MECHANISMS UNDERLYING POLYENE MACROLIDE MEDIATED RESCUE of GROWTH in ION CHANNEL DEFICIENT YEAST by JENNIFER HOU DISSERTATION Su

MECHANISMS UNDERLYING POLYENE MACROLIDE MEDIATED RESCUE OF GROWTH IN ION CHANNEL DEFICIENT YEAST

JENNIFER HOU

DISSERTATION

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry in the Graduate College of the University of Illinois at Urbana-Champaign, 2018

Urbana, Illinois

Doctoral Committee:

Professor Martin D. Burke, Chair Professor Robert B. Gennis Professor Peter Orlean Assistant Professor Erik Procko

ABSTRACT

Human diseases caused by missing or dysfunctional protein ion channels, known as channelopathies, affect several organs and systems of the body including that of the heart, lungs, kidneys, and nervous system. Channelopathies can result in devastating and potentially life-threatening diseases. To date, more than 30 human channelopathies are incurable. Although significant advances have been made in both gene therapy and protein replacement therapies, there is still a major need for new treatment strategies.

We questioned whether an imperfect small molecule mimic of a missing protein ion channel could be sufficient to restore physiology in protein-deficient systems.

Small molecules possess many qualities as potential therapeutics including that they are orally bio-available, cell permeable, and minimally immunogenic. Specifically for small molecule inhibitors, their primary mechanism of action is to bind to and block over- active proteins. However, in the case of unexpressed or degraded proteins where no target is available, this approach is futile. We hypothesized that through functionally interfacing with inherent networks of protein ion pumps and transporters, an imperfect small molecule ion channel could be enough to rescue physiology.

Amphotericin B (AmB) is a prime example of an ion channel forming small molecule. Clinically for more than 60 years, medical providers have utilized AmB to treat patients suffering from invasive, systemic fungal infections. Previously, AmB’s mode of cell killing was thought to be through ion channel formation. However, The Burke Group at The University of Illinois at Urbana-Champaign showed that AmB kills yeast through sterol extraction. Moreover, we found that AmB only caused cell death when the ratio of

AmB exceeded that of sterols; therefore, pre-complexation of AmB with sterols serves as

ii a strategy to ameliorate toxicity. Alternatively, to harness AmB’s ion channel forming activity, we employed this small molecule at low concentrations. Further characterization showed that AmB small molecule ion channels are unselective, wherein they conduct

+ + - - potassium (K ), sodium (Na ), chloride (Cl ), and bicarbonate (HCO3 ) ions.

We employed Saccharomyces cerevisiae (S.cerevisiae) as an outstanding model organism because this unicellular eukaryote is genetically tractable, well-annotated, and amenable to genetic deletion. Furthermore, yeast have been widely utilized to elucidate several, fundamental biological processes including cell cycle regulation, telomere maintenance, and vesicle trafficking, and to characterize the pathophysiology of human disease.

Yeast express two primary K+ transporters, Trk1 and Trk2 that are localized to the plasma membrane. Proton (H+)-ATPases Pma1 and V-ATPase utilize the energy from

ATP hydrolysis to drive H+ out of cells or into vacuoles respectively, thereby producing favorable electrochemical gradients. These gradients promote K+ flow through Trk1 and

Trk2 passive transporters into cells, to achieve cytosolic concentrations around 200-300 mM and cation storage into vacuoles. For cellular physiology, K+ is crucial for retaining intracellular pH, sustaining plasma membrane potential, and promoting enzyme activity.

Deficiencies of these integral K+ transporters result in a loss of cell growth under standard laboratory conditions (10-15 mM K+).

We found that an imperfect small molecule mimic of a missing protein ion channel, a.k.a. molecular prosthetic is sufficient to restore physiology in a protein-deficient model organism. We demonstrated that AmB both vigorously and sustainably restored physiology in K+ transporter deficient yeast (trk1Δtrk2Δ). Furthermore, we showed that

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AmB mediated rescue is ion channel dependent. Contributing to the lab’s molecular prostheses program, this thesis work has made four major contributions.

First, we predicted that small molecule mediated rescue is generalizable. Initially, we reconfirmed literature reports and demonstrated that Nystatin A1, Candicidin, and

Mepartricin permeabilize yeast. Then to test our prediction, we employed two assays: the disc diffusion and micro-broth dilution assay. We observed no rescue of growth with a

Natamycin control (does not form ion channels). In contrast, we observed restored cell growth for Nystatin A1 (optimal at 1 μM), Candicidin (8 nM), and Mepartricin (8 nM). These findings suggest the versatility of trk1Δtrk2Δ homeostatic mechanisms to functionally interface with different imperfect small molecule mimics, and thereby rescue physiology.

Second, we elucidated a mechanism by which imperfect small molecule ion channels rescue physiology in yeast. We hypothesized that small molecule mimics harness favorable electrochemical gradients to drive K+ into cells and back to physiological levels, thereby restoring cell growth in trk1Δtrk2Δ yeast. We have obtained evidence in support of each of these steps. Consistent with prior reports, trk1Δtrk2Δ yeast are hyperpolarized compared to that of WT. These gradients drive K+ into cells as demonstrated by radioactive rubidium (86Rb+, surrogate tracer for K+) influx assays. Next, we showed that through working together with the cell’s endogenous network of protein ion pumps, small molecule ion channels significantly restored total cellular K+ levels in trk1Δtrk2Δ back to that found in WT, in both a dose- and time-dependent manner. In contrast, Natamycin did not restore total cellular K+ content nor cell growth. Furthermore, chemical inhibition of H+-ATPases abrogated restoration of total cellular K+ content and

iv growth. Thus, these results provide mechanistic evidence for how small molecule ion channels rescue physiology.

Third, our findings suggest that imperfect small molecule mediated rescue of physiology is possible through functional collaboration with the cell’s inherent network of protein ion pumps and transporters. AmB and other polyene macrolide ion channels are unregulated, unrectified, unselective, and passive. However, we hypothesized that through working together with H+-ATPases (drivers) and transporters (correctors) that imperfect small molecule ion channels are sufficient to restore physiology. V-ATPase and

Pma1 generate favorable electrochemical gradients. These gradients promote K+ flux through Trk1 and Trk2 and into vacuoles. We predicted that inhibiting V-ATPase or Pma1 would abrogate small molecule mediated rescue of trk1Δtrk2Δ. To test our hypothesis, we employed three chemical inhibitors: Nocodazole (impedes microtubule dynamics),

Bafilomycin (blocks V-ATPase), and Ebselen (inhibits Pma1) against small molecule ion channel treated WT and trk1Δtrk2Δ. We observed exceptional sensitivity in small molecule rescued trk1Δtrk2Δ cells to Bafilomycin and to Ebselen blockage compared to that observed in WT. In contrast, minor differences were observed between these two treatment groups, to Nocodazole inhibition. Therefore, these results suggest that cells require active V-ATPase and Pma1, possibly to drive K+ through small molecule ion channels, for rescue of trk1Δtrk2Δ cell growth.

Next, we hypothesized that endogenous protein transporters are crucial for correcting the lack of ion selectivity of small molecule ion channels. Yeast express plasma membrane localized voltage gated K+ efflux pump Tok1, Na+(K+)/H+ antiporter Nha1, and

Na+-ATPase Ena1-5 that extrude excess K+ and Na+. We acquired a series of isogenic

v yeast mutants and conducted the liquid broth rescue assay. We observed that small molecule mimics did not recover growth in trk1Δtrk2Δnha1Δ yeast for any concentrations screened. Importantly, the nha1Δ mutant showed similar growth to that of WT. In contrast, deletion of either Tok1 (trk1Δtrk2Δtok1Δ) or Ena1-5 (trk1Δtrk2Δena1-5Δ) did not hamper small molecule mediated rescue. Thus, these findings suggest that Nha1 is important for expelling excess Na+ and/or K+ from small molecule treated trk1Δtrk2Δ.

Last, in collaboration with The Mitchell Group, we have identified several potential and uncharacterized small molecule polyene macrolides through biosynthetic gene cluster analysis. We hypothesized through interrogating the gene clusters of several, highly conserved domains including the aminotransferase, ketosynthase, etc. that we can identify novel producing organisms of polyene macrolides. Through utilizing a moderate throughput yeast screening platform, we have found several organisms that produce extracts that may contain novel polyene macrolides.

In summary, this thesis work culminates into four major findings: first, small molecule mediated rescue is generalizable to other polyene macrolide family members; second, a primary mechanism by which small molecule ion channels rescue cell growth in trk1Δtrk2Δ yeast is through restored cellular K+ content; third, small molecule mimics work in conjunction with the cell’s inherent network of protein ion pumps and transporters to restore physiology; and last, we have identified several potential and uncharacterized polyene macrolides through biosynthetic gene cluster analysis. In conclusion, these results demonstrate the potential for imperfect small molecule mimics to provide function in the case of protein ion channel deficiencies. Furthermore, these findings showcase an in-depth mechanistic pathway for how imperfect small molecule mimics restore cellular

vi physiology in protein-deficient yeast. Intriguingly, the same protein characters responsible for restoring physiology in yeast may also be at play in other eukaryotic systems.

