming less effective and most do not

effectively block transmission. This is compounded by the fact that resistance of the parasite to

antimalarial drugs, including artemisinins, and resistance of the mosquito vector to insecticides is increasing 1, 4, 5. A singular approach will not suffice to identify inhibitors that block the

transmission of this pathogen 6. In this project two potential targets are evaluated. Approach one

targets Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP), the sole aspartyl protease expressed in the parasite, and hence represents a unique opportunity for therapeutic intervention7,8. In addition, its amino acid homology with that of human AAPs is not substantial

providing an opportunity to find parasite specific inhibitors9. The second approach targets

transmission which involves uptake of mature gametocytes by the mosquito vector prior to

development into a fully functioning transmissible form. Hence this is another attractive target

10,11. Key variables are not only reagent acquisition (of enzyme and gametocytes) but, also to determine the druggability of the aforementioned targets 12. The Malaria Biology Laboratory

(Gardiner and Trenholme) at QIMR Berghofer Medical Research Institute has established current

in vitro technologies for recombinant PfM18AAP (rPfM18AAP) production and gametocyte culture, thereby paving the path to assay development, implementation and High Throughput

Screening (HTS) 13-14. The Scripps Research Institutes Molecular Screening Center affords the

unique and powerful capability to couple these two targets with ultra-high throughput screening

2 capability15. Both targets have successfully completed HTS. The AAP target has resulted in a published molecular probe, while the gametocyte target is undergoing refinement of the initial hits

to bonafied leads 9.

3

Declaration by author

This thesis is composed of my original work, and contains no material previously published or

written by another person except where due reference has been made in the text. I have clearly

stated the contribution by others to jointly-authored works that I have included in my thesis.

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award.

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made

available for research and study in accordance with the Copyright Act 1968 unless a period of

embargo has been approved by the Dean of the Graduate School.

I acknowledge that copyright of all material contained in my thesis resides with the copyright

holder(s) of that material. Where appropriate I have obtained copyright permission from the

copyright holder to reproduce material in this thesis and have sought permission from co-authors

for any jointly authored works included in the thesis.

4

Publications included in this thesis:

1. Spicer, Timothy P., Gardiner, Donald L., Schoenen, Frank, Roy, Sudenshna, Griffin, Patrick, Scampavia, Louis, Hodder, Peter and Trenholme, Katharine R. Identification of Anti-Malarial Inhibitors using Late Stage Gametocytes in a Phenotypic Live/Dead Assay. SLAS Discovery 2018 Aug 24, PMID: 30142014

2. Spicer, Timothy P., Fernandez-Vega, Virneliz, Chase, Peter, Scampavia, Louis, To, Joyce, Dalton, John P., Da Silva, Fabio L., Skinner-Adams, Tina S., Gardiner, Donald L., Trenholme, Katharine R., Brown, Christopher L., Ghosh, Partha, Porubsky, Patrick, Wang, Jenna L., Whipple, David A., Schoenen, Frank J., and Peter Hodder. Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High Throughput Screening. J Biomol Screen. 2014 Mar 11. PMID: 24619116. PMC: 4641816.

3. Schoenen FJ, Weiner WS, Baillargeon P, Brown CL, Chase P, Ferguson J, Fernandez- Vega V, Ghosh P, Hodder P, Krise JP, Matharu DS, Neuenswander B, Porubsky P, Rogers S, Skinner-Adams T, Sosa M, Spicer Timothy P., To J, Tower NA, Trenholme KR, Wang J, Whipple D, Aubé J, Rosen H, White EL, Dalton JP, Gardiner DL Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2013 Apr 15. PMID: 24479194. ML369

Submitted manuscripts included in this thesis

No other manuscripts related to this thesis have been submitted for publication

Other publications during candidature

I have >101 peer reviewed publications which can be found using the following link:

http://www.ncbi.nlm.nih.gov/sites/myncbi/timothy.spicer.1/bibliograpahy/47232264/public/?sort =date&direction=ascending

Peer reviewed publications since start of my Ph.D program (53 total; 23 either primary or

senior author):

5 1. Baillargeon P, Coss-Flores K, Singhera F, Shumate J, Williams H, DeLuca L, Spicer TP, Scampavia L. Design of Microplate-Compatible Illumination Panels for a Semiautomated Benchtop Pipetting System. SLAS Technology. 2019 January. PMID:30698997

2. Baillargeon P, Fernandez-Vega V, Sridharan B, Brown S, Griffin P, Rosen H, Cravatt B, Scampavia L, Spicer TP. The Scripps Molecular Screening Center and Translational Research Institute. SLAS Discovery. 2019 January. PMID:30682260

3. Spicer TP, Fernandez-Vega V, Scampvia L, Willetts L, Vessels M. A Novel 3D Culture System for High-Throughput Hepatoxicity Screening. BioProcess International. 2018 October.

4. Spicer, Timothy P., Gardiner, Donald L., Schoenen, Frank, Roy, Sudenshna, Griffin, Patrick, Scampavia, Louis, Hodder, Peter and Trenholme, Katharine R.. Identification of Anti-Malarial Inhibitors using Late Stage Gametocytes in a Phenotypic Live/Dead Assay. SLAS Discovery 2018 Aug 24, PMID: 30142014

5. Janovick JA, Spicer TP, Bannister TD, Smith E, Ganapathy V, Scampavia L. Chemical Validation and Optimization of Pharmacoperones Targeting Vasopressin Type 2 Receptor. Biochem J. 2018 August. PMID:30068530

6. Nieto A, Fernandez-Vega V, Spicer TP, Sturchler E, Adhikari P, Kennedy N, Mandat S, Chase P, Scampavia L, Bannister T, Hodder P, McDonald PH. Identification of Novel, Structurally Diverse, Small Molecule Modulators of GPR119. Assay Drug Dev Technol. 2018 July. PMID:30019946

7. Victor Quereda, Shurong Hou, Franck Madoux, Louis Scampavia, Timothy P. Spicer* and Derek Duckett. A Phenotypic 3 Dimensional-Spheroid High-Throughput Assay using Patient Derived Glioma Stem Cells. SLAS Discovery 2018 May. PMID:29750582 *Co- Communicated

8. Smitha Kota, Shurong Hou, William Guerrant, Franck Madoux, Scott Troutman, Virneliz Fernandez-Vega, Nina Alekseeva, Neeharika Madala, Louis Scampavia, Joseph Kissil and Timothy P. Spicer. A Novel 3-dimensional High Throughput Screening Approach Identifies Inducers of a Mutant KRAS Selective Lethal Phenotype. Oncogene. 2018 May. PMID:29743592

9. Shurong Hou, Hervé Triac, Banu Priya Sridharan, Louis Scampavia, Franck Madoux, Jan Seldin, Glauco R. Souza, Donald Watson, David Tuveson and Timothy Spicer. Advanced Development of Primary Pancreatic Organoid Tumor Models for High Throughput Phenotypic Drug Screening. SLAS Discovery. 2018 Apr. PMID:29673279

10. Brian S. Muntean, Dipak Patil, Franck Madoux, James Fossetta, Louis Scampavia, Timothy P. Spicer and Kirill A. Martemyanov. A High-Throughput HTRF Assay to Screen for Modulators of RGS7/Gβ5/R7BP Complex. Assay Drug Dev Technol. 2018 Apr. PMID:29658790

6

11. Justin Shumate, Pierre Baillargeon, Timothy P. Spicer and Louis Scampavia. IoT for Real-Time Measurement of High-Throughput Liquid Dispensing in Laboratory Environments. SLAS Technology 2018 Apr. PMID:29649373

12. Robert A. Wolff1, Andrea Wang-Gillam2, Hector Alvarez8, Hervé Tiriac, Dannielle Engle, Shurong Hou, Abigail F. Groff, Anthony San Lucas, Vincent Bernard, Kelvin Allenson, Jonathan Castillo, Dong Kim, Feven Mulu, Jonathan Huang, Bret Stephens, Ignacio I. Wistuba, Matthew Katz, Gauri Varadhachary, YoungKyu Park, James Hicks, Arul Chinnaiyan, Louis Scampavia, Timothy Spicer, Chiara Gerhardinger, Anirban Maitra, David Tuveson, John Rinn, Gregory Lizee, Cassian Yee and Arnold J. Levine. Dynamic changes during the treatment of pancreatic cancer. Oncotarget. Feb 13, 2018

13. Spicer T, Hubbs C, Vaissiere T, Collia D, Rojas C, Kilinc M, Vick K, Madoux F, Baillargeon P, Shumate J, Martemyanov K.A., Page D.T., Puthanveettil S, Hodder P, Davis R, Miller C.A., Scampavia L, Rumbaugh G. Improved Scalability of Neuron-Based Phenotypic Screening Assays for Therapeutic Discovery in Neuropsychiatric Disorders. Molecular Neuropsychiatry. 2017 Nov. PMID:29594133

14. Emery Smith, Kenneth Giuliano, Justin Shumate, Pierre Baillargeon, Brigid McEwan, Matthew D. Cullen, John P. Miller, Lawrence Drew, Louis Scampavia, Timothy P. Spicer. A Homogeneous Cell-Based Halide-Sensitive YFP Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel. Assay Drug Dev Technol. 2017 Nov. PMID:29172645

15. Singhera F, Cooper E, Scampavia L, Spicer T. Using bead injection to model dispensing of 3-D multicellular spheroids into microtiter plates. Talanta. 2017 Sept.

16. Kong J, Fang P, Madoux F, Spicer T, Scampavia L, Kim S, Guo M. High-Throughput Screening for Protein Synthesis Inhibitors Targeting Aminoacyl-tRNA Synthetases. SLAS Discovery. 2017 Oct. PMID:29020503

17. Collia D, Bannister T, Tan H, Jin S, Langaee T, Shumate J, Scampavia L, Spicer T. A Rapid Phenotypic Whole-Cell Screening Approach for the Identification of Small- Molecule Inhibitors That Counter B-Lactamase Resistance in Pseudomonas aeruginosa. SLAS Discovery. 2017 Aug. PMID:28850797

18. Janovick JA, Spicer TP, Scampavia L, Conn PM. Pharmacoperone rescue of vasopressin 2 receptor mutants reveals unexpected constitutive activity and coupling bias. PLoS One. 2017 Aug. PMID:28767678

19. Hou S, Madoux F, Scampavia L, Janovick JA, Conn PM, Spicer TP. Drug Library Screening for the Identification of Ionophores That Correct the Mistrafficking Disorder Associated with Oxalosis Kidney Disease. SLAS Discovery. 2017 Jan. PMID:28346094

7 20. Franck Madoux, Allison Tanner, Michelle Vessels, Lynsey Willetts, Shurong Hou, Louis Scampavia and Timothy P. Spicer. A 1,536-well 3D Viability Assay to Assess the Cytotoxic Effect of Drugs on Spheroids. SLAS Discovery. 2017 Jan. PMID:28346088

21. Wang J, Fang P, Chase P, Tshori S, Razin E, Spicer TP, Scampavia L, Hodder P, Guo M. Development of an HTS-Compatible Assay for Discovery of Melanoma-Related Microphthalmia Transcription Factor Disruptors Using AlphaScreen Technology. SLAS Discov. 2017 Jan;22(1):58-66

22. Madoux F, Dreymuller D, Pettiloud JP, Santos R, Becker-Pauly C, Ludwig A, Fields GB, Bannister T, Spicer TP, Cudic M, Scampavia LD, Minond D. Discovery of an enzyme and substrate selective inhibitor of ADAM10 using an exosite-binding glycosylated substrate. Sci Rep. 2016 Dec;6(1):11. Epub 2016 Dec 5

23. Janovick JA, Spicer TP†, Smith E, Bannister TD, Kenakin T, Scampavia L, Conn PM. Receptor antagonism/agonism can be uncoupled from pharmacoperone activity. Mol Cell Endocrinol. 2016 Jul 4. †Co-Communicated. PMID:27389877

24. Christina B. Cooley, Lars Plate, John J. Chen, Ryan J. Paxman, Ciara M. Gallagher, Franck Madoux, Joseph C. Genereux, Wesley Dobbs, Dan Garza, Timothy P. Spicer, Louis Scampavia, Steven J. Brown, Hugh Rosen, Evan T. Powers, Peter Walter, Peter Hodder, R. Luke Wiseman and Jeffery W. Kelly. Small Molecule Proteostasis Regulators that Transcriptionally Reprogram the Endoplasmic Reticulum and Reduce Extracellular Protein Aggregation. eLife July 2016; 5. PMID: 27435961

25. Smith E, Janovick JA, Bannister TD, Shumate J, Scampavia L, Conn PM, Spicer TP. Identification of Potential Pharmacoperones Capable of Rescuing the Functionality of Misfolded Vasopressin 2 Receptor Involved in Nephrogenic Diabetes Insipidus. J Biomol Screen. 2016 Jun 8. PMID: 27280550

26. Heather Ewing, Virneliz Fernández-Vega, Timothy P. Spicer*, Peter Chase, Steven Brown, Louis Scampavia, William Roush, Sean Riley, Hugh Rosen, and Michael H. Gelb. Fluorimetric High Throughput Screening Assay for Secreted Phospholipases A2 using Phospholipid Vesicles. J Biomol Screen. 2016 May 4. [Epub ahead of print] PMID: 27146384. *equal contributuion

27. Suzie Thenin-Houssier, Ian S. de Vera, Laura Pedro-Rosa, Angela Brady, Audrey Richard, Briana Konnick, Silvana Opp, Cindy Buffone, Jakob Fuhrmann, Smitha Kota, Blase Billack, Magdalena Pietka-Ottlik, Timothy Tellinghuisen, Hyeryun Choe, Timothy Spicer, Louis Scampavia, Felipe Diaz-Griffero, Douglas Kojetin, and Susana Valente. Ebselen, a small molecule capsid-inhibitor of HIV-1 replication. Antimicrob Agents Chemother. 2016 Mar 25;60(4):2195-208. PMCID: PMC4808204

28. Enrique Jambrina, Rok Cerne, Emery Smith, Louis Scampavia, Maria Cuadrado, Jeremy Findlay, Michael J. Krambis, Mark Wakulchik, Peter Chase, Michael Brunavs, Kevin Burris, Peter Gallagher, Timothy P. Spicer† and Daniel Ursu1†.An Integrated Approach

8 for Screening and Identification of Positive Allosteric Modulators of N-Methyl-D- Aspartate Receptors. J Biomol Screen. 2016 Feb 2. †Co-Communicated PMID: 26838761.

29. Chase, Peter; Enogieru, Imarhia; Madoux, Franck; Bishop, Eric; Beer, Jacob; Scampavia, Louis; and Spicer, Timothy. An Automated Miniaturized Method to Perform and Analyze Antimicrobial Drug Synergy Assays. Assay Drug Dev Technol. 2015 Dec 15. PMID: 26669516

30. Palde PB, Bhaskar A, Pedró Rosa LE, Madoux F, Chase P, Gupta V, Spicer T, Scampavia L, Singh A, Carroll KS. First-In-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M. tuberculosis. ACS Chem Biol. 2015 Nov 2. PMID: 26524379

31. Conn PM, Spicer TP, Scampavia L, Janovick JA. Assay strategies for identification of therapeutic leads that target protein trafficking. Trends Pharmacol Sci. 2015 Jun 8. PMID:26067100

32. Chang JW, Zuhl AM, Speers AE, Niessen S, Brown SJ, Mulvihill MM, Fan YC, Spicer TP, Southern M, Scampavia L, Fernandez-Vega V, Dix MM, Cameron MD, Hodder PS, Rosen H, Nomura DK, Kwon O, Hsu KL, Cravatt BF. Selective inhibitor of platelet- activating factor acetylhydrolases 1b2 and 1b3 that impairs cancer cell survival. ACS Chem Biol. 2015 Apr 17. PMID:25602368

33. Emery Smith, Peter Chase, Colleen M. Niswender, Thomas J. Utley, Douglas J. Sheffler, Michael R. Wood, P. Jeffrey Conn, Craig W. Lindsley, Franck Madoux, Mary Acosta, Louis Scampavia, Timothy Spicer*, and Peter Hodder. Application of Parallel Multiparametric Cell-Based FLIPR Detection Assays for the Identification of Modulators of the Muscarinic Acetylcholine Receptor 4 (M4). J Biomol Screen. 2015 Apr 15. *communicating author

34. Franck Madoux, Jo Ann Janovick, David Smithson, Sonia Fargue, Christopher J. Danpure, Louis Scampavia, Yih-Tai Chen, Timothy P. Spicer, and P. Michael Conn. Development of a Phenotypic High-Content Assay to Identify Pharmacoperone Drugs for the Treatment of Primary Hyperoxaluria Type 1 by High-Throughput Screening. Assay and Drug Development Technologies. Assay Drug Dev Technol. 2015 Jan-Feb;13(1):16-24.

35. Chang J, Zuhl A, Speers A, Niessen S, Brown S, Mulvihill M, Spicer TP, Southern M, Scampavia L, Fernandez-Vega V, Ferguson J, Dix M, Hodder P, Rosen H, Nomura D, Kwon O, Hsu K-L, Cravatt B. A selective inhibitor of platelet-activating factor acetylhydrolases 1b2 and 1b3 that impairs cancer cell survival. ACS Chem Biol. 2015 Jan 20.

36. Zhao, N., Darby, C., Small, J., Bachovchin, D., Jiang, X., Burns-Huang, K., Botella, H., Ehrt, S., Boger, D., Anderson, E., Cravatt, B., Speers, A., Fernandez-Vega, V., Rosen, H., Spicer, T., Nathan, C. A Target-Based Screen Against Mycobacterial Acid Resistance

9 Protease Implicates an Additional Periplasmic Serine Protease in Regulation of Intrabacterial pH Homeostasis in Mycobacterium tuberculosis. ACS Chemical Biology, Dec 2014 ahead of print, PMID: 25457457

37. Kadakkuzha B, Spicer TP*, Chase P, Richman J, Hodder P, Puthanveettil S. High- Throughput Screening for Small Molecule Regulators of Motor Protein Kinesin. ASSAY and Drug Development Technologies. October 2014, 12(8): 470-480. *equal contribution

38. Bannister TD, Yue, Z, Pedro-Rosa L, Hodder P, Cameron M, Brown S, Buckner FS, Hol WG, Gillespie JR, Ranade RM, Fan E, Koh CY, Zhang Z, Jian TY, Verlinde CL, Scampavia L, Spicer T. Small Molecule Inhibitors of Methionyl tRNA Synthetase (MetRS) from the Pathogenic Protozoan Trypanosoma brucei. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); June 4th 2014.

39. Spicer TP, Jiang J , Taylor A , Choi J-Y, Hart PJ , Roush W, Fields G, Hodder P , Minond D. Characterization of Selective Exosite-Binding Inhibitors of Matrix Metalloproteinase 13 That Prevent Articular Cartilage Degradation In Vitro. J Med Chem. 2014 Nov 26;57(22):9598-611

40. Pedró-Rosa L, Buckner F, Ranade R, Eberhart C, Madoux F, Gillespie R, Koh C-Y, Brown S, Lohse J, Verlinde C, Fan E, Bannister T, Scampavia L, Hol W, Spicer TP * and Hodder P. Identification of Potent Inhibitors of the Trypanosoma brucei Methionyl-tRNA Synthetase via High Throughput Orthogonal Screening. J Biomol Screen. 2015 Jan;20(1):122-30 PMID: 25163684 *communicating author

41. Madoux, F., Spicer, T., Scampavia, L., Fields, G., Becker-Pauly, C., Minond, D. HTS Assay Development and LOPAC Screen for Inhibitors of Meprin α and β. Biopolymers. 2014 Sep;102(5):396-406.

42. Sturchler E, Chen W, Spicer, T, Hodder P, McDonald P, Duckett D. Development of an HTS-Compatible Assay for the Discovery of ASK1 Signalosome Inhibitors Using AlphaScreen Technology. ASSAY and Drug Development Technologies. May 2014;12(4):229-37. PMID: 24831789

43. Conn P. Michael, Smith Emery, Spicer, T, Chase Peter, Scampavia Louis, and Janovick Jo Ann. A Phenotypic High Throughput Screening Assay for the Identification of Pharmacoperones for the Gonadotropin Releasing Hormone Receptor. ASSAY and Drug Development Technologies. May 2014, 12(4): 238-246. PMID: 24831790

44. Spicer, Timothy, Fernandez-Vega, Virneliz, Chase, Peter, Scampavia, Louis, To, Joyce, Dalton, John P., Da Silva, Fabio L., Skinner-Adams, Tina S., Gardiner, Donald L., Trenholme, Katharine R., Brown, Christopher L., Ghosh, Partha, Porubsky, Patrick, Wang, Jenna L., Whipple, David A., Schoenen, Frank J., and Peter Hodder. Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl

10 Aminopeptidase (PfM18AAP) of Human Malaria via High Throughput Screening. J Biomol Screen. 2014 Mar 11. PMID: 24619116

45. Miguel Guerreroa, Ramulu Poddutooria, Xuemei Peng, Peter Hodder, Spicer TP, Peter Chase, Marie-Therese Schaeffer, Steve Brown, Pedro J. Gonzalez-Cabrera, Stephan C. Schürer, Hugh Rosen, Edward Roberts, Discovery, Design and Synthesis of selective 3,5- diaryl-oxadiazoles S1P1 agonists. Bioorg Med Chem Lett. 2013 Dec 1;23(23):6346-9

46. Hydroxyquinoline-derived compounds and analoging of selective Mcl-1 inhibitors using a functional biomarker. Richard DJ, Lena R, Bannister T, Blake N, Pierceall WE, Carlson NE, Keller CE, Koenig M, He Y, Minond D, Mishra J, Cameron M, Spicer T, Hodder P, Cardone MH. Bioorg Med Chem. Volume 21, Issue 21, 1 November 2013, Pages 6642– 6649.

47. Syamalima Dube, Nitin Saksena, Timothy Spicer, Jayne Healey, Patricia Benz, Dipak K Dube and Bernard J Poiesz. Delayed seroconversion to STLV-1 infection is associated with mutations in the pol and rex genes. Virology Journal 2013, 10:282.

48. Pei W. Thomas, Timothy Spicer, Michael Cammarata, Jennifer S. Brodbelt, Peter Hodder, Walter Fast. An altered zinc-binding site confers resistance to a covalent inactivator of New Delhi metallo-beta-lactamase-1 (NDM-1) discovered by high-throughput screening. Bioorg Med Chem. 2013 Jun 1;21(11):3138-46.

49. Karapetyan YE, Sferrazza GF, Zhou M, Ottenberg G, Spicer TP, Chase P, Fallahi M, Hodder P, Weissmann C, Lasmézas CI. Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents. Proc Natl Acad Sci U S A. 2013 Apr 23;110(17):7044-9.

50. Yeung KS, Qiu Z, Yin Z, Trehan A, Fang H, Pearce B, Yang Z, Zadjura L, D'Arienzo CJ, Riccardi K, Shi PY, Spicer TP, Gong YF, Browning MR, Hansel S, Santone K, Barker J, Coulter T, Lin PF, Meanwell NA, Kadow JF. Inhibitors of HIV-1 attachment. Part 8: The effect of C7-heteroaryl substitution on the potency, and in vitro and in vivo profiles of indole-based inhibitors. Bioorg Med Chem Lett. 2013 Jan 1;23(1):203-8

51. Nagano JM, Hsu KL, Whitby LR, Niphakis MJ, Speers AE, Brown SJ, Spicer TP, Fernandez-Vega V, Ferguson J, Hodder P, Srinivasan P, Gonzalez TD, Rosen H, Bahnson BJ, Cravatt BF. Selective inhibitors and tailored activity probes for lipoprotein-associated phospholipase A(2). Bioorg Med Chem Lett. 2013 Feb 1;23(3):839-43.

52. Dreyton CJ, Jones JE, Knuckley BA, Subramanian V, Anderson ED, Brown SJ, Fernandez- Vega V, Eberhart C, Spicer T, Zuhl AM, Ferguson J, Speers AE, Wang C, Boger DL, Thompson P, Cravatt BF, Hodder P, Rosen H. Optimization and characterization of a pan protein arginine deiminase (PAD) inhibitor. Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. 2012 Dec 17.

11 53. Bannister TD, Nair R, Spicer T, Fernandez Vega V, Eberhart C, Mercer BA, Cameron M, Schurer S, Amundsen SK, Karabulut A, Londoño LM, Smith GR, Hodder P. A Novel Dual Inhibitor of Bacterial AddAB and RecBCD Helicase-nuclease DNA Repair Enzymes. Probe Reports from the NIH Molecular Libraries Program. Bethesda (MD): National Center for Biotechnology Information (US); 2010-. 2012 Dec 17

Conferences attended, talk/lectures given, poster presentations (>30 total):

Highlight Presentations

Mount Sinai School of Medicine, 2018 Scaling, Adapting, and CRISPR editing Human iPSC-derived Neurons for High Throughput and High Content Screening Stony Brook University, 2018 3D Platforms in High Throughput Formats to Assess the Cytotoxic Effect of Drugs on Cancer SLAS-Society of Laboratory Automation and Screening, 2018 Conference Co-Chair->6,000 attendees 3D Tissues Models, Oncology, 2018 Boston MA-Invited Speaker and Workshop Presenter A Comparative Analysis of 3D Platforms in High Throughput Formats to Assess the Cytotoxic Effect of Drugs for Discovery Taras Oceanographic Institute, Jupiter Florida-March 2018 3 Dimensional Approaches to Identify Potential Cancer Therapies SLAS-Society of Laboratory Automation and Screening, 2018 Hepatotoxicity Determination Using 3D Primary Human Hepatocyte Culture Systems Versus Large Approved Drug Libraries NCI-IMAT Program PI Retreat, 2017 Advanced Development and Validation of 3D Spheroid Culture of Primary Cancer Cells using Nano3D Technology International Conference on Diabetes and Its Complications, ICDC 2017 Identification of Potential Pharmacoperones Capable of Rescuing the Functionality of Misfolded Vasopressin 2 Receptor Involved in Nephrogenic Diabetes Insipidus Scripps Kellogg’s School of Graduate Studies, 2017 TSRI-Drug Discovery Graduate Course-Spicer Lecture Mount Sinai School of Medicine, 2017 3D Primary Tumor Cell Organoid Models for Phenotypic High-throughput Screening University of Florida Department of Pharmacotherapy and Translational Research, 2017 A Scripps Florida and University of Florida Collaboration Aimed at countering beta-lactam resistance in Pseudomonas aeruginosa SLAS-Society of Laboratory Automation and Screening, 2017 Speaker, 3-Dimensional Cell Culture Systems, Novel Technologies, and Their Use at the Laboratory Bench Compound and Sample Management Summit, 2016 Keynote Speaker, Philadelphia: Examining How the HTS Storage & Microplate Industry has Effected Translational Science & Precision Medicine SLAS-Society of Laboratory Automation and Screening, 2016 Track Chair-Automation and HTS Technologies

12 Scripps Department of Molecular Medicine, 2015 Live/dead Assays for the Purpose of Discovering Molecular Probes that Inhibit Plasmodium falciparum Gametocytes SLAS-Society of Laboratory Automation and Screening, 2015 Utility of the MaxCyte at the SRIMSC- presented at SLAS users forum SLAS-Society of Laboratory Automation and Screening, 2014 Associate Track Chair-Automation and HTS Technologies SLAS-Society of Laboratory Automation and Screening, 2012 Biotix Tips vs. OEM: Case Scenarios for Compounds Transfers and for Ca++ Signaling Assays on the FLIPR Tetra

Contributions by others to the thesis:

Other than mentoring and guidance, as typical for the obtainment of a higher degree, by Drs.

Trenholme, Gardiner and Hodder, no one else has contributed significantly to this work.

Statement of parts of the thesis submitted to qualify for the award of another degree

No works submitted towards another degree have been included in this thesis.

Research Involving Human or Animal Subjects

No animal or human subjects were involved in this research.

Acknowledgements

I formally acknowledge:

Pierre Baillargeon and Lina DeLuca (Scripps Florida) for compound management.

Financial support

International Postgraduate Research Scholarship (IPRS) including full tuition fee award from the

UQ Graduate School starting July 2, 2012 attending as a part time international student for Doctor

of Philosophy degree due semester 2, 2020.

13 NIH Funding 1 R33 CA206949-01(PI:Spicer) 08/01/16 - 07/31/19 NIH/NCI Advanced Development and Validation of 3 Dimensional Spheroid Culture of Primary Cancer Cells using Nano3D Technology

R33CA206949-02 (PI:Spicer) 02/01/2018- 07/31/2019 NIH/NCI PA-17-143 (Administrative Supplement to the R33 CA206949) Title: Integration of 3D primary tumor drug-profiling with patient-specific drug gene networks for recommending targeted cancer therapies

R01 MH113648 Rumbaugh/Scampavia/ Spicer (MPI) 07/01/17 – 03/31/20 NIH/NIMH A Scalable Neuron-Based High-Throughput Screening Platform for the Discovery of Compounds that Restore Protein Expression Caused by Genetic Haploinsufficiency

1 R01 DA046204-01 Sanna/Spicer 04/01/18 - 03/31/23 NIH/NDA Identification of small molecules for neurological complications of HIV and substance abuse comorbidity

Co-Investigator on at least 9 other funded NIH initiatives. Principal Investigator 6 current Special Funding Proposals (SFPs) = >1 million dollars

Patents The Scripps Research Institute-U.S. Provisional Patent Application No. 62/657,581 “UBIQUITIN LIGASE AGNOISTS, PHARMACEUTICAL COMPOSITIONS, AND RELATED METHODS OF USE” The Scripps Research Institute-Patent application on RecBCD/AddAB inhibitors- USSN 61/613,367; March 15, 2013.

NIH Study Sections: NCI-IMAT-2018-10 NCI-IMAT-2018-05-012-013 SBIR Contracts Phase I - Topic 015

Invited Reviewer, Nature Communications, 2017 Invited Reviewer, Journal of Biomolecular Screening, 2014,15,16,17,18 Invited Reviewer, PLOS One, 2016-2017 Invited Reviewer, AIDs Research and Human Retroviruses, 2014,2015, 2016, 2017 Invited Reviewer, ASSAY and Drug Development Technologies, 2014, 15,16, 17 Invited Reviewer, Journal of Molecular Medicine, 2018

Keywords

malaria, Plasmodium falciparum, aspartyl aminopeptidase, rPfM18AAP, parasite, exopeptidase,

1536 well, QFRET, gametocyte, High Throughput Screening (HTS)

14 Australian and New Zealand Standard Research Classifications (ANZSRC)

This research belongs with ANZSRC code: 060199, Biochemistry and Cell Biology not elsewhere

classified, 100%

Fields of Research (FoR) Classification

FoR code: 0601, Biochemistry and Cell Biology, 80% and 0699, Other Biological Sciences, 20%

15

Dedications:

This work is dedicated to my wife Cathy and family including Tara, Jordan and Anna. I also thank

Don Gardiner and Katharine Trenholme. Without their mentoring, their support, their patience and advice, this wouldn’t have begun nor gotten off the ground and certainly wouldn’t have been completed. Peter Hodder who hired me in the first place and subsequently made the connection to

Don Gardiner and Katharine Trenholme who then encouraged me to undertake this HDR with them. I thank Dr. Pat Griffin who serves as my department chair and is a leader that I have always looked up to. Finally a special thanks to Drs. Joe Kissil, Jonathan O’Connell and Chris Brown; the best thesis committee anyone could ever have.

16

Table of Contents Main Text of the Thesis ...... 20

Aims, Hypotheses and Objectives ...... 31

Thesis Chapters ...... 20

Chapter 1 –Introduction and Literature Review ...... 20

Chapter 2-Targeting rPfM18AAP ...... 33

Chapter 3-Mode of Actions for Inhibitors of rPfM18AAP ...... 49

Chapter 4-Targeting Gametocytes ...... 95

Chapter 5-Conclusions ...... 112

References ...... 116

Appendices ...... 124

Milestones Achieved ...... 124

(A) PfM18AAP Publication ...... 125

(B) Gametocyte Publication ...... 155

End ...... 194

List of Figures and Tables in order of where they are found within the thesis: Figure 1. A world map showing malaria endemic areas ...... 21

Figure 2. Stage progression of gametocytes in culture as viewed using giemsa stain and bright field microscopy...... 24

Figure 3. Life-cycle of the Plasmodium falciparum parasite ...... 25

Figure 4. rPfM18AAP enzymatic assay principle ...... 36

Figure 5. rPfM18AAP and CTSL1 primary HTS assay performance ...... 39

17 Figure 6. Lineweaver-Burk analysis of potent and selective inhibitors rPfM18APP ...... 41

Table 1. rPfM18AAP uHTS Campaign Summary and Results ...... 43

Figure 7. Comparison of rPfM18AAP and CTSL1 primary screening results ...... 45 Figure 8. Clustering results of potent rPfM18AAP hits ...... 47 Figure 9. The probe ML369 ...... 51 Table 2. List of all Assay Identifiers and Metadata Associated to Each Stage ...... 53 Figure 10. Critical Path for Primary Screening of rPfM18AAP ...... 56 Figure 11. rPfM18AAP primary HTS assay performance ...... 62 Table 3. Top 5 compounds of interest from the primary screening and hit validation studies. .... 66 Figure 12. Concentration-response Curves for the Probe (ML369) ...... 67 Table 4. Activity for CID 23724194, the starting point for SAR optimization ...... 70 Figure 13. SAR optimization divided the compound into three domains ...... 71 Table 5. Round 1 SAR and Changes to the Heterocycle Domain ...... 72 Table 6. Round 1 SAR and Changes to the Linker Domain ...... 73 Table 7. Round 1 SAR and Changes to the Putative Zinc-binding Domain ...... 74 Table 8. Round 1 SAR and Simultaneous Changes to the Heterocycle and Linker Domains ..... 75 Table 9. Round 1 SAR and Final Catechol-containing and Penultimate O,O-Dimethyl Catechol- containing Compounds ...... 77 Table 10. Round 2 SAR and Constrained N-Atom/Linker Analogues ...... 82 Table 11. Round 2 SAR and Final Catechol and Penultimate O,O-Dimethyl Catechol Compounds ...... 83 Table 12. Probe Candidates (Catechol Compounds Only) from Round 1 and 2 SAR ...... 85 Table 13. Comparison of the Observed Probe Properties to the Probe Criteria ...... 88 Figure 14. Reported Inhibitors of rPfM18AAP ...... 89 Figure 15. Example Future SAR Studies ...... 90 Figure 16. Lifecycle of Plasmodium falciparum ...... 97 Table 14. Stepwise Protocol for the 1536-Well Plate Live/Dead Gametocyte Assay ...... 105 Figure 17. Scatterplot of the data from the primary HTS campaign ...... 106 Table 15. Summary of the Ultra-HTS campaign to Identify Inhibitors of Late Stage Plasmodium falciparum Gametocytes ...... 107 Figure 18. Structure-activity relationship of the active molecules in the Gametocyte HTS ...... 108

18

List of Abbreviations Used in the Text: World Health Organization (WHO); Plasmodium (P. falciparum); Signal to Basal (S:B); PfM18 aspartyl aminopeptidase (PfM18AAP); recombinant PfM18AAP (rPfM18AAP); 7-amino-4-methylcoumarin (NHMec); L-Glutamic Acid 7-amino-4- methylcoumarin (H-Glu-NHMec); Compound Identifier (CID); Molecular Libraries Small Molecule Repository (MLSMR); Pan Assay Interference Compounds (PAINs); cathepsin L1 (CTSL1); Bovine Serum Albumin (BSA); Relative Fluorescence Units (RFU); unique Assay Identifier (AID); Lysotracker Red (LTR); PubMed Identifier (PMID); High Throughput Screening (HTS)

19 Main Text of the Thesis

Chapter 1-Introduction and Literature Review

Introduction:

According to the World Health Organization (WHO) up to 200 million people per year become infected with malaria resulting in an estimated 0.6 million fatalities. The young are primarily at risk with the majority of deaths from malaria occurring in those that are under five years of age and strikingly, in parts of Africa results in up to 15% of deaths for the same age range. Malaria accounts for roughly 1 out of 100 deaths worldwide even though it is typically only found in areas of tropical climate. While the rate of malaria transmission has leveled off and even slightly declined in the past few years in a regionally dependent manner, drugs and methods to block transmission are failing. Distribution and affordability of current therapies remains an obstacle due to the low socioeconomic status of regions were malaria is prevalent. Approximately 40% of the world’s populations live in areas where the risk of malaria transmission is high, typically the tropic zones located closer to the equator. In addition to the young, the most affected by malaria are elderly folks, pregnant women and foreign born travelers.

Currently there are only a limited number of drug choices for the treatment of malaria, the majority of which target the asexual blood stage parasite. These include chloroquine phosphate, atovaquone, mefloquine, quinine, quinidine, tetracycline, piperaquine, lumenfantrine, pyronaridine and clindamycin as designated by the World Health Organization. So there is a need for better more efficacious drugs that target either asexual or sexual stages of the parasite life cycle.

20

Literature Review:

Significance of Malaria

According to the World Health Organization (WHO) up to 200 million people per year become infected with malaria and this results in an estimated 0.6 million fatalities 1. The young are

primarily at risk with the majority of deaths from malaria occurring in those that are five years

Figure 1. A world map showing malaria endemic areas. Shading reflects the % of population at risk. Red=80-100%; Orange=60-80%; Yellow=40-60%; Blue=20-40%; Green=0-20%; Grey=Not applicable. Adapted from malariavacccine.org old or younger and strikingly, in parts of Africa, results in up to 15% of deaths for the same age

range. Malaria accounts for roughly 1 out of 100 deaths worldwide even though it is typically only

found in areas of tropical climate 16. While the rate of malaria transmission has leveled off and

even slightly declined in recent years in a regionally dependent manner, drugs and methods to

block transmission are failing17,18. Distribution and affordability of current therapies remains an obstacle due to the low socioeconomic status of regions were malaria is prevalent. Approximately

40% of the world’s populations live in areas where the risk of malaria transmission is high,

21 typically the tropic zones located closer to the equator (Figure 1.). In addition to the young, the

most affected by malaria are elderly folks, pregnant women and foreign born travelers.

Malaria is a very ancient disease and has troubled tropical civilizations throughout history. The

symptoms of malaria have a rapid onset with recurrent cycles of fever and chills, muscle aches,

headaches, vomiting and jaundice but, may be extended to 8-10 months after the initial infected

mosquito bite occurs 19.

It was not until the late 1800’s when the underlying cause of malaria was discovered 20. Until then

most believed malaria came from foul smelling swampy areas 21 22. In 1878 Dr. Alphonse Laveran,

a French physician, identified the causative agent of malaria. Initially, malaria was believed to be

caused by a bacterium 20. Laveran was the first to observe what we now call gametocytes, the

crescent shaped bodies, in the blood of a patient infected with Plasmodium falciparum 23. While

initial uptake of his discovery took some time, eventually Laveran was awarded the Nobel Prize

for Medicine in 1907. Following this discovery, in the late 1890’s Ronald Ross, while studying

avian malaria, identified the mosquito as the vector of P. falciparum for which he was also awarded the Nobel Prize.

Global distribution of Plasmodium

Plasmodium, a parasitic protozoon, has a complex life cycle involving both a mosquito vector and vertebrate host (Figure 3). There are six species that infect humans including P. falciparum, P. vivax, P. ovale(P. curtisi and P. wallikeri), P. knowlesi and P. malariae 24. There are many other

species which infect non-human primates and interspecies transmission is evidently possible25. Of

the six species, all but P. falciparum and P. vivax are relatively benign upon infection and of the

two highly pathogenic species P. falciparum is responsible for the highest mortality rates.

Importantly, the parasite lifecycle relies on the female mosquito which predicates it to be near

22 water and in the tropics or subtropics as mosquitoes require water for reproduction and cannot

survive cold temperatures. Temperature is also an important factor in the development rate of the

parasite extrinsically and is a key driver of vector (mosquito) development, bite rate, and survival

within the mosquito all of which are decreased dramatically in colder climate26. Hence the major areas of infectious malaria are in an around the tropics. P. vivax is an outlier in this case as it has genetically adapted to survive in lower temperature environments and can be found outside the tropic and subtropical regions27.

Life cycle of Plasmodium falciparum

The life cycle starts essentially at the point when a susceptible host is bitten by an infected

female Anopheles mosquito. At that point the host is inoculated with sporozoites (Figure 3) which, are highly motile and quickly enter the host blood stream ultimately arriving at the

liver. Here, the sporozoites invade the hepatocytes and, over the course of 5-21 days, mature

to form exoerythrocytic schizonts each containing ~10,000 merozoites28. When mature the

schizont ruptures and the merozoites are released into the blood stream where they invade

erythrocytes. During this intra-erythrocytic stage they undergo a cycle of asexual

multiplication where each merozoite produces up to 20 new merozoites every two- three

days. The rupture of red blood cells and merozoite release is associated with the characteristic fever and chill symptoms of malaria29. This multiplication results in increased

parasitaemia within the host which ultimately cause the pathogenic response elicited

including fever, chills, vomiting, diarrhea, arrhythmia as well as multi-organ damage

including renal failure 30.

Gametocytes are the only stage of the parasite able to mediate transmission from the host to the

mosquito vector. The trigger for sexual development of gametocytogenesis is unclear but a small

23 population of parasites invades red blood cells and switch to a pathway of sexual development. In

the case of P. falciparum gametocytes take 10 – 12 days to become mature and once they are

mature can survive in an infected individuals blood stream for a period of time that isn’t precisely

known but spans at least 7-14 days and may persist for weeks; even up to 50 days 31, 32.

P. falciparum gametocytes go through five distinct stages ( I-V) as they mature, based on light and electron microscopy (Figure2 ) Mature stage V gametocytes have a characteristic crescent shape and only mature gametocytes are infectious to the mosquito vector 33, 34.

When mature gametocytes are taken up by a female Anopheles mosquito, they are released from

the red blood cell and undergo gametogenesis and the haploid gametes fuse to become diploid

completing the sexual stage of the parasite's lifecycle. Over the course of 10-18 days the parasite

goes through a number of development stages and maturation steps, resulting in the production of

infectious haploid sporozoites that are ready for transmission to a new host when the mosquito

next takes a blood meal.

Stage I Stage II Stage III Stage IV Stage V Stage V

Figure 2. Stage progression of gametocytes in culture as viewed using Giemsa stain and bright field microscopy. Courtesy of Alice Butterworth.

24

Figure 3. Life-cycle of the Plasmodium falciparum parasite, the etiological agent of human malaria. There are multiple stages of malaria infection as described in the text above. Notably the reproduction stage and transmission of the parasite from host to host involves the transfer of mature sexually differentiated gametocytes into the mosquito.

Current Treatments

Currently there are only a handful of drug choices for the treatment of malaria, the majority of which target the asexual blood stage parasite. As mentioned these include chloroquine phosphate, atovaquone, mefloquine, quinine, quinidine, tetracycline, piperaquine, lumenfantrine, pyronaridine and clindamycin as designated by the World Health Organization 35, 35b. In addition combination therapy is now more common and includes piperaquine, lumenfantrine and pyronaridine which are all are currently in use as fixed combination partners with artemisinin compounds. Optimally one would design a drug that defeats the malaria parasite resistance and

25 renders it incapable of further transmission. During the sexual stage of the parasite life cycle the

gametocytes are transferred from host to vector where sexual reproduction and maturation renders the parasite infectious to the next round of hosts. So targeting via anti-gametocidal development,

while likely having very little effect to reduce the parasite burden to the affected patient, may

potentially prevent transmission.

However, many genetic and metabolic changes occur during gametocyte maturation and while these are not fully understood it is clear that these impact on drug efficacy. Targeting mature

gametocytes is problematic as they are refractory to most currently available antimalarial drugs 36,

37.

Primaquine is the only currently available antimalarial drug that is effective against stage V gametocytes, but there are problems associated with its safety in some populations. In particular, patients afflicted with Glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency) are already predisposed to spontaneous destruction of red blood cells. While this can be a genetic trait that protects some people from malaria infection, those that do get infected cannot take primaquine as it is known to cause haemolysis in these patients thereby acutely exasperating the condition.

Tafenoquine is less toxic but is still only clinically useful for G6PD patients at a lower dose again justifying the need for better anti-gametocidal drugs38.

Parasite resistance to current drugs

One of the clearest reasons to develop novel drugs is the emergence of parasite resistance to the current regimen of anti-malarial drugs. Genetic pressure on the parasite to evolve resistance to currently available drugs, aided in part by inappropriate therapeutic intervention, has created resistance to virtually all drugs and sadly now including the artimenisins5. Recently the outcomes

of the RTS,S phase 3 clinical trials for treatment with a malaria vaccine were published and

26 generally, efficacy was found in ~30% of the cases but, only in older infants when given multiple

boosters 39. However, a truly effective vaccine is not expected to be available for another decade

40.

Importantly, drug resistance is defined as a right shift in the effective concentration response

required for blocking malaria replication41. More directly, patients with increasing resistance

would result in less inhibition of parasite multiplication and a concomitant decrease in the

effectiveness of the drugs. Even further, patients with increasing resistance would result in less

inhibition of parasite multiplication and a concomitant decrease in the effectiveness of the drugs.

Rarely is there a direct correlation of in vivo to in vitro resistance which probably only exists in

the case of chloroquine.42 In other cases testing artemisinin-resistance with quinine co-resistance emerging in P. falciparum malaria under in vivo artesunate pressure resulted in controversial experimental resistance which evolved in a rodent model system, the genetic correlates of which are not understood 43. While this complicates current use of prescribed therapies it also

leaves opening to more easily test and determine if small molecule therapies maybe effective by

testing in vitro. New leads can thus be expected to affect the target parasite so long as they are

clinically safe for humans.

Targets for Molecular Intervention

PfM18AAP (protein, structure, localization and function)

So there is a need for better more efficacious drugs that target either asexual or sexual stages of the parasite life cycle. Some options are to target enzymes or proteins particular to only malaria

parasites. One such target is the aspartal aminopeptidase enzyme classified as M18, or PfM18AAP.

The PfM18AAP protease was previously identified as a potentially druggable therapeutic target by Trenholme et al and prior to this effort there were no known small-molecule

27 inhibitors of PfM18AAP. Previous reports have identified the phosphinic and phosphonic

acid analogues of glutamate and aspartate, GluP and AspP, as modest amino acid–derived

inhibitors of PfM18AAP in vitro. As such experimental confirmation of its potential as a drug

target has yet to be shown.

There are at least eight aminopeptidases expressed by the clinically significant malaria parasites7.

The specific role of malaria aminopeptidases are to cleave N-termini amino acids of short peptides

as part of the final stages of hemoglobin digestion in the parasite lifecycle; thought to create a

reservoir or nutrient pool of amino acids necessary for parasite biogenesis44. The amino acid

substrate used varies from methionine to leucine to aspartate. Some aminopeptidases have already

been targeted for drug intervention; in particular the methionine aminopeptidases45, so headway

has been made to identify inhibitors of these further substantiating them as druggable targets.

PfM18AAP protein is expressed in all phases of the intra-erythrocytic stage of the parasite life

cycle but, more so during the ring stage13. It has been demonstrated as a functional protease

obtained from the parasitic cytosol and has been shown to be exported to the parasitophorous

vacuole indicating it may also have a role in addition to Hb degradation13. Finally, Teuscher et al.

2007, also demonstrated direct inhibition of recombinant M18AAP and parasite cytosolic M18

protease in biochemical assays and using antisense inhibitors.

Targeting the asexual and gametocyte stages

New target strategies have been recently elucidated using novel modeling techniques, which predict four targets that have never been taken into account for anti-malarial drug design. These include modulating merozoite invasion of RBCs, increasing their transformation into gametocytes, decreasing the immune response to gametocytes, or a combined diminishment of the immune response while decreasing the recycling rate of the red blood cells46,47 .

28 The point of initiation for sexual differentiation starts at an early stage of asexual development;

likely the ring or trophozoite stage. To elucidate further, during the erythrocytic ring stage, trophozoites mature into schizonts which eventually rupture releasing merozoites, each of those now maturing into a gametocyte. The trigger for the parasite to undergo sexual differentiation to form gametocytes is still under investigation but appears to be controlled by a transcriptional switch that may be regulated by a DNA binding protein called PfAP2-G48. Commitment to

sexually differentiated gametes is essential for the maintenance of the parasite lifecycle which is

predicated by the uptake of such by the mosquito vector. New drugs or molecular probes, i.e. a

‘tool’ used to provide data or report on a biological or chemical target, usually within a biological

or chemical assay, that either effectively and specifically kill gametocytes or block their sexual

maturation would ultimately halt the parasites ability to replicate. So anti-gametocidals are indeed

a viable path for drug discovery 49.

Drug development pipelines

The last two decades have spurned much in the way of drug discovery targeting multiple stages of

malaria which have been supported in large part by the NIH and the Gates foundation. Still with

resistance to the standard of care drugs, i.e. of the artesunate and quinine variety, arising in

Africa there is an urgent need to fill the pipeline of drugs facilitating new treatments. To that end

there appears to be about 13 drugs in clinical development almost all of which are targeting the

schizont stage of uncomplicated malaria.50,51 Note that 9 of these are in phase 2 development as

of last year with a predicted chance of ~30-40% of making through the full regulatory approval.

Ideally new treatments would be potent, fast acting, cost effective and present a broad safety profile

for use even in children and pregnant women. In addition they would represent a high barrier to

developing resistance. To this end researchers and pharma have been developing inhibitors that

29 act at various points of the parasite replication life cycle with an eye towards using them in

combination with other existing or novel drugs. Briefly, a partial list of novel drugs, their target

and point of inhibition are: 1) DSM265 from Takeda, Dihydroorotate dehydrogenase inhibitor,

schizonticide 2) AQ-13, Immtech, modified 4-aminoquinoline, schizonticide 3) Methylene Blue

by University of Heidelberg, glutathione reductase, schizonticide and gametocidal 4) Sevuparin

(DF02) from Dilaforette, anti-adhesive, merozoite invasion 5) MMV 390048 from MMV and

University of Cape Town, K inhibitor, schizonticide 6) P218 from MMV and Jannsen, DHFR,

schizonticide 7) CDRI 97/78 from IPCA, trioxane, schizonticide 8) M5717 from MMV-Merck and

University of Dundee, blocks protein synthesis by inhibiting EF2, multi-stage 9) SJ733 from

MMV-University of Kentucky and Eisai, ATP4, schizonticide 10) ACT-451840 from Actelion, unknown mechanism of action, schizonticide.52

Challenges with currently available drug screening methods

So as you can see from the above the current status of drug discovery for antimalarials is in a decent state yet challenges in how we find new inhibitors are profound as well. For example, most drug libraries have been trending towards identifying compounds with blood-stage activity but more recently there has been progress in the discovery of agents capable of preventing transmission using dedicated screening technologies such as those described in my thesis which specifically target gametogenesis. Other more universal challenges towards screening and drug discovery are also applicable here such as the high cost of scaling and performing large high throughput screening (HTS) efforts, the scalability and availability of reagents, the risk of not finding any inhibitors and generally the post HTS chemistry initiatives being lengthy and also costly.

30 Aims, hypotheses and objectives

Two strategies of particular interest that are a focus of this thesis are to use a rational protein target based approach to screen the PfM18AAP and a more empirical approach to screen for inhibitors of mature late-stage gametocytes. These targets should be druggable from a small molecule perspective and represent a chance to determine if HTS is a viable path to find appropriate inhibitors that may become leads.

The primary aims of my research are:

1. To adapt, implement and develop the currently available low throughput techniques of the

current assays targeting rPfM18AAP and mature late-stage gametocytes into robust HTS

amenable assays.

2. To perform large scale compound screening on divesified molecular collections against the

targets of aim 1.

3. To complete the evaluation of the outcomes of aim 2 via concentration response analysis

of the most promising HTS hits including testing in post HTS specificity assays.

4. To take lead like molecule through early medicinal chemistry efforts and develop novel

molecular probes.

My research hypotheses are:

1. Targeting PfM18AAP for drug discovery, an aspartyl protease involved in the asexual

erythrocytic stage of the malaria lifecycle which is critical for parasite replication will be

done using good HTS practices, assay implementation and miniaturization. This will lead

to high throughput screening that will identify potent and tractable hits thereby proving

rPfM18AAP can be targeted for small molecule drug discovery. These hits will be further

pursued and characterized in terms of specificity towards the target and chemically

31 modified such that, if successful, a molecular probe will be elucidated.

2. Assay development to allow for phenotypic screening of whole cell late stage gametocytes,

which are essential for sexual reproduction of the malaria parasite within the mosquito

vector, will be scalable to 1536 well HTS. Completion of a large scale HTS campaign will

identify reproducible hits thereby validating this approach as a viable method for small

molecule drug discovery.

So in line with my aims and hypotheses, this project is divided into 2 different approaches to define or uncover new drugs active against either PfM18AAP or late stage gametocytes:

1. Develop, optimize, miniaturize and screen and identify small molecule inhibitors the

specifically effect the activity of PfM18AAP, an essential protein in the malaria life cycle.

2. Develop, optimize, miniaturize and screen and to identify specific inhibitors against P.

falciparum gametocytes.

The first approach is investigated in Chapter 2 which describes the rational target based approach to identify compounds that inhibit the activity of PfM18AAP by high-throughput screening (HTS) of compound libraries and which yielded several series of potent compounds that demonstrate selectivity to the enzyme. Chapter 3 covers the subsequent effort of probe development of small molecules for the PfM18AAP initiative that includes mechanism of actions studies aimed specifically at this target. Chapter 4 summarizes a feasibility study and HTS using more emperical phenotypic approach incorporating a live dead assay in high density microtiter plates for the purpose of arresting transmission of Plasmodium falciparum via inhibition of gametocytes.

32 Chapter 2

Targeting the Plasmodium falciparum protease –PfM18AAP

Chapter 2 is directly derived from my manuscript demonstrating how I found compounds that inhibit the activity of rPfM18AAP by high-throughput screening.

Please see Spicer, et.al.. Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High Throughput

Screening. J Biomol Screen. 2014 Mar 11. PMID: 24619116. PMC: 4641816. (Appendix 1).

My contribution to the authorship includes a substantive contribution to the following activities:

1) conception and design of the project as well as execution of the experiments;

2) analysis and interpretation of the research data on which the publication is based;

3) drafting the publication as well as completing its submission as the communicating author.

Summary:

There are several potentially vulnerable points in the parasite lifecycle particularly during the

blood stage as invasion of erythrocytes and subsequent parasite growth and development is critical

for parasite survival. It is during this stage that P. falciparum uses protein-protein interactions and

enzymatically driven processes to allow entry into, growth within, and escape from the human

erythrocyte. One such enzyme is PfM18AAP, a ~67-kDa metallo-aminoipeptidase that

oligomerizes and is the sole aspartyl aminopeptidase (AAP) present in the malaria parasite 8.

PfM18AAP is thought to play an important role in the process of protein degradation and, working

together with other exo-aminopeptidases, is believed to digest host hemoglobin, an important

33 source of amino acids for the parasite 53, 54. PfM18AAP localises to both the parasite and the

parasitophorus vacuole, suggesting that it may play a role in addition to haemoglobin degradation.

Data from several laboratories suggest that the enzyme could also perform additional functions in

the parasitophorus vacuole, the erythrocyte cytosol and at the infected erythrocyte membrane

skeleton.PfM18AAP may also interact with the erythrocyte membrane protein, spectrin,

presumably to regulate the integrity of the infected erythrocyte membrane skeleton during parasite growth, allowing for host cell expansion and parasite exit 55.

Therapeutic approach to PfM18AAP inhibitors

Antisense RNA knockdown experiments have shown that inhibition of intraerythrocytic

PfM18AAP debilitates the malaria parasite, making it a promising drug target13. Inhibitors of

PfM18AAP will thus provide new tools to further our understanding of this enzyme’s function and

increase the potential to develop therapies against P. falciparum transmission. Currently, there are

no known small-molecule inhibitors of PfM18AAP. Previous reports have identified the

phosphinic and phosphonic acid analogues of glutamate and aspartate, GluP and AspP, as modest

amino acid–derived inhibitors of PfM18AAP in vitro. However, these amino acid derivatives do

not reduce malaria growth in culture when tested at concentrations up to 100 uM 13. Thus, the

objective of this research program was to identify compounds that inhibit the activity of

rPfM18AAP. Described here is my approach to discovering such inhibitors by high-throughput

screening (HTS) of compound libraries, which yielded several series of potent compounds that,

demonstrate selectivity to the enzyme. We identified two compounds that are noncompetitive

inhibitors of rPfM18AAP and also inhibit the growth of P. falciparum in vitro.

Methodology

34 Functionally active purified recombinant PfM18AAP was prepared at the University of

Technology Sydney as previously described13. The Molecular Libraries Small Molecule

Repository (MLSMR) library was provided by the National Institutes of Health’s Molecular

Libraries Initiative. Details regarding compound selection for this library can be found online

(http://mli.nih.gov/mli/compound-repository/mlsmrcompounds/).

Briefly, the MLSMR library is a highly diversified collection of small molecules (more than 50%

of compounds exhibit molecular weights between 350 and 410 g/mol) comprising both synthetic

and natural products, from either commercial or academic sources, that can be grouped into the

three following categories: specialty sets of know bioactive compounds such as drugs and toxins,

focused libraries aimed at specific target classes, and diversity sets covering a large area of

chemical space 56. The enzymatic assay was originally developed by Dalton & Gardiner in a low

throughput format and uses a fluorogenic peptide substrate (H-Glu-NHMec), which is incubated

with purified recombinant PfM18AAP in the presence of test compounds. The assay was

subsequently transferred to me, at Scripps Florida, was first recapitulated and subsequently

implemented and optimized for 1536 well ultra-high throughput screening. In this assay, cleavage

of the substrate by rPfM18AAP liberates the 7-amino-4-methylcoumarin fluorogenic leaving

group (NHMec) from the peptide, leading to increased fluorescence (Figure 4). Enzymatic inhibitors block rPfM18AAP mediated cleavage of H-Glu-NHMec and liberation of the NHMec leaving group from the substrate, resulting in decreased fluorescence as measured at 340 nm excitation and 450 nm emission. To reduce the number of compounds that optically interfere with the measurement, initial (T0) and 90-min (T90) measurements of plate fluorescence were taken

after addition of substrate. Test compounds were assayed in singlicate at a final nominal

concentration of 7uM. A stepwise assay protocol is presented in Supplemental Table S1. Further

35 details of this assay can be found at the PubChem AID 1822

(http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1822).

340nm Excitation 340nm Excitation 450nm Emmision

Low fluorescence

Me Me

O

H2N N O O H N O O H 2 7-amino-4- methylcoumarin (NHMec)

OH O L-Glutamic acid 7-amino-4-methylcoumarin (H-Glu-NHMec)

Figure 4: rPfM18AAP enzymatic assay principle. rPfM18AAP activity is determined by measuring the release of the 7-amino-4-methylcoumarin fluorogenic group (NHMec) from a peptide substrate (H-Glu-NHMec). Represented by the wavy line, cleavage of H-Glu-NHMec’s glutamic acid group by PfM18AAP liberates NHMec from the peptide, leading to increased well fluorescence. As designed the screening assay identifies compounds that inhibit rPfM18AAP- mediated cleavage, resulting in decreased well fluorescence.

rPfM18AAP HTS Campaign

Following the miniaturization and optimization of the assay for 1536 well HTS, the first stage involved robotic validation and stability testing on a fully automated HTS platform. This was followed by singlicate screening against rPfM18AAP, the primary screen, and Cathepsin L1

36 (CTSL1) the primary counterscreen, at a final compound test concentration of 7 uM or 6 uM,

respectively (see PubChem AIDs 1822 and 1906 for protocol details). The counterscreen

incorporated for this assay used Cathepsin L1 which ultimately if inhibited may lead to host cell

toxicity. Like the HTS for rPfM18AAP, this assay also exploits fluorogenic enzyme substrates.

The final DMSO concentration was 0.7% (v/v) for the rPfM18AAP assay or 0.6% for the CTSL1

assay. Well fluorescence was measured with a ViewLux plate reader (PerkinElmer, Waltham,

MA), and the percent inhibition of each test compound was calculated on a per-plate basis as

further described below. The numerical cutoff used to qualify active (“hit”) compounds was

calculated as the average percentage inhibition of all compounds tested plus three times their

standard deviation57. The confirmation and counterscreens were run on selected hits in the same

conditions as the primary screens, except that plates were assessed in triplicate and results for each

compound were reported as the average percentage inhibition of the three measurements, plus or

minus the associated standard deviation (PubChem AIDs 2170 and 2178). For titration

experiments, assay protocols were identical to those described above, with the exception that

compounds were prepared in 10-point, 1:3 serial dilutions starting at a nominal test concentration

of 74 uM and assessed in triplicate (PubChem AIDs 2195 and 2196). To confirm activity of the

best inhibitors, compounds were purchased as powder samples and tested in various secondary

assays, including Ki determination, a malaria cell lysate assay and a P. falciparum parasite growth assay.

Results

The rPfM18AAP primary assay and CTSL1 counterscreen assay were implemented at a final volume of 5 microliters per well in 1536-well plates. The assays were screened against the entire

MLSMR collection; 277,728 unique compounds were tested in both assays. All compounds were

37 screened at 7 uM for rPfM18AAP and 6 uM for CTSL1. For the rPfM18AAP screening assay, an

IC100 of Zn(II) was used as a positive control for enzyme inhibition. Zinc has previously been shown to sufficiently suppress the enzyme activity here. The median of the wells containing test compounds was used as a negative control (i.e., IC0) where as the negative control wells contained enzyme and substrate along with vehicle only. Using these controls (Figure 5 ), the rPfM18AAP assay demonstrated robust screening statistics. It had an average signal-to-background ratio (S/B) of 3.42 +/- 0.89 and a Z′ of 0.84 +/- 0.04 (n = 241 plates)

38 Figure 5 : rPfM18AAP (left panel) and CTSL1 (right panel) primary HTS assay performance. Positive control wells are shown as inverted green triangles (IC100 of ZnCl2 or Z-Phe-Ala-diazomethylketone). Results of compound wells (black triangles) and negative control wells (white squares) are also graphed. Calculated hit-cutoffs are indicated via dashed lines. Due to the high degree of compound activity found in the PfM18AAP assay, data for both HTS campaigns was normalized to the median of the compound wells (IC0) and the median of the respective IC100. Hence, the noticeable shift below 0% inhibition for rPfM18AAP assay.

The CTSL1 assay was similarly robust; using Z-Phe-Ala diazomethylketone and the median of the wells containing test compounds as

positive and negative controls, respectively, yielded an S/B of 3.61 +/- 0.26 and a Z′ of 0.88 +/- 0.04 over 247 plates. Any compound

found active against rPfM18AAP but not active in the CTSL1 assay was prioritized for follow-up in the next round of testing. To further

39 enrich the data set for downstream follow-up studies, fresh aliquots of 2378 hit compounds demonstrating selective inhibition of rPfM18AAP were tested in triplicate against both assays. To confirm activity and selectivity, the primary HTS hit activity cutoffs were reapplied to each set of screening results. This yielded 125 compounds that confirmed selective activity against rPfM18AAP (i.e., their measured %inhibition was above the rPfM18AAP primary HTS activity cutoff and less than the CTSL1 counterscreen HTS activity cutoff). Fresh aliquots of these compounds were obtained for further characterization in titration (IC50) assays. All compounds

yielded IC50 values of <10 uM in the primary assay and >10 uM in the counterscreen assay were

considered active. From the 125 compounds that demonstrated potent and selective inhibition of

rPfM18AAP, a subset of compounds most tractable for structure-activity relationship (SAR) studies was purchased or resynthesized as powder samples. These powder samples were retested to confirm inhibition of purified recombinant PfM18AAP, as well as in malaria lysates, and also tested for their ability to inhibit P. falciparum parasite growth via 3H hypoxanthine incorporation.

Two efficacious compounds were identified from this effort: CID 6852389, (S)-(+)-apomorphine hydrochloride hydrate, and CID 23724194, the hydrochloride salt of 4-[2-(acridin-9-

ylamino)ethyl]benzene-1,2-diol. CID 6852389 was found active in the P. falciparum lysate assay

(84% efficacy at ≤5 uM; CID 23724194 was unavailable for testing). In the 3H hypoxanthine

incorporation assay, CID 6852389 and CID 23724194 yielded 87% inhibition and 96% inhibition

at 10 uM test concentrations, respectively. All compounds that inhibited parasite growth by >50%

were titrated and retested as concentration-response curves in the same experiment. CID 6852389

and CID 23724194 had the highest potency in these assays, with IC50 values of 4 uM and 1.3 uM,

respectively. CID 6852389 and CID 23724194 were also assayed enzymatically to assess their

mode of inhibition. Results of nonlinear regression analysis for Ki values were 3.39 +/- 0.36 uM

40 for CID 6852389 and 1.35 +/- 0.15 uM for CID 23724194. Mode of inhibition was determined by software-based comparison of fits method using competitive, noncompetitive, uncompetitive, and mixed models of inhibition. The preferred fit was the noncompetitive model of inhibition in all cases. In addition, the mode of inhibition was confirmed by steady-state velocity plots, semilog

scale plots, and Lineweaver-Burke plots. As determined by these methods, both behave as

noncompetitive inhibitors of rPfM18AAP (Figure 6).

CID 6852389 CID 23724194 20000 15000 0 µM 0 µM 1 µM 1 µM 15000 µ 5 M 10000 5 µM 10 µM 10 µM 10000 50 µM 50 µM Velocity Velocity 5000 5000

0 0 0 200 400 600 0 200 400 600 [S], µM [S], µM

20000 15000 0 µM 0 µM 1 µM 1 µM 15000 5 µM 10000 5 µM 10 µM 10 µM 10000 50 µM 50 µM Velocity Velocity 5000 5000

0 0 1 10 100 1000 1 10 100 1000 µ [Substrate], µM [Substrate], M 0.12 0.4 0 µM 0 uM 1 µM 1 uM 0.09 0.3 5 µM 5 uM 10 µM 10 uM 0.2 0.06 1/v 1/v 50 µM 50 uM

0.03 0.1

0.00 0.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 1/[S] 1/[S]

Figure 6: Determination of modes of inhibitor interaction using untransformed data analysis (first set of panels), semilog scale analysis (2nd set of panels) and Lineweaver-Burk analysis (bottom set

41 of panels) of potent and selective inhibitors rPfM18APP. Both compounds exhibit non- competitive inhibition.

Results from previous primary screening assays were used to better understand possible off-target

activities and liabilities of CID 6852389 and CID 23724194. When tested in similarly formatted

enzymatic inhibition assays run at the Scripps Research Institute Molecular Screening Center

(SRIMSC), these compounds were devoid of activity (cf. PubChem AIDs 1859, 1931, 651959).

Similarly, where tested, they were also inactive in mammalian cytotoxicity (cf. PubChem AIDs

1486, 1825) and bacterial viability (cf. PubChem AIDs 449731, 651606) screens, as well >100

other primary screens run at the screening center. Remarkably, both compounds were found to be

inactive in all cell-based screens tested at the SRIMSC, except for two serotonin receptor screens

(cf. PubChem AIDs 612 and 504916). In summary, in silico evaluation of the activity of CID

6852389 and CID 23724194 in a variety of cell-based and biochemical primary screening assays,

including screens that used similar detection methodologies to the rPfM18AAP primary assay,

indicated that the activity of these two compounds was specific to rPfM18AAP.

Described here is my effort to identify small-molecule inhibitors of rPfM18AAP via an HTS approach. As formatted, both the rPfM18AAP primary screen and the CTSL1 counterscreen were

highly amenable to automated screening in 1536-well microtiter plates, with a throughput of

~20,000 compounds tested per hour. As indicated by Z′ and positive control IC50 values, excellent assay windows and consistent pharmacology were demonstrated throughout the HTS effort, which enabled facile identification of selective rPfM18AAP inhibitors. This screening approach identified 125 hit compounds with potent, selective inhibition of rPfM18AAP. Two of these compounds, CID 6852389 and CID 23724194, were found to be noncompetitive inhibitors of rPfM18AAP, with low-uM Ki values. As described previously58, I applied a “parallel” screening

approach for the rPfM18AAP HTS, where the CTSL1 counterscreen was used to triage hits at each

42 stage of the rPfM18AAP campaign (Table 1). This was implemented since both the PfM18AAP and CTSL1 assays shared similar assay protocols, including the use of the same coumarin-based fluorophore for measurement of activity. CTSL1, a cysteine metalloproteinase, would be expected to be affected by divalent metal ions such as zinc or chelators of such. Divalent metal ions were not added as part of the reaction mix during the counterscreen assay, so identifying chelators is less likely, but similar in principle to rPfM18AAP, compounds that may coordinate metal atoms would be expected to affect the activity of CTSL1 and may be identified via this counterscreen.

Table 1: rPfM18AAP uHTS Campaign Summary and Results.