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To Eddie

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ACKNOWLEDGEMENTS

This thesis work shows the wealth of support and resources that I was fortunate enough to have available to me during my graduate training. I thank each of you for your help and time throughout my journey.

I would like to first and foremost thank my advisor Professor Martin D. Burke for giving me the opportunity to pursue this thesis work. I thank Marty for his guidance and encouragement throughout my graduate career and scientific development. Thank you for always believing in me and challenging me to pursue difficult scientific questions and to continue to “push the boundaries” of scientific thinking forward. I greatly admire his genuine zest for science and innovation, and hope to continue his spirit of discovery one day.

Many thanks to my thesis committee Professor Robert B. Gennis, Professor James

H. Morrissey, Professor Peter Orlean, and Assistant Professor Erik Procko for your support and invaluable advice during my departmental seminars, and informal correspondences throughout the years. I really appreciated the chance to be able to ask for your suggestions and input when I was facing roadblocks in my research and to brainstorm ways to address them.

I am grateful to have the chance to work together with so many dedicated and talented undergraduate and graduate students, and post-docs in The Burke Group. I especially would like to thank Alex for training me in many lab techniques when I first started in the group. I have enjoyed working together with Alex, Tony, and Katrina on the

AmB rescue story in yeast. And more recently, it has been exciting to team up with Alice,

Rajeev, Jiabao, and Arun and to start a collaboration with Xiao Rui and Professor Douglas

Mitchell on the polyene macrolide discovery project.

I would also like to give a special thank you to Dr. Junqi Li. I valued our informal scientific discussions and having a fellow scientist to bounce around ideas with. Thank you so much to Calgary for all your help with coordinating schedules, planning, and for keeping the lab highly organized. I thank all my lab mates for making the lab an exciting and energetic environment. Each of you have helped me tremendously during my graduate years through providing feedback and suggestions during lab meetings, taking the time to edit manuscript drafts, training me on new techniques, and lending a hand.

Much of the careful planning and logistics could not have been possible without the dedication of our Biochemistry Department staff and faculty. Special thanks to Jeff

Goldberg, Cara Day, Marita Romine, and Sherry Unkraut for all your support, for your help with graduation timelines, and for your assistance with scheduling seminars and my defense. A tremendous thank you for all the behind the scenes planning that takes place, to give rise to engaging scientific seminars and talks from visiting scientists from all over the world. The chance to participate in student luncheons with invited speakers has been very enriching.

A sincere thank you to the UIUC College of Medicine staff and faculty for helping me to navigate the waters during both my graduate and medical training. I thank Dr. Jim

Slauch, Dr. Jim Hall, Dr. Nora Few, Heather Wright, and Jenni Crum for all your guidance and helpful discussions. Thank you for your advice during my advisory meetings and on planning for courses.

I would like to thank the staff members of The Institute for Genomic Biology and

The Microanalysis Lab for their suggestions and troubleshooting advice when we were first developing the viability study assays and potassium content analysis, respectively.

Thank you to Dr. Mayandi Sivaguru for his extensive knowledge on confocal microscopy that greatly aided this work. I would also like to give a big thank you to Dr. Kiran Subedi,

Crislyn Lu, and Elizabeth Eves for all their help with the ICP-MS analysis. I thank Dr.

Barbara Pilas for her assistance in developing the flow cytometry protocols. A special thank you to Dr. Sandra McMasters for her helpfulness during many aspects of my project. Thank you all for your tireless efforts and commitment to these projects.

Last, a very special thank you goes out to my friends and family. I could not have done it without you. Thank you for your unwavering support every step of the way. I will always cherish the memories that we have made here in Urbana-Champaign and hope that our paths will continue to cross where ever the future takes us.

TABLE OF CONTENTS

ABBREVIATIONS ...... xiii

CHAPTER 1: GENERAL INTRODUCTION ...... 1

CHAPTER 2: IMPERFECT SMALL MOLECULE MIMICS RESTORE CELL GROWTH IN YEAST ...... 6

CHAPTER 3: SMALL MOLECULE ION CHANNELS RESTORE PHYSIOLOGICAL ION CONTENT AND THEREBY CELL GROWTH ...... 45

CHAPTER 4: SMALL MOLECULE ION CHANNELS FUNCTIONALLY INTERFACE WITH ENDOGENOUS PROTEIN ION PUMPS AND TRANSPORTERS ...... 66

CHAPTER 5: IDENTIFICATION OF NOVEL SMALL MOLECULE MIMICS ...... 97

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ABBREVIATIONS

AmB Amphotericin B

AmdeB Amphoteronolide B

C35deOAmB C35 deoxyAmphotericin B

ANOVA analysis of variance

ASL airway surface liquid

ATP adenosine triphosphate

BGC biosynthetic gene cluster

Can. Candicidin cDNA complementary deoxyribonucleic acid

CFU colony-forming unit

DEPC diethyl pyrocarbonate

DIC differential interference contrast

DiSC3(3) 3,3-dipropylthiacarbocyanine iodide

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

EC50 half maximal effective concentration

Erg ergosterol

EtOH ethanol

FDR false discovery rate

GuBc glycerol, sucrose, beef extract, casamino acids

HPLC high-performance liquid chromatography

ICP-MS inductively coupled plasma-mass spectrometry

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ISP International Streptomyces Project

ITC isothermal titration calorimetry kME module membership

LUV large unilamellar vesicles

MβCD methyl-β-cyclodextrin

Mep. Mepartricin

MeOH methanol

MIC minimum inhibitory concentration

MS mannitol soy

Nat. Natamycin

NB nitrogen base

ND not determined

NMR nuclear magnetic resonance

NS not significant

Nys./Nyst. Nystatin A1

OD600 optical density at 600 nm

ORF open reading frame

PABA p-amino benzoic acid

PBS phosphate buffered saline

PKS polyketide synthase

POPC 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine

PPM parts per million

RODEO rapid ORF description and evaluation online

xiv

R2A Reasoner’s 2A

RNA ribonucleic acid

RPM revolutions per minute

RT-qPCR real time-quantitative polymerase chain reaction

SAR structure activity relationship

SEm standard error measurement

SSNMR solid state nuclear magnetic resonance

TEA tetraethylammonium

TEM transmission electron microscopy

UV-VIS ultraviolet-visible spectroscopy

WGCNA weighted gene co-expression network

WT wild-type

YNB yeast nitrogen base

YPAD yeast extract adenine peptone dextrose

YPD yeast extract peptone dextrose

YEAST STRAIN ABBREVIATIONS

BY4741 wild-type yeast WT

BYT3 Tok1 mutant yeast (Δ3)

BYT4 Nha1 mutant yeast (Δ4)

BYT5 Ena1-5 mutant yeast (Δ5)

BYT12 Trk K+ transporter deficient yeast (trk1Δtrk2Δ)

BYT123 Trk K+ transporter and Tok1 triple mutant (trk1Δtrk2Δtok1Δ)

BYT124 Trk K+ transporter and Nha1 triple mutant (trk1Δtrk2Δnha1Δ)

BYT125 Trk K+ transporter and Ena1-5 triple mutant (trk1Δtrk2Δena1-5Δ)

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CHAPTER 1: GENERAL INTRODUCTION

Channelopathies represent one subset of human diseases caused by the dysfunction of and/or lack of expression of protein ion channels.1,2 They can affect different organs and systems of the body including that of the heart, kidneys, lungs, and nervous system, and manifest in debilitating and/or life-threatening diseases including

Long QT syndrome, Bartter syndrome, Cystic Fibrosis, episodic ataxia, and chronic pain.3,4 Currently, more than 30 human channelopathies remain incurable,5 thus demonstrating the need for new therapeutic strategies.

Most pharmacological treatments use small molecules that bind to and either block,6 or turn on protein activity.7 However, these strategies will not work in cases where there is no protein to target. Significant advances have been made in gene editing with

CRISPR/Cas9 technology including promising outcomes in murine models of Duchenne muscular dystrophy,8,9 hereditary tyrosinemia,10 cataracts,11 and many others. Although

CRISPR/Cas9 shows tremendous potential to provide therapeutic benefit, several challenges remain including the need for more effective methods to deliver viral vectors,12 undesired activation of the immune system,13 and concerns over the lack of long-term safety data.14 An alternative strategy is to employ small molecules that autonomously albeit imperfectly perform protein function.15,16,17,18,19 Small molecules have several advantageous features including that they effectively penetrate cells, are less likely to trigger an immune response, and their synthesis is scalable to metric ton quantities.20 We questioned whether imperfect small molecule mimics of protein ion channels might be enough to restore physiology in a protein ion channel deficient system?