Step Screen type Target Number of Selection Number of PubChem Assay statistics compounds criteria compounds AID Z’ S/B tested selected 1 Primary PfM18AAP 291,944 >28.03% 3,522 1822 0.84 ± 0.04 3.42 ± 0.89 1 Primary CTSL1 302,759 inhibition 1,481 1906 0.88 ± 0.04 3.61 ± 0.26 Counterscreen >16.63% inhibition 2 Confirmation PfM18AAP 2,378 >28.03% 661 2170 0.87 ± 0.03 1.57 ± 0.04 inhibition 3 Counterscreen CTSL1 2,378 >16.63% 7 2178 0.79 ± 0.01 3.53 ± 0.20 inhibition 4 Titrations PfM18AAP 125 IC50<10µM 125 2195 0.88 ± 0.02 2.03 ± 0.05

CTSL1 125 IC50<10µM 0 2196 0.84 ± 0.03 4.12 ± 0.04 5 Powder testing PfM18AAP 76 >50% 22 492974 NA NA inhibition

Malaria Cell 60 >50% 28 492975 NA NA Lysate inhibition

6 Follow-up Parasite 76 >50% 5 489015 NA NA Growth @ inhibition 10µM Parasite 5 IC50<5uM 2 489011 NA NA Growth IC50

*NA=Not Applicable

To prioritize viable rPfM18AAP inhibitors, compounds that were active in both assays were triaged with the assumption that they optically interfered with the measurement of fluorescence in microtiter plates or were promiscuous inhibitors. The validity of this assumption is partially supported by the results of the in silico analysis of CID 6852389 and CID 23724194; these

43 compounds were inactive not only in the CTSL1 counterscreen but also in all other similarly

formatted coumarin based enzymatic assays run in the screening laboratory. At the stage of

potency assays, recapitulating this parallel approach enabled facile identification of selective

rPfM18AAP inhibitors in the HTS data sets, allowing prioritization of compounds for labor-

intensive mechanistic assays. Following the HTS stage (i.e., step 4 in Table 1), 125 compounds

remained that appeared to be selective inhibitors. At this stage, a chemical triage was applied and

76 compounds were procured for testing in the lysate and parasite in vitro growth assay. Not all

compounds were available in sufficient quantity for testing in both assays, in which case preference

for usage was given to the parasite growth assay. Compounds that were active in both the parasite growth assay and the lysate assay (if available), were progressed for IC50 determination in the

parasite growth assay. Of those five compounds, 6852389 and CID 23724194 were found to be

the most potent. Although the CTSL1 results were used to identify selective inhibitors for the rPfM18AAP HTS effort, it is important to note several MLSMR compounds also selectively inhibited CTSL1 (see Figure 7). Alternatively, it is reasonable to assume that the criteria for prioritizing viable leads in the HTS effort may have removed genuine rPfM18AAP inhibitors.

44 CID 6852389 CID 23724194

OH

OH

HN

N % inhibition in CTSL1 inhibitionscreen % in CTSL1

% inhibition in PfM18AAP screen

Figure 7: Comparison of rPfM18AAP and CTSL1 primary screening results. The results of 277,728 compounds (grey circles) are shown. The horizontal dashed line represents the CTSL1 activity cutoff while the vertical dashed line represents the rPfM18AAP activity cut-off. Green circles represent the two “hit” compounds that were identified as part of the primary HTS. Arrows indicate the screening results for CID 6852389 and CID 23724194.

For this reason, all results for the rPfM18AAP and CTSL1 screening assays have been placed in a publicly available database, PubChem (https://pubchem.ncbi.nlm.nih.gov/), which enables the reexamination and identification of compounds with apparent activity in either target’s screening assay. Cheminformatic analysis of the 125 hit compounds provides insight on the possible pharmacology of the hits identified from the HTS effort. These hits were subjected to analysis using the maximum common substructure hierarchical method of cluster analysis which I used to generate Figure 8. This helped define molecules that are more appropriate for further pursuit in mechanism of action studies that will be discussed and described in Chapter 3. As represented by the cluster 9, catechols and catecholamines are well-known stimulants 59.

45 Derivatives of a 1,2,4 triazol-4-yl urea, found in cluster 3, have been shown to possess anti-

inflammatory and antibacterial activities 60. Cluster 6 and its 1,2,4 triazole derivatives have been

associated with a wider range of pharmacology, including anti-inflammatory, antibacterial,

antifungal, anticancer, analgesic, and antidepressant 61. Finally, cluster 1 and its benzamide scaffold have derivatives with antagonistic activities against various receptors such as dopamine

D2, 5-HT2, and 5-HT362. Both CID 6852389 and CID 23724194 are nominally members of the

“catechol” cluster 9. While catechols, in general, have been described as pan assay interference

compounds (PAINS),63 there is contradicting evidence that this is the case here in which we search

our entire database and found no activity in any of the >100 assays tested in our laboratory. Initial

analysis of the catechols found here demonstrates a relatively flat SAR, which really only obviates

that the catechol is important for activity. Figure 8 also demonstrates that most compounds from the 125 tested for IC50 associate into cluster 9, so clearly the catechol component must be important

for activity. In addition, the nearly identical activity of CID 2215 and CID 6852389 clearly

demonstrates that similar structures have reproducible activity, yet stereoselectivity cannot be fully

ascertained due to lack of informative compound registration.

46

Cluster #9 Cluster #3 Cluster #6 Cluster #1

33 14 10 9

3 26 2 2 2 5 7 2 2 6 3 2 4

Figure 8: Clustering results of potent rPfM18AAP hits. Presented are the most common substructure, and respective second tier derivatives for the four most populous clusters. Cluster #9 (33 compounds total) is represented by a 1,2 catechol moiety; Cluster #3 (14 total) is represented by a 1,2,4 triazol-4-yl-urea moiety; Cluster #6 (10 total) is represented by a (1,2,4 triazol-4-yl) acetamide moiety; and Cluster #1 (9 total) is represented by a benzamide moiety. CID 6852389 and CID 23724194 both belong to Cluster #9.

47

While these observations do not clarify whether the biological activity was simply due to

“interference” (in other words, the catechol compounds acting as PAINS or that the catechol

compounds might be coordinating the metal atoms that the enzyme requires to function, thus

inhibiting the enzyme), we do know that rPfM18AAP reacts differently to various different metal

ions. The enzyme is inhibited by zinc (>1 mM), which was employed as a positive control in

screening assays but conversely is enhanced by cobalt and manganese. Therefore, the relationship

of metal ion binding is complex64. While the catechol group can chelate metal ions, which is

important for their rPfM18AAP inhibitory activity, the compound structure is essential for

specificity as the selected probes did not inhibit other malaria exometallo- aminopeptidases such

as rPfM17LAP and rPfM1AAP (J. P. Dalton, unpublished data). Interestingly, another screening

center has also reported that both CID 2215 and CID 6852389 inhibit a cell-based luciferase

reporter assay to identify inhibitors of P. falciparum growth in vitro (http://

pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=504834) with IC50 values of similar potency. Still,

taken together, there are clearly some additional and important determinations to be made to better

understand how the molecules described in this study, in particular catechols, affect not only

rPfM18AAP but also P. falciparum. Combined with other relevant biological data presented here, this provides a basis for future rPfM18AAP probe development.

The rPfM18AAP inhibitors detailed here, CID 6852389 and CID 23724194, yield potent, reproducible results across laboratories in both biochemical and whole-cell studies. In addition, these heterocyclic compounds contain a basic nitrogen atom, a physicochemical property that is suspected to encourage lysosomotropism65. They are the basis of continuing efforts to identify efficacious small molecule probes of rPfM18AAP, which are the subject of Chapter 3.

48

Chapter 3

Subsequent Mechanism of Action Studies Targeting PfM18AAP Aimed at the Treatment and Prevention of Plasmodium falciparum

Introduction

Since there are several small molecule inhibitors of PfM1MAA and PfM17LAP, and very few small molecule inhibitors of PfM18AAP, we set out to identify small molecule inhibitors of PfM18AAP. Recall that rPfM18AAP is a target of interest as described previously in chapter 2 and in the summary following this introduction. High throughput screening of the biochemical assay employing rPfM18AAP was also described in Chapter 2 and this is a continuing effort that includes small molecule testing and chemical optimization using other biochemical assays.

Chapter 3 is directly derived from the manuscript describing how post HTS efforts, i.e. those described in Chapter 2, including mechanism of action studies and medicinal chemistry optimization, lead to the development and subsequent acceptance of a molecular probe that inhibits the activity of rPfM18AAP. Note a molecular probe is any molecule that achieves superior potency, i.e. inhibition than any other molecule identified as part of another scientifically published or patented prior art. In order for a molecule to be deemed a probe it must meet or surpass all probe criteria as approved by the National Institute of Health. Probe criteria for this target included the following: 1) rPfM18AAP potency demonstrating an IC50<10uM; 2) extracted PfM18AAP potency demonstrating and IC50<500nM; 3) 10X selectivity over rPfM1AAP; 4) 10X selectivity over PfM17LAP; 5) preferably < 1uM inhibition in the Pf parasite growth inhibition assay; 6)

>50uM IC50 in the Vero cell cytotoxicity assay and 7) sufficient aqueous solubility at pH 7.4 and room temperature. Upon meeting these criteria one can assume a probe is a well validated lead molecule that is appropriate for transition into early phase clinical studies.

49 My contribution to the authorship includes a substantive contribution to the following activities:

1) conception and design of the project as well as execution of a the medicinal chemistry

support experiments including rPfM18AAP, rPfM17AAP, rPfM1AAP and the mammalian cell cytotoxicity assays

2) analysis and interpretation of the research data on which the publication is based;

3) drafting the portions of the publication for the results that I am responsible for and are described below9.

Summary:

Malaria is one of the most prevalent human parasitic diseases and is a global health issue accounting for >600,000 deaths annually. For survival, the Plasmodium falciparum (Pf) malaria

parasite requires the action of a number of metallo-aminopeptidases that each display restricted

amino acid specificities, including PfM1MAA (membrane alanine aminopeptidase), PfM17LAP

(leucine aminopeptidase), and PfM18AAP (aspartyl aminopeptidase), which are thought to act in

concert to degrade host erythrocyte hemoglobin that the parasite uses as a source of amino acid

building blocks for the synthesis of its own proteins. Since there are several small molecule

inhibitors of PfM1MAA and PfM17LAP, and very few small molecule inhibitors of PfM18AAP,

we set out to identify small molecule inhibitors of PfM18AAP. High throughput screening of the

biochemical assay employing rPfM18AAP was described in Chapter 2 and this effort now includes

small molecule testing and chemical optimization using other biochemical assays. These include

native PfM18AAP, recombinant Fasciola hepatica cathepsin L1 (rFhCTSL1), rPfM1MAA,

rPfM17LAP, and rhM18, and cell-based parasite growth inhibition and cytotoxicity assays which

were used to identify CID 23724194 (from the NIH MLSMR) as a viable starting point for

medicinal chemistry optimization. Two rounds of structure-activity relationship studies were

50 performed to generate the probe ML369 (CID 56846691) (Figure 9). The probe is the best-in-class small molecule inhibitor of PfM18AAP; however, certain liabilities discussed in detail in the probe report limit its usefulness. When the probe is used as recommended, the probe is “fit-for-purpose” and should be useful for advancing the field.

Figure 9. The probe ML369

Methodology

Recombinant enzymes

Recombinant PfM18AAP, PfM17LAP, PfM1AAP, and human M18 were prepared at the

University of Technology, Sydney, as described previously13,66. Recombinant cathepsin L of the parasitic helminth Fasciola hepatica was prepared in the same laboratory as described previously67.

Native PfM18AAP enzyme

51 Parasite extracts containing PfM18AAP were prepared at the University of Technology, Sydney,

as described previously13.

Assays

A list of the relevant assays and corresponding PubChem assay identifier numbers (AIDs) is

provided in Table 2-

(https://www.ncbi.nlm.nih.gov/books/NBK179825/table/ml369.t13/?report=objectonly) which is

also represented below:

52 Table 2: List of all Assay Identifiers and Metadata Associated to Each Stage

Test Compound Performance PubChem Number of Compound Assay Format Assay Target Concentration Site Stage AID No. Samples Tested Range (µM)

rPfM18 inhibitor 1855 Summary NA NA SRIMSC NA project

rPfM18 inhibition 1822 Biochemical 7.35 291,944 SRIMSC Primary Screen assay

rCTSL1 inhibition 1906 Biochemical 5.96 302,759 SRIMSC Primary Screen assay

rPfM18 inhibition 2170 Biochemical 7.35 2,378 SRIMSC Primary Screen assay

rCTSL1 inhibition 2178 Biochemical 5.96 2,378 SRIMSC Primary Screen assay

rPfM18 inhibition 2195 Biochemical 0.0037-73.5 125 SRIMSC Primary Screen assay

rCTSL1 inhibition 2196 Biochemical 0.003-59.6 125 SRIMSC Primary Screen assay

rPfM18 inhibition 492974 Biochemical 5 76 Dalton Lab Hit Validation assay

ePfM18 inhibition 492975 Biochemical 5 60 Dalton Lab Hit Validation assay

Pf growth 489015 Cell-based 10 76/TBD Gardiner Lab Hit Validation/SAR inhibition assay

53 Test Compound Performance PubChem Number of Compound Assay Format Assay Target Concentration Site Stage AID No. Samples Tested Range (µM)

Pf growth 489011 Cell-based 1-25 8/TBD Gardiner Lab Hit Validation/SAR inhibition assay

rPfM18 inhibition 588678 Biochemical 0.19-100 63 SRSBSC SAR assay

rPfM18 inhibition 602222 Biochemical 0.19-100 10 SRSBSC SAR assay

rPfM18 inhibition 624177 Biochemical 0.19-100 45 SRSBSC SAR assay

rPfM1 inhibition 588680 Biochemical 0.19-100 63 SRSBSC SAR assay

rPfM1 inhibition 602219 Biochemical 0.19-100 10 SRSBSC SAR assay

rPfM1 inhibition 624176 Biochemical 0.19-100 45 SRSBSC SAR assay

rPfM17 inhibition 588679 Biochemical 0.19-100 63 SRSBSC SAR assay

rPfM17 inhibition 602220 Biochemical 0.19-100 10 SRSBSC SAR assay

rPfM17 inhibition 624175 Biochemical 0.19-100 45 SRSBSC SAR assay

rhM18 inhibition 588696 Biochemical 0.19-100 63 SRSBSC SAR assay

54 Test Compound Performance PubChem Number of Compound Assay Format Assay Target Concentration Site Stage AID No. Samples Tested Range (µM)

rhM18 inhibition 602221 Biochemical 0.19-100 10 SRSBSC SAR assay

rhM18 inhibition 624174 Biochemical 0.19-100 45 SRSBSC SAR assay

Vero cytotoxicity 588714 Cell-based 0.19-100 63 SRSBSC SAR assay

Vero cytotoxicity 602225 Cell-based 0.19-100 15 SRSBSC SAR assay

Vero cytotoxicity 624205 Cell-based 0.19-100 TBD SRSBSC SAR assay NA = not applicable

The following flowchart (Figure 10) summarizes the results for the high-throughput screening campaign that was described in chapter

2 and is pertinent to the assays described in the first 7 AIDs shown in table 2.

55

Figure 10. Critical Path for Primary Screening of rPfM18AAP

Please see the (https://www.ncbi.nlm.nih.gov/books/NBK179825/#ml369.app2) for the detailed

assay protocols which are also detailed below. Also note the definition of EC50 and IC50 is

technically different with EC50 being the effective concentration that yields fifty percent activity

and IC50 is the inhibitory concentration that yields fifty percent activity. For the purpose of this

thesis they may at times be used interchangeably. For brevity and because the detailed protocols

for AIDs 1855 -2196 are already described in chapter 2, only details for the protocols associated

to the probe development work in this chapter are shown below (AIDs 492974 through 624205

56 from table 2 above).

57 Additional Mode of Action, Probe Synthesis and Analytical Quality Control Activities.

The work described below in sections 2.1.7 through section 2.2, while critical to the successful probe development effort of ML369, was not performed by me and hence is included to demonstrate knowledge around the subject matter but, is limited in detail because it is not a result of my direct efforts. However complete information is easily attained using the listed pubchem

AIDs and also in the download of the full probe report.

Fluorescence-based rPfM18AAP Confirmatory Biochemical Assay (Hit Validation Assay

AID No. 492974)

The purpose of this assay is to determine inhibitory activity of powder samples of compounds for recombinant P. falciparum M18AAP. This assay was performed in the labs of the assay provider,

John P. Dalton

Fluorescence-based Malarial Cell Lysate PfM18AAP Confirmatory Biochemical Assay (Hit

Validation Assay AID No. 492975)

The purpose of this assay is to determine inhibitory activity of powder samples of compounds for

PfM18AAP in a malarial cell lysate. This assay was performed in the labs of the assay provider,

John P. Dalton.

Radiolabel-based Pf Parasite Growth Cell-based Assay (Hit Validation and SAR Assays AID

No. 489015)

The purpose of this assay is to determine the ability of powder samples of inhibitor compounds to inhibit the growth of Plasmodium falciparum in its asexual erythrocytic stage. For the purposes of hit validation and SAR optimization, this assay was performed in the labs of the assay provider,

Donald Gardiner.

Radiolabel-based Pf Parasite Growth Cell-based Assay (Hit Validation and SAR Assays AID

58 No. 489011)

The purpose of this assay is to determine the potency of a powder sample of an inhibitor compound

identified in a previous assay to inhibit the growth of Plasmodium falciparum in its asexual

erythrocytic stage. For the purposes of hit validation and SAR optimization, this assay was

performed in the labs of the assay provider, Donald Gardiner.

rPfM18AAP QFRET-based Concentration-response Biochemical High Throughput Assay

(SAR Assay AID Nos. 588678, 602222, 624177)

The purpose of this assay is to determine concentration response curves for compounds to support

on-target SAR studies. This assay was performed at the Southern Research Specialized

Biocontainment Screening Center.

rPfM1MAA QFRET-based Countersceeen Concentration-response Biochemical High

Throughput Assay (Counterscreen Assay AID Nos. 588680, 602219, 624176)

The purpose of this assay is to determine concentration response curves for compounds to support

off-target-selectivity SAR studies. This assay was performed at the Southern Research Specialized

Biocontainment Screening Center.

rPfM17LAP QFRET-based Countersceeen Concentration-response Biochemical High

Throughput Assay (Counterscreen Assay AID Nos. 588679, 602220, 624175)

The purpose of this assay is to determine dose response curves for compounds to support off- target-selectivity SAR studies. This assay was performed at the Southern Research Specialized

Biocontainment Screening Center. rhM18 QFRET-based Countersceeen Concentration-response Biochemical High

Throughput Assay (Counterscreen Assay AID Nos. 588696, 602221, 624174)

The purpose of this assay is to determine dose response curves for compounds to support off-

59 target-selectivity SAR studies. This assay was performed at the Southern Research Specialized

Biocontainment Screening Center.

Vero Cell-based Concentration-response Cytotoxicity Assay (Counterscreen Assay AID Nos.

588714, 602225, 624205)

The purpose of this assay is to determine dose response curves for compounds to determine

cytotoxicity associated to mammalian cells, in this case Vero cells, for use in determining which

compounds to proceed with in SAR studies. This assay was performed at the Southern Research

Specialized Biocontainment Screening Center.

Probe Chemical Characterization

All of the probe synthesis and chemical characterization was done by the Kansas University

Specialized Chemistry Screening Center. Specifically ML369 was synthesized in two steps from

commercially available materials as described in

(https://www.ncbi.nlm.nih.gov/books/NBK179825/#ml369.app3) and its spectral data are in

provided in (https://www.ncbi.nlm.nih.gov/books/NBK179825/#ml369.app5). This effort

included aqueous solubility testing and the probe compound ML369 was found to have a solubility

of 2.9 micrograms per milliliter, or 9 uM, under assay buffer conditions, which is 2X and 7X the

IC50 values in the biochemical and cell-based rPfM18AAP and PfM18AAP inhibition assays,

respectively. Aqueous stability and thiol stability was also assessed in 1:1 acetonitrile:PBS (no

antioxidants or other protectants, DMSO concentration 1%, room temperature). The probe

compound ML369 was found to be moderately stable in 1:1 acetonitrile:PBS, whereby 75% of the

compound remained after 48 hours, which is the timeframe for the radiolabel-based Pf parasite growth cell-based inhibition assay

Submission of the Probe and Analogues to the NIH MLSMR

60 Samples of the probe and five analogues were submitted to the NIH MLSMR compound collection

on March 21, 2013.

Results

Summary of Screening Results

Assay validation and high-throughput screening for the project were carried out as collaboration between the Scripps Research Institute Molecular Screening Center, and the assay providers, John

P. Dalton and Donald L. Gardiner. Professor Dalton and his team produced purified, and functionally characterized the recombinant Plasmodium falciparum (rPfM18AAP) and

recombinant cathepsin L (rCTSL1) enzymes which were used by the SRIMSC team. The flowchart

shown above summarizes the results for the high-throughput screening campaign (Figure 10).

The approximately 300k compounds in the NIH Molecular Libraries Small Molecule Repository

(MLSMR) were screened in singlicate at 7.35 uM. The results and statistics for the screen are described schematically in the following figure (Figure 11)

Figure 11: rPfM18AAP primary HTS assay performance. Positive control wells are shown as green circles (IC100 of ZnCl2 or Z-Phe-Ala-diazomethylketone). Results of compound wells (black) and negative control wells (blue) are also graphed. Calculated hit-cutoff is indicated via dashed

61 lines. Due to the high degree of compound activity found in the rPfM18AAP assay, data was normalized to the median of the compound wells (IC0) and the median of the respective IC100. Hence, the noticeable shift below 0% inhibition for rPfM18AAP assay.

62 Of the approximately 300k compounds screened for rPfM18AAP inhibition at 7.35 uM, 3,522

compounds showed inhibition >28% and were considered to be active. These screening results are

captured in PubChem AID 1822. Of the approximately 300k compounds screened for cathepsin

L1 inhibition in singlicate at 5.96 uM, 1,481 compounds showed inhibition >16% and were

considered to be active. These screening results are captured in PubChem AID 1906.

The most active 2,500 compounds from the primary screen for rPfM18AAP inhibition, which were

considered to be inactive in the primary counterscreen against capthepsin L1, were ordered from

the NIH MLSMR as DMSO stock solutions, and 2,378 of the 2,500 compounds were available

and were delivered to the SRIMSC for confirmatory screening.

Of the 2,378 compounds screened for rPfM18AAP inhibition in triplicate at 7.35 uM, 661

compounds showed inhibition >28% and were considered to be active. These screening results are

captured in PubChem AID 2170. Of the 2,378 compounds screened against cathepsin L1 in

triplicate at 5.96 uM, 7 compounds showed inhibition >16% and were considered to be active.

These screening results are captured in PubChem AID 2178.

The most active 128 compounds from the confirmatory, single-concentration-in-triplicate screen for rPfM18AAP inhibition, which were inactive in the confirmatory, single-concentration-in- triplicate counterscreen for cathepsin L1 inhibition, were ordered from the NIH MLSMR as

DMSO stock solutions, and 125 of the 128 compounds were available, and were delivered to the

SRIMSC for concentration-response confirmatory screening.

Of the 125 compounds screened for rPfM18AAP inhibition in 10-point concentration-response between 0.0037 and 73.5 uM, all 125 showed IC50 values < 10 uM, and were considered to be

active. These screening results are captured in the PubChem AID 2195. Of the 125 compounds

screened against cathepsin L1 in 10-point concentration-response between 0.003 and 59.6 uM,

63 none of the 125 showed IC50 values < 10 uM and all were considered to be inactive. These assay

results are captured in the PubChem AID 2196.

Hit validation for the project using compound samples from the solid physical state was carried out as collaboration between the chemistry center, KU SCC, and the assay providers, John P.

Dalton and Donald L. Gardiner. In parallel, the Dalton team performed percent-inhibition biochemical assays using recombinant PfM18AAP and PfM18AAP in soluble extracts of the malaria parasite, while the Gardiner team performed percent-inhibition and concentration- response parasite growth inhibition cell-based assays.

All of the 125 compounds considered active against rPfM18AAP and inactive against cathepsin

L1 were suggested for follow-up hit validation using compound samples from the solid physical state. While many of the 125 compounds are considered PAINS68, 63, the assay providers suggested

that many compounds used to treat malaria might be considered PAINS and that PAINS should

not be excluded from hit validation. Of the 125 compounds, 76 compounds were purchased or

synthesized, purified, analyzed, and shipped by the University Of Kansas Specialized Chemistry

Center to the assay providers, John P. Dalton and Donald L. Gardiner.

Of the 76 compounds screened for confirmatory activity against the recombinant PfM18AAP in

singlicate at 5 uM, 22 compounds showed inhibition >50% and were considered to be active. These

screening results are captured in the PubChem AID 492974. Of the 60 compounds screened for

confirmatory activity against the PfM18AAP in soluble extracts of malaria in singlicate at 5 uM,

28 compounds showed inhibition >50% and were considered active. These screening results are

captured in PubChem AID 492975.

Of the 76 compounds screened for parasite growth inhibition in triplicate at 10 uM, 8 compounds

showed inhibition >50% and were considered to be active. These screening results are captured in

64 the PubChem AID 489015. Of the 8 compounds screened for parasite growth inhibition in 5-point

concentration-response between 1 and 25 uM, 5 showed IC50 values < 5 uM, and were considered to be active. These screening results are captured in PubChem AID 489011.

The top 5 compounds of interest resulting from the primary screening and hit validation studies are shown in the Table 3. In selecting a compound chemotype for medicinal chemistry optimization, the aqueous solubility and biological promiscuity (i.e., lack of biological promiscuity, as determined from a survey of PubChem) for the top 5 compounds were considered in addition to the requirement for selectivity and reasonable activity in the biochemical and cell-

based assays. Ultimately, the compound CID 23724194 (Entry 1) was selected by the greater team

for medicinal chemistry optimization. This served as the starting point for SAR optimization which is detailed in tables 4-13 below; ultimately leading to the identification of the molecular probe

ML-369. One structural feature worth noting, common to all of the top compounds of interest, is a putative zinc-binding domain (i.e., catechol or other), consistent with the nature of PfM18AAP as a metallo-aminopeptidase.

65 Table 3 Top 5 compounds of interest from the primary screening and hit validation studies.

66 Figure 12. Concentration-response Curves for the Probe (ML369). ML369 (CID 56846691, SID 135378316) was tested across a range of concentrations up to 100 μM in the primary and several secondary assays. Concentration response data was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC50 values were calculated. (A) rPfM18AAP biochemical assay (AID 624177), IC50 = 4.6 μM; (B) rPfM1MAA biochemical assay (AID 624176), IC50 = 74.9 μM; (C) rPfM17LAP biochemical assay (AID 624175), IC50 = 37.7 μM; (D) rhM18 biochemical assay (AID 624174) IC50 = 4.43 μM; (E) Vero cell cytotoxicity assay (AID 624205) EC50 > 50 μM

67 Dose Response Curves for Probe

As shown in Figure 12, ML369 (CID 56846691, SID 135378316) was tested across a range of

concentrations up to 100 uM in the primary and several secondary assays. Concentration response data was analyzed using a four parameter logistic fit to the data (Excel Fit equation 205) with the maximum and minimum locked at 100 and 0. From these curves IC50 values were calculated. (A)

rPfM18AAP biochemical assay (AID 624177), IC50 = 4.6 uM; (B) rPfM1MAA biochemical assay

(AID 624176), IC50 = 74.9 uM; (C) rPfM17LAP biochemical assay (AID 624175), IC50 = 37.7

uM; (D) rhM18 biochemical assay (AID 624174) IC50 = 4.43 uM; (E) Vero cell cytotoxicity assay

(AID 624205) EC50 > 50 uM

Scaffold/Moiety Chemical Liabilities

As a reminder please note a molecular probe is any molecule that achieves superior potency, i.e.

inhibition than any other molecule identified as part of another scientifically published or patented

prior art. In order for a molecule to be deemed a probe it must meet or surpass all probe criteria as

approved by the National Institute of Health including sufficient aqueous solubility at pH 7.4 and

room temperature. Upon meeting these criteria one can assume a probe is a well validated lead

molecule that is appropriate for transition into early phase clinical studies.

The aqueous solubility for ML369 was determined to be 2.9 µg/mL (9.1 uM) in phosphate buffered

saline (PBS, pH 7.4) containing 1% DMSO, which is 2X and 7X the IC50 values in the biochemical

and cell-based rPfM18AAP and PfM18AAP inhibition assays, respectively. The probe is stable in

1:1 acetonitrile:PBS, with 100% remaining after 8 hours and 75% remaining after 48 hours, which is the timeframe for the radiolabel-based Pf parasite growth cell-based inhibition assay. The probe

was determined to be moderately stable to thiol (DTT), with 70% remaining after 8 hours. While

the catechol functional group may be a liability due to its perceived toxicity, a number of currently

68 prescribed drugs contain the catechol functional group, and methods of circumventing the toxicity

of catecholics have been suggested59.

SAR Tables

SAR optimization for the project was carried out as collaboration between the chemistry center, the KU SCC, the assay providers, John P. Dalton and Donald L. Gardiner, and the screening center, the Southern Research Specialized Biocontainment Screening Center. In parallel, the Gardiner team performed percent inhibition and concentration-response parasite growth inhibition cell- based assays, while the SRSBSC team performed the biochemical rPfM18AAP, rPfM1MAA, rPfM17LAP and hM18 assays, and the Vero cell cytotoxicity assays.

During the primary screening and compound hit validation stages of the project, the main drivers for compound prioritization were activity against the rPfM18AAP and ePfM18AAP in biochemical assays, selectivity against cathepsin L1 in a biochemical assay, activity against the

malaria parasite in a red blood cell cell-based assay, and, finally, acceptable aqueous solubility and biological promiscuity. Based on these considerations, the catechol-containing compound CID

23724194 was chosen for medicinal chemistry optimization. The main project drivers during the

SAR optimization stage of the project were activity against the rPfM18AAP in a biochemical assay, selectivity against rPfM1MAA, rPfM17LAP and rhM18 in biochemical assays, activity against the malaria parasite in a red blood cell cell-based assay, and lack of activity in a Vero cell cytotoxicity assay. While achieving 10-fold selectivity for the rPfM18AAP versus the rPfM1MAA and rPfM17LAP was considered to be quite likely, achieving selectivity against the rhM18 was considered unlikely. Although there is only a low level of amino acid sequence identity between the rPfM18AAP enzyme and the human orthologue (18%), suggesting that selectivity of compound binding for the malaria enzyme might be achieved, significant conservation exists

69 between the active sites of the malaria and human enzymes. For example, three histidine residues

(His-94, His-170, and His-440) which are predicted from site-directed mutagenesis studies to be critical for enzymatic activity and another (His-352) essential for stabilization of the quaternary structure of human M18 are conserved in the malaria enzyme. In fact, when the compound CID

23724194/CID 53464134 was tested across the suite of assays that would be used to drive SAR, the above predictions were substantiated, as modest selectivity was observed for the inhibition of rPfM18AAP versus rPfM1MAA and rPfM17LAP, and poor selectivity was observed versus the human M18 orthologue (Table 4).

Table 4. Activity for CID 23724194, the starting point for SAR optimization.

From this starting point, the team's goal was to improve upon CID 23724194 over two rounds of

SAR optimization. For the purpose of SAR optimization, the team divided the compound into three domains: (i) a putative zinc-binding domain, (ii) a linker domain, and (iii) a heterocycle domain

(Figure 13).

70 Figure 13

For the first round of SAR the team chose to survey the effect of gross structural changes to these three domains, in isolation, and in combination. As shown in Table 5, while changes to the heterocycle domain were tolerated, none of the changes led to increased activity against rPfM18AAP. Although the selectivity against rPfM1MAA and rPfM17LAP was largely unchanged, the selectivity against rhM18 was observed to increase quite dramatically for alternative heterocycles (Table 5, entries 1 and 5).

71

Table 5. Round 1 SAR and Changes to the Heterocycle Domain. The parent compound is included (top table) for comparison.

72

As shown in Table 6, changes to the length of the linker domain had a profound effect on activity across the biochemical assays.

Shortening the linker by one carbon atom abolished activity across the biochemical assays (Table 6, entry 1) and lengthening the linker by one carbon atom diminished the activity across the biochemical assays (Table 6, entry 2).

Table 6. Round 1 SAR and Changes to the Linker Domain. The parent compound is included (top table) for comparison.

73 For the compounds listed in Table 7, changes to the putative zinc-binding domain were explored. Exchanging the catechol for a phenol resulted in complete loss of activity across the biochemical assays (Table 7, entry 1). Replacing the catechol moiety using the 3-hydroxy-

4-pyridone moiety, a zinc-binding group popularized by Cohen69, also resulted in complete loss of activity across the biochemical assays

(Table 7, entry 2). Replacing the catechol moiety with the carboxyl group resulted in complete loss of activity across the biochemical

assays, too (Table 7, entries 3 and 4).

Table 7. Round 1 SAR and Changes to the Putative Zinc-binding Domain. The parent compound is included (top table).

74 As shown in Table 8, as was the case for changes to the heterocycle domain only (Table 5), while simultaneous changes to the heterocycle

and linker domains were tolerated, none of the changes led to increased activity against rPfM18AAP. Although the selectivity against

rPfM1MAA and rPfM17LAP was largely unchanged, the selectivity against hM18 was observed to increase quite dramatically (versus

the initial compound hit) in some cases (Table 8, entries 3 and 5).

Table 8. Round 1 SAR and Simultaneous Changes to the Heterocycle and Linker Domains.

A curious discrepancy is apparent from inspection of the data in the Tables 5, 6, 7, and 8, which is that there does not appear to be a very good correlation between the activity of the compounds against rPfM18AAP and their activity in the parasite growth inhibition

assay. One striking example is entry 2 in the Table 7, which shows no activity across the various biochemical assays and no cytotoxicity

75 against Vero cells, yet has an EC50 of 6 uM in the parasite growth inhibition assay. In general, across the larger set of compounds in

these tables, this discrepancy might be explained by potential off-target activity. With respect to entry 2, Table 7, specifically, it is

possible that the activity observed for this compound might result from the off-target iron-chelating ability of the 3-hydroxy-4-pyridone

moiety65.

The curious discrepancy between the activity of some compounds against rPfM18AAP and their activity in the parasite growth inhibition

assay was further highlighted by the purposeful circumstance whereby the Gardiner team performed percent inhibition and

concentration-response parasite growth inhibition cell-based assays on compounds independent from the SRSBSC team, which performed the biochemical rPfM18AAP, rPfM1MAA, rPfM17LAP and hM18 assays, and the Vero cell cytotoxicity assays. In other words, all compounds, even compounds that were inactive across the biochemical assays, were tested purposefully for inhibition of parasite growth. Table 3.4.6 lists the biological activity for target compounds containing the catechol functional group and the respective penultimate target compounds containing the O,O-dimethyl catechol functional group from which the final compounds were prepared.

In a number of cases, specifically for entries 5, 8, and 14, Table 9, even though the compounds were inactive across the various biochemical assays and non-toxic to Vero cells, the compounds showed significant activity in the parasite growth inhibition assay. With respect to these entries, it is possible that the activity observed for these compounds might result from off-target hemozoin capping,

much as is the case for chloroquine, which shares the 4-aminoquinoline heterocycle with these compounds70.

76 Table 9. Round 1 SAR and Final Catechol-containing and Penultimate O,O-Dimethyl Catechol-containing Compounds.

Table 9. Continued

77

Table 9. Continued

78

Table 9. Continued

79

80

On the other hand and most importantly, compounds from the first round of SAR, specifically,

entries 1 and 5, Table 5, and entry 5, Table 8, showed the expected correlation between activity in

the biochemical assay and activity in the parasite growth inhibition assay (see Table 9 and compare

the activity for entries 2, 6, and 16 to the activities for their respective O,O-dimethyl catechol

counterparts, listed in the rows immediately above each entry).