To probe this question, we selected Saccharomyces cerevisiae (S.cerevisiae) as an outstanding model organism because this unicellular eukaryote is well-annotated,21 genetically tractable22,23,24,25 with well-established techniques for constructing gene deletion strains,26 and amenable to cultivation in the lab. The yeast model organism has been widely utilized to characterize the pathophysiology of human diseases.27 Last, studies in yeast have illuminated several fundamental biological processes important to human health including cell cycle regulation,28 telomere maintenance,29 and vesicle trafficking.30,31

We predicted that an imperfect small molecule ion channel that is unregulated, unselective, and unrectified might work through functionally interfacing the cells’ robust homeostatic mechanisms.32 Cells possess highly regulated and sophisticated mechanisms for maintaining ion homeostasis33 including the generation of electrochemical gradients through ATP-driven protein ion pumps,33 selective gating of ion channels,34 efflux of ions through voltage-gated channels,35 and sequestration of ions into vacuoles.36 Given this inherent and robust grid of ion regulatory proteins, we hypothesized that an imperfect small molecule mimic could work in concert with these endogenous proteins to restore physiology in a protein-deficient system (Figure 1). Consistent with our hypothesis, we found that an imperfect small molecule ion channel mimic,

Amphotericin B (AmB) vigorously and sustainably restored physiology in a potassium (K+) transporter deficient yeast model system.37 Furthermore, our pharmacological and genetic studies suggest that small molecule mediated rescue depends on the contributing activities of protein ion pumps, Pma1 and V-ATPase to generate favorable driving forces for ion movement through passive AmB channels.37

Figure 1. Small Molecule Mimics Functionally Interface with Endogenous Networks of Protein Ion Pumps to Restore Physiology. A. In wild-type (WT) yeast, regulated networks of protein ion pumps Pma1 and V-ATPase power the movement of protons (H+) across cellular membranes, to generate favorable electrochemical gradients. These gradients promote secondary active transport of ions including potassium (K+) into cells. B. However, when a critical protein channel is not expressed or dysfunctional, this can lead to ion dysregulation and pathophysiology. C. An imperfect small molecule mimic of the missing protein ion channel could work in concert with the cell’s endogenous network of protein ion pumps, and thereby provide sufficient ion transport function to restore physiology.

These findings hold exceptional promise for the betterment of human health, as we could envision applying this strategy for the treatment of many different types of protein deficiencies. The Online Mendelian Inheritance in Man (OMIM)38 states that there are 3,825 genes with a known “phenotype-causing mutation.” Coupled with our small molecule based strategy, we could potentially find new treatment options for patients suffering from deficiencies of scaffolding proteins, enzymes, and others. Furthermore, we can gain a better fundamental understanding of the primary functions of these proteins by employing small molecules as chemical probes. Thus, through both rigorous chemical and biological studies, we hope to contribute to our fundamental understanding of how small molecules functionally interface with cellular systems.

References

1 Jentsch TJ, Hubner CA, Fuhrmann JC. (2004). Ion channels: functions unraveled by dysfunction. Nat. Cell Biol. 6(11): 1039-1047. 2 Ashcroft FM. (2006). From molecule to malady. Nature. 440(7083): 440-447. 3 Jim JB. (2014). Channelopathies. Korean J. Pediatr. 57(1): 1-18. 4 Raouf R, Quick K, Wood JN. (2010). Pain as a channelopathy. Journal of Clinical Investigation. 120(11): 3745-3752. 5 Hubner CA, Jentsch TJ. (2002). Ion channel diseases. Hum. Mol. Genet. 11(30): 2435- 2445. 6 Fabian MA, et al. (2005). A small molecule-kinase interaction map for clinic kinase inhibitors. Nature Biotechnology. 23: 329-336. 7 Wolan DW, Zorn JA, Gray DC, Wells JA. (2009). Small-molecule activators of a proenzyme. Science. 326(5954): 853-858. 8 Long C, et al. (2014). Prevention of muscular dystrophy in mice by CRISPR/Cas9- mediated editing of germline DNA. Science. 345(6201): 1184-1188. 9 Bengtsson NE, et al. (2016). Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy. Nature Communications. 8(14454): 1-10. 10 Yin H, et al. (2014). Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nature Biotechnology. 32(6): 551-553. 11 Wu Y. et al. (2013). Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 13(6): 659-662. 12 Cai L. et al. (2016). CRISPR-mediated genome editing and human disease. Genes & Diseases. 3(4): 244-251. 13 Cox DBT, PlatT RJ, Zhang F. (2015). Therapeutic genome editing: prospects and challenges. Nature Medicine. 21(2): 121-131. 14 Sander JD and Joung JK. (2014). CRISPR-Cas systems for editing, regulating and targeting genomes. Nature Biotechnology. 32(4): 347-355. 15 Ermishkin LN, Kasumov KM, Potzeluyev VM (1976). Single ionic channels induced in lipid bilayers by polyene antibiotics amphotericin B and nystatine. Nature. 262(5570): 698- 699. 16 Sakai S and Matile S. (2013). Synthetic ion channels. Langmuir. 29(29): 9031-9040. 17 Fyles TM. (2013). How do amphiphiles form ion-conducting channels in membranes? Lessons from linear oligoesters. Acc. Chem. Res. 46(12): 2847-2855. 18 Gokel GW and Negin S. (2013). Synthetic ion channels: from pores to biological applications. Acc. Chem. Res. 46(12): 2824-2833. 19 Shen B, Li X, Wang F, Yao X, Yang D. (2012). A synthetic chloride channel restores chloride conductance in human cystic fibrosis epithelial cells. PLoS One. 7(4): e34694. 20 Subramanian G. 2012. Biopharmaceutical Production Technology. Weinheim, Germany: Wiley. 884 p. 21 Giaever G et al. (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature. 418(6896): 387-391. 22 Forsburg SL. (2001). The art and design of genetic screens: yeast. Nat. Rev. Genet. 2(9): 659-668. PMID11533715.

23 Arino J, Ramos J, and Sychrova H. (2010). Alkali metal cation transport and homeostasis in yeast. Microbiol. Mol. Biol. Rev. 74(1): 95-119. 24 Giaever G, Nislow C. (2014). The yeast deletion collection: a decade of functional genomics. Genetics. 197(2): 451-465. 25 Eide DJ, Clark S, Nair TM, Gehl M, Gribskov M, Guerinot ML, Harper JF. (2005). Characterization of the yeast ionome: a genome-wide analysis of nutrient mineral and trace element homeostasis in Saccharomyces cerevisiae. Genome Biol. 6(9): R77. 26 Gelis S, Curto M, Valledor L, Gonzales A, Arino J, Jorrin J, Ramos J. (2012). Adaptation to potassium starvation of wild-type and K+-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach. Microbiologyopen. 1(2): 182-193. 27 Foury F. (1997). Human genetic diseases: a cross-talk between man and yeast. Gene. 195(1): 1-10. 28 Hartwell LH, Mortimer RK, Culotti J, Culotti M. (1973). Genetic control of cell division cycle in yeast: v. genetic analysis of cdc mutants. Genetics. 74(2): 267-286. 29 Blackburn EH, Greider CW, Szostak JW. (2006). Telomeres and telomerase: the path from maize, Tetrahymena and yeast to human cancer and aging. Nat. Med. 12(10): 1133- 1138. 30 Novick P, Field C, Schekman R. (1980). Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 21(1): 205- 215. 31 Novick P, Ferro S, Schekman R. (1981). Order of events in the yeast secretory pathway. Cell. 25(2): 461-469. 32 Wagner A. (2007). Robustness and evolvability in living systems. Princeton, N.J.: Princeton University Press. 384 p. 33 Cyert MS and Philpott CC. (2013). Regulation of cation balance in Saccharomyces cerevisiae. Genetics. 193(3): 677-713. 34 Dubyak GR. (2004). Ion homeostasis, channels, and transporters: an update on cellular mechanisms. Advances in Physiology Education. 28(4): 143-154. 35 Maresova L, Urbankova E, Gaskova D, Sychrova H. (2006). Measurements of plasma membrane potential changes in Saccharomyces cerevisiae cells reveal the importance of Tok1 channel in membrane potential maintenance. FEMS Yeast Research. 6(7): 1039- 1046. 36 Simm C. et al. (2007). Saccharomyces cerevisiae vacuole in zinc storage and intracellular zinc distribution. Eukaryot. Cell. 6(7): 1166-1177. 37 Cioffi AG, Hou J, Grillo AS, Diaz KA, Burke MD (2015) Restored physiology in protein- deficient yeast by a small molecule channel. J. Am. Chem. Soc. 137(22): 10096-10099. 38 Amberger JS. et al. (2015). OMIM.org: Online Mendelian Inheritance in Man (OMIM), an online catalog of human genes and genetic disorders. Nucleic Acids Res. 43: D789- D798.

CHAPTER 2: IMPERFECT SMALL MOLECULE MIMICS RESTORE CELL GROWTH IN YEAST

Introduction

To date more than 30 human channelopathies remain incurable,1 demonstrating the need for new therapeutic approaches. Although phenomenal efforts and advances have been made in the fields of gene therapy2 and protein replacement therapies,3 treatment options for channelopathies are still limited. One untested strategy is to employ small molecule ion channels to partially perform the functions of these missing proteins.

Small molecules possess several advantageous qualities including that they effectively penetrate cells, are less likely to trigger an immune response, and their synthesis is scalable to metric ton quantities.4 Given the potential to impact the health of many individuals world-wide, we asked whether imperfect small molecule mimics of missing protein ion channels are sufficient to restore physiology in protein ion channel deficient cells.

One prime example of an ion channel forming small molecule is the natural product

Amphotericin B (AmB, Figure 2A and Figure 2B).5 For more than 60 years, medical providers have employed AmB as an effective and life-saving anti-fungal agent for patients suffering from invasive, systemic fungal infections.6 However, one major drawback of this pharmaceutical is its severe toxicity to the kidneys.7 Previously, many researchers had supported the hypothesis that AmB’s primary mechanism of yeast cell killing was through cell permeabilization (Figure 2C)8 and disruption of ion gradients.9

Figure 2. The Small Molecule AmB Associates with Sterols to Form Ion Channels or Membranous Aggregates.10 A. Chemical structures of AmB and yeast sterol, ergosterol. B. Two potential models of AmB small molecule ion channels. On the left, eight AmB molecules associate into a channel that pinches and spans the membrane. On the right, two AmB channels associate to span the membrane. C. Two hypotheses for AmB’s mechanism of cell killing, either through ion channel formation or sterol extraction. [This image was reproduced from Gray KC et al. (2012). Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. USA. 109(7): 2234-9.]

However, The Burke Group at The University of Illinois at Urbana-Champaign proposed an alternative hypothesis wherein AmB exerts its cell killing activity through sterol binding and extraction. With the goal of improving the therapeutic utility of AmB as an antifungal agent, the Burke Group sought to test this hypothesis. First, graduate students Kaitlyn Gray, Daniel Palacios, Ian Dailey, and Matthew Endo hypothesized that

7 sterol binding is critical for yeast cell killing. In yeast, the major sterol ergosterol plays a role in several critical processes for physiology including endocytosis, stabilization of membrane proteins,11 pheromone signaling, and vacuole fusion.12

To test this first hypothesis, they employed three chemical probes

Amphoteronolide (AmdeB), Natamycin (Nat.), and Nat. aglycone (Figure 3A). AmdeB is a derivative of AmB that lacks the mycosamine sugar and was predicted to abolish sterol binding. Nat. is a known polyene macrolide that kills yeast but does not permeabilize cells.13 Deletion of the mycosamine sugar in Nat., also known as Nat. aglycone was predicted to eliminate cell killing activity.

Second, they hypothesized that loss of ion channel activity in AmB would not impact its antifungal activity. Taking note of modeling experiments that proposed the essential role of the C35 oxygen atom in AmB's capacity to form ion channels,14,15 they synthesized an AmB derivative that lacks an oxygen atom at the C35 position

(C35deOAmB, Figure 3A). With these chemical probes in hand, they investigated AmB’s major mechanism of cell killing.

Figure 3. AmB Kills Yeast through Sterol Binding.10 A. Chemical structures of AmB, yeast sterol ergosterol, Amphoteronilide (AmdeB), Natamycin (Nat.), Nat. aglycone, and channel inactivated C35deOxyAmB (C35deOAmB). B. Isothermal titration calorimetry assays showed significant differences in the total exotherm for AmB to ergosterol- containing LUVs vs. that of sterol-free LUVs. The same trend was observed in the case

Figure 3. (continued) of Nat. and for C35deOAmB. No differences were found in AmdeB to sterol-free LUVs vs. ergosterol-containing LUVs, nor for Nat. aglycone to sterol-free LUVs vs. ergosterol- containing LUVs. C. Next, to investigate the permeabilizing capacitities of each of these small molecules, K+ efflux assays were performed in wild-type yeast. AmB promoted pronounced permeabilzation. No permeabilization was observed with any of the other small molecules. D. Miniminum inhibitory concentration (MIC) assays showed that in contrast to AmB, loss of the mycosamine sugar (as in AmdeB) led to a dramatic reduction in the potency of this derivative for cell killing against both Saccharomyces cerevisiae (S.cerevisiae) and Candida albicans (C.albicans). A similar effect was observed in the case of Nat., where removal of the mycosamine sugar (Nat. aglycone) led to a drastic increase in the MIC (i.e. loss of potency). Last, C35deOAmB still retained cell killing activity, albeit moderately reduced. E. At the MIC of AmB, the ratio of AmB to ergosterol was found to be higher in both S.cerevisiae and in C.albicans. [This image was reproduced from Gray KC et al. (2012). Amphotericin primarily kills yeast by simply binding ergosterol. Proc. Natl. Acad. Sci. USA. 109(7): 2234-9.].

First, the authors investigated the sterol binding interactions of each of their small molecule probes. They employed isothermal titration calorimetry (ITC) assays which revealed significant binding interactions of AmB, Nat., and C35deOAmB to ergosterol (in ergosterol-containing LUVs) compared to that of the sterol-free LUVs (Figure 3B). In contrast, AmdeB and Nat. aglycone showed no differences in the total exotherm for ergosterol-containing vs. sterol-free LUVs (Figure 3B). These results demonstrate that the mycosamine sugar is critical for sterol binding activity. Furthermore, these findings show that channel inactivated C35deOAmB can still effectively bind ergosterol.

Next, they investigated how the loss of the C35 oxygen atom would affect channel formation and permeabilizing activity. Through performing K+ efflux assays in wild-type

(WT) yeast (Figure 3C), they observed that AmB led to vigorous K+ efflux. In contrast, loss of the C35 oxygen atom caused a complete loss of AmB’s permeabilizing capacity when tested at 3 μM and even at a 10 X higher dose of 30 μM. Furthermore, control compounds Nat. and Nat. aglycone (lacking the mycosamine sugar) showed no K+ efflux.

To evaluate for antifungal efficacy, the authors challenged each chemical probe against Saccharomyces cerevisiae (S.cerevisiae) and Candida albicans (C.albicans).

They observed that in contrast to AmB which showed potent cell killing around 0.5 μM for

S.cerevisiae and 0.25 μM for C.albicans (Figure 3D); cleavage of the mycosamine sugar

(AmdeB) caused a dramatic reduction in the potency of AmB. Loss of the mycosamine sugar in AmdeB abrogated sterol binding and thereby yeast cell killing activity. A similar effect was observed in the case of Nat. Removal of the mycosamine sugar as in Nat. aglycone, led to a dramatic reduction in potency; the Nat. aglycone showed an MICs >500

μM, in contrast to around 2-4 μM for Nat. Deletion of the C35 oxygen atom did not drastically impair its antifungal activity, wherein an MIC was observed around 3-4 μM.

Thus, this channel inactivated derivative C35deOAmB still retained antifungal activity.

AmB killed cells only when the amount of AmB surpassed that of sterols16 (Figure

3E). These results shed light on the importance of the ratio of AmB: sterol, for either ion channel or sterol binding activity. Through utilizing AmB at low doses below the threshold for cell-killing, we posit that we will primarily harness AmB’s ion channel activity. At higher doses, AmB binds sterols to promote yeast cell killing. Furthermore, pre-complexation of

AmB with sterols may alleviate potential toxicity.17 In summary, sterol binding is critical for yeast cell killing as demonstrated by the dramatic loss of potency in the aglycone derivatives.

A second finding of this study was that the length of the polyene region impacted the ability for small molecule polyene macrolides to form ion channels. Nat. which contains four double bonds in the polyene region (tetraene) showed no cell permeabilization. In contrast, AmB which contains seven double bonds (heptaene)

11 exhibited substantial K+ efflux. Potentially, the core framework of tetraene, Nat. is insufficient to span the cell membrane for permeabilization. Current efforts are geared towards understanding the structure activity relationship (SAR) between polyene length and ion channel activity.

To further delve into elucidating AmB’s mechanism of yeast cell killing, graduate students Thomas Anderson, Mary Clay, Alex Cioffi, and Katrina Muraglia investigated how AmB binding and sequestration of sterols would impact cell physiology. Solid state nuclear magnetic resonance (SSNMR) studies revealed the localization of AmB outside of lipid membranes.16 Furthermore, transmission electron microscope (TEM) images showed the association of AmB aggregates to ergosterol containing monounsaturated 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes (Figure 4B).16 These structural studies demonstrated the propensity of AmB to self-aggregate and associate with lipid membranes.