It should be noted, however, that the correlation between activity in the biochemical assays and

the activity in the parasite growth inhibition assay may be confounded by the selective

accumulation of appropriately basic inhibitor compounds in the low-pH 71 parasitophorous vacuole of the parasite65, the site in which PfM18AAP is believed to function (the pH for the red blood cell

cytoplasm and parasite cytoplasm is estimated as 7.0-7.2 and 6.8-7.0, respectively 72).

With respect to the second round of SAR, the results for only a small subset of the compounds

synthesized will be highlighted, where the results augment (as opposed to reproduce) the SAR

story from the first round.

For the second round of SAR the team chose to build on the observation from the first round that

selectivity against rhM18 was observed to increase quite dramatically for alternative

heterocycles (Table 5, entries 1 and 5) and for simultaneous changes to the heterocycle and

linker domains (Table 8, entries 3 and 5). Armed with this specific information and the general

precedent that conformational constraint may influence selectivity, compounds where the N-

atom of the linker domain was joined to a carbon atom of the linker domain were targeted for

synthesis. Examples of these compounds are shown in the Table 10. In fact, across the three

compounds shown in Table 10, the selectivity across the biochemical assays is improved,

relative to the starting compound hit (Table 4).

81

Table 10. Round 2 SAR and Constrained N-Atom/Linker Analogues. Parent compound included (top table).

82 The discrepancy between the activity of some compounds against rPfM18AAP and their activity in the parasite growth inhibition assay continued for this set of compounds, as may be seen in Table 11 (Parent compound included (top table).

Table 11. Round 2 SAR and Final Catechol-containing and Penultimate O,O-Dimethyl Catechol-containing Compounds.

83 Based on these two rounds of SAR optimization, the compounds in Table 12 were considered as probe candidates (catechol-containing

compounds, only). While the activity and selectivity for the compounds varies across the biochemical assays, one constant is that none

of the catechol-containing compounds is cytotoxic, as judged by the results for the Vero cell assay, and, more importantly, for three of

the four compounds, the biochemical activity correlates relatively well with the activity observed in the cell-based parasite growth

inhibition assay, suggesting that the activity for these compounds is on-target. Ultimately, the compound high-lighted in this table (CID

56846691) was nominated as the probe (and assigned the NIH Molecular Libraries Initiative probe number ML369), based on the good activity against rPfM18AAP, good selectivity against the rPfM1MAA and rPfM17LAP enzymes, and lack of cytotoxicity, even though the compound showed no selectivity against the rhM18.

84 Table 12. Probe Candidates (Catechol-containing Compounds Only) from Round 1 and 2 SAR.

85 Cellular Activity

The malaria parasite in vitro growth inhibition assay and the Vero cell cytotoxicity assay are cell-

based assays. The majority of compounds screened were active in the malaria parasite killing assay

and were inactive in the Vero cell cytotoxicity assay. The cellular permeability (PAMPA) for

ML369 and three of the supporting analogues was measured and was determined to be good. The

cellular permeability for the probe (PAMPA) was determined to be approximately 1400 × 10-6

cm/s, 900 × 10-6 cm/s, and 120 × 10-6 cm/s at pH 7.4, 6.2, and 5.0, respectively.

Profiling Assays

The probe was submitted to Eurofins Panlabs for screening in the LeadProfilingScreen, a screen profiling binding to 68 protein targets of therapeutic or toxicological interest, and the results for this screen are listed in detail in Appendix H. In summary, not surprisingly, the probe was observed to be particularly active against a few of the GPCR biological targets, such as the dopamine transporter and norepinephrine transporter, for which it shares structural similarity to the known ligands, dopamine and norepinephrine, respectively. In addition, significant activity was observed against the hERG potassium channel. While the potential for GPCR and ion channel activity would be most important if the probe were recommended for use in in vivo studies, this activity is much less important with respect to the more limited recommended uses for ML369 (i.e., biochemical and cell-based assays; electron microscopy studies; biotin, photo-affinity, or fluorescent conjugates for chemical biology; co-crystallography with rPfM18AAP; modeling with

PfM18AAP; and physical chemistry to study lysosomotropic properties). In any case, as the SAR surrounding ML369 is expanded to include bioisosteric replacements for the putative catechol zinc-binding domain, testing for off-target effects should include GPCR and ion channel targets.

The probe compound and supporting analogues were submitted to the Sanford-Burnham Medical

86 Research Institute for various levels of in vitro pharmacology profiling (i.e., aqueous solubility in

Pion's buffer (pH 5.0, 6.2, and 7.4), aqueous solubility in 1x PBS, cell permeability, (PAMPA),

plasma stability (human and mouse), plasma protein binding (human and mouse), hepatic

microsome stability (human and mouse), and toxicity towards Fa2N-4 immortalized human

hepatocytes), and the results are provided in Table J1, Appendix J. Overall, the probe showed

moderate aqueous solubility that increases as pH decreases, very good permeability that decreases

as pH decreases, poor/very poor stability to human/mouse plasma, high binding to human/mouse

plasma proteins, very poor/poor stability to human/mouse hepatic microsomes, and no significant

toxicity toward immortalized human hepatocytes. See the Appendix D for the experimental

procedures for the various in vitro PK measurements.

Discussion

At the outset, the aim of this project was to identify inhibitors of PfM18AAP that were selective

versus PfM1MAA and PfM17LAP which also inhibited cell-based parasite growth and were also

non-toxic to Vero cells. Subsequent to the HTS campaign, the previously unreported human

recombinant M18 became available (Dalton Lab), and was incorporated into the list of anti-targets.

After screening a compound library of almost 300,000 members and two subsequent rounds of

SAR optimization, compound ML369 was identified, and, for the first time, a correlation between inactivation of the PfM18MAAP and malaria parasite killing was demonstrated. In general, the non-toxicity of this compound against mammalian cells augurs wells for the development of anti- malaria drugs that do not exhibit toxic off-target effects, although this must be examined in vivo in the future.

While the probe shows no selectivity against the human rM18, it also did not show toxicity against mammalian cells. The lack of toxicity against mammalian cells could suggest that the compound

87 is taken into parasite-infected RBCs via a mechanism that is not active in mammalian cells. It is

well known that malaria parasites insert various transport channels into the membrane of the RBC,

which facilitates the active up-take of nutrients and other compounds (e.g. amino acids) form the

external milieu.

The probe ML369 meets the probe criteria for the project, which are listed in the Table 13.

Table 13. Comparison of the Observed Probe Properties to the Probe Criteria.

Comparison to existing art and how the new probe is an improvement

The prior art was investigated by searching the Chemical Abstracts database using the SciFinder

software. Abstracts were obtained for all references returned from the search and were analyzed

for relevance to the current project. For all references that were deemed relevant, the articles were

analyzed and the results are summarized. These search results were current as of April 4, 2013.

The only existing prior art was reported from the labs of the assay providers Dalton and Gardiner13.

Dalton and Gardiner prepared phosphinic and phosphonic acid derivatives of aspartic and glutamic

acid, the N-terminal acidic amino acids for which the specificity of the aspartyl aminopeptidase is restricted, for which the Ki's against rPfM18AAP ranged from a high of >2000 uM to a low of

0.34 uM (Figure 14). While a number of these inhibitors showed moderate inhibition of rPfM18AAP, none of the inhibitors showed any significant inhibition of the growth of P. falciparum D10 parasites in culture (even at 100 uM final concentration).

88

Figure 14. Reported Inhibitors of rPfM18AAP which are analogues of the target amino acids Asp and Glu.. The selectivity for these compounds toward rPfM1MAA, rPfM17LAP, and rhM18, and their

cytotoxicity against Vero cells was not reported. The current probe is in an improvement over this

prior art, in that, it is the first small-molecule inhibitor for rPfM18AAP that is moderately selective

against rPfM1MAA and rPfM17LAP and exhibits good blocking of parasite growth in culture (e.g.

lower than the broad-range neutral aminopeptidase inhibitor bestatin, IC50 10 uM), and, strikingly, is not toxic to Vero cells. The studies support that PfM18AAP is a viable target for anti-malarial

drug discovery as the studies suggest the first link between its enzymatic inhibition and parasite

killing via small-molecule compounds.

Mechanism of Action Studies

The assays used in this project included a combination of on- and off-target biochemical and cell- based assays. Recombinant PfM18AAP was used for the biochemical target assay, while recombinant FhCTSL1, PfM1MAA, PfM17LAP, and human M18 were used for the biochemical anti-target assays. Malaria parasite growth inhibition in red blood cells and cytotoxicity against

Vero cells were used as the cell-based target and anti-target assays, respectively. While the probe

89 ML369 and a handful of analogues listed in Table 12 show activity across this suite of assays that

is consistent with an on-target mechanism of action, some ambiguity does remain, in this regard.

Future studies, including additional rounds of SAR, the measurement of IC50 values against the native PfM18AAP, PfM1MAA, and PfM17LAP, and activity-based protein profiling using derivatives of the current, or improved, probes could address this ambiguity. In addition, one could imagine performing an experiment to help clarify the mechanism of action, whereby, PfM18AAP is overexpressed in the cell-based assay, and if the probe and analogues are acting on target their activity might be reduced.

Planned Future Studies

SAR Studies

Additional rounds of SAR could combine the structural features of the compounds listed in Table

12. For example, the first and third compound structures or the first and fourth compound structures could be combined to afford the compounds on the left and right, respectively, in the

Figure 15.

Figure 15. Example Future SAR Studies.

Bioisosteric replacements for the catechol moiety, such as those popularized by Cohen, should also be revisited 63. While many such catechol replacements were earmarked for synthesis, only a

few were prepared due to the challenges associated with their preparation.

Mechanism of Action Studies

90 The mechanism of action for the probe or analogues could be explored using affinity-based protein

profiling. This chemical biology strategy using bestatin-based probes has been used to illuminate

the distinct roles for malaria PfM1MAA and PfM17LAP73. While the activity for the current probe

is at the upper-end of what might be considered useful for such a probe, ideally, additional SAR

studies should lead to significantly more active analogues, with activities more appropriate for

such studies. The SAR studies to date suggest that the region of the inhibitor structure that is distal

to the catechol moiety is a likely candidate for conjugation to biotin, photo-affinity, or fluorescent

labels.

Co-Crystallization Studies using rPfM18AAP and the Probe or Analogues

Recently, the 3-D structure of the malaria recombinant PfM18AAP was resolved and showed that

the enzyme exists as a large complicated multimeric structure64. Because of the lack of availability of a potent inhibitor, no structure for an inhibitor-enzyme complex was obtained. However, the activity and solubility of the current probe, and its analogues, should now fill this gap. One of the probe analogues, in particular, CID 53308676 (Table 8, entry 3), that shows potency in the low single-digit micromolar range (IC50 against the rPfM18AAP) is quite soluble in aqueous solution

across the pH range from 5.0 to 7.4 (>0.5 mM). Co-crystallization studies should allow a better understanding of the manner in which these compounds bind and interact with the malaria enzyme.

Fluorescent and Electron Microscopy Studies

Fluorescent and electron microscopy studies could be used to examine the capacity for the probe and analogues to disrupt the intracellular architecture of the malaria cell. For instance, as reported by Trenholme et al., one could envision the development of an assay utilizing a green fluorescent protein chimera of PfM18AAP as a marker for analysis of parasites via fluorescence activated cell sorting which would allow for the accurate assessment of PfM18AAP production and when

91 changes are apparent within the malaria cell.14

Studies on the Physical Properties of the Probe and Analogues and Their Selective

Accumulation in the Parasitophorous Vacuole

The uptake and intracellular distribution properties of a compound are fundamentally important variables in achieving a potent and desirable biological response. The ability of compounds to cross cellular lipid bilayers is a necessary step in this process but rarely is it a sufficient one. This is because cells are highly compartmentalized entities with over 50% of their total volume comprised of membrane-bound organelles/compartments that can provide barriers between a probe and its intended target molecule.

Malaria parasite enzymes that interfere with hemoglobin digestion/metabolism are often presumed to be localized, at least partially, within the acidic digestive vacuole. Interestingly, PfM18AAP has been shown to be secreted into the parasitophorous space 13, which is also acidic due to the action of the V-H+-ATPase localized at the plasma membrane of the parasite 74. Being relatively acidic, these compartments have the propensity to significantly concentrate weakly basic compounds according to an ion trapping-type mechanism. Accordingly, we hypothesize that compounds that have ideal structural and physicochemical properties that promote ion trapping (i.e., lysosomotropism) will have enhanced interactions with PfM18AAP relative to non- lysosomotropic counterparts. It is important to note that the presence or absence of lysosomotropic properties should not influence probe interactions with PfM18AAP in such assays. However, the lysosomotropic propensity of probes will be expected to favor interactions with PfM18AAP in the anti-malarial red blood cell cell-based assay since intracellular proton gradients are actively maintained. Interestingly, lysosomotropic compounds show enhanced potency in anti-malarial assays65.

92 For next-generation probe analogues we will rationally incorporate functional groups that will

impart increasing degrees of lysosomotropic properties and evaluate them using in vitro enzyme

inhibition and RBC-based anti-malarial assays as described, previously. There are two key physicochemical properties of drugs that influence the degree of ion trapping in acidified intracellular compartments that will be systematically modified. The first parameter is the pKa value of the conjugate acid of the weak base75. The second is the ratio of the permeabilities of the

base in the ionized versus unionized form76. We will experimentally estimate the ratio of permeabilities by evaluating octanol/water partition coefficients as a function of pH as previously described76. The pKa values of probes will be estimated using spectrophotometric approaches. In

addition, we will experimentally determine the lysosomotropic characteristics of the probes using

a recently developed approach that has been validated using the fluorescent lysosomotropic probe

Lysotracker Red (LTR). In this approach we comparatively evaluate the total cell uptake in cells

before and after treatment with ionophores nigericin and monensin. The ionophores dissipate the

lysosome-to-cytosol pH gradient that provides the driving force for ion trapping, and cells treated

with ionophores have markedly reduced lysosomal sequestration of LTR. Using this data we can

mathematically arrive at the percentage of total cell uptake that is driven by an ion trapping-based

mechanism. For lysosomotropic molecules such as LTR, greater than 76% of its total cell uptake

can be attributed to ion trapping. This measure of lysosomotropic potential will be evaluated for

all probe analogues. Using these approaches, together with optimization of pKa and permeability

ratios of probe analogues, we expect that next-generation probes will have greatly enhanced

intracellular distribution properties and therefore fully optimized anti-malarial activity if the

lysosomotropic hypothesis is correct.

93 Pros and Cons Ways of Improving Screening Methods for On-target Effects

Again, while we identified a novel molecular probe ML369 that shows activity across this suite of

assays that is consistent with an on-target mechanism of action, some ambiguity does remain, in

this regard. It would be beneficial to screen initially against the native proteins PfM18AAP,

PfM1MAA, and PfM17LAP. The difficulty in doing so is in the scalability in obtaining sufficient

quantities of native enzyme which is a constant fundamental problem in large scale HTS which

typically requires liters of material which aren’t always easy to obtain nor cost effective. Another

approach which we could apply is an activity-based protein profiling methodology using derivatives of the current probe which could also help address this ambiguity. In this sense the probe, ML369, would be chemically conjugated to a fluorophore such as rhodamine and then applied either a gel based ABPP determination or fluorescence polarization readout. The complication in this approach is the labeled probe would increase the size of the current probe dramatically which may or may not decrease its potency.

94

Chapter 4 Targeting Plasmodium falciparum Gametocytes

Chapter 4 is directly derived from my manuscript demonstrating how I found compounds that inhibit the activity of Plasmodium falciparum Gametocytes by high-throughput screening.

Please see Spicer, et.al.. Identification of Antimalarial Inhibitors Using Late-Stage Gametocytes in a Phenotypic Live/Dead Assay. SLAS Discovery. 2018 August. PMID:30142014. (Appendix

A).

My contribution to the authorship includes a substantive contribution to the following activities:

1) conception and design of the project as well as execution of the experiments;

2) analysis and interpretation of the research data on which the publication is based;

3) drafting the publication as well as completing its submission as the communicating author.

Summary:

According to the World Health Organization (WHO) the parasites responsible for causing human malaria, Plasmodium falciparum, P. vivax, P. ovale P. malariae and P. knowlesi infect up to 216 million people per year resulting in an estimated 445,000 fatalities77. Current antimalarial regimens are not fully effective due to emerging drug resistance and their inability to prevent transmission17,78. Approximately 40% of the world’s population lives in areas where the risk of

malaria transmission is high, typically the tropic zones located closer to the equator27.

The WHO along with charitable organization such as Gates Foundation and the global ministries from highly affected countries, are now driving renewed efforts toward the eradication of malaria.

While vector control strategies, including impregnated bed nets and drug therapies such as

artemisinin combination therapies have helped tremendously their efficacy may have plateaued.

Additionally, while at least one vaccine, Mosquirix or RTS,S exists, it appears to have limited

95 efficacy in inducing either robust antigenic responses or long-lasting immunity, thus leaving some vaccinated individuals unprotected. It remains under study to determine just how effective it is79,39,40. Vector control and case management by chemotherapy remain the primary means of

control for malaria. In the best scenario, while we may be able to achieve low levels of infection,

additional measures are still necessary for complete eradication. This is true, in part, because drug

resistant parasite strains are evolving thus providing a reservoir for transmission80. In other words,

the route to complete elimination of malaria resides in our ability to not only block both the hepatic

and erythrocytic stages of parasite replication within the human host but, also to block transmission

or the parasite between the human host and the mosquito vector.

Hence, we are focused on identifying inhibitors that act on this late-stage of the parasite’s lifecycle.

Gametocytes are part of the sexual phase of the malaria parasite life cycle and are essential for transmission from one host to another via the mosquito. They are produced in the human host and once mature remain in a state of arrested cell development until ingested by a feeding female

Anopheles mosquito where they undergo further development (Figure 16A)33,34.

96

Figure 16: (A) Lifecycle of Plasmodium falciparum. Gametocytes are transmitted to the mosquito vector during a blood feed. The point in the lifecycle where transmission-blocking therapies may be effective is annotated with the large red X. (1B) the five stages of gametocyte development of which this HTS effort targets stages IV and V.

P. falciparum gametocytes require 10-12 days to reach maturity, passing through five distinct stages (I-V) along the way (Figure 16B)31. As the gametocytes mature to stage V they become

relatively metabolically inert until they are taken up by a feeding mosquito33 and they can remain

in the host circulation for significant periods of time at subpatent levels32. This lack of metabolic

activity correlates to less druggable targets making them insensitive to almost all commonly used

antimalarial agents and our own recent findings confirm and extend these results81,12. However,

97 some reports have found that late stage gametocytes have increased lipid requirements which is divergent from early stage asexual parasites making fatty acid metabolism a possible target82. Still, our group has confirmed that late-stage gametocytes (stage IV-V) are largely speaking, refractory to treatment by all the classes of antimalarial agents tested. This data indicates that even clinically effective antimalarial treatment of the host may not lead to the prevention of transmission. The metabolite of 8-aminoquinoline primaquine (PQ) is currently the only licensed antimalarial drug that is effective against late stage gametocytes83. Unfortunately, there are side effects to treatment with PQ which decrease its usefulness. It is also known to cause acute hemolysis in patients with

G6PD deficiency, for whom PQ is contraindicated. G6PD deficiency is highly prevalent in malaria endemic areas and severely limits the use of PQ. Early reports that Tafenoquine has activity against gametocytes have not been confirmed and it appears to have no in vitro activity against stage IV/V gametocytes38. It too is contraindicated in patients with G6PD deficiency. Therefore, for both primaquine and tafenoquine, in vitro testing with the parent compound may not be a good guide to in vivo activity (for any parasite stage) as the metabolites generated by host enzymes, that are probably the anti-parasite active compounds, remain little understood. Therefore, blocking transmission of malaria by targeting late-stage gametocytes remains a top priority.

Traditionally, HTS campaigns use one of two approaches to identify new agents against a given disease; cell based or target based. In contrast to the asexual stages, because late-stage gametocytes are essentially terminally differentiated, the effect of compounds on this stage cannot be monitored using cell multiplication as a marker. In addition, working with gametocytes is technically challenging and until very recently, methods for production of large numbers of gametocytes were not available14,49. Consequently, we have little information with respect to the differences in protein expression and metabolism between late-stage gametocytes and asexually replicating

98 parasites. As a result, neither a cell- or target-based HTS approach to identifying compounds with

activity against gametocytes was feasible. However, as a result of improved gametocyte

production methods and the development of tools for downstream analysis, by our group and

others, large-scale anti-gametocidal HTS approaches are now feasible84,85,86.

Herein, I describe the miniaturization and completion of a large-scale screening agenda using a

phenotypic approach incorporating a live/dead assay which monitors ATP, and, hence, metabolic

activity of gametocytes. Late-stage gametocytes were used in a rapid, cost effective, highly

sensitive luminescent detection assay in 1536-well format for detection of phenotypic gametocyte

inhibitors. I also report the results of a mammalian cell cytotoxicity assay which was employed as

a counterscreen.

Methodology:

BacTiter-Glo microbial cell viability assay detection reagents were purchased from Promega

Corp., Madison Wisconsin, and Part # G8231. The BacTiter-Glo microbial cell viability assay

detection reagents detect the amount of ATP present in a live gametocyte. Therefore if a

gametocyte is alive and metabolically active it will produce ATP which when detected using

BacTiter-Glo, will result in a high luminescence signal. When a gametocyte is killed or becomes metabolically inactive then ATP is rapidly degraded and results in a massively reduced or low

luminescence signal. This assay is designed to identify inhibitors of whole gametocyte activity and

hence we are trying to identify compounds that achieve a low overall luminescence signal

compared to vehicle treated wells. Late-stage (Stage V) gametocytes were isolated and

cryopreserved as previously described.14 These were subsequently shipped by overnight courier

frozen on dry ice, to TSRI Florida where they were stored at -130 °C until the time of use.

Cryopreserved gametocytes were thawed and returned to culture via a dropwise saline solution revitalization procedure followed by resuspension at 62,500 gametocytes per milliliter in RPMI

99 1640 (Life Technologies, 15750) supplemented with 10% human serum (BioWorld, 30611043-1),

0.2% sodium bicarbonate and 10ug/ml of gentamycin (Life Technologies, 15750). Four microliters

of gametocyte cell suspension were then dispensed into each well of 1536-well microtiter plates

(250 cells per well) (Corning, part 7254) using the Flying Reagent Dispenser (FRD-Aurora

Biosciences Corp). Next, 44 nanoliters of test compound in DMSO, low control (DMSO alone,

1.08% final concentration) or high control (media only, no gametocytes) were added to the appropriate wells using the GNF pintool (GNF Systems). Low controls in this sense are “vehicle only” treated wells and will elicit an uninhibited luminescence response equivalent to the amount of viable gametocytes within the well whereas high control wells have no gametocytes and elicit no appreciable luminescence which is anticipated to be corollary to a compound with maximal inhibitory effect. Plates were incubated for 24 hours at 37 °C, 95% relative humidity and 5% CO2, whereupon 4 microliters of BacTiter-Glo solution was added to all wells. Plates were centrifuged at 1000 RPM for 1 minute and luminescence was measured using a 30-second read on the ViewLux microplate reader (PerkinElmer Lifesciences).

Compounds.

The Molecular Libraries Small Molecule Repository library was provided by the NIH’s Molecular

Libraries Initiative. Details regarding compound selection for this library can be found online

(http://mli.nih.gov/mli/compound-repository/mlsmr-compounds/). Briefly, the MLSMR library is a highly diversified collection of small molecules (more than 50% of compounds exhibit molecular weights between 350 and 410 grams per mole) comprising both synthetic and natural products, from either commercial or academic sources, that can be grouped into the three following categories: specialty sets of known bioactive compounds such as drugs and toxins, focused libraries aimed at specific target classes, and diversity sets covering a large area of chemical space.

100 Solid samples (powders) were purchased by the University of Kansas Specialized Screening

Center from various commercial sources. Purity of all final compounds was confirmed by RP

HPLC/MS analysis and determined to be > 95%. 1H and 13C NMR spectra were recorded on a

Bruker AM 400 spectrometer (operating at 400 and 101 MHz, respectively) or a Bruker AVIII

spectrometer (operating at 500 and 126 MHz, respectively). NMR spectra for the 13 purchased

compounds used in the structure-activity relationship (SAR) analysis are shown in the

supplemental materials.

Data acquisition and analysis

All screening assays were run on a Kalypsys/GNF robotic platform in 1,536-well microtiter plates.

Luminescence was measured by the ViewLux plate reader using a luminescence protocol that

utilized a 30 seconds exposure time. Raw data were uploaded into an institutional HTS database

(Symyx Technologies, Santa Clara, CA) for further processing. Activity of each well was

normalized on a per-plate basis using the following equation:

The percent inhibition of each test compound was calculated as follows:

% Inhibition= 100 *((Test Compound- Median Low Control))/ ((Median High Control- Median

Low Control))

Where:

Test Compound is defined as wells treated with test compound. Low Control is defined as wells

treated with DMSO only. High Control is defined as wells treated with media only and no

gametocytes.

Each assay plate underwent a quality control check; a Z’ value greater than 0.5 was required for

acceptance of data87. Any assay plate for which the Z’ value did not exceed 0.5 was rescheduled

101 for another robotic procedure until an acceptable Z’ value was observed.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary

screen. Percent inhibition is calculated for each individual well including control wells using the

expression included above and applied here to determine an interval-based cut-off which was applied to take into account assay noise and general variability within compound activity to help preserve and identify more hits at the primary HTS phase88. Four values were calculated: (1) the

average percent inhibition of all high controls tested plus three times the standard deviation of the

high controls, (2) the average percent inhibition of all low controls tested minus three times the

standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values,

(3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity than the cutoff parameter was declared active.

For titration experiments, triplicate percent inhibition values were plotted against compound concentration. A four-parameter equation describing a sigmoidal concentration-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx). Concentration- response curves and IC50 values presented in this manuscript were generated by Prism (GraphPad

Software, San Diego, CA). In cases where the highest concentration tested did not result in greater

than 50% inhibition, the IC50 was determined manually as greater than the highest concentration.

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50

equal to or less than 10 uM were considered active.

Counterscreen

The purpose of this cell-based assay is to determine if compounds that originally were found to inhibit viability of late stage P. falciparum gametocyte in a primary HTS assay, also are cytotoxic

102 to mammalian cells, an undesirable feature. In the past, counterscreening utilizing this method

when screening for other parasite targets has been extremely helpful in ruling out non-specific

cytotoxic compounds, and, hence we sought to apply the same principle here24.

This assay employs Jurkat cells, a human T-cell line originally isolated from an adolescent male

with T cell leukemia. The cells are grown in suspension which facilitates tissue culture and

preparation for dispensing in the assay. The end-point assay presented here employed a similar

reagent to the primary assay, CellTiter-Glo luminescent reagent (Promega Corp. Part G7573),

which contains luciferase enzyme to catalyze the oxidation of beetle luciferin to oxyluciferin and

light in the presence of ATP, in this case from Jurkat cells. Since metabolically active cells produce

ATP, an increase in the number of dead or dying cells will correlate with a reduction in ATP levels.

As designed, compounds that inhibit cell viability and reduce intracellular ATP will reduce the catalytic conversion of luciferin into oxyluciferin, resulting in decreased luciferase activity and well luminescence. This assay included doxorubicin as a positive control, an antibiotic used as an anti-cancer drug which elicits an appropriate anti-proliferative effect on Jurkat cells.25

Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nominal test concentration of 83.3 uM.

Jurkat cells (clone E6.1; ATCC Cat# TIB-152) were routinely cultured in suspension in T-175

0 standing flasks at 37 C in 95% relative humidity (RH) at 5% CO2. Media consisted of RPMI-1640

containing 10% dialyzed fetal bovine serum, 0.1 mM NEAA, 1 mM Sodium Pyruvate, 25 mM

HEPES, 5 mM L Glutamine, and 1x antibiotic.

Prior to the start of the assay, cells were suspended to a concentration of 100,000 cells/ml in media.

To start the assay, 5 microliters of media was dispensed into the first two columns of a 1536 well plate and 5 microliters of cell suspension was dispensed to the remaining wells (500 cells per well).

103 The assay was started immediately by dispensing 42 nanoliters of test compound in DMSO,

Doxorubicin (8 uM final concentration) or DMSO alone (0.6% final concentration) to the

0 appropriate wells. The plates were then incubated for 48 hours at 37 C, 5% CO2 and 95% RH).

Following the two-day incubation, plates were equilibrated to room temperature for 10 minutes

and 5 microliters of CellTiter-Glo reagent was added to each well. Plates were centrifuged and incubated at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader. The percent inhibition for each compound was calculated exactly as it was for the primary HTS assay using the following controls: Test Compound is defined as wells containing test compound, Low Control is defined as wells containing DMSO only (0% inhibition), and High

Control is defined as wells containing 8 uM doxorubicin (100% inhibition).

Results

Since all HTS at Scripps is done in 1536-well format the former 96-well method was further miniaturized to optimize conditions based on Z’ value and reproducible response to methylene blue, a known inhibitor of malaria (Table 14)12, 89. The purpose of the methylene blue titration in

this assay is to control for the day to day response of a known pharmacologic inhibitor of

gametocyte viability. In a good whole cell based HTS campaign an IC50 value for a well validated control should not vary by more than two to three fold. In this case the value achieved was well within this tolerance at an average calculated IC50 of 14.7±6.6 uM.

Table 14. Stepwise Protocol for the 1536-Well Plate Live/Dead Gametocyte Assay Order Step Condition Comments Revitalization of Dilute to 62,500 gametocytes per milliliter in RPMI 1640 supplemented Thaw frozen stock late-stage with 10% human serum, 0.2% sodium bicarbonate and 10ug/ml of 1 gametocytes gentamycin 2 Dispense gametocytes 4μL/well 250 cells/well into 1536-well plates (Corning, part 7254) 3 Pin compounds/controls 44nL Compounds pinned at ~10µM final concentration in ~1% DMSO 4 Incubation 24 hours 37 °C, 95% relative humidity and 5% CO2

104 5 Dispense BacTiter-Glo 4µl/well Luminescence substrate is stable at RT RT incubation allows for plates to equilibrate to alleviate temperature Incubation 10 minutes 6 gradient prior to reading Plates were centrifuged and luminescence was measured on aViewLux Read Luminescence microplate reader (PerkinElmer, Turku, Finland) using a luminscence 7 protocol that utilized a 30 seconds exposure time

bDue to the novelty of this target, compounds with any observable activity in the Concentration- Response assay, and not in the counterscreen, were of interest for follow-up.

In particular, the number of gametocytes necessary per well to remain in the linear range of the

detection modality was optimized, as was the incubation time, and further validated for HTS in

the context of the fully automated screening system. This resulted in using 250 gametocytes per

well at >90% viability, immediately adding test compound in 75:25 DMSO:H2O, and incubating

for 24hrs prior to adding BacTiter-Glo luminescence detection reagent. These conditions led to

reproducible Z’ factor averaging 0.53±0.1 over the course of 124 assay test plates. After testing

>153,000 compounds at ~11 uM, 100 actives were identified using a cut-off set at >38% inhibition

(Figure 17).

105

Figure 17: (A) Scatterplot of the data from the primary HTS campaign. Wells were treated with either test compounds (black), no gametocytes (high control=red) or gametocytes only (low control=green). The hit cutoff is shown as an orange line at 38% inhibition. Titration results of the known anti-gametocyte cytotoxic compound methylene blue, N=4 separate experiments, N=16 wells per replicate point, error bars are shown. The average calculated IC50 was 14.7±6.6 uM.

106 This effectively sets the hit cut-off such that it approaches the level of the sample field. Still, the

hit rate was very low, observed as 0.1% of the compounds. This is further reflected by the average

Z score assessed over the same course of plates equal to 0.60±0.05. Here Z factor is calculated

using the method to generate Z’ but incorporates the use of the sample field in the expression used in place of the low controls. As such, achieving lower Z scores, such as those <0.5, would represent higher activity of the samples tested, which isn’t the case here.

Considering the low number of actives identified, the project proceeded immediately to the

concentration-response curve (CRC) determination phase, avoiding the expenditure of time and

cost associated with the typical confirmation and counterscreen phase. Since this biological target

is of major significance, difficult to hit, and the NIH repository allowed for up to 275 compounds

to proceed to CRCs the hit list was expanded to include the top 275 molecules for testing. Of those,

244 were available and subsequently tested in both the primary assay and counterscreen assay for

CRC determination. Of the 244 molecules, only two compounds were identified with moderate

potency and selectivity (see Table 15).

Table 15: Summary of the Ultra-HTS campaign to Identify Inhibitors of Late Stage Plasmodium falciparum Gametocytes

These two molecules, SR-01000751192 and SR-01000471307 were found to have potencies <10 uM in the whole gametocyte assay (Figure 18).

107

Figure 18: Structure-activity relationship of the top active molecules identified as hits in this HTS campaign. Compounds are ranked with most potent first. Data has been normalized to “no gametocytes” as 100% inhibition vs. “DMSO and gametocytes” only. Data is plotted as log M on the x-axis versus percent inhibition on the Y-axis.

108 Only SR-01000751192 exhibited minimal cytotoxicity when tested in the Jurkat T-cell toxicity assay (>83 uM), an example of an HTS qualified assay for cytotoxicity profiling15.

After validation of the hit compounds from dry powders that were originally identified from the

high-throughput screening conditions and prior to moving into serious SAR optimization where

analogs are typically obtained via laboratory synthesis, a routine procedure of analog-by-catalog

(ABC) is usually performed to streamline the process. In this step, structurally close analogs are

purchased from different commercially available sources that provide a basic idea to map the

structural changes and their correlate with inhibitory activities. Often, this process provides useful

insights towards which directions the structural changes should be performed to improve the

potency before the onset of SAR optimization using synthetic efforts. In this project, a quick ABC

process was carried out by purchasing analogs that are close structural analogs of the hit compound

SR-01000751192 with single-, double-, and triple-point changes (as summarized in the attached

files). Note that SR-01000751192 provided batch-to-batch reproducibility as identified from

samples from the solid physical state. A raw 4 parameter curve fit for this compound yield nearly

identical IC50 values (7.1uM -batch2 vs. 8 uM-original hit). A small set of analogs were obtained

as fresh samples from the solid physical state. The late-stage SAR assay performed well with Z’

values averaging better than 0.6, and the methylene blue control worked as expected. The

counterscreen assay also behaved as expected, with consistent Z’ scores, and response to the

control doxorubicin. Unfortunately, improved anti-gametocydial activity was not observed for any of the 13 compounds purchased for initial SAR, although the compound sample from the solid physical state for the original compound hit did recapitulate the activity observed previously (see supplemental Table 1). Therefore, it is possible to conclude that changing with pyridyl substituent at the position 3 of the pyrazole with phenyl, substituted phenyl, and cyclopropyl groups led to

109 diminished activities. It was also observed that changing the thiazolyl structure by adding

substituents, or additional heteroatom wasn’t tolerated.