Figure 4. Transmission Electron Microscope Images of AmB Aggregates.16 A. Monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes were prepared in a 10:1 ratio of POPC to ergosterol. B. Association of AmB aggregates to a POPC liposome (10:1 ratio of POPC to ergosterol). C. AmB aggregates. [Image from Anderson TM, et al. (2014). Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat. Chem. Biol. 10(5): 400-406.]

Last, they examined how AmB-aggregates led to yeast cell killing. These aggregates not only associated with lipid membranes but extracted sterols from whole yeast cells (Figure 5A) within 30 minutes compared to that of AmdeB which does not bind sterols. In addition, the sterol extraction diminished the number of yeast colony forming units (CFUs, Figure 5B), demonstrating that sterol sequestration is sufficient to cause yeast cell killing. Furthermore, methyl-β-cyclodextrin (MβCD) which is a characterized sterol extractor led to significant sterol extraction and yeast cell killing

(Figure 5C-D), in contrast to treatment with AmB-ergosterol complexes.

Figure 5. AmB Aggregates Promote Yeast Cell Killing through Sterol Extraction.16 A. Within 30 minutes, AmB removed ergosterol (Erg) from whole WT yeast. In contrast,

Figure 5. (continued) AmdeB did not extract ergosterol. B. AmB mediated ergosterol extraction led to yeast cell killing, while no effect was observed in AmdeB treated yeast. C. Methyl-β-cyclodextrin (MβCD) extracted ergosterol from whole WT yeast, while treatment with AmB-ergosterol complexes (AmB/Erg) did not. D. Positive control MβCD caused yeast cell killing, while ergosterol and AmB-ergosterol complex (AmB/Erg) showed no toxicity. [Image from Anderson TM, et al. (2014). Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat. Chem. Biol. 10(5): 400-406.]

Through determining that AmB kills yeast by binding and extracting their sterols,

The Burke Group distinguished this small molecule’s cell killing activity from its ion channel forming capability. Encouraged by this prospect that we could separate the two activities, we sought to investigate whether AmB and other ion channel forming polyene macrolides could serve as molecular mimics of dysfunctional or missing protein ion channels, to restore physiology.

Results

Polyene macrolides are diverse and complex natural products (Figure 6).18

Previous reports demonstrated differences in their lipid permeabilizing capacities, ion channel conductances, and ion selectivities.5,19 Encouraged by the prospect of harnessing AmB’s ion channel forming activity separate from its antifungal activity, we hypothesized that an imperfect small molecule ion channel mimic might be sufficient to restore physiology in a protein ion channel deficient system.

Figure 6. Small Molecule Polyene Macrolide Family Members. Chemical structures of polyene macrolide family members Natamycin (Nat.), Nystatin (Nyst.), AmB, Candicidin (Can.), and Mepartricin (Mep.). These polyene macrolides have been shown to bind sterols. Nat. is a polyene macrolide that does not form ion channels but retains antifungal activity against yeast. Nyst., AmB, Can., and Mep. form ion channels in vivo.

To test this hypothesis, we first investigated whether a low dose of AmB would restore growth in potassium (K+) transporter deficient (trk1Δtrk2Δ) yeast. We streaked agar plates containing standard amounts of K+ (approx. 10 mM KCl) with WT or trk1Δtrk2Δ yeast. Consistent with the genotypes of these yeast, we observed growth of

WT cells (Figure 7A, left panel) and no growth of the trk1Δtrk2Δ cells (Figure 7A, middle panel).20 However, upon supplementing the plates with a low dose of AmB (125 nM final), we observed vigorous rescue of trk1Δtrk2Δ cell growth (Figure 7A, right panel).20

Next, we asked whether AmB rescue of trk1Δtrk2Δ cell growth was concentration dependent. To probe this question, we performed a disc-diffusion assay wherein trk1Δtrk2Δ yeast were streaked onto agar plates. Then sterile paper discs containing either DMSO control or AmB were placed onto the plates. No growth was observed around the DMSO control disc (Figure 7B, left disc). Distal or proximal to the AmB disc,

15 we saw no growth (Figure 7B, right disc); these correspond to AmB concentrations that are too low or too high (toxic) for cell growth. However, at intermediate distances from the

AmB disc, where concentrations of this small molecule are moderate, we observed a zone of “functional complementation,” or rescued cell growth.

Next, we quantified the extent of small molecule mediated rescue by performing a liquid broth dilution assay. We observed poor growth of the DMSO control treated trk1Δtrk2Δ cells in contrast to the WT cells (Figure 7C). However, AmB vigorously restored growth of trk1Δtrk2Δ cells back to WT levels.20

Following, we investigated whether small molecule mediated rescue was sustainable. We iteratively cultured WT and trk1Δtrk2Δ cells between liquid broth and solid agar conditions (10 mM KCl final). We observed sustained AmB mediated rescue of trk1Δtrk2Δ cell growth for more than 40 days (Figure 7D). Importantly, removal of AmB from the trk1Δtrk2Δ culture led to a rapid loss of cell rescue, demonstrating the continued dependence of trk1Δtrk2Δ cells on AmB for ion transport.

Figure 7. Imperfect Small Molecule Ion Channel Mimic, AmB Vigorously and Sustainably Rescued Mutant Cell Growth.20 A. On agar plates containing standard concentrations of K+, WT cells grew while trk1Δtrk2Δ cells did not. However, supplementing a low dose of AmB to the plates rescued trk1Δtrk2Δ cell growth. B. Disc diffusion assay results showed no growth of the trk1Δtrk2Δ cells around the paper disc containing vehicle (DMSO). In contrast, a robust zone of cell growth was observed around the AmB containing disc. C. UV-VIS quantification of liquid cultures showed that in contrast to WT cells, trk1Δtrk2Δ cells grew poorly. However, the addition of a low dose of AmB restored mutant cell growth back to WT levels. D. Last, small molecule mediated rescue was sustainable over the course of more than 40 days, wherein WT and AmB rescued trk1Δtrk2Δ cells were cultured iteratively between both liquid cultures and on solid agar plates. [Image reproduced from Cioffi AG, et al. (2015). Restored physiology in protein-deficient yeast by a small molecule channel. J. Am. Chem. Soc. 137: 10096- 10099.]

After finding that small molecule mediated rescue is both vigorous and sustainable, we next probed whether AmB mediated rescue is ion channel dependent. Previously, our

17 lab synthesized a derivative of AmB wherein we deleted a single oxygen atom at the C35 position, C35deOAmB (Figure 8A, right). This oxygen atom was predicted to be important for ion channel formation. To test this prediction, we performed a broth dilution assay. We hypothesized that since C35deOAmB is unable to form ion channels, it would no longer rescue cell growth. Consistent with our hypothesis, we observed no rescue of cell growth in the C35deOAmB treated trk1Δtrk2Δ cells up to 1 μM tested (Figure 8B). In contrast,

AmB optimally rescued trk1Δtrk2Δ cell growth around 125 nM.

Figure 8. Small Molecule Mediated Rescue is Ion Channel Dependent.20 A. To test the hypothesis that ion channel function is essential for small molecule mediated rescue of trk1Δtrk2Δ growth, a single atom deletion variant of AmB, C35deOAmB was utilized. C35deOAmB was previously shown to not permeabilize yeast. B. AmB restored growth in trk1Δtrk2Δ yeast around 125 nM. In contrast, C35deOAmB did not rescue growth across all concentrations tested up to 1 μM. C. Inhibition of AmB ion channels with bulky tetraethylammonium (TEA) abrogated AmB mediated rescue of trk1Δtrk2Δ growth. In contrast, TEA had no impact on the growth of trk1Δtrk2Δ cultured under permissive conditions (100 mM KCl). D. Last, similar 86Rb+ influx (surrogate tracer for K+) was

Figure 8. (continued) observed for both the WT and AmB rescued trk1Δtrk2Δ cells. In contrast, C35deOAmB treated trk1Δtrk2Δ cells showed no improvements in 86Rb+ influx compared to that of the DMSO control. [Image reproduced from Cioffi AG, et al. (2015). Restored physiology in protein-deficient yeast by a small molecule channel. J. Am. Chem. Soc. 137: 10096- 10099.]

Next, we further interrogated this ion channel dependence through employing two chemical probes. First, tetraethylammonium (TEA) is a known blocker of K+ channels.21

We observed that in contrast to trk1Δtrk2Δ grown under permissive conditions of 100 mM

KCl, TEA dose-dependently blocked rescue of AmB treated trk1Δtrk2Δ cells when tested under K+ limiting conditions (Figure 8C). Second, 86Rb+ is a surrogate tracer for K+.22 We observed that both WT and AmB treated trk1Δtrk2Δ cells showed similar 86Rb+ influx. In contrast, minimal influx was observed for the DMSO and C35deOAmB treatment groups

(Figure 8D).

Encouraged by these results, we were fascinated by the possibility that this small molecule mediated approach could be generalizable to other small molecule ion channel formers. Many members of the polyene macrolide family have been shown to permeabilize lipid membranes.5,23 We sought to first reproduce the literature precedent and compare the permeabilizing capacities of several polyene macrolide candidates.

We employed a K+ selective probe and quantified the release of K+ from small molecule treated WT and trk1Δtrk2Δ cells. For each run, we recorded a 5-minute baseline before adding the DMSO control, or each small molecule compound. We noted minimal

K+ efflux in DMSO treated WT cells (Figure 9). This baseline was inherent to the experimental set-up, and we terminated the experiment if the drift exceeded 15-20%

19 efflux for the DMSO control. For our negative control Nat., we observed minimal efflux

(Figure 9).

Figure 9. K+ Efflux in WT Yeast. Separate samples of WT yeast were treated individually with DMSO control or 3 μM final of Nat., AmB, Nyst., Can., and Mep., at 5 minutes and monitored over time.

However, we observed moderate K+ efflux from AmB and Nyst. treated WT and trk1Δtrk2Δ cells (Figure 9 and Figure 10). Can. (Figure 9 and Figure 10) and Mep.

(Figure 9) promoted rapid efflux within 15 minutes. Valinomycin and Monensin showed minimal or no efflux respectively (Figure 10).

Figure 10. K+ Efflux in trk1Δtrk2Δ Yeast. Separate samples of trk1Δtrk2Δ yeast were treated individually with DMSO control or 3 μM final of AmB, Nyst., Can., Valinomycin, and Monensin at 5 minutes. The K+ efflux was monitored over time.

Having demonstrated that other polyene macrolide family members could permeabilize yeast, we hypothesized that these ion channel forming small molecules could restore physiology in trk1Δtrk2Δ yeast. To test this hypothesis, we performed both disc diffusion and liquid broth dilution assays.

For the disc diffusion assays, we utilized Nat. as a negative control since this small molecule does not permeabilize yeast. We observed no trk1Δtrk2Δ growth around the

Nat. containing disc (Figure 11A). In contrast, small molecule ion channel formers AmB

(Figure 11B), Nyst. (Figure 11C), Can. (Figure 11D), and Mep. (Figure 11E) all promoted rescue of trk1Δtrk2Δ cell growth; we observed rings of cell growth at intermediate distances from these small molecule containing discs.

Figure 11. Imperfect Small Molecule Ion Channel Mimics Restored Growth in trk1Δtrk2Δ Cells. Trk1Δtrk2Δ cells were streaked onto agar plates containing 10 mM KCl and sterile paper discs containing A. Nat., negative control at 4 mg/mL, B. AmB at 4 mg/mL, C. Nyst. at 4 mg/mL, D. Can. at 4 mg/mL, E. Mep. at 4 mg/mL were applied. No growth was observed around the DMSO nor 1 M KCl control discs. Furthermore, Nat. which does not permeabilize yeast, did not restore cell growth. In contrast, the other ion channel forming small molecules, showed robust zones of restored cell growth around each of the compound containing discs. Images were taken at 24 hours.

With these results in hand, we were interested in comparing the relative, optimal rescuing concentrations for each of these small molecule ion channel formers. Would

22 differences in the chemical structures of each of these small molecules impact the concentrations at which rescue of trk1Δtrk2Δ growth occurred? We predicted that the more potent permeabilizing small molecules Can. and Mep. would rescue growth at lower concentrations. First, as a negative control Nat. did not rescue trk1Δtrk2Δ cell growth under any concentration tested up to 3 μM, while toxicity in WT cells was observed above

2 μM (Figure 12A). AmB was previously shown to restore physiology in trk1Δtrk2Δ yeast at 125 nM (data not shown). Consistent with our hypothesis, the more permeabilizing small molecule ion channel formers Can. and Mep. restored cell growth in trk1Δtrk2Δ yeast at 8 nM and 8 nM respectively (Figure 12C and D). While Nyst. which moderately permeabilized yeast, required a higher concentration of 1 μM (Figure 12B) for optimal rescue of cell growth.

Figure 12. Predicted Ion Channel Forming Small Molecules Rescued Yeast at Distinct Concentrations. A. Consistent with Nat.’s inability to permeabilize yeast, no growth was observed in the trk1Δtrk2Δ yeast across all concentrations tested up to 3 μM,

Figure 12. (continued) at 24 hours. In contrast, WT cells exhibited robust growth under these conditions, where cell killing was observed above 2 μM, graph represents the average of N = 3 independent experiments. B. Ion channel former, Nyst. vigorously rescued trk1Δtrk2Δ cell growth around 1 μM, at 24 hours. Representative graph is shown of N = 3 independent experiments. C. and D. Both Can. and Mep. restored physiology in yeast at around 8 nM, at 24 hours. For Can., graph represents the average of N = 3 independent experiments. For Mep., representative graph is shown for N = 3 independent experiments.

To further test the hypothesis that ion channel function is critical for small molecule mediated rescue of trk1Δtrk2Δ cell growth, we investigated whether small molecule carriers and antiporters could be enough, to import adequate amounts of cations required for physiology. We found that other small molecule cation carriers Lasalocid (monovalent and divalent cation carrier)24,25 and Valinomycin (K+ selective carrier),26 and antiporters

Monensin (Na+/H+ antiporter)27 and Nigericin (K+/H+ antiporter)28 (Figure 13)

Figure 13. Chemical Structures of Small Molecule Carriers and Antiporters. Shown here are the chemical structures for carriers Lasalocid and Valinomycin, and antiporters Monensin and Nigericin.

did not rescue growth in trk1Δtrk2Δ cells (Figure 14) in our disc diffusion assay. These results suggest that ion channel flux is critical for rescue of trk1Δtrk2Δ physiology. Future

24 studies will be conducted in spheroplasts (yeast cells with enzymatically removed cell walls) to determine whether the lack of activity of these small molecules could be due to physical cell wall barriers.

Figure 14. Cation Carriers and Antiporters Did Not Restore Growth in trk1Δtrk2Δ Yeast. Neither cation carriers A. Lasalocid (163 μM) and B. Valinomycin (4 mg/mL) nor antiporters C. Monensin (4 mg/mL) and D. Nigericin (4 mg/mL) promoted growth in trk1Δtrk2Δ cells, by 24 hours.

To rule out the possibility of poor diffusion in agar, we also tested Valinomycin and

Nigericin in our liquid broth dilution assay. We observed no restoration of trk1Δtrk2Δ cell growth when tested up to the limits of solubility for both Valinomycin (Figure 15A) and for

Nigericin (Figure 15B). Furthermore, other alkali metal ion transporters and ionophores did not promote rescue in trk1Δtrk2Δ cells (Figure 17); these findings provide further

25 evidence that ion channel mediated flux is critical for restoring physiology in this yeast system.

Figure 15. Valinomycin and Nigericin Did Not Restore Physiology in trk1Δtrk2Δ Yeast. Neither A. Valinomycin nor B. Nigericin promoted trk1Δtrk2Δ growth by 24 hours. N=1 independent experiment for each.

Last, one major challenge of utilizing AmB is its toxicity caused by sterol binding.

We hypothesized that pre-complexation of small molecules with host-sterols would ameliorate toxicity. To test this hypothesis, we prepared 5:1 ergosterol to small molecule complexes16 and evaluated these complexes for yeast cell killing. To quantify the reduction in toxicity, we performed a liquid broth dilution assay. In contrast to AmB

(minimum inhibitory concentration, MIC = 0.5 μM), pre-complexation of ergosterol to AmB led to a dramatic 32-fold reduction in yeast cell killing (MIC = 16 μM, data not shown).20

Discussion

We discovered that an imperfect small molecule mimic of a missing protein ion channel is enough to rescue physiology in a protein-deficient system. Furthermore, small molecule mediated rescue is vigorous, sustainable, and ion channel dependent. In addition, our small molecule mediated approach is generalizable to other polyene macrolide family members. These findings demonstrate the potential of employing

26 imperfect small molecule mimics to provide physiological benefit in cases of dysfunctional or missing protein ion channels in patients.

Future studies investigating the structure activity relationships of diverse small molecule ion channels and their ion conducting capacities and selectivities could enable the rational design of derivatives that favor the transport of cation or anion species.

Furthermore, why does the presence of a p-amino benzoic acid (PABA) motif on both

Can. and Mep. appear to enhance ion conductance, wherein we observed rescue of trk1Δtrk2Δ yeast at 8 nM compared to that of 125 nM for AmB (structurally similar compound that does not contain a PABA motif). Further studies probing the binding activity of these small molecules to sterols could shed light on potential differences in how these small molecules interact with cellular membranes.