Outcomes- Targeting Plasmodium falciparum Gametocytes

This work represents the first report of a large-scale HTS campaign completed in 1536 well format against late-stage P. falciparum gametocytes. Most HTS efforts targeting P. falciparum are directed at the liver and blood stages of the parasite life cycle (Figure 16). Some involve target based approaches such as isolation of purified proteins and screening for protease inhibitors that may block the active site for its respective substrate.8, 9 Others that have the potential to identify

transmission blocking molecule are low throughput and or non-homogenous in assay format

making them ill-suited for large scale HTS.86 As a critical biological target of unmet therapeutic

need, the bar was set low in terms of accepting any compound hit of appreciable activity to proceed

for further testing. This meant incorporating a conservative primary HTS cut-off followed by a

rather relaxed 10 uM IC50 cut-off in the late-stage analysis. While our efforts were in progress, a

few key hurdles were overcome. During the HTS of the gametocyte project there was an

unavoidable but, significant batch-to-batch difference in the gametocyte numbers and viability

which was overcome by pooling the batches at the point of HTS testing. This afforded an assay

with acceptable Z’ scores. Methylene blue, while not a good anti-malarial drug due to side effects

in humans90, was used as a pintool-transferred control on each plate and was also tested for anti-

gametocidal pharmacologic response as a CRC control during each experiment and during each

run. It is notable that the Z and Z’ values are nearly overlapping, which simply indicates the overall

activity of the test compound was very low. The initial hits, while low in efficacy, do consistently

reproduce activity in both labs (Scripps and QIMR Berghofer Medical Research Institute

(QIMRB). While it is unusual to obtain such a low hit rate against such a large number of diverse

110 compounds tested, namely the Molecular Library Probe Production Centers Network (MLPCN)

library which has yielded multiple clinical drugs,91 this observation highlights the complexity and natural resistance, or refractiveness, of the gametocytes to the cytotoxic effects of small molecules, similar to what has been observed by others to date. Incorporating a cytotoxicity counterscreen was effective in focusing attention on SR-01000751192 and SR-01000599986, but with the limited hit set may not be as useful at this stage as originally planned. Ultimately, the HTS campaign was successfully completed against this difficult target and there were no insurmountable challenges encountered.

This work involved a multi-institute and multi-content initiative that was supported by the

MLPCN. One of the mains goals of the MLPCN was to identify novel molecular probes; i.e. ligands that supersede the activity of all prior art in all assays of relevance to support such an achievement. So the goal was to obtain a series of small molecules that met these criteria. Based on this outcome, a probe was not identified for this project which would have been defined as a compound the elicited an anti-gametocidal effect < 500nM IC50 versus the primary assays and

which produced reasonable potency in the low-throughput mechanism-of-action assays, such as the in vitro assay performed at QIMRB.

An alternative approach that has shown promise by others incorporated fragment based screening against biochemical targets in tandem with phenotypic assays92,93. Perhaps, armed with a

biochemical assay directly targeting gametocytes, this hybrid approach would have helped here

but, at the time of writing none such exist that are HTS amenable. It can be concluded that this method of HTS provides a path forward for future HTS, perhaps testing larger more diverse libraries for anti-gametocidal inhibitor identification and development.

111

Chapter 5

Conclusion of Thesis

Progress toward the original aims

• I have adapted, implemented, optimized and developed each target into robust HTS

amenable assays including their respective confirmation and counter screens.

• I completed large scale compound screening (>150K compounds) on diversified molecular

collections for each target using a fully automated robotic screening system followed by

secondary analysis and subsequently concentration response analysis of the most

promising HTS hits including testing in late stage specificity assays.

• At the conclusion of the HTS campaigns I took lead like molecules through early medicinal

chemistry efforts and in the case of the rPfM18AAP developed and submitted novel

molecular probes to the NIH via the PubChem database which can be found at

http://www.ncbi.nlm.nih.gov/books/NBK179825/ (ID: 3070092) and represents a 4.6nM

effective inhibitor of this target with >16X selectivity over the anti-target rPfM1MAA.

• Finally I drafted, submitted and obtained publications in a peer reviewed journal that is

germane to HTS for the PfM18AAP effort and also the gametocyte. Both are published in

SLAS Discovery.

Problems encountered

During the course of this work the biggest issue was with the gametocyte effort directly related to

the scalability of the assay. This required multiple batches of late stage whole cell live gametocytes

to be prepared in Australia and delivered to Scripps in the USA. This required each batch to be

112 tested separately, for appropriate efficacy, and subsequently pooled during the HTS effort in order

to produce sufficient S:B and Z’. While adding an unexpected burden it was ultimately successful.

Final Outcome

At the conclusion of my thesis work the original goals of this work were all well achieved. A good example is shown in the outcomes of the PfM18AAP HTS campaign which resulted in two

publications and the identification of a novel molecular probe inhibitor proving for the first time

this protein can be targeted for small molecule drug discovery.

The large scale screen against the whole cell late stage gametocytes was also successful which,

identified reproducible activity of the best inhibitors through medicinal chemistry, both of which

had never before been done on such scale nor in a 1536 well HTS. This work also resulted in a

third publication accepted in the foremost leading journal for the publication of HTS results.

However, not all the work went easily. In this sense the gametocyte scalability was the biggest

concern. Scalability of cells in HTS is always a primary consideration and can be daunting if not well planned. Gametocytes fall into this category. To achieve the proper number of gametocytes

required production of multiple batches of late stage whole cell live gametocytes to be prepared in

Australia and delivered to Scripps in the USA. In HTS, like other areas of science, reproducibility

of the day to day results is paramount. Typically I use one batch or pooled batch of cells to achieve

the robustness and reproducibility required. Hence each batch of gametocytes had to be tested

separately, for appropriate efficacy, followed by subsequently pooling during the HTS effort in

order to produce sufficient S:B and Z’. This work encumbered multiple shipments and hundreds

of vials of gametocytes produced from dozens of batches. All told this was a bit of an unexpected

burden but ultimately I was successful.

Malaria research has clearly benefitted from the work presented in my thesis albeit there is much

113 more that must be done in order to create effective cures. Funding remains the primary hurdle for

future work to proceed. My greatest need in this regard would be to be able to fund and further

investigate more compounds to find better starting points for medicinal chemistry initiatives

targeting gametocytes. Leveraging HTS in this sense was limited to testing only 150K compounds

but, if I could interrogate the Scripps Drug Discovery library in its entirety that would include

665K molecules never before tested in the whole cell gametocyte assay. An effort of this

magnitude would be costly and still presents significant risk but it would afford us new

opportunities and directions to go in.

Alternative approaches targeting the transmission of malaria via anti-gametocidals have more

recently focused on distinguishing male and female gametocyte formation using sensitive reports to help identify which a small molecule is targeting which is perceived to help understand the transmission blocking capabilities of such molecules. This approach combined with the suite of assays including some of those described in this thesis may ultimately lead investigators to the next generation of anti-malarial inhibitors and perhaps even block transmission which would lead to a cure94,95.

So in conclusion, my thesis work has advanced significant findings towards filling gaps in public

health and knowledge related to early drug discovery directed at malaria. Prior to this work no

one had successfully completed large scale screening on neither PfM18AAP nor whole cell

gametocytes. The peer reviewed publication and probe report detailing the identification of the

initial hit and subsequent chemical optimization and characterization of ML369 has at a

minimum provided the field with a molecular control for future drug discovery efforts. In

addition this has provided a well planned and executable road map for anti-malarial assay

development, optimized and miniaturized HTS, post HTS medicinal chemistry support, and

114 small molecule lead development. It also provides the starting point and chemical basis for others to refine their own work towards lead identification and potential drug development.

Finally, while a chemical probe was not identified for the anti-gametocidal effort, it did shine a spotlight on how to pursue early drug discovery related to preventing malaria transmission; something that both the NIH and the world sees as a critical goal and unmet need which if effective will save millions of lives in a very short period of time. It also clearly provides proof of principal for screening and early drug discovery against a very tricky, finicky target. This has affected the malarial research community in numerous ways in that we now know that the hit rate and ability to kill whole cell gametocytes is not trivial but, possible. Future work by our group will included more whole cell gametocyte screening against a diversified chemical library such as the Scripps Drug Discovery Library. This library is unique from the library I screened

(MLPCN NIH library) and has been shown to be < than 15% overlapping. Finally, this innovative work has been exploited at the international level in which others have used the exact same methods to screen their small molecule; a major accomplishment in its own right.

115

References:

1. World Health Organization. Factsheet on the World Malaria Report 2014. World Malaria Report 2014, 1. 2. Crawley, J.; Chu, C.; Mtove, G.; Nosten, F., Malaria in children. Lancet 2010, 375 (9724), 1468-81. 3. Crawley, J.; Waruiru, C.; Mithwani, S.; Mwangi, I.; Watkins, W.; Ouma, D.; Winstanley, P.; Peto, T.; Marsh, K., Effect of phenobarbital on seizure frequency and mortality in childhood cerebral malaria: a randomised, controlled intervention study. Lancet 2000, 355 (9205), 701-6. 4. Winzeler, E. A.; Manary, M. J., Drug resistance genomics of the antimalarial drug artemisinin. Genome biology 2014, 15 (11), 544. 5. Fairhurst, R. M.; Dondorp, A. M., Artemisinin-Resistant Plasmodium falciparum Malaria. Microbiol Spectr 2016, 4 (3). 6. Shanks, G. D.; Edstein, M. D.; Jacobus, D., Evolution from double to triple-antimalarial drug combinations. Transactions of the Royal Society of Tropical Medicine and Hygiene 2014. 7. Skinner-Adams, T. S.; Stack, C. M.; Trenholme, K. R.; Brown, C. L.; Grembecka, J.; Lowther, J.; Mucha, A.; Drag, M.; Kafarski, P.; McGowan, S.; Whisstock, J. C.; Gardiner, D. L.; Dalton, J. P., Plasmodium falciparum neutral aminopeptidases: new targets for anti- malarials. Trends in biochemical sciences 2010, 35 (1), 53-61. 8. Spicer, T.; Fernandez-Vega, V.; Chase, P.; Scampavia, L.; To, J.; Dalton, J. P.; Da Silva, F. L.; Skinner-Adams, T. S.; Gardiner, D. L.; Trenholme, K. R.; Brown, C. L.; Ghosh, P.; Porubsky, P.; Wang, J. L.; Whipple, D. A.; Schoenen, F. J.; Hodder, P., Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening. Journal of biomolecular screening 2014, 19 (7), 1107-1115. 9. Schoenen, F. J.; Weiner, W. S.; Baillargeon, P.; Brown, C. L.; Chase, P.; Ferguson, J.; Fernandez-Vega, V.; Ghosh, P.; Hodder, P.; Krise, J. P.; Matharu, D. S.; Neuenswander, B.; Porubsky, P.; Rogers, S.; Skinner-Adams, T.; Sosa, M.; Spicer, T.; To, J.; Tower, N. A.; Trenholme, K. R.; Wang, J.; Whipple, D.; Aube, J.; Rosen, H.; White, E. L.; Dalton, J. P.; Gardiner, D. L., Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase. In Probe Reports from the NIH Molecular Libraries Program, Bethesda (MD), 2010. 10. Peatey, C. L.; Skinner-Adams, T. S.; Dixon, M. W.; McCarthy, J. S.; Gardiner, D. L.; Trenholme, K. R., Effect of antimalarial drugs on Plasmodium falciparum gametocytes. The Journal of infectious diseases 2009, 200 (10), 1518-21. 11. Barnes, K. I.; White, N. J., Population biology and antimalarial resistance: The transmission of antimalarial drug resistance in Plasmodium falciparum. Acta tropica 2005, 94 (3), 230-40.

116 12. Peatey, C. L.; Spicer, T. P.; Hodder, P. S.; Trenholme, K. R.; Gardiner, D. L., A high- throughput assay for the identification of drugs against late-stage Plasmodium falciparum gametocytes. Molecular and biochemical parasitology 2011, 180 (2), 127-31. 13. Teuscher, F.; Lowther, J.; Skinner-Adams, T. S.; Spielmann, T.; Dixon, M. W.; Stack, C. M.; Donnelly, S.; Mucha, A.; Kafarski, P.; Vassiliou, S.; Gardiner, D. L.; Dalton, J. P.; Trenholme, K. R., The M18 aspartyl aminopeptidase of the human malaria parasite Plasmodium falciparum. The Journal of biological chemistry 2007, 282 (42), 30817-26. 14. Dixon, M. W.; Peatey, C. L.; Gardiner, D. L.; Trenholme, K. R., A green fluorescent protein-based assay for determining gametocyte production in Plasmodium falciparum. Molecular and biochemical parasitology 2009, 163 (2), 123-6. 15. Pedro-Rosa, L.; Buckner, F. S.; Ranade, R. M.; Eberhart, C.; Madoux, F.; Gillespie, J. R.; Koh, C. Y.; Brown, S.; Lohse, J.; Verlinde, C. L.; Fan, E.; Bannister, T.; Scampavia, L.; Hol, W. G.; Spicer, T.; Hodder, P., Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening. Journal of biomolecular screening 2015, 20 (1), 122-30. 16. World Health Organization. The top 10 causes of death. 2012. World Malaria Report 2012, 1. 17. Feachem, R. G.; Phillips, A. A., Malaria: 2 years in the fast lane. Lancet 2009, 373 (9673), 1409-11. 18. Feachem, R. G., Shrinking the Malaria Map: A Prospectus on Malaria Elimination. Lancet 2011, 376, (9752), 1566-78. 19. Emilio V Perez-Jorge, M., FACP, Malaria Clinical Presentation. 2014. 20. Cox, F. E., History of the discovery of the malaria parasites and their vectors. Parasites & vectors 2010, 3 (1), 5. 21. Carter, R.; Mendis, K. N., Evolutionary and historical aspects of the burden of malaria. Clinical microbiology reviews 2002, 15 (4), 564-94. 22. Neghina, R.; Neghina, A. M.; Marincu, I.; Iacobiciu, I., Malaria, a journey in time: in search of the lost myths and forgotten stories. The American journal of the medical sciences 2010, 340 (6), 492-8. 23. Bruce-Chwatt, L. J., Alphonse Laveran's discovery 100 years ago and today's global fight against malaria. Journal of the Royal Society of Medicine 1981, 74 (7), 531-6. 24. Calderaro, A.; Piccolo, G.; Gorrini, C.; Rossi, S.; Montecchini, S.; Dell'Anna, M. L.; De Conto, F.; Medici, M. C.; Chezzi, C.; Arcangeletti, M. C., Accurate identification of the six human Plasmodium spp. causing imported malaria, including Plasmodium ovale wallikeri and Plasmodium knowlesi. Malaria journal 2013, 12, 321. 25. Prugnolle, F.; Durand, P.; Neel, C.; Ollomo, B.; Ayala, F. J.; Arnathau, C.; Etienne, L.; Mpoudi-Ngole, E.; Nkoghe, D.; Leroy, E.; Delaporte, E.; Peeters, M.; Renaud, F., African great apes are natural hosts of multiple related malaria species, including Plasmodium falciparum. Proceedings of the National Academy of Sciences of the United States of America 2010, 107 (4), 1458-63.

117 26. Blanford, J. I.; Blanford, S.; Crane, R. G.; Mann, M. E.; Paaijmans, K. P.; Schreiber, K. V.; Thomas, M. B., Implications of temperature variation for malaria parasite development across Africa. Sci Rep 2013, 3, 1300. 27. Gething, P. W.; Elyazar, I. R.; Moyes, C. L.; Smith, D. L.; Battle, K. E.; Guerra, C. A.; Patil, A. P.; Tatem, A. J.; Howes, R. E.; Myers, M. F.; George, D. B.; Horby, P.; Wertheim, H. F.; Price, R. N.; Mueller, I.; Baird, J. K.; Hay, S. I., A long neglected world malaria map: Plasmodium vivax endemicity in 2010. PLoS neglected tropical diseases 2012, 6 (9), e1814. 28. Crutcher, J. M.; Hoffman, S. L., Malaria. In Medical Microbiology, th; Baron, S., Eds. Galveston (TX), 1996. 29. Wipasa, J.; Elliott, S.; Xu, H.; Good, M. F., Immunity to asexual blood stage malaria and vaccine approaches. Immunology and cell biology 2002, 80 (5), 401-14. 30. Greenwood, B. M.; Fidock, D. A.; Kyle, D. E.; Kappe, S. H.; Alonso, P. L.; Collins, F. H.; Duffy, P. E., Malaria: progress, perils, and prospects for eradication. The Journal of clinical investigation 2008, 118 (4), 1266-76. 31. Bousema, T.; Drakeley, C., Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clinical microbiology reviews 2011, 24 (2), 377-410. 32. Gebru, T.; Lalremruata, A.; Kremsner, P. G.; Mordmuller, B.; Held, J., Life-span of in vitro differentiated Plasmodium falciparum gametocytes. Malaria journal 2017, 16 (1), 330. 33. Hawking, F., The clock of the malaria parasite. Scientific American 1970, 222 (6), 123-31. 34. Sinden, R. E., Gametocytogenesis of Plasmodium falciparum in vitro: an electron microscopic study. Parasitology 1982, 84 (1), 1-11. 35. Trenholme, K. R.; Brown, C. L.; Skinner-Adams, T. S.; Stack, C.; Lowther, J.; To, J.; Robinson, M. W.; Donnelly, S. M.; Dalton, J. P.; Gardiner, D. L., Aminopeptidases of malaria parasites: new targets for chemotherapy. Infectious disorders drug targets 2010, 10 (3), 217-25. 36. Spicer, T. P.; Gardiner, D. L.; Schoenen, F. J.; Roy, S.; Griffin, P. R.; Chase, P.; Scampavia, L.; Hodder, P.; Trenholme, K. R., Identification of Antimalarial Inhibitors Using Late-Stage Gametocytes in a Phenotypic Live/Dead Assay. SLAS Discov 2018, 2472555218796410. 37. (a) Visser, B. J.; van Vugt, M.; Grobusch, M. P., Malaria: an update on current chemotherapy. Expert opinion on pharmacotherapy 2014, 15 (15), 2219-54; (b) Vale, N.; Moreira, R.; Gomes, P., Primaquine revisited six decades after its discovery. European journal of medicinal chemistry 2009, 44 (3), 937-53. 38. Delves, M.; Plouffe, D.; Scheurer, C.; Meister, S.; Wittlin, S.; Winzeler, E. A.; Sinden, R. E.; Leroy, D., The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites. PLoS medicine 2012, 9 (2), e1001169.

118 39. Delves, M. J.; Ruecker, A.; Straschil, U.; Lelievre, J.; Marques, S.; Lopez-Barragan, M. J.; Herreros, E.; Sinden, R. E., Male and female Plasmodium falciparum mature gametocytes show different responses to antimalarial drugs. Antimicrobial agents and chemotherapy 2013, 57 (7), 3268-74. 40. Llanos-Cuentas, A.; Lacerda, M. V.; Rueangweerayut, R.; Krudsood, S.; Gupta, S. K.; Kochar, S. K.; Arthur, P.; Chuenchom, N.; Mohrle, J. J.; Duparc, S.; Ugwuegbulam, C.; Kleim, J. P.; Carter, N.; Green, J. A.; Kellam, L., Tafenoquine plus chloroquine for the treatment and relapse prevention of Plasmodium vivax malaria (DETECTIVE): a multicentre, double-blind, randomised, phase 2b dose-selection study. Lancet 2014, 383 (9922), 1049-58. 41. Moorthy, V. S.; Okwo-Bele, J. M., Final results from a pivotal phase 3 malaria vaccine trial. Lancet 2015. 42. Rts, S. C. T. P., Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet 2015. 43. Mockenhaupt, F. P.; May, J.; Bergqvist, Y.; Ademowo, O. G.; Olumese, P. E.; Falusi, A. G.; Grossterlinden, L.; Meyer, C. G.; Bienzle, U., Concentrations of chloroquine and malaria parasites in blood in Nigerian children. Antimicrobial agents and chemotherapy 2000, 44 (4), 835-9. 44. White, N. J., Antimalarial drug resistance. The Journal of clinical investigation 2004, 113 (8), 1084-92. 45. Umotong, A. B.; Ezedinachi, E. N.; Okerengwo, A. A.; Usanga, E. A.; Udo, J. J.; Williams, A. I., Correlation between in vivo and in vitro response of chloroquine-resistant Plasmodium falciparum in Calabar, south-eastern Nigeria. Acta tropica 1991, 49 (2), 119- 25. 46. Tyagi, R. K.; Gleeson, P. J.; Arnold, L.; Tahar, R.; Prieur, E.; Decosterd, L.; Perignon, J. L.; Olliaro, P.; Druilhe, P., High-level artemisinin-resistance with quinine co-resistance emerges in P. falciparum malaria under in vivo artesunate pressure. BMC Med 2018, 16 (1), 181. 47. McGowan, S.; Oellig, C. A.; Birru, W. A.; Caradoc-Davies, T. T.; Stack, C. M.; Lowther, J.; Skinner-Adams, T.; Mucha, A.; Kafarski, P.; Grembecka, J.; Trenholme, K. R.; Buckle, A. M.; Gardiner, D. L.; Dalton, J. P.; Whisstock, J. C., Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases. Proceedings of the National Academy of Sciences of the United States of America 2010, 107 (6), 2449-54. 48. Mistry, S. N.; Drinkwater, N.; Ruggeri, C.; Sivaraman, K. K.; Loganathan, S.; Fletcher, S.; Drag, M.; Paiardini, A.; Avery, V. M.; Scammells, P. J.; McGowan, S., Two-pronged attack: dual inhibition of Plasmodium falciparum M1 and M17 metalloaminopeptidases by a novel series of hydroxamic acid-based inhibitors. Journal of medicinal chemistry 2014, 57 (21), 9168-83. 49. Prabhu, P.; Patravale, V., Novel targets for malaria therapy. Current drug targets 2011, 12 (14), 2129-43.

119 50. Santos, G.; Torres, N. V., New targets for drug discovery against malaria. PloS one 2013, 8 (3), e59968. 51. Kafsack, B. F.; Rovira-Graells, N.; Clark, T. G.; Bancells, C.; Crowley, V. M.; Campino, S. G.; Williams, A. E.; Drought, L. G.; Kwiatkowski, D. P.; Baker, D. A.; Cortes, A.; Llinas, M., A transcriptional switch underlies commitment to sexual development in malaria parasites. Nature 2014, 507 (7491), 248-52. 52. Peatey, C. L.; Dixon, M. W.; Gardiner, D. L.; Trenholme, K. R., Temporal evaluation of commitment to sexual development in Plasmodium falciparum. Malaria journal 2013, 12, 134. 53. Lew, V. L.; Macdonald, L.; Ginsburg, H.; Krugliak, M.; Tiffert, T., Excess haemoglobin digestion by malaria parasites: a strategy to prevent premature host cell lysis. Blood cells, molecules & diseases 2004, 32 (3), 353-9. 54. Rosenthal, P. J., Hydrolysis of erythrocyte proteins by proteases of malaria parasites. Current opinion in hematology 2002, 9 (2), 140-5. 55. Lauterbach, S. B.; Coetzer, T. L., The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro. Malaria journal 2008, 7, 161. 56. Smith, E.; Chase, P.; Niswender, C. M.; Utley, T. J.; Sheffler, D. J.; Noetzel, M. J.; Lamsal, A.; Wood, M. R.; Conn, P. J.; Lindsley, C. W.; Madoux, F.; Acosta, M.; Scampavia, L.; Spicer, T.; Hodder, P., Application of Parallel Multiparametric Cell-Based FLIPR Detection Assays for the Identification of Modulators of the Muscarinic Acetylcholine Receptor 4 (M4). Journal of biomolecular screening 2015. 57. Hodder, P.; Cassaday, J.; Peltier, R.; Berry, K.; Inglese, J.; Feuston, B.; Culberson, C.; Bleicher, L.; Cosford, N. D.; Bayly, C.; Suto, C.; Varney, M.; Strulovici, B., Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system. Analytical biochemistry 2003, 313 (2), 246-54. 58. Brothers, S. P.; Saldanha, S. A.; Spicer, T. P.; Cameron, M.; Mercer, B. A.; Chase, P.; McDonald, P.; Wahlestedt, C.; Hodder, P. S., Selective and brain penetrant neuropeptide y y2 receptor antagonists discovered by whole-cell high-throughput screening. Molecular pharmacology 2010, 77 (1), 46-57. 59. Yang, D. P.; Ji, H. F.; Tang, G. Y.; Ren, W.; Zhang, H. Y., How many drugs are catecholics. Molecules 2007, 12 (4), 878-84. 60. Dimova, V. R., K.; Kakurinov, V, QSAR of Some N-1- Aryl/Heteroarylaminomethyl/Ethyl-1,2,4-Triazoles Part II: Antimicrobial Activity against Bacillus subtilis. Int. J. Mol. Sci 2006, 7, 119-129. 61. Lipinski, C. A.; LaMattina, J. L.; Hohnke, L. A., Pseudosymmetry and bioisosterism in biaryl pyridyl competitive histamine H2-receptor antagonists. Journal of medicinal chemistry 1985, 28 (11), 1628-36. 62. Turconi, M.; Donetti, A.; Schiavone, A.; Sagrada, A.; Montagna, E.; Nicola, M.; Cesana, R.; Rizzi, C.; Micheletti, R., Pharmacological properties of a novel class of 5-HT3 receptor antagonists. European journal of pharmacology 1991, 203 (2), 203-11.

120 63. Baell, J. B., Observations on screening-based research and some concerning trends in the literature. Future medicinal chemistry 2010, 2 (10), 1529-46. 64. Sivaraman, K. K.; Oellig, C. A.; Huynh, K.; Atkinson, S. C.; Poreba, M.; Perugini, M. A.; Trenholme, K. R.; Gardiner, D. L.; Salvesen, G.; Drag, M.; Dalton, J. P.; Whisstock, J. C.; McGowan, S., X-ray crystal structure and specificity of the Plasmodium falciparum malaria aminopeptidase PfM18AAP. Journal of molecular biology 2012, 422 (4), 495-507. 65. Dehkordi, L. S.; Liu, Z. D.; Hider, R. C., Basic 3-hydroxypyridin-4-ones: potential antimalarial agents. European journal of medicinal chemistry 2008, 43 (5), 1035-47. 66. Stack, C. M.; Lowther, J.; Cunningham, E.; Donnelly, S.; Gardiner, D. L.; Trenholme, K. R.; Skinner-Adams, T. S.; Teuscher, F.; Grembecka, J.; Mucha, A.; Kafarski, P.; Lua, L.; Bell, A.; Dalton, J. P., Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. The Journal of biological chemistry 2007, 282 (3), 2069-80. 67. Collins, P. R.; Stack, C. M.; O'Neill, S. M.; Doyle, S.; Ryan, T.; Brennan, G. P.; Mousley, A.; Stewart, M.; Maule, A. G.; Dalton, J. P.; Donnelly, S., Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells. The Journal of biological chemistry 2004, 279 (17), 17038-46. 68. Baell, J. B.; Holloway, G. A., New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. Journal of medicinal chemistry 2010, 53 (7), 2719-40. 69. Puerta, D. T.; Lewis, J. A.; Cohen, S. M., New beginnings for matrix metalloproteinase inhibitors: identification of high-affinity zinc-binding groups. J Am Chem Soc 2004, 126 (27), 8388-9. 70. Hempelmann, E., Hemozoin biocrystallization in Plasmodium falciparum and the antimalarial activity of crystallization inhibitors. Parasitol Res 2007, 100 (4), 671-6. 71. de Duve, C.; de Barsy, T.; Poole, B.; Trouet, A.; Tulkens, P.; Van Hoof, F., Commentary. Lysosomotropic agents. Biochemical pharmacology 1974, 23 (18), 2495-531. 72. Krogstad, D. J.; Schlesinger, P. H., A perspective on antimalarial action: effects of weak bases on Plasmodium falciparum. Biochemical pharmacology 1986, 35 (4), 547-52. 73. Harbut, M. B.; Velmourougane, G.; Dalal, S.; Reiss, G.; Whisstock, J. C.; Onder, O.; Brisson, D.; McGowan, S.; Klemba, M.; Greenbaum, D. C., Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases. Proceedings of the National Academy of Sciences of the United States of America 2011, 108 (34), E526-34. 74. Hayashi, M.; Yamada, H.; Mitamura, T.; Horii, T.; Yamamoto, A.; Moriyama, Y., Vacuolar H(+)-ATPase localized in plasma membranes of malaria parasite cells, Plasmodium falciparum, is involved in regional acidification of parasitized erythrocytes. The Journal of biological chemistry 2000, 275 (44), 34353-8.

121 75. Duvvuri, M.; Krise, J. P., A novel assay reveals that weakly basic model compounds concentrate in lysosomes to an extent greater than pH-partitioning theory would predict. Mol Pharm 2005, 2 (6), 440-8. 76. Duvvuri, M.; Gong, Y.; Chatterji, D.; Krise, J. P., Weak base permeability characteristics influence the intracellular sequestration site in the multidrug-resistant human leukemic cell line HL-60. The Journal of biological chemistry 2004, 279 (31), 32367- 72. 77. World Health Organization. Factsheet on the World Malaria Report 2014. WHO World Malaria Report. World Wide Web 2014, ISBN: 978 92 4 156483 0, 1-242. 78. Olotu, A.; Fegan, G.; Wambua, J.; Nyangweso, G.; Leach, A.; Lievens, M.; Kaslow, D. C.; Njuguna, P.; Marsh, K.; Bejon, P., Seven-Year Efficacy of RTS,S/AS01 Malaria Vaccine among Young African Children. N Engl J Med 2016, 374 (26), 2519-29. 79. Chang, H. H.; Childs, L. M.; Buckee, C. O., Variation in infection length and superinfection enhance selection efficiency in the human malaria parasite. Sci Rep 2016, 6, 26370. 80. Lang-Unnasch, N.; Murphy, A. D., Metabolic changes of the malaria parasite during the transition from the human to the mosquito host. Annual review of microbiology 1998, 52, 561-90. 81. Lamour, S. D.; Straschil, U.; Saric, J.; Delves, M. J., Changes in metabolic phenotypes of Plasmodium falciparum in vitro cultures during gametocyte development. Malaria journal 2014, 13, 468. 82. White, N. J.; Ashley, E. A.; Recht, J.; Delves, M. J.; Ruecker, A.; Smithuis, F. M.; Eziefula, A. C.; Bousema, T.; Drakeley, C.; Chotivanich, K.; Imwong, M.; Pukrittayakamee, S.; Prachumsri, J.; Chu, C.; Andolina, C.; Bancone, G.; Hien, T. T.; Mayxay, M.; Taylor, W. R.; von Seidlein, L.; Price, R. N.; Barnes, K. I.; Djimde, A.; ter Kuile, F.; Gosling, R.; Chen, I.; Dhorda, M. J.; Stepniewska, K.; Guerin, P.; Woodrow, C. J.; Dondorp, A. M.; Day, N. P.; Nosten, F. H., Assessment of therapeutic responses to gametocytocidal drugs in Plasmodium falciparum malaria. Malaria journal 2014, 13, 483. 83. Lucantoni, L.; Duffy, S.; Adjalley, S. H.; Fidock, D. A.; Avery, V. M., Identification of MMV malaria box inhibitors of Plasmodium falciparum early-stage gametocytes using a luciferase-based high-throughput assay. Antimicrobial agents and chemotherapy 2013, 57 (12), 6050-62. 84. Duffy, S.; Avery, V. M., Identification of inhibitors of Plasmodium falciparum gametocyte development. Malaria journal 2013, 12, 408. 85. Plouffe, D. M.; Wree, M.; Du, A. Y.; Meister, S.; Li, F.; Patra, K.; Lubar, A.; Okitsu, S. L.; Flannery, E. L.; Kato, N.; Tanaseichuk, O.; Comer, E.; Zhou, B.; Kuhen, K.; Zhou, Y.; Leroy, D.; Schreiber, S. L.; Scherer, C. A.; Vinetz, J.; Winzeler, E. A., High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission. Cell Host Microbe 2016, 19 (1), 114-26.

122 86. Zhang, J. H.; Chung, T. D.; Oldenburg, K. R., A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of biomolecular screening 1999, 4 (2), 67-73. 87. Smith, E.; Giuliano, K. A.; Shumate, J.; Baillargeon, P.; McEwan, B.; Cullen, M. D.; Miller, J. P.; Drew, L.; Scampavia, L.; Spicer, T. P., A Homogeneous Cell-Based Halide- Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel. Assay Drug Dev Technol 2017, 15 (8), 395-406. 88. Ehrhardt, K.; Davioud-Charvet, E.; Ke, H.; Vaidya, A. B.; Lanzer, M.; Deponte, M., The antimalarial activities of methylene blue and the 1,4-naphthoquinone 3-[4- (trifluoromethyl)benzyl]-menadione are not due to inhibition of the mitochondrial electron transport chain. Antimicrobial agents and chemotherapy 2013, 57 (5), 2114-20. 89. Schirmer, R. H.; Coulibaly, B.; Stich, A.; Scheiwein, M.; Merkle, H.; Eubel, J.; Becker, K.; Becher, H.; Muller, O.; Zich, T.; Schiek, W.; Kouyate, B., Methylene blue as an antimalarial agent. Redox Rep 2003, 8 (5), 272-5. 90. Schreiber, S. L.; Kotz, J. D.; Li, M.; Aube, J.; Austin, C. P.; Reed, J. C.; Rosen, H.; White, E. L.; Sklar, L. A.; Lindsley, C. W.; Alexander, B. R.; Bittker, J. A.; Clemons, P. A.; de Souza, A.; Foley, M. A.; Palmer, M.; Shamji, A. F.; Wawer, M. J.; McManus, O.; Wu, M.; Zou, B.; Yu, H.; Golden, J. E.; Schoenen, F. J.; Simeonov, A.; Jadhav, A.; Jackson, M. R.; Pinkerton, A. B.; Chung, T. D.; Griffin, P. R.; Cravatt, B. F.; Hodder, P. S.; Roush, W. R.; Roberts, E.; Chung, D. H.; Jonsson, C. B.; Noah, J. W.; Severson, W. E.; Ananthan, S.; Edwards, B.; Oprea, T. I.; Conn, P. J.; Hopkins, C. R.; Wood, M. R.; Stauffer, S. R.; Emmitte, K. A.; Team, N. I. H. M. L. P., Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes. Cell 2015, 161 (6), 1252-65. 91. Blaazer, A. R.; Orrling, K. M.; Shanmugham, A.; Jansen, C.; Maes, L.; Edink, E.; Sterk, G. J.; Siderius, M.; England, P.; Bailey, D.; de Esch, I. J.; Leurs, R., Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits. Journal of biomolecular screening 2015, 20 (1), 131-40. 92. Vu, H.; Roullier, C.; Campitelli, M.; Trenholme, K. R.; Gardiner, D. L.; Andrews, K. T.; Skinner-Adams, T.; Crowther, G. J.; Van Voorhis, W. C.; Quinn, R. J., Plasmodium gametocyte inhibition identified from a natural-product-based fragment library. ACS Chem Biol 2013, 8 (12), 2654-9. 93. Delves, M. J.; Miguel-Blanco, C.; Matthews, H.; Molina, I.; Ruecker, A.; Yahiya, S.; Straschil, U.; Abraham, M.; Leon, M. L.; Fischer, O. J.; Rueda-Zubiaurre, A.; Brandt, J. R.; Cortes, A.; Barnard, A.; Fuchter, M. J.; Calderon, F.; Winzeler, E. A.; Sinden, R. E.; Herreros, E.; Gamo, F. J.; Baum, J., A high throughput screen for next-generation leads targeting malaria parasite transmission. Nat Commun 2018, 9 (1), 3805. 94. Delves, M. J.; Angrisano, F.; Blagborough, A. M., Antimalarial Transmission-Blocking Interventions: Past, Present, and Future. Trends Parasitol 2018, 34 (9), 735-746. 95. Lauterbach, S. B.; Coetzer, T. L., The M18 aspartyl aminopeptidase of Plasmodium falciparum binds to human erythrocyte spectrin in vitro. Malar J 2008, 7 (1), 161.