Last, investigation of the relationship between the length of the polyene region and ion channel activity could lead to insights on how these small molecules assemble into ion channels across cellular membranes. Nat. which contains four double bonds in the polyene region (tetraene) does not permeabilize yeast. Recently, we found that Eurocidin

(contains five double bonds, pentaene) also does not promote trk1Δtrk2Δ rescue, suggesting a lack of channel activity. We are currently attempting to express a polyene macrolide with six double bonds in the polyene region (hexaene). We hypothesize that a polyene macrolide with six double bonds in the polyene region may be sufficient in length to form ion channels.

We are encouraged by our findings that pre-complexation of small molecules with host-sterols could prove as an effective strategy to reduce potential toxicity. These results greatly increase the utility of small molecule mimics as potential surrogates for missing

27 proteins, due to the larger range of therapeutic concentrations available. We are invested in better understanding the structures, binding strengths, and lipid membrane interactions of these sterol: small molecule complexes. Once in a living system, how stable are these complexes? Does exchange with endogenous sterols occur?

Given the complexity of protein ion channels, it is fascinating that an unrectified, poorly selective, and unregulated small molecule ion channel surrogate could functionally interface with the cell to restore cell growth. Future studies investigating which endogenous proteins in cells play a role in facilitating small molecule mediated rescue, either through generating favorable electrochemical gradients or though correcting for the lack of ion selectivity of small molecule channels, may reveal conserved features in higher eukaryotes.

Excitingly in a higher eukaryotic system, Katrina Muraglia and Rajeev Chorghade demonstrated that a low dose of AmB could restore physiology in cystic fibrosis transmembrane conductance regulator (CFTR) deficient primary human lung epithelia.29

This imperfect small molecule mimic rescued several hallmarks of lung physiology including airway surface liquid (ASL) pH, ASL antimicrobial activity, and viscosity.

Mechanistically, they discovered that AmB mediated bicarbonate (HCO3-) transport led to restored physiology. Current efforts are geared towards investigating whether aerosolized AmBisome, a FDA approved liposomal formulation of AmB, could restore physiology in CFTR deficient pigs.

It is intriguing to consider the possibility of restoring physiology in other protein ion channel deficiencies. For example, voltage gated Na+ and K+ channel deficiencies impair important neurological functions.30 Dr. Yu-Qing Cao, from The Department of

Anesthesiology and Pain Center at Washington University in St. Louis studies TRESK K+ channels in primary afferent neurons. Through collaborative efforts, we are interested in investigating whether imperfect small molecules ion channels could potentially restore K+ conductance in these neurons and corresponding mouse models, for the treatment of chronic pain.

Last, we posit that this small molecule based approach to restoring protein deficiency could expand beyond protein ion channel dysfunction. The next horizon would be to identify small molecules that mimic diverse protein functions. To date, The Online

Mendelian Inheritance in Man (OMIM) Morbid Map reports 3,825 genes with a known

“phenotype-causing mutation.” 31 In cases where protein replacement therapy and gene therapy are insufficient to provide therapeutic improvement, a small molecule based approach could benefit patient outcomes. For example, known enzymatic dysfunction affects several physiological processes including metabolism, collagen production, steroid hormone generation, and lysosomal storage.32,33,34,35 These manifest in diseases including phenylketonuria (dysfunctional phenylalanine hydroxylase),36 Ehler Danlos syndrome (lack of functional enzymes involved in collagen production),33 congenital adrenal hyperplasia (mutated 21-hydroxylase),34 and Gaucher’s disease (mutated acid-

β-glucosidase).32

Summary

Here, we discovered that imperfect small molecule ion channel mimics are sufficient to restore physiology in a protein-deficient system. Furthermore, we observed that small molecule mediated rescue is vigorous, sustainable, and ion channel dependent. Exploration into other polyene macrolide ion channels showed that this small

29 molecule based approach is generalizable, wherein PABA containing polyene macrolides

Candicidin and Mepartricin showed effective permeabilization and rescued trk1∆trk2∆ cell growth at lower doses compared to that of non-PABA containing polyene macrolides AmB and Nystatin. Furthermore, pre-complexation of small molecules to native sterols proved to be an effective strategy to mitigate unwanted toxicity. It is intriguing to consider the possibility that imperfect small molecule mimics may be sufficient to restore physiology in other types of protein deficiencies.

Author Contributions

For scientific contributions, Alex Cioffi performed the growth rescue assay development and discovery of AmB mediated rescue in trk1Δtrk2Δ yeast (Figure 7) and demonstrated that AmB mediated rescue is ion channel dependent (Figure 8). Jenn Hou performed the K+ efflux studies (Figure 9 and Figure 10), executed studies demonstrating that small molecule mediated rescue is generalizable to other polyene macrolides (Figure 11, Figure 12, Figure 14, and Figure 15), investigated other potential small molecule candidates (Figure 17), ran HPLC purification of AmB (Figure 18 and

Figure 19), and determined the extinction coefficients of Nystatin A1 (Nyst.) and

Candicidin (Can.) (Figure 20).

Methods and Materials

Yeast Media Conditions

The media was prepared as follows. For 1 L of 10 mM KCl YPAD pH 5.0 medium, we added 20 g of bactopeptone, 10 g of yeast extract, 15 mg of adenine hemisulfate to approximately 700 mL of milli-Q H2O. The base components of this medium contain

30 approximately 9.44 mM KCl and for simplicity we refer to this medium as 10 mM KCl or

“normal” K+ medium. The medium was buffered with citric acid to a final pH of 4.9-5.0.

For 1 L of 100 mM KCl YPAD pH 5.0 medium, we added 20 g of bactopeptone, 10 g of yeast extract, 7.44 g of KCl, 15 mg of adenine hemisulfate to approximately 700 mL of milli-Q H2O. Then we buffered the medium with citric acid to attain a final pH of 4.9-5.0.

Next, we autoclaved the medium for 30 minutes. After, we added 50 mL of a 40% sterile dextrose solution per L of medium and filled the remaining volume with sterile milli-Q H2O to obtain a final volume of 1 L. For solid agar plates, 20 g per L of agar was added before autoclaving.

Yeast Culture

The yeast strains were maintained as frozen 15-20% glycerol stocks at -80°C. We streaked the yeast cell strains onto solid agar plates utilizing sterile inoculating loops.

Then we grew the plates upside down in a 30°C incubator for 24-48 hours until single colonies were visible by eye. The plates were then wrapped in parafilm and kept upside down in a 4°C fridge.

Yeast Disc Diffusion Assay

This protocol was modified from The National Committee of Clinical Laboratory

Standards document M2-A9.37 First to set up overnight cultures, we suspended 3-5 single colonies of WT and separately trk1Δtrk2Δ yeast into 1 mL each of 100 mM KCl YPAD 5.0 medium. We then transferred this suspension to sterile 250 mL Erlenmeyer flasks containing 50 mL of 100 mM KCl YPAD and cultured overnight at 30°C, 200 X RPM. To harvest the cells, we centrifuged the saturated cell suspension at 500 X G for 5 minutes

31 at 23°C. Then we removed the supernatant and resuspended the cells in an equivalent volume of sterile milli-Q H2O, vortexed to mix, and then recentrifuged. We repeated the wash step. Following, we added 10 mM KCl YPAD to each cell pellet and vortexed to mix.

Next, we obtained an optical density (absorbance at 600 nm, OD600) ranging between 0.090-0.110 as measured by an ultraviolet-visible (UV-VIS) spectrophotometer.

Following, we used sterile cotton applicators to apply each yeast strain onto 10 mM KCl

YPAD agar plates. To do so, we dipped a sterile cotton applicator into the yeast cell suspension and streaked the whole agar plate. Then the plate was rotated 60° and a new cotton applicator was dipped and applied. We repeated this step for a total of 3 times and let the plates rest for 2-3 minutes. Then 10 μL of either DMSO control or small molecule solution was pipetted onto sterile 8 mm paper (Whatman 4) discs. Then the discs were transferred onto the plates. Or we applied the discs directly to the plates, and then let the plates rest for 10 minutes before pipetting 10 μL of solution per disc. The plates were allowed to rest before placing them upside down in a 30°C incubator for 36-48 hours.

As a control experiment, we investigated whether alkali metal ion transporters and ionophores restore physiology in K+ transporter deficient trk1Δtrk2Δ yeast (Figure 16).

We observed no rescue with Beauvericin (alkali metal ion transporter),38 Nonactin

(ammonium transporter),39 A23187 (Ca2+ transporter),40 magnesium ionophores I and III, and zinc ionophores I and IV (Figure 17). These results suggest that small molecule mediated rescue in trk1Δtrk2Δ yeast is specific for K+.

Figure 16. Chemical Structures of Alkali Metal Ion Transporters and Ionophores. Shown are the chemical structures for alkali metal ion transporter Beauvericin, and diverse ionophores including Nonactin (ammonium transporter), A23187 (calcium ionophore), magnesium ionophores I and III, and zinc ionophores I and IV.