123 96. Roche, L.; Dowd, A. J.; Tort, J.; McGonigle, S.; MacSweeney, A.; Curley, G. P.; Ryan, T.; Dalton, J. P., Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae. Eur J Biochem 1997, 245 (2), 373-80. 97. Skinner-Adams, T. S.; McCarthy, J. S.; Gardiner, D. L.; Hilton, P. M.; Andrews, K. T., Antiretrovirals as antimalarial agents. J Infect Dis 2004, 190 (11), 1998-2000. 98. Mendivil-Perez, M.; Velez-Pardo, C.; Jimenez-Del-Rio, M., Doxorubicin induces apoptosis in Jurkat cells by mitochondria-dependent and mitochondria-independent mechanisms under normoxic and hypoxic conditions. Anticancer Drugs 2015, 26 (6), 583- 98.

Appendices

Milestones achieved: Task Year1 Year 2 Year 3 equivalent equivalent equivalent Literature Review X X X X Develop and test protocol for X X rPfM18AAP HTS rPfM18AAP primary screen X Completion of rPfM18AAP HTS X X X Campaign Develop and test protocol for Plasmodium X falciparum gametocyte HTS Pf gametocyte primary screen X X Completion of the Plasmodium falciparum X X X X X gametocyte HTS Confirmation of Candidature X X Mid Candidature Review X X PhD Candidature Thesis Review X X Thesis Preparation/Submission X X X X= completed; # = to be done

Supplementary files associated with peer reviewed publications:

124 Appendix A:

Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl

Aminopeptidase (PfM18AAP) of Human Malaria via High Throughput Screening

Spicer, Timothy*1; Fernandez-Vega, Virneliz*1; Chase, Peter1; Louis Scampavia1; To, Joyce4;

Dalton, John P.3,4; Da Silva, Fabio L.2 ; Skinner-Adams, Tina S.2; Gardiner, Donald L.2;

Trenholme, Katharine R.2; Brown, Christopher L.5; Ghosh, Partha6; Porubsky, Patrick6; Wang,

Jenna L.6; Whipple, David A.6; Schoenen, Frank J.6; and Peter Hodder1

1The Scripps Research Institute Molecular Screening Center, Scripps Florida, Jupiter, Florida;

2Malaria Biology Laboratory, The Queensland Institute of Medical Research, Brisbane, Australia;

3Institute of Parasitology, McGill University, Quebec, Canada, 4Institute for Biotechnology of

Infectious Diseases, University of Technology Sydney, Sydney, Australia, 5School of

Biomolecular and Physical Sciences, Griffith University, Brisbane, Australia, 6The University of

Kansas Specialized Chemistry Center, Lawrence, Kansas

*These authors had equal contributions.

Short title: An Enzymatic Screen yields PfM18 Aspartyl Aminopeptidase Inhibitors Address correspondence to: Peter Hodder Lead Identification Scripps Florida 130 Scripps Way #1A1 Jupiter, FL 33458 U.S.A. E-mail: [email protected] Word count: 5,297

ABSTRACT

The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present

in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-

125 exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP suggesting that it is a valid target for new anti-

malaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay

was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-

Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers

Network (MLPCN) collection of ~292,000 compounds (the Molecular Libraries Small Molecule

Repository (MLSMR)). A Cathepsin L1 (CTSL1) enzyme-based assay was developed and used as

a counterscreen to identify compounds with nonspecific activity. Enzymology and phenotypic

assays were used to determine mechanism of action and efficacy of selective and potent

compounds identified from HTS. Two structurally related compounds, CID 6852389 and CID

23724194, yield uM potency and are inactive in CTSL1 titration experiments (IC50 >59.6 µM). As

measured by Ki assay, both compounds demonstrate uM non-competitive inhibition in the

rPfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrate potency in

malaria growth assays (IC50 4 µM and 1.3 µM, respectively).

Key words: Malaria, Plasmodium falciparum, Aspartyl aminopeptidase, PfM18AAP, parasite,

exopeptidase, 1536 well, QFRET

INTRODUCTION

According to the World Health Organization (WHO) the parasites responsible for causing malaria,

Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae infect

up to 500 million people per year and result in an estimated 650,000 fatalities 77. Current

126 antimalarial regimens are not fully effective due to emerging drug resistance and lack of their

ability to prevent transmission (2). Approximately 40% of the world’s populations live in areas

where the risk of malaria transmission is high, typically the tropic zones located closer to the

equator. The symptoms of malaria have a rapid onset with recurrent cycles of fever and chills,

muscle aches, headaches, vomiting and jaundice, but may be extended to 8-10 months after the initial infected mosquito bite occurs (3).

The life cycle of Plasmodium falciparum, the most prevalent and severest etiologic agent of malaria, is complex. It is transmitted by the female Anopheles mosquito to its human host where

the parasites first enter the liver cells before emerging and invading erythrocytes. It is during this

stage that P. falciparum utilizes protein-protein interactions and enzymatically-driven processes to allow entry, growth and escape from the human erythrocyte. One such enzyme is PfM18AAP, a ~67kDa metallo-aminoipeptidase that oligomerizes and is the sole aspartyl aminopeptidase

(AAP) present in the malaria parasite (4). PfM18AAP is thought to play an important role in the

process of protein degradation, and working together with other exo-aminopeptidases is believed

to digest host hemoglobin, an important source of amino acids for the parasite (5,6). PfM18AAP

may also interact with the erythrocyte membrane protein, spectrin, presumably to regulate the

integrity of the infected erythrocyte membrane skeleton during parasite growth allowing for host

cell expansion and parasite exit 96. Antisense RNA knockdown experiments have shown that

inhibition of intra erythrocytic PfM18AAP debilitates the malaria parasite making it a promising

drug target (6). Inhibitors of PfM18AAP will thus provide new tools to further our understanding

this enzyme’s function and increase the potential to develop therapies against P. falciparum

transmission.

127 Currently there are no known small molecule inhibitors of PfM18AAP. Previous reports have

identified the phosphinic and phosphonic acid analogs of glutamate and aspartate, GluP and AspP,

as modest amino-acid-derived inhibitors of PfM18AAP in vitro. However, these amino acid

derivatives do not reduce malaria growth in culture when tested at concentrations up to 100 µM

13. Thus the objective of this research program was to identify compounds that inhibit the activity

of rPfM18AAP. Described here is our approach to discovering such inhibitors by an HTS of

compound libraries, which yielded several series of potent compounds that demonstrate selectivity

to the enzyme. Two of these compounds are non-competitive inhibitors of rPfM18AAP and also

inhibit growth of P. falciparum.

MATERIALS AND METHODS

Recombinant enzymes and assay reagents. Functionally active purified recombinant PfM18AAP

was prepared at University of Technology Sydney as previously described (8). Characterization

studies with this enzyme have demonstrated that it has comparable properties to that of native

PfM18AAP taken from cytosolic extracts (8). Recombinant cathepsin L was prepared in the same

laboratory as previously described 97. All other reagents were purchased or made from standard

laboratory supplies as listed: H-Glu-NHMec substrate (Bachem, part I-1180); Z-Leu-Arg-MCA

substrate (Peptides International, part MCA-3210-v); 1,536-well plates (Greiner, part 789176);

. Tris (Amresco, part 0497); CoCl2 6H20 (Univar, part D3247); DTT (Invitrogen, part 15508-013);

ZnCl2 (Sigma, part 208086); Z-Phe-Ala-diazomethylketone (Bachem, part N-1040); BSA

(Calbiochem, part 126609). rPfM18AAP assay buffer was prepared as 50 mM Tris HCl pH=7.5,

4mM CoCl2, 0.1% BSA (w/v). rPfM18AAP substrate buffer was prepared as 50 mM Tris HCl pH

8.8. CTSL1 assay buffer was prepared as 25mM Tris HCl pH 7.5, 1 mM DTT, 0.1% BSA (w/v).

128 Compounds. The Molecular Libraries Small Molecule Repository library was provided by the

NIH’s Molecular Libraries Initiative. Details regarding compound selection for this library can be

found online (http://mli.nih.gov/mli/compound-repository/mlsmr-compounds/). Briefly, the

MLSMR library is a highly diversified collection of small molecules (more than 50% of compounds exhibit molecular weights between 350 and 410 g/mol) comprising both synthetic and natural products, from either commercial or academic sources, that can be grouped into the three following categories: specialty sets of know bioactive compounds such as drugs and toxins, focused libraries aimed at specific target classes, and diversity sets covering a large area of chemical space. Solid samples (powders) of CID 6852389 (SID 11532952/87693049) and CID

23724194 (SID 47200698/92117383) were purchased from Sigma Chemical Company (Catalog #

D043) and Vitas-M Laboratory (Catalog # STK091533), respectively.

rPfM18AAP screening assay. This enzymatic assay utilizes a fluorogenic peptide substrate (H-

Glu-NHMec), which is incubated with purified recombinant PfM18AAP in the presence of test

compounds. Cleavage of the substrate by PfM18AAP liberates the 7-amino-4-methylcoumarin fluorogenic leaving group (NHMec) from the peptide, leading to increased fluorescence (Figure

1). Enzymatic inhibitors block rPfM18AAP-mediated cleavage of H-Glu-NHMec and liberation

of the NHMec leaving group from the substrate, resulting in decreased fluorescence as measured

at 340 nm excitation and 450 nm emission. In order to reduce the number of compounds that

optically interfere with the measurement, initial (T0) and 90 minute (T90) measurements of plate

fluorescence were taken after addition of substrate. Test compounds were assayed in singlicate at

a final nominal concentration of 7 µM. A stepwise assay protocol is presented in Supplemental

Table 1. Further details of this assay can be found at the PubChem AID 1822

129 (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=1822). The performance of the assay at

various stages of the HTS campaign is listed in Table 1.

Cathepsin L1 (CTSL1) screening assay. CTSL1 is a functionally active recombinant form of a

cysteine endopeptidase derived from the worm parasite Fasciola hepatica. Similar in principle to

the rPfM18AAP assay, this enzymatic assay utilizes a fluorogenic peptide substrate (Z-Leu-Arg-

NHMec) that is incubated with purified recombinant CTSL1 in the presence of test compound.

This assay serves as a useful counterscreen because it also measures the level of the released

coumarin moiety to monitor enzyme activity, albeit using a completely different enzyme. Hence,

compounds that inhibit both assays are either non-specifically inhibiting the enzymes or possibly

behaving as fluorescence quenching artifacts. Either outcome is undesirable and leads to a

definitive path to remove unwanted compounds. Final assay conditions are shown in

Supplemental Table 1. Methods for data analysis and hit selection were identical to those used

for the rPfM18AAP HTS assay. This biochemical assay served as the counterscreen for each step

of the rPfM18AAP HTS campaign; compounds that were active against this target and

rPfM18AAP were not pursued at subsequent HTS campaign stages (Table 1). rPfM18AAP HTS campaign. Details relative to each step of the rPfM18AAP & CTSL1 screening assays are shown Table 1. Briefly, the first stage involved singlicate screening against PfM18AAP

(the primary screen) and CTSL1 (the primary counterscreen) at a final compound test concentration of 7 µM or 6 µM, respectively (see PubChem AIDs 1822 and 1906 for protocol details). The final DMSO concentration was 0.7% (v/v) for the PfM18AAP assay or 0.6% for the

CTSL1 assay. Well fluorescence was measured with a ViewLux plate reader (Perkin Elmer), and the percent inhibition of each test compound was calculated on a per-plate basis as further

described below. The numerical cutoff used to qualify active (“hit”) compounds was calculated

130 as the average percentage inhibition of all compounds tested plus three times their standard

deviation (10). The confirmation and counter screens were run on selected hits in the same

conditions as the primary screens, except that plates were assessed in triplicate and results for each

compound were reported as the average percentage inhibition of the three measurements, plus or

minus the associated standard deviation (PubChem AIDs 2170 and 2178). For titration

experiments, assay protocols were identical to those described above, with the exception that

compounds were prepared in 10 point, 1:3 serial dilutions starting at a nominal test concentration

of 74 µM, and assessed in triplicate (PubChem AIDs 2195 and 2196). To confirm activity of the

best inhibitors, compounds were purchased as powders samples and tested in various secondary

assays including Ki determination and two other assays including a malaria cell lysate assay and a

P. falciparum parasite growth assay (see below).

Screening data acquisition, normalization, representation and analysis. All screening assays

were run on a Kalypsys/GNF robotic platform in 1,536-well microtiter plates. Fluorescence was

measured by ViewLux plate reader (Perkin Elmer). Raw data were uploaded into an institutional

HTS database (Accelrys, San Ramon, CA) for further processing. As a first calculation, T0 was

subtracted from T90 for each individual well (“delta RFU”). Activity of each well was normalized

on a per-plate basis using the following equation:

Percent inhibition = (test_compound_delta RFU - negative_control_ delta

RFU)/(positive_control_ delta RFU-negative_control_deltaRFU)*100

Where “test_compound” is defined as wells containing test compound, “negative_control” is defined as the median of the wells containing test compounds, and “positive_control” is defined

131 as the median of the wells containing ZnCl2 or Z-Phe-Ala-diazomethylketone for rPfM18AAP or

CTSL1, respectively (n = 24 wells).

Each assay plate underwent a quality control check; a Z’ value greater than 0.5 was required for

acceptance of data (11). For titration experiments, triplicate percent inhibition values were plotted against compound concentration. A four-parameter equation describing a sigmoidal concentration- response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys).

Concentration-response curves and IC50 values presented in this manuscript were generated by

Prism (GraphPad Software, San Diego, CA).

Malaria cell lysate assay. This assay utilized soluble freeze-thaw extracts of parasites and the substrate H-Glu-NHMec as previously published. Briefly, in vitro infected human red blood cells were harvested, washed with PBS and parasites were then released using saponin. The released parasites were subjected to three rounds of freeze-thaw and extracts were harvested as supernatants

(8). Activity of test compounds versus PfM18AAP extracts was assessed by incubating the compounds with the extracts in the presence of CoCl2 and H-Asp NHMec. Vehicle and background

controls were included in the assay. Test compounds that inhibited extract activity decreased

cleavage of the substrate, which was measured as a decrease in fluorescence proportional to the inhibitory activity of the test compound.

Parasite growth assay. The whole-cell malaria growth assay was used to identify compounds that

inhibit P. falciparum growth in red blood cells, i.e. P. falciparum’s asexual erythrocytic stage as

previously published 98. Briefly, compounds were incubated with P. falciparum infected red blood cells (RBC) in hypoxanthine-free media. 3H-hypoxanthine was added to treated cultures. After 48

132 hours 3H incorporation was determined. Vehicle and background (RBC) controls (DMSO ≤ 1%)

were included on each plate. As designed, compounds that inhibit the growth of P. falciparum in

RBC decreased the level of 3H incorporated.

Ki determination and mode of inhibition. CID 6852389 and CID 23724194 powder samples were subjected to Ki determination versus purified recombinant PfM18AAP. Reaction buffer consisted

of 50 mM Tris-HCl pH 7.5 + 0.1 mM CoCl2. Reactions were run a 100 µL assay volume for 1

hour with 60 second measurement intervals. The substrate Glu-NHMec was tested in a the range

of 0.5 µM, 1 µM, 10 µM, 100 µM, 250 µM, and 500 µM while the inhibitor concentrations were

tested 1 µM, 5 µM, 10 µM, and 50 µM. Enzyme and inhibitors were incubated 30 minutes with

reaction buffer before substrate addition. GraphPad Prism software was used for graphical

(Lineweaver-Burk) and quantitative (non-linear regression) determination of inhibition mode and

Ki.

Cheminformatics: A Maximum Common Substructure hierarchical clustering (ChemAxon

LibraryMCS 5.10.1) was used to identify the scaffolds of active compound families from the 125

active compounds found from the PfM18AAP HTS (13-16). In addition to clustering analysis, physical properties of the clustered compounds were calculated (ChemAxon Instant JChem 5.9.4).

Calculated results included molecular mass, topological polar surface area, chiral atoms, H-bond

acceptors/donors, ring count, and rotatable bonds.

RESULTS

Implementation of the rPfM18AAP & CTSL1 screening assays. To help identify potent and

selective inhibitors we implemented a screening strategy that uses a counterscreen to triage non-

selective hits and assay artifacts at each HTS stage; in this case, recombinant CTSL1, a cysteine

133 peptidase derived from parasitic helminth F. hepatica, was selected as the counterscreen for rPfM18AAP compound hits. The rPfM18AAP & CTSL1 screening assays were both designed to

measure the inhibition of recombinant enzyme by monitoring the fluorescence of a

methylcoumarin-labeled peptide substrate (H-Glu-NHMec and Z-Leu-Arg-NHMec, respectively).

The measured fluorescence signal increases as enzyme cleaves the substrate and releases aminomethylcoumarin, whereas enzymatic inhibitors leave the substrate intact, resulting in low amounts of measured fluorescence. The assay principle is illustrated for the rPfM18AAP screening assay in Figure 1; the CTSL1 counterscreen assay was similarly formatted with an appropriate fluorogenic substrate (Z-Leu-Arg-NHMec). As shown in Figure 2A the positive control inhibitor for the rPfM18AAP assay, Zn(II), yielded an IC50 of 662nM ± 314nM (N=33 plates).

Concentration-response analysis of the CTSL1 inhibitor, Z-Phe-Ala diazomethylketone, resulted

in an average IC50= 15.45 nM ± 2.66 nM (N=22 plates).

The rPfM18AAP primary assay and CTSL1 counterscreen assay were implemented at a final

volume of 5 µL/well in 1,536-well plates. The assays were screened against the entire available

MLSMR collection; 277,728 unique compounds were tested in both assays. All MLSMR

compounds were screened at 7 µM for rPfM18AAP and 6uM for CTSL1. For the rPfM18AAP

screening assay, an IC100 of Zn(II) was used as a positive control for enzyme inhibition. The

median of the wells containing test compounds were used as negative controls (i.e. IC0). Using

these controls (Figure 3), the rPfM18AAP assay demonstrated robust screening statistics. It had

an average signal-to-background ratio (S/B) of 3.42 ± 0.89 and a Z’ of 0.84 ± 0.04 (n= 241 plates).

The CTSL1 assay was similarly robust; using Z-Phe-Ala diazomethylketone and the median of the

wells containing test compounds as positive and negative controls, respectively, yielded a S/B of

3.61 ± 0.26 and a Z’ of 0.88 ± 0.04 over 247 plates.

134 Selection of hits. The excellent screening statistics dictated our ability to select active compounds from singlicate data (11). Due to an update in the MLSMR collection between the two screening campaigns, rPfM18AAP was tested against 291,944 compounds and CTSL1 was tested against

302,759 compounds; this resulted in 277,728 compounds being tested in both assays. For these compounds, the CTSL1 assay results were used as a counterscreen for the rPfM18AAP screening assay. That is, any compound found active against rPfM18AAP but not active in the CTSL1 assay was prioritized for follow-up in the next round of testing (Table 1). A graphical representation of these selection criteria is shown in Figure 4. As graphed in the correlation plot, compounds that are to the right of the rPfM18AAP inhibition cutoff (vertical dashed line) were considered active

compounds in the rPfM18AAP screen (“hits”); hits that fall below the CTSL1 inhibition cutoff

(horizontal dashed line) were considered selective inhibitors of rPfM18AAP.

In order to further enrich the data set for downstream follow-up studies, fresh aliquots of 2,378 hit compounds demonstrating selective inhibition of rPfM18AAP were tested in triplicate against both assays. To confirm activity and selectivity, the primary HTS hit activity cutoffs were reapplied to each set of screening results. This yielded 125 compounds that confirmed selective activity against rPfM18AAP, i.e. their measured %inhibition was above the rPfM18AAP primary HTS activity cutoff and less than the CTSL1 counterscreen HTS activity cutoff. Fresh aliquots of these compounds were obtained for further characterization in titration (IC50) assays. All compounds yielded IC50 values of <10µM in the rPfM18AAP titration assays; none of these demonstrated

potency (defined as IC50 <10µM) against CTSL1.

Cheminformatics results: Maximum Common Substructure clustering of the 125 compounds analyzed resulted in 30 cluster groups being identified. However, 21 of these clusters had 3 or less members, in which 15 were singletons. In contrast, the four largest clusters (clusters: 1, 3, 6, 9)

135 encompassed ~53% of the 125 compounds evaluated. As shown in Figure 5, these four clusters

contain catechol, triazole and benzamide moieties, which are found in compounds with known pharmacology (17-20). The largest grouping of compounds was in cluster #9, containing 33

compounds (~26%) with a methylated catechol moiety. This cluster of compounds has distinguishing properties that include a moderate level of hydrophobicity (ALogP ~2.2) as well as greater enrichment in H-bond acceptors/donors number with respect to the other clusters. Cluster

#3 is the second largest grouping with 14 compounds represented by a 1,2,4 triazol-4-yl urea scaffold. These compounds have distinguishing properties that include greater hydrophilicity

(ALogP ~0.36), smaller mass and a lack of chiral centers. Cluster #6 is the third largest grouping with 10 compounds represented by a 1, 2, 4 triazol-4-yl acetamide scaffold, similar to cluster-3 in its structural and physical properties. Cluster #1 is the fourth largest grouping with 9 benzamides.

The physical properties generally straddle the values found within the other three clusters. A summary of physical parameters for each cluster is presented in Figure 5. Clustering results and calculated physical properties for all compounds are shown in Supplemental Table #2 as well as

Supplemental Table #3.

Mechanistic assay results. From the 125 compounds that demonstrated potent and selective inhibition of rPfM18AAP, a subset of compounds most tractable for SAR studies were purchased or resynthesized as powder samples. These powder samples were retested to confirm inhibition of purified recombinant PfM18AAP, as well as in malaria lysates, and also tested for their ability to inhibit P. falciparum parasite growth via 3H hypoxanthine incorporation. Two efficacious

compounds were identified from this effort: CID 6852389, (S)-(+)-Apomorphine hydrochloride

hydrate, and CID 23724194, the hydrochloride salt of 4-[2-(acridin-9-ylamino)ethyl]benzene-1,2- diol. CID 6852389 was found active in the P. falciparum lysate assay (84% efficacy at ≤5µM; CID

136 23724194 was unavailable for testing). In the 3H hypoxanthine incorporation assay, CID 6852389

and CID 23724194 yielded 87% inhibition and 96% inhibition at 10 µM test concentrations,

respectively.

All compounds that inhibited parasite growth by >50% were titrated and retested as concentration response curves in the same experiment. CID 6852389 and CID 23724194 had the highest potency in these assays, with IC50 values of 4µM and 1.3µM, respectively. CID 6852389 and CID

23724194 were also assayed enzymatically to assess their mode of inhibition. Results of non-linear

regression analysis for Ki values were 3.39 ± 0.36µM for CID 6852389 and 1.35 ± 0.15 µM for

CID 23724194. Mode of inhibition was determined by software-based comparison of fits method using competitive, non-competitive, uncompetitive and mixed models of inhibition. The preferred fit was the non-competitive model of inhibition in all cases. Additionally, the mode of inhibition was confirmed by steady state velocity plots, semilog scale plots, as well as Lineweaver-Burke plots. As determined by these methods, both behave as non-competitive inhibitors of rPfM18AAP

(Supplemental figure 1).

Results from previous primary screening assays were used to better understand possible off-target activities and liabilities of CID 6852389 and CID 23724194. When tested in similarly formatted enzymatic inhibition assays run at Scripps Research Institute Molecular Screening Center

(SRIMSC), these compounds were devoid of activity (cf. PubChem AIDs 1859, 1931, 651959).

Similarly, where tested, they were also inactive in mammalian cytotoxicity (cf. PubChem AIDs

1486, 1825) and bacterial viability (cf. PubChem AIDs 449731, 651606) screens, as well >100 other primary screens run at the screening center. Remarkably both compounds were found to be

inactive in all cell-based screens tested at the SRIMSC, except for two serotonin receptor screens

137 (cf. PubChem AIDs 612 and 504916). In summary, in silico evaluation of the activity of CID

6852389 and CID 23724194 in a variety of cell based and biochemical primary screening assays,

including screens that used heterologous target enzymes and similar detection methodologies to

the rPfM18AAP primary assay, indicated that the activity of these two compounds was specific to

rPfM18AAP.

DISCUSSION

Described here is our effort to identify small-molecule inhibitors of rPfM18AAP via an HTS

approach. As formatted, both the rPfM18AAP primary screen and the CTSL1 counterscreen were

highly amenable to automated screening in 1,536 well microtiter plates, with a throughput of

~20,000 compounds tested per hour. As indicated by Z’ and positive control IC50 values, excellent assay windows and consistent pharmacology were demonstrated throughout the HTS effort, which enabled facile identification of selective rPfM18AAP inhibitors. This screening approach identified 125 hit compounds with potent, selective inhibition of rPfM18AAP. Two of these compounds, CID 6852389 and CID 23724194, were found to be non-competitive inhibitors of

PfM18AAP, with low-uM Ki values.

As described previously (21-24), we applied a “parallel” screening approach for the rPfM18AAP

HTS, where the CTSL1 counterscreen was used to triage hits at each stage of the rPfM18AAP

campaign (Table 1). This was implemented since both the rPfM18AAP and CTSL1 assays shared

similar assay protocols, including the use of the same coumarin-based fluorophore for

measurement of activity. CTSL1, a cysteine metalloproteinase, would be expected to be affected

by divalent metal ions such as zinc, or chelators of such. Divalent metal ions were not added as

part of the reaction mix during the counterscreen assay so identifying chelators is less likely but,

138 similar in principle to rPfM18AAP, compounds that may coordinate metal atoms would be expected to affect the activity of CTSL1 and may be identified via this counterscreen. To prioritize viable rPfM18AAP inhibitors, we triaged compounds that were active in both assays with the assumption that they optically interfered with the measurement of fluorescence in microtiter plates, or were promiscuous inhibitors. The validity of this assumption is partially supported by the results of our in silico analysis of CID 6852389 and CID 23724194; not only were these compounds inactive in the CTSL1 counterscreen, but also in all other similarly formatted coumarin-based enzymatic assays run in our screening laboratory.

At the stage of potency assays, recapitulating this parallel approach enabled facile identification of selective rPfM18AAP inhibitors in the HTS datasets, allowing us to prioritize compounds for labor-intensive mechanistic assays. Following the HTS stage, i.e. step 4 in table 1, 125 compounds remained that appeared to be selective inhibitors. At this stage a chemical triage was applied and

76 compounds were procured for testing in the lysate and parasite growth assay. Not all compounds were available in sufficient quantity for testing in both assays in which case preference for usage was given to the parasite growth assay. Compounds that were active in lysate assay, if tested and parasite growth assay were progressed for Ic50 determination in the parasite growth assay. Of those five compounds, 6852389 and CID 23724194 were found to be the most potent.

Although the CTSL1 results were used to identify selective inhibitors for the rPfM18AAP HTS effort, it is important to note several MLSMR compounds also selectively inhibited CTSL1 (see

Figure 4). Alternatively, it is reasonable to assume that our criteria for prioritizing viable leads in the HTS effort may have triaged genuine rPfM18AAP inhibitors. For this reason, all results for

139 the rPfM18AAP and CTSL1 screening assays have been placed in a publically available database,

PubChem (https://pubchem.ncbi.nlm.nih.gov/), which enables the re-examination and identification of compounds with apparent activity in either target’s screening assay.

Cheminformatic analysis of the 125 hit compounds provides insight on the possible pharmacology of the hits identified from the HTS effort. As represented by the Cluster #9, catechols and catecholamines are well known stimulants (17). Derivatives of a 1, 2, 4 triazol-4-yl urea, found in cluster #3, have been shown to possess anti-inflammatory and antibacterial activities (18).

Cluster #6 and its 1,2,4 triazole derivatives have been associated with a wider range of pharmacology including anti-inflammatory, antibacterial, antifungal, anticancer, analgesic and antidepressants (18,19). Finally, cluster #1 and its benzamide scaffold have derivatives with antagonistic activities against various receptors such as dopamine D2, 5-HT2 and 5-HT3 receptors

(20). Both CID 6852389 and CID 23724194 are nominally members of the “catechol” cluster #9.

While catechols, in general, have been described as pan assay interference compounds (PAINS)

(25), there is contradicting evidence that this is the case, here, such as lack of activity versus >100 assays tested in our lab. Initial analysis of the catechols shown in Supplemental Table 3 demonstrates a relatively flat SAR which really only obviates that the catechol is important for activity. Figure 5 also demonstrates most compounds from the 125 tested for Ic50 associate into cluster 9 so clearly the catechol component must be important for activity. In addition, the nearly identical activity of CID 2215 and CID 6852389 clearly demonstrate that similar structures have reproducible activity, yet stereo selectivity cannot be fully ascertained due to lack of informative compound registration. While these observations do not clarify whether the biological activity was simply due to, “interference”, in other words, the catechol compounds acting as PAINS or that the

140 catechol compounds might be coordinating the metal atoms that the enzyme requires to function,

thus inhibiting the enzyme, we do know that rPfM18AAP reacts differently to various different

metal ions. The enzyme is inhibited by zinc (> 1 mM) which, was employed as our positive control in our screening assays but, conversely is enhanced by cobalt and manganese. Therefore the relationship of metal ion binding is complex (26). While the catechol group can chelate metal ions and we believe this is important for their rPfM18AAP inhibitory activity, the compound structure is essential for specificity as the selected probes did not inhibit other malaria exo-metallo- aminopeptidases such as PfM17LAP and PfM1AAP (Dalton, unpublished). Interestingly, another

screening center has also reported that both CID 2215 and CID 6852389 inhibit a cell based

luciferase reporter assay to identify inhibitors of P. falciparum growth in-vitro

(http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=504834) with Ic50 values of similar

potency. Still, taken together there are clearly some additional and important determinations to be

made to better understand how the molecules described in this manuscript, in particular catechols,

affect not only PfM18AAP but, also P. falciparum. Combined with other relevant biological data

presented here, this provides a basis for future PfM18AAP probe development.

The rPfM18AAP inhibitors detailed here, CID 6852389 and CID 23724194, yield potent,

reproducible results across laboratories in both biochemical and whole-cell studies. In addition, these heterocyclic compounds contain a basic nitrogen atom, a physicochemical property that is suspected to encourage lysosomotropism (27). They are the basis of continuing efforts to identify efficacious small molecule probes of rPfM18AAP, which will be the subject of future reports.

ACKNOWLEDGEMENTS

141 This work was supported by the National Institutes of Health’s Roadmap Initiative through grants R03MH084103 (DG, CB, KT, JPD), U54HG005031 awarded to Professor Jeffrey Aubé (PG, PP, JLW, DAW, FJS) U54MH084512 (TS, VFV, PC, LS, PH). JPD was also supported by the National Institute for Health Research (NIHR, Australia) and Canada Institute for Health Research (CIHR) Tier 1 Canada Research Chair. We thank Pierre Baillargeon and Lina DeLuca (Lead Identification, Scripps Florida) for compound management.

REFERENCES

1. WHO 2011, World Malaria Report.

http://www.who.int/malaria/world_malaria_report_2011/WMR2011_factsheet.pdf

2. Yamey, G. Roll Back Malaria: a failing global health campaign. Brit. Med. J. 2004,

328, 1086.

3. Schlitzer, M. Malaria chemotherapeutics part I: History of antimalarial drug

development, currently used therapeutics, and drugs in clinical development. Chem.

Med. Chem. 2007, 2, 944-986.

4. Thivierge, K.; Mathews, R.T.; Nsangou, D.; et al. Anti-malaria drug development

targeting the M1 alanyl aminopeptidases and M17 leucyl aminopeptidases. ARKIVOC

2012 (iv): 330-346.

5. Lew, V. L.; Macdonald, L.; Ginsburg, H.; et al. Excess haemoglobin digestion by

malaria parasites: a strategy to prevent premature host cell lysis. Blood Cells Mol. Dis.

2004, 32, 353-359.

6. Rosenthal, P. J.. Hydrolysis of erythrocyte proteins by proteases of malaria parasites.

Curr. Opin. Hematol., 2002, 9, 140-145.

7. Lauterbach, S. B.; Coetzer. T. L.. The M18 aspartyl aminopeptidase of Plasmodium

falciparum binds to human erythrocyte spectrin in vitro. Malar. J. 2008, 7, 161.

142 8. Teuscher, F.; Lowther, J.; Skinner-Adams, T.; et al. The M18 aspartyl aminopeptidase

of the human malaria parasite Plasmodium falciparum. J. Biol. Chem. 2007, 282,

30817-30826.

9. Collins, P.; Stack, C.; O’Neill, S.; et al. Cathepsin L1, the major protease involved in

liver fluke (Fasciola hepatica) virulence: propeptide cleavage sites and autoactivation

of the zymogen secreted from gastrodermal cells. J. Biol. Chem. 2004, 279, 17038-

17046.

10. Hodder P.; Cassaday J.; Peltier R.; et al. Identification of metabotropic glutamate

receptor antagonists using an automated high-throughput screening system. Anal.

Biochem. 2003, (313), 246-254.

11. Zhang J.; Chung T.; Oldenburg K. A Simple Statistical Parameter for Use in Evaluation

and Validation of High Throughput Screening Assays. J. Biomol. Screen. 1999, 4(2),

67-73.

12. Skinner-Adams, T.; McCarthy, J.; Gardiner, D.; et al. Antiretrovirals as antimalarial

agents. J. Infect. Dis. 2004 190, 1998-2000.

13. Krier, M.; Bret, G.; Rognan, D.; Assessing the scaffold diversity of screening libraries.

Journal Of Chemical Information And Modeling 2006, 46(2), 512-524.

14. Lisurek, M.; Rupp, B.; Wichard, J.; et al.; Design of chemical libraries with potentially

bioactive molecules applying a maximum common substructure concept. Molecular

Diversity 2010, 14(2), 401-408.

15. Ijjaali, I.; Dubus, E.; Bourinet, E.; et al. Assessing the chemical and biological diversity

of an ion channels knowledge database. Channels 2007, 1(4), 291-299.

143 16. Vargyas M.; Csizmadia F.; Hierarchical clustering of chemical structures by maximum

common substructures: Poster at the 8th International Conference on Chemical

Structures P-17, June 1-5, 2008 Noordwijkerhout, Netherlands.

17. Yang, D.-P.; Ji, J.F.; Tang, G.-Y.; et al. How many drugs are catecholics. Molecules

2007, 12, 878-885.

18. Dimova, V.; Ragenovic, K.; Kakurinov, V. QSAR of some N-1-

aryl/heteroarylaminomethyl/ethyl-1,2,4-triazoles part II: Antimicrobial activity against

Bacillus subtilis. International Journal Of Molecular Sciences 2006, 7(4), 119-129.

19. Lipinski, C.; Lamattina, J.; Hohnke, L. Pseudosymmetry and bioisosterism in biaryl

pyridyl competitive histamine H2-receptor antagonists. J. Med. Chem. 1985 28(11),

1628-1636.

20. Turconi, M.; Donetti, A.; Schiavone, A.; et al. Pharmacological Properties of a Novel

Class of 5-T-HT3 Receptor Antagonists. European Journal of Pharmacology 1991,

203(2), 203-211.

21. Grimm, C.; Jörs, S.; Saldanha, S.; et al. Small molecule activators of TRPML3. Chem

Biol. 2010, 17(2), 135-48.

22. Brothers, S.; Saldanha, S.; Spicer, T.; et al. Selective and brain penetrant neuropeptide

y y2 receptor antagonists discovered by whole-cell high-throughput screening. Mol.

Pharmacol. 2010, 77(1), 46-57.

144 23. Madoux, F.; Li, X.; Chase, P.; et al. Potent, selective and cell penetrant inhibitors of

SF-1 by functional ultra-high-throughput screening. Mol. Pharmacol. 2008, 73(6),

1776-84.

24. Madoux, F.; Simanski, S.; Chase, P.; et al. An ultra-high throughput cell-based screen

for wee1 degradation inhibitors. J. Biomol. Screen. 2010, 15(8), 907-17.

25. Baell, J. Observations on screening-based research and some concerning trends in the

literature. Future Med. Chem. 2010, 2(10), 1529–1546.

26. Sivaraman, K.; Oellig, C.; Huynh, K.; et al. X-ray Crystal Structure and Specificity of

the Plasmodium falciparum Malaria Aminopeptidase PfM18AAP. J. Mol. Biol. 2012

422, 495–507.

27. Dehkordi, L.; Liu, Z.; Hider, R. Basic 3-hydroxypyridin-4-ones: potential antimalarial

agents. Eur. J. Med. Chem. 2007, 43, 1035-1047.

145

Table 1: rPfM18AAP uHTS Campaign Summary and Results.

Screen type Target Number of Selection criteria Number of PubChem Assay statistics compounds compounds AID tested selected Z’ S/B 1 Primary PfM18AAP 291,944 >28.03% inhibition 3,522 1822 0.84 ± 0.04 3.42 ± 0.89 1 Primary CTSL1 302,759 >16.63% inhibition 1,481 1906 0.88 ± 0.04 3.61 ± 0.26 Counterscreen 2 Confirmation PfM18AAP 2,378 >28.03% inhibition 661 2170 0.87 ± 0.03 1.57 ± 0.04

3 Counterscreen CTSL1 2,378 >16.63% inhibition 7 2178 0.79 ± 0.01 3.53 ± 0.20 4 Titrations PfM18AAP 125 IC50<10µM 125 2195 0.88 ± 0.02 2.03 ± 0.05

CTSL1 125 IC50<10µM 0 2196 0.84 ± 0.03 4.12 ± 0.04 5 Powder testing PfM18AAP 76 >50% inhibition 22 492974 NA NA

Malaria Cell Lysate 60 >50% inhibition 28 492975 NA NA

6 Follow-up Parasite Growth @ 76 >50% inhibition 5 489015 NA NA 10µM Parasite Growth 5 IC50<5uM 2 489011 NA NA IC50

*NA=Not Applicable

146 340nm Excitation 340nm Excitation 450nm Emmision

Low fluorescence

Me Me

O H N Active 2 E N O O H N O O H 2 7-amino-4- methylcoumarin (NHMec)

OH O L-Glutamic acid 7-amino-4-methylcoumarin (H-Glu-NHMec)

Figure 1: rPfM18AAP enzymatic assay principle. rPfM18AAP activity is determined by measuring the release of the 7-amino- 4-methylcoumarin fluorogenic group (NHMec) from a peptide substrate (H-Glu-NHMec). Represented by the wavy line, cleavage of H-Glu-NHMec’s glutamic acid group by rPfM18AAP liberates NHMec from the peptide, leading to increased well fluorescence. As designed the screening assay identifies compounds that inhibit rPfM18AAP-mediated cleavage, resulting in decreased well fluorescence.

147

A. B. 120 120

100 100

80 80

60 60

40 40 % Inhibition % Inhibition

20 20

0 0 -8 -7 -6 -5 -4 -3 -11 -10 -9 -8 -7 -6 -5 -4 Log [ZnCl2], M Log [Z-Fa-Dk], M

Figure 2: Performance of positive controls in the rPfM18AAP and CTSL1 screening assays. (A) Zn(II) was used as the positive control for the rPfM18AAP screening assay. It yielded average IC50 = 662 ± 314nM for N=33 in the 1,536 well plates. (B) Z-Phe-Ala-diazomethylketone (Z-Fa-Dk) was used as the positive control throughout the CTSL1

HTS assay yielding an average IC50 = 15.45 ± 2.66nM for N=22. Note: Both curves were fitted using non linear regression analysis and the variable slope log (inhibitor) vs. response method provided by GraphPad Prism.

148

Figure 3: rPfM18AAP (left panel) and CTSL1 (right panel) primary HTS assay performance. Positive control wells are shown as inverted grey triangles (IC100 of ZnCl2 or Z-Phe-Ala-diazomethylketone). Results of compound wells (black triangles) and negative control wells (white squares) are also graphed. Calculated hit-cutoffs are indicated via dashed lines. Due to the high degree of compound activity found in the rPfM18AAP assay, data for both HTS campaigns was normalized to the median of the compound wells (IC0) and the median of the respective IC100. Hence, the noticeable shift below 0% inhibition for rPfM18AAP assay.

149 CID 6852389 % inhibition in CTSL1 OH CID 23724194

OH HO

OH N H HN CH3

N

% inhibition in rPfM18AAP screen

Figure 4: Comparison of rPfM18AAP and CTSL1 primary screening results. The results of 277,728 compounds (grey circles) are shown. The horizontal dashed line represents the CTSL1 activity cutoff while the vertical dashed line represents the rPfM18AAP activity cut-off. Green circles represent the two “hit” compounds that were identified as part of the primary HTS. Arrows indicate the screening results for CID 6852389 and CID 23724194.

150

Cluster #9 Cluster #3 Cluster #6 Cluster #1

33 14 10 9

3 26 2 2 2 5 7 2 2 6 3 2 4

Figure 5: Clustering results of potent rPfM18AAP hits. Presented are the most common substructure, and respective second tier derivatives for the four most populous clusters. Cluster #9 (33 compounds total) is represented by a 1,2 catechol moiety; Cluster #3 (14 total) is represented by a 1,2,4 triazol-4-yl-urea moiety; Cluster #6 (10 total) is represented by a (1,2,4 triazol-4-yl) acetamide moiety; and Cluster #1 (9 total) is represented by a benzamide moiety. CID 6852389 and CID 23724194 both belong to Cluster #9. Cluster numbers are assigned by the clustering algorithm, all results are provided in the supplemental section.

151

CID 6852389 CID 23724194 20000 15000 0 µM 0 µM 1 µM 1 µM 15000 µ 5 M 10000 5 µM 10 µM 10 µM 10000 50 µM 50 µM Velocity Velocity 5000 5000

0 0 0 200 400 600 0 200 400 600 [S], µM [S], µM

20000 15000 0 µM 0 µM 1 µM 1 µM 15000 5 µM 10000 5 µM 10 µM 10 µM 10000 50 µM 50 µM Velocity Velocity 5000 5000

0 0 1 10 100 1000 1 10 100 1000 µ [Substrate], µM [Substrate], M

0.12 0.4 0 µM 0 uM 1 µM 1 uM 0.09 0.3 5 µM 5 uM 10 µM 10 uM 0.2 0.06 1/v 1/v 50 µM 50 uM

0.03 0.1

0.00 0.0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 1/[S] 1/[S]

Supplemental Figure 1: Determination of modes of inhibitor interaction using untransformed data analysis, semilog scale analysis and Lineweaver-Burk analysis of potent and selective inhibitors rPfM18APP. Both compounds exhibit non-competitive inhibition.

152

Supplemental TABLE 1.

Step Amount of Volume PfM18AAP HTS Assay Conditions CTSL1 Assay Conditions or Time

Enzyme dispensing 2.5 µL/well Final concentration: 2.5 µg/mL of enzyme in 50mM Tris Final concentration: 0.75 µg/mL CTSL1 in 1mM Tris pH

pH 7.5, 0.05% BSA and 2mM CoCl2 7.5, 0.1% BSA and 1mM DTT

Compound addition 37 nL/well Final concentration : 7 µM Final concentration : 6 µM

Final DMSO concentration: 0.7% Final DMSO concentration: 0.6%

Pre-incubation 30 minutes Room temperature Room temperature

Substrate mix dispensing 2.5 µL/well Final concentrations: 50 µM H-Glu-AMC in 50mM Tris Final concentrations: 50 µM Z-Leu-Arg-MCA in 25 mM pH 8.8 Tris HCl, pH 7.5, 1mM DTT

Incubation 90 minutes Room temperature Room temperature

Record Fluorescence Ex = 340nm, Em = Read at T0 and T90 Read at T0 and T90 450nm

153

Supplemental Table 2: Shown are the average chemical properties for all compounds found within a given cluster grouping.

154 Appendix B:

Identification of Anti-Malarial Inhibitors using Late Stage Gametocytes in a Phenotypic Live/Dead Assay

Timothy P. Spicer1,4*, Donald L. Gardiner4, Frank J. Schoenen2, Sudeshna Roy2, Patrick R.

Griffin1, Peter Chase1†, Louis Scampavia1, Peter Hodder1†, and Katharine R. Trenholme3,4

1 The Scripps Research Institute, Department of Molecular Medicine, Scripps Florida

2The University of Kansas Specialized Chemistry Center, Lawrence, Kansas

3Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute,

Brisbane, Queensland, Australia.

4School of Medicine, University of Queensland, Herston, Queensland, Australia.

†Current affiliations: SR is now at The University of Mississippi, MS. PC is now at BMS,

Hopewell, NJ. PH is now at Amgen, Inc. Thousand Oaks, CA.

*Address correspondence to:

Timothy P. Spicer

Department of Molecular Medicine

The Scripps Research Institute, Scripps Florida,

130 Scripps Way, Jupiter, FL 33458 (USA)

Fax: (+1) 561-228-2150

E-mail: [email protected]

155 Abstract:

Malaria remains a major cause of morbidity and mortality worldwide with ~3.3 billion people at

risk of contracting malaria and an estimated 450 thousand deaths each year. While tools to reduce the infection prevalence to low levels are currently under development, additional efforts will be required to interrupt transmission. Transmission between human host and vector by the malaria parasite involves gametogenesis in the host and uptake of gametocytes by the mosquito vector.

This stage is a bottleneck for reproduction of the parasite making it a target for small-molecule

drug discovery. Targeting this stage, we used whole P. falciparum gametocytes from in-vitro

culture and implemented them into 1536 well plates to create a live-dead phenotypic anti- gametocyte assay. Using specialized equipment and upon further validation, we screened

~150,000 compounds from the NIH repository currently housed at Scripps Florida. We identified

100 primary screening hits which were tested for concentration response. Additional follow-up

studies to determine specificity, potency and increased efficacy of the anti-gametocyte candidate

compounds resulted in a starting point for initial medicinal chemistry intervention. From this, 13

chemical analogs were subsequently tested as de-novo powders which confirmed original activity from the initial analysis and now provide a point of future engagement.

Key words: Malaria, gametocyte, Plasmodium falciparum, parasite, 1536 well

Introduction

156 According to the World Health Organization (WHO) the parasites responsible for causing human

malaria, Plasmodium falciparum, P. vivax, P. ovale P. malariae and P. knowlsei infect up to 216

million people per year resulting in an estimated 445,000 fatalities.77 Current antimalarial regimens are not fully effective due to emerging drug resistance and their inability to prevent transmission.17,78 Approximately 40% of the world’s population lives in areas where the risk of

malaria transmission is high, typically the tropic zones located closer to the equator.27

The WHO along with charitable organization such as Gates Foundation and the global ministries

from highly affected countries, are now driving renewed efforts toward the eradication of malaria.

While vector control strategies, including impregnated bed nets and drug therapies such as

artemisinin combination therapies have helped tremendously their efficacy may have plateaued.

Additionally, while at least one vaccine, Mosquirix or RTS,S exists, it appears to have limited

efficacy in inducing either robust antigenic responses or long-lasting immunity, thus leaving some vaccinated individuals unprotected. It remains under study to determine just how effective it is.79,39,40 Vector control and case management by chemotherapy remain the primary means of

control for malaria. In the best scenario, while we may be able to achieve low levels of infection,

additional measures are still necessary for complete eradication. This is true, in part, because drug resistant parasite strains are evolving thus providing a reservoir for transmission.80 In other words,

the route to complete elimination of malaria resides in our ability to not only block both the hepatic

and erythrocytic stages of parasite replication within the human host but, also to block transmission

or the parasite between the human host and the mosquito vector.

Hence, we are focused on identifying inhibitors that act on this late-stage of the parasite’s lifecycle.

Gametocytes are part of the sexual phase of the malaria parasite life cycle and are essential for transmission from one host to another via the mosquito. They are produced in the human host and

157 once mature remain in a state of arrested cell development until ingested by a feeding female

Anopheles mosquito where they undergo further development (Figure 1A).33,34 P. falciparum

gametocytes require 10-12 days to reach maturity, passing through five distinct stages (I-V) along

the way (Figure 1B).31 As the gametocytes mature to stage V they become relatively metabolically

inert until they are taken up by a feeding mosquito and they can remain in the host circulation for

significant periods of time at subpatent levels. This lack of metabolic activity correlates to less

druggable targets making them insensitive to almost all commonly used antimalarial agents and

our own recent findings confirm and extend these results.81,12 However, some reports have found

that late stage gametocytes have increased lipid requirements which is divergent from early stage

asexual parasites making fatty acid metabolism a possible target.82 Still, we have confirmed that

late-stage gametocytes (stage IV-V) are largely speaking refractory to treatment by all the classes

of antimalarial agents tested. This data indicates that even clinically effective antimalarial

treatment of the host may not lead to the prevention of transmission. The metabolite of 8-

aminoquinoline primaquine (PQ) is currently the only licensed antimalarial drug that is effective

against late stage gametocytes.83 Unfortunately, there are side effects to treatment with PQ which

decrease its usefulness. It is also known to cause acute hemolysis in patients with G6PD deficiency,

for whom PQ is contraindicated. G6PD deficiency is highly prevalent in malaria endemic areas

and severely limits the use of PQ. Early reports that Tafenoquine has activity against gametocytes

have not been confirmed and it appears to have no in vitro activity against stage IV/V

gametocytes.38 It too is contraindicated in patients with G6PD deficiency. Therefore, blocking

transmission of malaria by targeting late-stage gametocytes remains a top priority.

158 Traditionally, HTS campaigns use one of two approaches to identify new agents against a given

disease; cell based or target based. In contrast to the asexual stages, because late-stage gametocytes

are essentially terminally differentiated, the effect of compounds on this stage cannot be monitored

using cell multiplication as a marker. In addition, working with gametocytes is technically

challenging and until very recently, methods for production of large numbers of gametocytes were

not available.14,49 Consequently, we have little information with respect to the differences in

protein expression and metabolism between late-stage gametocytes and asexually replicating

parasites. As a result, neither a cell- or target-based HTS approach to identifying compounds with

activity against gametocytes was feasible. However, as a result of improved gametocyte

production methods and the development of tools for downstream analysis, by our group and

others, large-scale anti-gametocidal HTS approaches are now feasible.84,85,86

Herein, we describe the miniaturization and completion of a large-scale screening agenda using a phenotypic approach incorporating a live/dead assay which monitors ATP, and, hence, metabolic activity of gametocytes. Late-stage gametocytes were used in a rapid, cost effective, highly sensitive luminescent detection assay in 1536-well format for detection of phenotypic gametocyte inhibitors. We also report the results of a mammalian cell cytotoxicity assay which was employed

as a counterscreen.

Materials and Methods

Assay reagents.

BacTiter-Glo microbial cell viability assay detection reagents were purchased from Promega

Corp., Madison Wisconsin, Part# G8231. Late-stage (Stage V) gametocytes were isolated and cryopreserved as previously described.14 These were subsequently shipped by overnight courier frozen on dry ice, to TSRI Florida where they were stored at -130 °C until the time of use.

159 Cryopreserved gametocytes were thawed and returned to culture via a dropwise saline solution revitalization procedure followed by resuspension at 62,500 gametocytes per milliliter in RPMI

1640 (Life Technologies, 15750) supplemented with 10% human serum (BioWorld, 30611043-1),

0.2% sodium bicarbonate and 10ug/ml of gentamycin (Life Technologies, 15750). Four µL of gametocyte cell suspension were then dispensed into each well of 1536-well microtiter plates (250 cells per well) (Corning, part 7254) using the Flying Reagent Dispenser (FRD-Aurora Biosciences

Corp). Next, 44 nL of test compound in DMSO, low control (DMSO alone, 1.08% final concentration) or high control (media only, no gametocytes) were added to the appropriate wells using the GNF pintool (GNF Systems). Low controls in this sense are “vehicle only” treated wells and will elicit an uninhibited luminescence response equivalent to the amount of viable gametocytes within the well whereas high control wells have no gametocytes and elicit no appreciable luminescence which is anticipated to be corollary to a compound with maximal inhibitory effect. Plates were incubated for 24 hours at 37 °C, 95% relative humidity and 5% CO2,

whereupon 4 µL of BacTiter-Glo solution was added to all wells. Plates were centrifuged at 1000

RPM for 1 minute and luminescence was measured using a 30-second read on the ViewLux

microplate reader (PerkinElmer Lifesciences).

Compounds.

The Molecular Libraries Small Molecule Repository library was provided by the NIH’s Molecular

Libraries Initiative. Details regarding compound selection for this library can be found online

(http://mli.nih.gov/mli/compound-repository/mlsmr-compounds/). Briefly, the MLSMR library is a highly diversified collection of small molecules (more than 50% of compounds exhibit molecular weights between 350 and 410 g/mole) comprising both synthetic and natural products, from either commercial or academic sources, that can be grouped into the three following categories: specialty

160 sets of known bioactive compounds such as drugs and toxins, focused libraries aimed at specific

target classes, and diversity sets covering a large area of chemical space. Solid samples (powders)

were purchased by the University of Kansas Specialized Screening Center from various

commercial sources. Purity of all final compounds was confirmed by RP HPLC/MS analysis and

determined to be > 95%. 1H and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer

(operating at 400 and 101 MHz, respectively) or a Bruker AVIII spectrometer (operating at 500

and 126 MHz, respectively). NMR spectra for the 13 purchased compounds used in the structure- activity relationship (SAR) analysis are shown in the supplemental materials.

Data acquisition and analysis.

All screening assays were run on a Kalypsys/GNF robotic platform in 1,536-well microtiter plates.

Luminescence was measured by the ViewLux plate reader using a luminescence protocol that utilized a 30 seconds exposure time. Raw data were uploaded into an institutional HTS database

(Symyx Technologies, Santa Clara, CA) for further processing. Activity of each well was normalized on a per-plate basis using the following equation:

The percent inhibition of each test compound was calculated as follows:

( ) % = 100 ( ) 𝑇𝑇𝑇𝑇𝑇𝑇𝑇𝑇 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶− 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝐿𝐿𝐿𝐿𝐿𝐿 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝐼𝐼𝐼𝐼ℎ𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 ∗ 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝐻𝐻𝐻𝐻𝐻𝐻ℎ 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶− 𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀𝑀 𝐿𝐿𝐿𝐿𝐿𝐿 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 Where:

Test Compound is defined as wells treated with test compound. Low Control is defined as wells

treated with DMSO only. High Control is defined as wells treated with media only and no

gametocytes.

161 Each assay plate underwent a quality control check; a Z’ value greater than 0.5 was required for

acceptance of data.87 Any assay plate for which the Z’ value did not exceed 0.5 was rescheduled

for another robotic procedure until an acceptable Z’ value was observed.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary

screen. Percent inhibition is calculated for each individual well including control wells using the

expression included above and applied here to determine an interval-based cut-off which was applied to take into account assay noise and general variability within compound activity to help preserve and identify more hits at the primary HTS phase.88 Four values were calculated: (1) the

average percent inhibition of all high controls tested plus three times the standard deviation of the

high controls, (2) the average percent inhibition of all low controls tested minus three times the

standard deviation of the low controls, (3) the average percent inhibition of all compounds tested

between (1) and (2), and (4) three times their standard deviation. The sum of two of these values,

(3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater %

inhibition/activity than the cutoff parameter was declared active.

For titration experiments, triplicate percent inhibition values were plotted against compound

concentration. A four-parameter equation describing a sigmoidal concentration-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx). Concentration- response curves and IC50 values presented in this manuscript were generated by Prism (GraphPad

Software, San Diego, CA). In cases where the highest concentration tested did not result in greater than 50% inhibition, the IC50 was determined manually as greater than the highest concentration.

Compounds with an IC50 greater than 10 µM were considered inactive. Compounds with an IC50

equal to or less than 10 µM were considered active.

Counterscreen

162 The purpose of this cell-based assay is to determine if compounds that originally were found to

inhibit viability of late stage P. falciparum gametocyte in a primary HTS assay, also are cytotoxic

to mammalian cells, an undesirable feature. In the past, counterscreening utilizing this method

when screening for other parasite targets has been extremely helpful in ruling out non-specific

cytotoxic compounds, and, hence we sought to apply the same principle here.15

This assay employs Jurkat cells, a human T-cell line originally isolated from an adolescent male with T cell leukemia. The cells are grown in suspension which facilitates tissue culture and preparation for dispensing in the assay. The end-point assay presented here employed a similar reagent to the primary assay, CellTiter-Glo luminescent reagent (Promega Corp. Part G7573), which contains luciferase enzyme to catalyze the oxidation of beetle luciferin to oxyluciferin and light in the presence of ATP, in this case from Jurkat cells. Since metabolically active cells produce

ATP, an increase in the number of dead or dying cells will correlate with a reduction in ATP levels.

As designed, compounds that inhibit cell viability and reduce intracellular ATP will reduce the catalytic conversion of luciferin into oxyluciferin, resulting in decreased luciferase activity and well luminescence. This assay included doxorubicin as a positive control, an antibiotic used as an anti-cancer drug which elicits an appropriate anti-proliferative effect on Jurkat cells.99 Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nominal test concentration of 83.3 µM.

Jurkat cells (clone E6.1; ATCC Cat# TIB-152) were routinely cultured in suspension in T-175

0 standing flasks at 37 C in 95% relative humidity (RH) at 5% CO2. Media consisted of RPMI-1640

containing 10% dialyzed fetal bovine serum, 0.1 mM NEAA, 1mM Sodium Pyruvate, 25mM

HEPES, 5mM L Glutamine, and 1x antibiotic.

163 Prior to the start of the assay, cells were suspended to a concentration of 100,000 cells/ml in media.

To start the assay, 5 µL of media was dispensed into the first two columns of a 1536 well plate

and 5 µL of cell suspension was dispensed to the remaining wells (500 cells per well). The assay

was started immediately by dispensing 42 nL of test compound in DMSO, Doxorubicin (8 µM

final concentration) or DMSO alone (0.6% final concentration) to the appropriate wells. The plates

0 were then incubated for 48 hours at 37 C, 5% CO2 and 95% RH).

Following the two-day incubation, plates were equilibrated to room temperature for 10 minutes

and 5 µL of CellTiter-Glo reagent was added to each well. Plates were centrifuged and incubated

at room temperature for 10 minutes. Well luminescence was measured on the ViewLux plate reader. The percent inhibition for each compound was calculated exactly as it was for the primary

HTS assay using the following controls: Test Compound is defined as wells containing test

compound, Low Control is defined as wells containing DMSO only (0% inhibition), and High

Control is defined as wells containing 8 µM doxorubicin (100% inhibition).

Structure

Results

Since all HTS at Scripps is done in 1536-well format our former 96-well method was further

miniaturized to optimize conditions based on Z’ value and reproducible response to methylene

blue, a known inhibitor of malaria (Table 1).12,89 In particular, we optimized the number of gametocytes necessary per well to remain in the linear range of the detection modality, the incubation time, and further validated for HTS in the context of our fully automated screening system. This resulted in using 250 gametocytes per well at >90% viability, immediately adding test compound in 75:25 DMSO:H2O, and incubating for 24hrs prior to adding BacTiter-Glo

164 luminescence detection reagent. These conditions led to reproducible Z’ factor averaging 0.53±0.1

over the course of 124 assay test plates. After testing >153,000 compounds at ~11 µM, we identified 100 actives using a cut-off set at >38% inhibition (Figure 2). This effectively sets the hit cut-off such that it approaches the level of the sample field. Still, the hit rate was very low, observed as 0.1% of the compounds. This is further reflected by the average Z score assessed over the same course of plates equal to 0.60±0.05. Here Z factor is calculated using the method to generate Z’ but incorporates the use of the sample field in the expression used in place of the low controls. As such, achieving lower Z scores, such as those <0.5, would represent higher activity of

the samples tested, which isn’t the case here.

Considering the low number of actives identified, we proceeded immediately to the concentration-

response curve (CRC) determination phase, avoiding the expenditure of time and cost associated with the typical confirmation and counterscreen phase. Since this biological target is of major significance, difficult to hit, and the NIH repository allowed for up to 275 compounds to proceed to CRCs we expanded the hit list to include the top 275 molecules for testing. Of those, 244 were available and subsequently tested in both the primary assay and counterscreen assay for CRC determination. Of the 244 molecules, only two compounds were identified with moderate potency and selectivity (see Table 2). These two molecules, SR-01000751192 and SR-01000471307 were found to have potencies <10 µM in the whole gametocyte assay (Figure 3). Only SR-01000751192 exhibited minimal cytotoxicity when tested in the Jurkat T-cell toxicity assay (>83 µM), an example of an HTS qualified assay for cytotoxicity profiling.15

After validation of the hit compounds from dry powders that were originally identified from the

high-throughput screening conditions and prior to moving into serious SAR optimization where

analogs are typically obtained via laboratory synthesis, a routine procedure of analog-by-catalog

165 (ABC) is usually performed to streamline the process. In this step, structurally close analogs are

purchased from different commercially available sources that provide a basic idea to map the

structural changes and their correlate with inhibitory activities. Often, this process provides useful

insights towards which directions the structural changes should be performed to improve the

potency before the onset of SAR optimization using synthetic efforts. In this project, we carried

out a quick ABC process by purchasing analogs that are close structural analogs of the hit

compound SR-01000751192 with single-, double-, and triple-point changes (as summarized in the

attached files). Note that SR-01000751192 provided batch-to-batch reproducibility as identified

from samples from the solid physical state. A raw 4 parameter curve fit for this compound yield nearly identical IC50s (7.1µM -batch2 vs. 8 µM-original hit). A small set of analogs (13) were obtained as fresh samples from the solid physical state. The late-stage SAR assay performed well with Z’ values averaging better than 0.6, and the methylene blue control worked as expected. The counterscreen assay also behaved as expected, with consistent Z’ scores, and response to the control doxorubicin. Unfortunately, improved anti-gametocydial activity was not observed for any of the 13 compounds purchased for initial SAR, although the compound sample from the solid physical state for the original compound hit did recapitulate the activity observed previously (see supplemental Table 1). We therefore conclude that changing with pyridyl substituent at the position 3 of the pyrazole with phenyl, substituted phenyl, and cyclopropyl groups led to diminished activities. We also observed that changing the thiazolyl structure by adding substituents, or additional heteroatom wasn’t tolerated.

Discussion

This work represents the first report of a large-scale HTS campaign completed in 1536 well format against late-stage P. falciparum gametocytes. Most HTS efforts targeting P. falciparum are

166 directed at the liver and blood stages of the parasite life cycle (Figure 1). Some involve target based approaches such as isolation of purified proteins and screening for protease inhibitors that may block the active site for its respective substrate.8, 9 Others that have the potential to identify transmission blocking molecule are low throughput and or non-homogenous in assay format making them ill-suited for large scale HTS.86 As a critical biological target of unmet therapeutic need, we set the bar high in terms of accepting any compound hit of appreciable activity to proceed for further testing. This meant incorporating a conservative primary HTS cut-off followed by a rather relaxed 10 µM IC50 cut-off in the late-stage analysis. While our efforts were in progress, we overcame a few key hurdles. During the HTS of the gametocyte project there was an unavoidable but, significant batch-to-batch difference in the gametocyte numbers and viability which was overcome by pooling the batches at the point of HTS testing. This afforded us with acceptable Z’ scores. Methylene blue, while not a good anti-malarial drug due to side effects in humans, was used as a pintool-transferred control on each plate and was also tested for anti-gametocidal pharmacologic response as a CRC control during each experiment and during each run. It is notable that the Z and Z’ values are nearly overlapping, which simply indicates the overall activity of the test compound was very low. The initial hits, while low in efficacy, do consistently reproduce activity in both labs (Scripps and QIMR Berghofer Medical Research Institute (QIMRB). While it is unusual to obtain such a low hit rate against such a large number of diverse compounds tested, namely the Molecular Library Probe Production Centers Network (MLPCN) library which has yielded multiple clinical drugs,91 this observation highlights the complexity and natural resistance, or refractiveness, of the gametocytes to the cytotoxic effects of small molecules, similar to what has been observed by others to date. Incorporating a cytotoxicity counterscreen was effective in focusing our attention on SR-01000751192 and SR-01000599986, but with the limited hit set may

167 not be as useful at this stage as originally planned. Ultimately, we were successful in completing

the HTS campaign against this difficult target and there were no insurmountable challenges

encountered.

This work involved a multi-institute and multi-content initiative that was supported by the

MLPCN. One of the mains goals of the MLPCN was to identify novel molecular probes; i.e.

ligands that supersede the activity of all prior art in all assays of relevance to support such an

achievement. So our goal was to obtain a series of small molecules that met these criteria. Based

on this outcome, a probe was not identified for this project which would have been defined as a

compound the elicited an anti-gametocidal effect < 500nM IC50 versus the primary assays and

which produced reasonable potency in the low-throughput mechanism-of-action assays, such as

the RBC infectivity assay performed at QIMRB.

An alternative approach that has shown promise by others incorporated fragment based screening against biochemical targets in tandem with phenotypic assays.92,93 Perhaps, armed with a

biochemical assay directly targeting gametocytes, this hybrid approach would have helped here

but, to our knowledge none such exist that are HTS amenable. We conclude that this method of

HTS provides a path forward for future HTS, perhaps testing larger more diverse libraries for anti-

gametocidal inhibitor identification and development.

Acknowledgements

We thank Pierre Baillargeon and Lina DeLuca (Scripps Florida) for compound management. This

work was supported by the Molecular Library Probe Production Centers Network (MLPCN) Grant

Proposal Number 1R21NS075609-01 and MLPCN funding for the University of Kansas

Specialized Chemistry Center (U54HG005031).

168 References

1. WHO. World Malaria Report 2017. ISBN: 978 92 4 156552 3. http://www.who.int/malaria/publications/world-malaria-report-2017/report/en/

2. Feachem, R. G.; Phillips, A. A. Malaria: 2 years in the fast lane. Lancet 2009, 373 (9673), 1409-11.

3. Group, M. E. Shrinking the Malaria Map: A Prospectus on Malaria Elimination. 2011.

4. Gething, P. W.; Elyazar, I. R.; Moyes, C. L.; et al. A long neglected world malaria map: Plasmodium vivax endemicity in 2010. PLoS neglected tropical diseases 2012, 6 (9), e1814.

5. Olotu, A.; Fegan, G.; Wambua, J.; et al. Seven-Year Efficacy of RTS,S/AS01 Malaria Vaccine among Young African Children. N Engl J Med 2016, 374 (26), 2519-29.

6. Moorthy, V. S.; Okwo-Bele, J. M. Final results from a pivotal phase 3 malaria vaccine trial. Lancet 2015, 386(9988), 5-7.

7. Rts, S. C. T. P. Efficacy and safety of RTS,S/AS01 malaria vaccine with or without a booster dose in infants and children in Africa: final results of a phase 3, individually randomised, controlled trial. Lancet 2015, 386(9988), 31-45.

8. Chang, H. H.; Childs, L. M.; Buckee, C. O. Variation in infection length and superinfection enhance selection efficiency in the human malaria parasite. Sci Rep 2016, 6, 26370.

9. Hawking, F. The clock of the malaria parasite. Scientific American 1970, 222 (6), 123-31.

10. Sinden, R. E. Gametocytogenesis of Plasmodium falciparum in vitro: an electron microscopic study. Parasitology 1982, 84 (1), 1-11.

11. Bousema, T.; Drakeley, C. Epidemiology and infectivity of Plasmodium falciparum and Plasmodium vivax gametocytes in relation to malaria control and elimination. Clinical microbiology reviews 2011, 24 (2), 377-410.

12. Lang-Unnasch, N.; Murphy, A. D. Metabolic changes of the malaria parasite during the transition from the human to the mosquito host. Annual review of microbiology 1998, 52, 561-90.

13. Peatey, C. L.; Spicer, T. P.; Hodder, P. S.; et al. A high-throughput assay for the identification of drugs against late-stage Plasmodium falciparum gametocytes. Molecular and biochemical parasitology 2011, 180 (2), 127-31.

14. Lamour, S. D.; Straschil, U.; Saric, J.; et al. Changes in metabolic phenotypes of Plasmodium falciparum in vitro cultures during gametocyte development. Malaria journal 2014, 13, 468.

15. White, N. J.; Ashley, E. A.; Recht, J.; et al. Assessment of therapeutic responses to

169 gametocytocidal drugs in Plasmodium falciparum malaria. Malaria journal 2014, 13, 483.

16. Llanos-Cuentas, A.; Lacerda, M. V.; Rueangweerayut, R.; et al. Tafenoquine plus chloroquine for the treatment and relapse prevention of Plasmodium vivax malaria (DETECTIVE): a multicentre, double-blind, randomised, phase 2b dose-selection study. Lancet 2014, 383 (9922), 1049-58.

17. Dixon, M. W.; Peatey, C. L.; Gardiner, D. L.; et al. A green fluorescent protein-based assay for determining gametocyte production in Plasmodium falciparum. Molecular and biochemical parasitology 2009, 163 (2), 123-6.

18. Peatey, C. L.; Dixon, M. W.; Gardiner, D. L.; et al. Temporal evaluation of commitment to sexual development in Plasmodium falciparum. Malaria journal 2013, 12, 134.

19. Lucantoni, L.; Duffy, S.; Adjalley, S. H.; et al. Identification of MMV malaria box inhibitors of plasmodium falciparum early-stage gametocytes using a luciferase-based high- throughput assay. Antimicrobial agents and chemotherapy 2013, 57 (12), 6050-62.

20. Duffy, S.; Avery, V. M. Identification of inhibitors of Plasmodium falciparum gametocyte development. Malaria journal 2013, 12, 408.

21. Plouffe, D. M.; Wree, M.; Du, A. Y.; et al. High-Throughput Assay and Discovery of Small Molecules that Interrupt Malaria Transmission. Cell Host Microbe 2016, 19 (1), 114-26.

22. Zhang, J. H.; Chung, T. D.; Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of biomolecular screening 1999, 4 (2), 67-73.

23. Smith, E.; Giuliano, K. A.; Shumate, J.; et al. A Homogeneous Cell-Based Halide-Sensitive Yellow Fluorescence Protein Assay to Identify Modulators of the Cystic Fibrosis Transmembrane Conductance Regulator Ion Channel. Assay Drug Dev Technol 2017, 15 (8), 395-406.

24. Pedro-Rosa, L.; Buckner, F. S.; Ranade, R. M.; et al. Identification of potent inhibitors of the Trypanosoma brucei methionyl-tRNA synthetase via high-throughput orthogonal screening. Journal of biomolecular screening 2015, 20 (1), 122-30.

25. Mendivil-Perez, M.; Velez-Pardo, C.; Jimenez-Del-Rio, M. Doxorubicin induces apoptosis in Jurkat cells by mitochondria-dependent and mitochondria-independent mechanisms under normoxic and hypoxic conditions. Anticancer Drugs 2015, 26 (6), 583-98.

26. Ehrhardt, K.; Davioud-Charvet, E.; Ke, H.; et al. The antimalarial activities of methylene blue and the 1,4-naphthoquinone 3-[4-(trifluoromethyl)benzyl]-menadione are not due to inhibition of the mitochondrial electron transport chain. Antimicrobial agents and chemotherapy 2013, 57 (5), 2114-20.

27. Spicer, T.; Fernandez-Vega, V.; Chase, P.; et al. Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening. Journal of biomolecular screening 2014, 19 (7), 1107-

170 1115.

28. Schoenen, F. J.; Weiner, W. S.; Baillargeon, P.; et al. Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase. In Probe Reports from the NIH Molecular Libraries Program; Bethesda (MD), 2010.

29. Schreiber, S. L.; Kotz, J. D.; Li, M.; et al. Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes. Cell 2015, 161 (6), 1252-65.

30. Blaazer, A. R.; Orrling, K. M.; Shanmugham, A.; et al. Fragment-based screening in tandem with phenotypic screening provides novel antiparasitic hits. Journal of biomolecular screening 2015, 20 (1), 131-40.

31. Vu, H.; Roullier, C.; Campitelli, M.; et al. Plasmodium gametocyte inhibition identified from a natural-product-based fragment library. ACS Chem Biol 2013, 8 (12), 2654-9.

Table 1. Stepwise Protocol for the 1536-Well Plate Live/Dead Gametocyte Assay

Order Step Condition Comments

Revitalization of Dilute to 62,500 gametocytes per milliliter in RPMI 1640 supplemented with Thaw frozen stock late-stage 10% human serum, 0.2% sodium bicarbonate and 10ug/ml of gentamycin 1 gametocytes

2 Dispense gametocytes 4μL/well 250 cells/well into 1536-well plates (Corning, part 7254)

3 Pin compounds/controls 44nL Compounds pinned at ~10µM final concentration in ~1% DMSO

4 Incubation 24 hours 37 °C, 95% relative humidity and 5% CO2

5 Dispense BacTiter-Glo 4µl/well Luminescence substrate is stable at RT

RT incubation allows for plates to equilibrate to alleviate temperature Incubation 10 minutes 6 gradient prior to reading

Plates were centrifuged and luminescence was measured on aViewLux Read Luminescence microplate reader (PerkinElmer, Turku, Finland) using a luminscence 7 protocol that utilized a 30 seconds exposure time

Table 2: Summary of the Ultra-HTS campaign to Identify Inhibitors of Late Stage Plasmodium falciparum Gametocytes

Number of Number of Selected PubChem Compounds Selection Compounds (Hit Step Screen Type Readout Target AID Tested Criteria Rate) Z' S/B

a 1a Primary Luminescence Gametocytes 743093 153,343 38.12% 100 (0.1%) 0.53±0.15 28±8

171 2a Dose-response Luminescence Gametocytes 1117278 244 IC50 < 10µM 2 (1.0%) 0.80±0.02 48±1

2b Dose-response Luminescence Jurkat T-Cells 1117277 244 IC50 < 10µM 5 (2.0%) 0.87±0.02 31±2

b 3a MedChem Luminescence Gametocytes 1159507 13 Chemist 0 (0%) 0.88±0.03 14±1

aThe primary screen hit cutoff was calculated using the interval-based cutoff. bDue to the novelty of this target, compounds with any observable activity in the Concentration- Response assay, and not in the counterscreen, were of interest for follow-up.

Figure Legends:

Figure 1: (A) Lifecycle of Plasmodium falciparum. Gametocytes are transmitted to the mosquito vector during a blood feed. The point in the lifecycle where transmission-blocking therapies may be effective is annotated with the large red X. (1B) the five stages of gametocyte development of which this HTS effort targets stages IV and V.

Figure 2: (A) Scatterplot of the data from the primary HTS campaign. Wells were treated with either test compounds (black), no gametocytes (high control=red) or gametocytes only (low control=green). The hit cutoff is shown as a dashed line at 38% inhibition. Titration results of the known anti-gametocyte cytotoxic compound methylene blue, N=4 separate experiments, N=16

wells per replicate point, error bars are shown. The average calculated IC50 was 14.7±6.6µM.

Figure 3: Structure-activity relationship of the top active molecules identified as hits in this HTS campaign. Compounds are ranked with most potent first. Data has been normalized to “no gametocytes” as 100% inhibition vs. “DMSO and gametocytes” only. Data is plotted as log M on the x-axis versus percent inhibition on the Y-axis.

172 Figure 1:

A.

B.

I - II II-III III-IV V Female V Male

Early stage gametocytes Mature Stage

173

Figure 2:

A.

High Control

Compounds

Single point % Inhibition 350 B. Low Control Methylene Blue 300 250 200

RLU 150 100 50 0 -7 -6 -5 -4 Well Number [Log M]

174

Figure 3:

175 Analytical Experiments for 13 analogs tested post HTS for the Gametocidal Project

General Experimental Section

Purity of all final compounds was confirmed by HPLC/MS analysis and determined to be > 90%. 1H and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126 MHz respectively) in CDCl3 (residual internal standard CHCl3 = δ 7.26), DMSO-d6 (residual internal standard CD3SOCD2H = δ 2.50), or acetone-d6 (residual internal standard CD3COCD2H = δ 2.05). The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, bs = broad singlet, bm = broad multiplet = doublet, t = triplet, q = quartet, p = pentuplet, dd = doublet of doublet, ddd = doublet of doublet of doublet, dt = doublet of triplet, td = triplet of doublet, tt = triplet of triplet, and m = multiplet.

HPLC/MS analysis was carried out with gradient elution (5% CH3CN to 100% CH3CN) on an Agilent 1200 RRLC with a photodiode array UV detector and an Agilent 6224 TOF mass spectrometer (also used to produce high resolution mass spectra). Automated preparative RP HPLC purification was carried out by Mass Directed Fractionation with gradient elution (a narrow CH3CN gradient was chosen based on the retention time of the target from LCMS analysis of the crude sample) on an Agilent 1200 instrument with photodiode array detector, an Agilent 6120 quadrupole mass spectrometer, and a HTPAL LEAP autosampler. Fractions were triggered using an MS and UV threshold determined by HPLC/MS analysis of the crude sample. One of two column/mobile phase conditions were chosen for both analysis and purification to promote the targets neutral state: 0.02% formic acid with Waters Atlantis T3 5um, 19 x 150mm (Prep scale), Waters Atlantis T3 1.7um, 2.1 x 50mm (Analytical Scale); pH 9.8 NH4OH with Waters XBridge C18 5um, 19 x 150mm (Prep scale), Waters BEH C-18 1.7um, 2.1 x 50mm (Analytical Scale).

All the following compound analogs were commercially available. Compound purity was measured on the basis of peak integration (area under the curve) from UV/vis absorbance (at 214 nm), and compound identity was determined on the basis of high resolution mass analysis and 1H NMR spectroscopy. S O N N H N N

KSC-439-001 1-phenyl-N-(thiazol-2-yl)-3-(p-tolyl)-1H-pyrazole-4-carboxamide: Physical state: white solid; 1 Purity: 99%; H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 9.29 (s, 1H), 7.93–7.84 (m, 2H), 7.74–7.67 (m, 2H), 7.64–7.55 (m, 2H), 7.54 (d, J = 3.6 Hz, 1H), 7.46–7.37 (m, 1H), 7.31–7.17 (m, + 3H), 2.37 (s, 3H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C20H17N4OS 361.1118; Found 361.1111.

176 KSC-439-001.1.fid in DMSO at 400.23 MHz 12.3418 9.2911 7.9054 7.9025 7.9008 7.8883 7.8862 7.8836 7.8809 7.7194 7.7149 7.7037 7.6990 7.6113 7.6091 7.6060 7.5980 7.5926 7.5895 7.5850 7.5759 7.5734 7.5712 7.5434 7.5345 7.4388 7.4360 7.4332 7.4211 7.4175 7.4156 7.4139 7.4017 7.3988 7.3961 7.2807 7.2789 7.2773 7.2755 7.2733 7.2641 7.2589 7.2499 3.3325 HDO 2.5095 DMSO 2.5048 DMSO 2.5003 DMSO 2.4956 DMSO 2.4909 DMSO 2.3669

S O N NH H NMR (400 MHz, DMSO- d ) δ 12.34 (s, 1H), 9.29 (s, 1H), 7.93 – 7.84 N 1 (m, 2H), 7.74 – 7.67 (m, 2H), 7.64 – 7.556 (m, 2H), 7.54 (d, J = 3.6 Hz, 1H), N 7.46 – 7.37 (m, 1H), 7.31 – 7.17 (m, 3H), 2.37 (s, 3H).

H C 3 1.00 0.98 1.99 2.00 2.01 1.01 1.01 2.96 2.99

3.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

177 S O N N H N N N

KSC-439-002 1-phenyl-3-(pyridin-3-yl)-N-(thiazol-2-yl)-1H-pyrazole-4-carboxamide:

1 Physical state: white solid; Purity: 100%; H NMR (400 MHz, DMSO-d6) δ 12.43 (s, 1H), 9.42 (s, 1H), 8.98 (dd, J = 2.3, 0.9 Hz, 1H), 8.63 (dd, J = 4.8, 1.7 Hz, 1H), 8.21 (dt, J = 7.9, 1.9 Hz, 1H), 7.96–7.75 (m, 2H), 7.69–7.57 (m, 2H), 7.55 (d, J = 3.5 Hz, 1H), 7.52 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 7.49–7.37 (m, 1H), 7.27 (d, J = 3.6 Hz, 1H); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C18H14N5OS 348.0914; Found 348.0910.

KSC-439-002.1.fid in DMSO at 400.23 MHz 2.4911 DMSO 2.4956 DMSO 2.5002 DMSO 2.5049 DMSO 2.5095 DMSO 3.3358 HDO 7.2630 7.2719 7.4263 7.4291 7.4320 7.4439 7.4477 7.4515 7.4633 7.4662 7.4691 7.4991 7.5014 7.5111 7.5134 7.5188 7.5210 7.5308 7.5330 7.5459 7.5548 7.5929 7.5971 7.6114 7.6144 7.6280 7.6328 7.8948 7.8975 7.9003 7.9025 7.9116 7.9160 7.9192 8.1907 8.1952 8.2005 8.2103 8.2154 8.2203 8.6244 8.6287 8.6365 8.6407 8.9740 8.9763 8.9797 8.9819 9.4181 12.4288

S O N NH

N

N N

H NMR (400 MHz, DMSO- d ) δ 12.43 (s, 1H), 9.42 (s, 1H), 1 8.98 (dd, J = 2.3, 0.9 Hz, 1H), 8.63 (dd,6 J = 4.8, 1.7 Hz, 1H), 8.21 (dt, J = 7.9, 1.9 Hz, 1H), 7.96 – 7.75 (m, 2H), 7.69 – 7.57 (m, 2H), 7.55 (d, J = 3.5 Hz, 1H), 7.52 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 7.49 – 7.37 (m, 1H), 7.27 (d, J = 3.6 Hz, 1H). 1.03 1.05 1.09 0.97 2.06 2.05 1.05 1.00 0.96 1.03 1.00 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0

178 S O N N H N N

KSC-439-003 3-cyclopropyl-1-phenyl-N-(thiazol-2-yl)-1H-pyrazole-5-carboxamide: Physical state: white 1 solid; Purity: 100%; H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H), 7.84–7.71 (m, 2H), 7.65– 7.56 (m, 2H), 7.55–7.47 (m, 2H), 7.27 (d, J = 3.5 Hz, 1H), 6.82 (d, J = 0.7 Hz, 1H), 1.95–1.74 (m, 1H), 1.07–0.93 (m, 2H), 0.86–0.72 (m, 2H); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C16H15N4OS 311.0961; Found 311.0956.

KSC-439-003.1.fid in DMSO at 400.23 MHz 0.7922 0.8031 0.8048 0.8089 0.8157 0.8185 0.8215 0.8314 0.9782 0.9880 0.9939 0.9989 1.0048 1.0088 1.0147 1.0257 1.0281 1.8794 1.8812 1.8874 1.8891 1.9002 1.9020 1.9128 1.9146 1.9210 2.4910 DMSO 2.4956 DMSO 2.5003 DMSO 2.5049 DMSO 2.5095 DMSO 3.3310 HDO 6.8202 6.8219 7.2650 7.2738 7.4913 7.5045 7.5093 7.5108 7.5147 7.5251 7.5292 7.5380 7.5794 7.5807 7.5822 7.5847 7.5940 7.5970 7.6004 7.6021 7.6041 7.6068 7.6141 7.6187 7.6209 7.7647 7.7711 7.7737 7.7766 7.7785 7.7868 7.7916 7.7952 7.7984 12.1111

S H NMR (400 MHz, DMSO- d ) δ 12.11 (s, 1H), 7.84 – 7.71 (m, 2H), O 1 7.65 – 7.56 (m, 2H), 7.55 – 7.47 (m, 2H),6 7.27 (d, J = 3.5 Hz, 1H), N NH 6.82 (d, J = 0.7 Hz, 1H), 1.95 – 1.74 (m, 1H), 1.07 – 0.93 (m, 2H), 0.86 – 0.72 (m, 2H). N N 2.01 2.15 1.03 1.00 0.95 2.00 2.02 2.05 1.00

12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

179 S O N N H N N

F

KSC-439-004 3-(4-fluorophenyl)-1-phenyl-N-(thiazol-2-yl)-1H-pyrazole-4-carboxamide: Physical state: 1 white solid; Purity: 98%; H NMR (400 MHz, DMSO-d6) δ 12.38 (s, 1H), 9.34 (s, 1H), 8.01–7.75 (m, 4H), 7.67–7.55 (m, 2H), 7.54 (d, J = 3.6 Hz, 1H), 7.50–7.38 (m, 1H), 7.36–7.25 (m, 2H), 7.26 + (d, J = 3.6 Hz, 1H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C19H14FN4OS 365.0867; Found 365.0865.

KSC-439-004.1.fid in DMSO at 400.23 MHz 2.4909 DMSO 2.4956 DMSO 2.5001 DMSO 2.5048 DMSO 2.5095 DMSO 3.3311 HDO 7.2542 7.2631 7.2764 7.2840 7.2893 7.2988 7.3007 7.3063 7.3118 7.3139 7.3234 7.3287 7.3363 7.4081 7.4108 7.4134 7.4254 7.4275 7.4292 7.4328 7.4450 7.4478 7.4506 7.5401 7.5490 7.5731 7.5784 7.5807 7.5832 7.5921 7.5959 7.5974 7.5999 7.6053 7.6132 7.6164 7.6185 7.6232 7.8684 7.8740 7.8777 7.8828 7.8857 7.8888 7.8908 7.8994 7.9031 7.9048 7.9079 7.9120 9.3414 12.3756

S O

N NH

N

N

F

H NMR (400 MHz, DMSO- d ) δ 12.38 (s, 1H), 9.34 (s, 1H), 8.01 1 – 7.75 (m, 4H), 7.67 – 7.55 (m, 2H), 7.546 (d, J = 3.6 Hz, 1H), 7.50 – 7.38 (m, 1H), 7.36 – 7.25 (m, 2H), 7.26 (d, J = 3.6 Hz, 1H). 1.00 2.10 1.06 0.97 2.09 4.20 1.04 1.00

13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

180 S O N N H N N

Cl

KSC-439-005

3-(4-chlorophenyl)-1-phenyl-N-(thiazol-2-yl)-1H-pyrazole-4-carboxamide: Physical state: 1 white solid; Purity: 100%; H NMR (400 MHz, DMSO-d6) δ 12.40 (s, 1H), 9.35 (s, 1H), 7.94– 7.85 (m, 2H), 7.90–7.83 (m, 2H), 7.65–7.55 (m, 2H), 7.58–7.49 (m, 3H), 7.48–7.38 (m, 1H), 7.26 + (d, J = 3.6 Hz, 1H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C19H14ClN4OS 381.0571; 381.0569.

KSC-439-005.1.fid in DMSO at 400.23 MHz 2.4907 DMSO 2.4953 DMSO 2.5001 DMSO 2.5048 DMSO 2.5094 DMSO 3.3309 HDO 7.2593 7.2682 7.4123 7.4151 7.4179 7.4296 7.4336 7.4375 7.4493 7.4522 7.4550 7.5220 7.5286 7.5335 7.5419 7.5451 7.5505 7.5566 7.5750 7.5803 7.5827 7.5851 7.5946 7.5987 7.6017 7.6072 7.6152 7.6203 7.6253 7.8520 7.8571 7.8685 7.8735 7.8799 7.8836 7.8862 7.8890 7.8911 7.9000 7.9049 7.9082 7.9121 9.3453 12.3995

S O N NH H NMR (400 MHz, DMSO- d ) δ 12.40 (s, 1H), 9.35 (s, N 1 1H), 7.94 – 7.85 (m, 2H), 7.90 – 7.83 (m,6 2H), 7.65 – 7.55 N (m, 2H), 7.58 – 7.49 (m, 3H), 7.48 – 7.38 (m, 1H), 7.26 (d, J = 3.6 Hz, 1H). Cl 0.96 1.03 2.96 2.05 1.97 2.01 1.02 1.00

13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

181 S O N N H N N

O KSC-439-006 3-(4-methoxyphenyl)-1-phenyl-N-(thiazol-2-yl)-1H-pyrazole-4-carboxamide: Physical state: 1 white solid; Purity: 93%; H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 9.31 (s, 1H), 7.95–7.87 (m, 2H), 7.85–7.73 (m, 2H), 7.65–7.55 (m, 2H), 7.55 (d, J = 3.5 Hz, 1H), 7.48–7.37 (m, 1H), 7.27 (d, J = 3.6 Hz, 1H), 7.08–6.96 (m, 2H), 3.83 (s, 3H); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C20H17N4O2S 377.1067; Found 377.1071.

KSC-439-006.1.fid in DMSO at 400.23 MHz 12.3413 7.6234 7.6213 7.6183 7.6103 7.6047 7.6021 7.5987 7.5882 7.5856 7.5834 7.5782 7.5587 7.5498 7.4490 7.4463 7.4435 7.4314 7.4277 7.4258 7.4240 7.4119 7.4091 7.4063 7.2724 7.2635 7.0588 7.0514 7.0461 7.0345 7.0293 7.0218 3.8311 3.3463 HDO 2.5235 DMSO 2.5188 DMSO 2.5141 DMSO 2.5095 DMSO 2.5049 DMSO 9.3058 7.9207 7.9168 7.9137 7.9119 7.9087 7.8998 7.8976 7.8948 7.8922 7.8209 7.8135 7.8083 7.7967 7.7913 7.7841 7.6285

S O N NH

N N

H3C O

H NMR (400 MHz, DMSO- d ) δ 12.34 (s, 1H), 9.31 (s, 1H), 7.95 – 7.87 1 (m, 2H), 7.85 – 7.73 (m, 2H), 7.65 – 7.556 (m, 2H), 7.55 (d, J = 3.5 Hz, 1H), 7.48 – 7.37 (m, 1H), 7.27 (d, J = 3.6 Hz, 1H), 7.08 – 6.96 (m, 2H), 3.83 (s, 3H). 1.00 0.98 2.00 1.91 2.02 0.97 1.01 0.95 1.95 2.88

13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

182 S O N N H N N

KSC-439-007 1,3-diphenyl-N-(thiazol-2-yl)-1H-pyrazole-5-carboxamide: Physical state: white solid; Purity: 1 96%; H NMR (400 MHz, DMSO-d6) δ 12.34 (s, 1H), 7.56 (d, J = 3.6 Hz, 1H), 7.50–7.43 (m, 3H), 7.43–7.34 (m, 5H), 7.33 (s, 1H), 7.31–7.23 (m, 3H); HRMS (ESI-TOF) m/z: [M + H]+ Calcd for C19H15N4OS 347.0961; Found 347.0962.

KSC-439-007.1.fid in DMSO at 400.23 MHz 12.3396 7.5638 7.5548 7.4894 7.4847 7.4822 7.4766 7.4737 7.4712 7.4654 7.4631 7.4601 7.4575 7.4543 7.4501 7.4471 7.4433 7.4178 7.4116 7.4066 7.4048 7.4014 7.3975 7.3937 7.3899 7.3865 7.3845 7.3341 7.3039 7.3008 7.2950 7.2893 7.2857 7.2820 7.2799 7.2766 3.3322 HDO 2.5095 DMSO 2.5048 DMSO 2.5001 DMSO 2.4956 DMSO 2.4909 DMSO

S O N NH H NMR (400 MHz, DMSO- d ) δ 12.34 (s, 1H), 7.56 (d, J = 1 3.6 Hz, 1H), 7.50 – 7.43 (m, 3H), 7.43 –6 7.34 (m, 5H), 7.33 (s, N N 1H), 7.31 – 7.23 (m, 3H). 1.00 1.01 3.03 5.06 1.04 3.00 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.

183 S O N N H N N N

KSC-439-008 1-phenyl-3-(pyridin-4-yl)-N-(thiazol-2-yl)-1H-pyrazole-4-carboxamide: Physical state: white 1 solid; Purity: 98%; H NMR (400 MHz, DMSO-d6) δ 12.48 (s, 1H), 9.39 (s, 1H), 8.83–8.48 (m, 2H), 7.95–7.88 (m, 2H), 7.87–7.75 (m, 2H), 7.66–7.56 (m, 2H), 7.56 (d, J = 3.5 Hz, 1H), 7.50– + 7.38 (m, 1H), 7.28 (d, J = 3.6 Hz, 1H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C18H14N5OS 348.0914; Found 348.0913.

KSC-439-008.1.fid in DMSO at 400.23 MHz 12.4785 9.3851 8.6878 8.6837 8.6767 8.6726 7.9274 7.9243 7.9227 7.9192 7.9104 7.9082 7.9054 7.9028 7.8454 7.8413 7.8341 7.8300 7.6352 7.6331 7.6300 7.6220 7.6166 7.6144 7.6129 7.6088 7.5999 7.5976 7.5952 7.5624 7.5535 7.4764 7.4736 7.4708 7.4590 7.4551 7.4511 7.4395 7.4367 7.2851 7.2761 3.3336 HDO 2.5094 DMSO 2.5048 DMSO 2.5001 DMSO 2.4954 DMSO 2.4908 DMSO

S O N NH H NMR (400 MHz, DMSO- d ) δ 12.48 (s, 1H), 9.39 (s, 1H), 8.83 1 N – 8.48 (m, 2H), 7.95 – 7.88 (m, 2H), 7.876 – 7.75 (m, 2H), 7.66 – N 7.56 (m, 2H), 7.56 (d, J = 3.5 Hz, 1H), 7.50 – 7.38 (m, 1H), 7.28

N (d, J = 3.6 Hz, 1H). 1.00 1.01 2.07 2.04 2.03 2.03 1.00 1.03 0.98

13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

184 S O N N H N N N

KSC-439-009 N-(4,5-dihydrothiazol-2-yl)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxamide: Physical 1 state: white solid; Purity: 95%; H NMR (400 MHz, DMSO-d6) δ 8.98 (dd, J = 2.3, 0.9 Hz, 1H), 8.92 (s, 1H), 8.60 (dd, J = 4.8, 1.7 Hz, 1H), 8.23 (dt, J = 8.0, 1.9 Hz, 1H), 7.96–7.85 (m, 2H), 7.58– 7.52 (m, 2H), 7.48 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 7.43–7.37 (m, 1H), 3.63 (t, J = 7.9 Hz, 2H), + 3.22 (t, J = 7.9 Hz, 2H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C18H16N5OS 350.1070; Found 350.1075.

KSC-439-009.1.fid in DMSO at 400.23 MHz 8.9869 8.9848 8.9813 8.9791 8.9224 8.6035 8.5992 3.6319 3.6122 8.5915 8.5872 8.2412 8.2364 8.2314 8.2214 8.2164 8.2115 7.9424 7.9391 7.9347 7.9254 7.9234 7.9205 7.9177 7.5748 7.5699 7.5564 7.5535 7.5396 7.5351 7.5293 7.4961 7.4938 7.4841 7.4819 7.4764 7.4741 7.4644 7.4621 7.4223 7.4194 7.4167 7.4050 7.4010 7.3972 7.3853 7.3824 7.3796 3.6517 3.3339 HDO 3.2419 3.2219 3.2025 2.5094 DMSO 2.5048 DMSO 2.5002 DMSO 2.4956 DMSO 2.4909 DMSO

S O N NH

N

N N H NMR (400 MHz, DMSO- d ) δ 8.98 (dd, J = 2.3, 0.9 Hz, 1H), 1 8.92 (s, 1H), 8.60 (dd, J = 4.8, 1.76 Hz, 1H), 8.23 (dt, J = 8.0, 1.9 Hz, 1H), 7.96 – 7.85 (m, 2H), 7.58 – 7.52 (m, 2H), 7.48 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 7.43 – 7.37 (m, 1H), 3.63 (t, J = 7.9 Hz, 2H), 3.22 (t, J = 7.9 Hz, 2H). 0.94 1.03 1.00 1.02 2.10 2.13 1.17 1.16 2.02 2.18 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0

185 N O S N H N N

KSC-439-010 1-phenyl-N-(thiazol-2-yl)-3-(m-tolyl)-1H-pyrazole-4-carboxamide: Physical state: white solid; 1 Purity: 96%; H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 9.30 (s, 1H), 7.96–7.85 (m, 2H), 7.65–7.57 (m, 4H), 7.55 (d, J = 3.6 Hz, 1H), 7.46–7.40 (m, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.28– + 7.20 (m, 2H), 2.38 (s, 3H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C20H17N4OS 361.1118; Found 361.1123.

KSC-439-010.1.fid in DMSO at 400.23 MHz 2.3830 2.5049 DMSO 2.5096 DMSO 2.5141 DMSO 2.5188 DMSO 2.5235 DMSO 3.3476 HDO 7.2452 7.2480 7.2501 7.2519 7.2529 7.2552 7.2591 7.2647 7.2680 7.2715 7.2737 7.3373 7.3562 7.3752 7.4135 7.4163 7.4191 7.4310 7.4330 7.4348 7.4385 7.4504 7.4532 7.4561 7.5468 7.5557 7.5821 7.5874 7.5898 7.5922 7.5957 7.5964 7.5989 7.6005 7.6060 7.6087 7.6148 7.6164 7.6177 7.6193 7.6221 7.6275 7.6307 7.6324 7.6341 7.6368 7.8970 7.9028 7.9054 7.9081 7.9102 7.9191 7.9238 7.9273 7.9312 9.3045 12.3607

N O S NH

N CH 3 N H NMR (400 MHz, DMSO- d ) δ 12.36 (s, 1H), 9.30 (s, 1H), 7.96 – 7.85 1 (m, 2H), 7.65 – 7.57 (m, 4H), 7.55 (d, 6 J = 3.6 Hz, 1H), 7.46 – 7.40 (m, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.28 – 7.20 (m, 2H), 2.38 (s, 3H). 3.16 2.05 1.09 1.09 1.07 4.18 2.19 1.07 1.00 13.5 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0

186 S O N N H N N

KSC-439-011 N-(4,5-dihydrothiazol-2-yl)-1,3-diphenyl-1H-pyrazole-5-carboxamide: Physical state: white 1 solid; Purity: 98%; H NMR (400 MHz, DMSO-d6) δ 9.80 (s, 1H), 7.47 – 7.40 (m, 3H), 7.38 – 7.33 (m, 3H), 7.33 – 7.28 (m, 2H), 7.27 – 7.20 (m, 2H), 7.02 (s, 1H), 3.65 (t, J = 7.9 Hz, 2H); + HRMS (ESI-TOF) m/z: [M + H] Calcd for C19H17N4OS 349.1118; Found 349.1121.

KSC-439-012.1.fid in DMSO at 400.23 MHz 2.4909 DMSO 2.4955 DMSO 2.5001 DMSO 2.5048 DMSO 2.5095 DMSO 3.2313 3.2504 3.2709 3.3292 HDO 3.6319 3.6519 3.6717 7.0182 7.2341 7.2379 7.2398 7.2421 7.2445 7.2477 7.2514 7.2546 7.2584 7.2903 7.2941 7.2963 7.2998 7.3041 7.3093 7.3146 7.3484 7.3516 7.3535 7.3565 7.3597 7.3644 7.4055 7.4087 7.4149 7.4179 7.4227 7.4278 7.4323 7.4384 7.4420 9.8049

S O N NH

N N

H NMR (400 MHz, DMSO- d ) δ 9.80 (s, 1 1H), 7.47 – 7.40 (m, 3H), 7.38 – 7.33 (m,6 3H), 7.33 – 7.28 (m, 2H), 7.27 – 7.20 (m, 2H), 7.02 (s, 1H), 3.65 (t, J = 7.9 Hz, 2H). 2.35 2.25 1.00 2.22 2.18 3.28 3.30 1.06 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.

187 N O S N H N N N

KSC-439-012 N-(5-methylthiazol-2-yl)-1-phenyl-3-(pyridin-3-yl)-1H-pyrazole-4-carboxamide: Physical 1 state: white solid; Purity: 96%; H NMR (400 MHz, DMSO-d6) δ 12.24 (s, 1H), 9.38 (s, 1H), 8.97 (dd, J = 2.2, 0.9 Hz, 1H), 8.63 (dd, J = 4.8, 1.7 Hz, 1H), 8.19 (ddd, J = 7.9, 2.2, 1.7 Hz, 1H), 7.94– 7.76 (m, 2H), 7.65–7.55 (m, 2H), 7.51 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 7.49–7.38 (m, 1H), 7.28– + 7.11 (m, 1H), 2.36 (d, J = 1.3 Hz, 3H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C19H16N5OS 362.1070; Found 362.1074.

KSC-439-011.1.fid in DMSO at 400.23 MHz 2.3570 2.3603 2.4909 DMSO 2.4955 DMSO 2.5001 DMSO 2.5048 DMSO 2.5094 DMSO 3.3382 HDO 7.1991 7.2020 7.2054 7.2082 7.4194 7.4222 7.4250 7.4368 7.4408 7.4446 7.4566 7.4594 7.4622 7.4895 7.4918 7.5017 7.5039 7.5093 7.5115 7.5213 7.5236 7.5858 7.5882 7.5905 7.5995 7.6035 7.6049 7.6071 7.6125 7.6207 7.6236 7.6258 7.8839 7.8896 7.8922 7.8951 7.8972 7.9060 7.9092 7.9111 7.9142 7.9179 8.1798 8.1841 8.1856 8.1897 8.1996 8.2041 8.2054 8.2095 8.6177 8.6219 8.6297 8.6339 8.9666 8.9689 8.9722 8.9744 9.3799

H C N 3 O S NH

N

N N

H NMR (400 MHz, DMSO- d ) δ 12.24 (s, 1H), 9.38 (s, 1 1H), 8.97 (dd, J = 2.2, 0.9 Hz, 1H),6 8.63 (dd, J = 4.8, 1.7 Hz, 1H), 8.19 (ddd, J = 7.9, 2.2, 1.7 Hz, 1H), 7.94 – 7.76 (m, 2H), 7.65 – 7.55 (m, 2H), 7.51 (ddd, J = 7.9, 4.9, 0.9 Hz, 1H), 7.49 – 7.38 (m, 1H), 7.28 – 7.11 (m, 1H), 2.36 (d, J = 1.3 Hz, 3H). 3.11 0.98 1.18 1.13 2.13 2.14 1.06 1.02 0.97 1.08 1.00 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

188 S N O N N H N N

KSC-439-013 1,3-diphenyl-N-(1,3,4-thiadiazol-2-yl)-1H-pyrazole-5-carboxamide: Physical state: white 1 solid; Purity: 96%; H NMR (400 MHz, DMSO-d6) δ 9.19 (s, 1H), 7.51–7.44 (m, 3H), 7.43–7.38 + (m, 5H), 7.33 (s, 1H), 7.31–7.26 (m, 2H); HRMS (ESI-TOF) m/z: [M + H] Calcd for C18H14N5OS 348.0914; Found 348.0919.

KSC-439-013.1.fid in DMSO at 400.23 MHz 2.4909 DMSO 2.4956 DMSO 2.5001 DMSO 2.5048 DMSO 2.5094 DMSO 3.3345 HDO 7.2773 7.2810 7.2830 7.2864 7.2904 7.2953 7.2978 7.3016 7.3326 7.3836 7.3864 7.3908 7.3926 7.3951 7.4001 7.4037 7.4095 7.4153 7.4437 7.4471 7.4515 7.4549 7.4592 7.4612 7.4645 7.4668 7.4710 7.4731 7.4758 7.4782 7.4911 9.1855

S N O N NH

N N

H NMR (400 MHz, DMSO- d ) δ 9.19 (s, 1 1H), 7.51 – 7.44 (m, 3H), 7.43 – 7.38 (m,6 5H), 7.33 (s, 1H), 7.31 – 7.26 (m, 2H). 2.19 1.12 5.57 3.48 1.00 13.0 12.5 12.0 11.5 11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

189 Supplemental Table 1:

190 Supplemental Table 2:

191

192

193

194