Figure 17. Alkali Metal Ion Transporters and Ionophores Did Not Restore Physiology in trk1Δtrk2Δ Yeast. No growth rescue was observed with A. Beauvericin (4 mg/mL), B. Nonactin (4 mg/mL), C. calcium ionophore A23187 (4 mg/mL), D.

Figure 17. (continued) magnesium ionophore I (4 mg/mL), E. magnesium ionophore 3 (4 mg/mL), F. zinc ionophore I (4 mg/mL), and G. zinc ionophore IV (4 mg/mL).

Yeast Liquid Broth Dilution Assay

We followed the method reported in The Clinical and Laboratory Standards

Institute document M27-A341 with the following changes. First, we utilized 10 mM KCl

YPAD pH 5.0 for the medium source. Second, WT and trk1Δtrk2Δ yeast were treated with

DMSO control or final concentrations of small molecules. The experiments terminated at

24 hours and growth (optical density at 600 nm, OD600) was evaluated by UV-VIS.

Tetraethylammonium (TEA) Blocking

We adapted the previously described yeast liquid broth dilution assay with the following modifications. First, trk1∆trk2∆ + 125 nM AmB yeast in 10 mM KCl YPAD, were treated with increasing doses of tetraethylammonium (TEA). Second, we also treated trk1∆trk2∆ yeast under nonlimiting K+ conditions (100 mM KCl YPAD) with increasing doses of TEA.

86Rb+ Influx Assay

We made modifications to the protocol reported by Mulet et al.42 The day before the experiment, we prepared an overnight yeast culture. We harvested cells via centrifugation, decanted the supernatant, and washed the cells with sterile Milli-Q H2O.

Following, we performed the wash step twice. Next, we subjected the cells to uptake buffer, (40 mL of 50 mM succinic acid, 2% glucose, adjusted to pH 5.5 with Tris) for three hours. Afterwards, we rinsed the cells twice with sterile Milli-Q H2O and transferred them in uptake buffer (1 mL). We quantified the cell counts with a hemocytometer (INCYTO)

35 and adjusted the cell density to 3 X 107 cells/mL. We transferred 0.7 mL of cells to an eppendorf tube and let the samples rest for 5 minutes in uptake buffer. We dosed cells with vehicle (DMSO), 3 μM of AmB, or 3 μM of C35deOAmB. Per tube, we spiked in 1.1

μCi of 86RbCl and then thoroughly mixed the samples.

We quenched the reaction by combining 100 μL of sample with 10 mL of 20 mM

MgCl2 (cold). We rapidly filtered the samples onto nitrocellulose membranes (0.45 μm,

Millipore HAWP). We rinsed the cells twice with 15 mL of 20 mM MgCl2 (cold). Then we placed the membranes into scintillation vials. We determined the radioactivity (counts per minute, Perkin Elmer Wizard2 automatic ɣ counter) and the graph depicts averages from three biological samples.

K+ Efflux Assay

This protocol was modified from Palacios et al. (2011).43 Nonisogenic WT and trk1Δtrk2Δ yeast were cultured in YPD medium for 12 – 24 hours, at 200 X RPM, 30°C.

+ 10, 100, and 1000 ppm K standards (KCl) were prepared in Milli-Q H2O. Then 400 μL of

5 M NaCl was added to 20 mL of each standard. 4 mL of each standard was aliquoted into 7 mL glass vials and small stir bars were added to each vial. We carefully rinsed out the inside of the K+ selective probe with the 1000 ppm standard (no NaCl added) and made sure not to puncture the valinomycin coated membrane. Following, we placed the

K+ probe into a vial containing 1000 ppm K+ and equilibrated at 30°C, 150 RPM (with a stir bar). The 10, 100, and 1000 ppm K+ standards were also equilibrated for at least one hour. We prepared fresh 300 μM small molecule stock solutions in DMSO for Nat., AmB,

Nyst., Can. (TOKU-E), and Mep.

Then about 20 mL of saturated cell suspension was centrifuged at 300 x G, 23°C, for 5 minutes. We poured off the supernatant and then resuspended the cells in 20 mL of

Milli-Q H2O. We vortexed to fully re-suspend the cell pellet. We repeated the wash step.

Following, we re-suspended the cell pellet in 150 mM NaCl, 5 mM Hepes, pH 7.4 to an

OD600 between 1.4 to 1.6. Afterwards, we transferred 3 mL each of cell suspension into separate 7 mL glass vials and equilibrated for at least 15 minutes at 30°C, 150 X RPM.

After equilibrating the cell suspensions and standards and calibrating the instrument, we first ran a DMSO control run. The probe was placed into a vial containing the yeast cell suspension and data points were recorded every 30 seconds for the first 6 minutes. Following, we rapidly pipetted 30 μL of DMSO into the sample vial. We recorded the next 30 minutes of data. Next, to obtain maximum efflux from the cells, we injected

30 μL of a 1% (w/v) digitonin solution; we recorded the following 15 minutes of data. Once the DMSO control drift was determined to be less than 10-15% of the total K+ efflux, we went ahead with the remaining samples and made sure to rinse the probe with Milli-Q

H2O between each sample.

To determine the % relative efflux over time, we employed the following equation:

[K]    t 1S 100 [K]0  

[K]t = potassium concentration at time t [K]0 = potassium concentration at time 0 S = scaling factor wherein we determined the scaling factor by taking [K]t as the ppm value 15 minutes post- digitonin treatment and [K]0 as the ppm value 1 minute after starting the run. Next, we applied this scaling factor to determine the % relative efflux over time.

High-Performance Liquid Chromatography (HPLC) of AmB

This method was based on our in-house protocol. The evening before HPLC purification, at least 8 - 10 L of acetonitrile and 8 – 10 L of 15 mM ammonium acetate were filtered through 0.2 μm filters. The day of the purification, we loaded the HPLC ammonium acetate, acetonitrile gradient method (MME_JH_Method) and rinsed the

HPLC SunFire Prep C18 OBD column (30 x 150 mm) for at least 15 minutes. During this time, we allowed a bottle of AkScientific Amphotericin warm to room temperature and then prepared a 3 mg/mL solution in DMSO. We sonicated and vortexed the solution to fully suspend the Amphotericin. The solution was a dark yellow-orange color. The

Amphotericin solution was filtered through iso-disc syringe tip filters and transferred to

HPLC vials.

For the first run, we programmed the instrument to inject 400 μL of DMSO to clean the column of any residual impurities. Once we observed that the spectra were clean at

406 nm and 383 nm, then we injected 400 μL of Amphotericin stock solution. There was typically about a one minute separation between Amphotericin A and Amphotericin B

(AmB) peaks. We manually collected the second large peak containing AmB (typically around 15.8 – 16.25 minutes, but this varied day to day). Following, we programmed the instrument to auto-inject, and we manually collected the AmB peak. We typically ran 10

– 14 injections and collections per day to obtain a worthwhile amount of AmB per purification.

After the last HPLC run, we then pulled off the acetonitrile and ammonium acetate by rotary evaporation. The remaining yellowish solution (almost paint-like) was resuspended in about 20-30 mL of Milli-Q H2O and toluene (from the solvent dispenser

38 system) to azeotrope the Amphotericin. This solution was roto-vaped. We repeated the azeotroping step two more times. For the last step, the AmB was resuspended in dimethyl sulfoxide-d6, transferred into pre-weighed 1.5 mL glass vials, and frozen. Then the samples were lyophilized for at least 24 hours or until completely dry. The AmB vials

(Figure 18) were purged with either nitrogen or argon, wrapped with parafilm, stored in a container with desiccant, protected from light, and then stored at -20°C.

Figure 18. HPLC Purified AmB. After lyophilization, AmB appeared as a sunflower yellow powder that was consistent in texture and not hygroscopic. Samples were aliquoted to avoid unnecessary exposure to atmospheric air.

Analytical HPLC of AmB

The analysis of the purity of AmB was determined on an analytical HPLC (Figure

19) with a SunFire C18 column (5 μm). The same gradient system was used as in the purification of AmB. A control standard of previously purified AmB was utilized to determine the retention time. For biological assays, we utilized purified AmB that had ≥

85% purity by analytical HPLC.

Figure 19. Representative Analytical HPLC Analysis of Purified AmB. Analytical HPLC analysis showed a single major peak of purified AmB for both 406 nm (top) and 383 nm (bottom). The retentions times were found to match those of a known AmB standard (data not shown).

Determination of Nystatin and Candicidin Extinction Coefficients

This protocol was modified from Palacios et al. (2011).43 First, we determined the weight of the glass vials to be used. To do so, 4 mL glass vials were carefully rinsed with hexanes and then acetone, and oven dried at 120°C for several hours. Next, we allowed the glass vials to rest on the bench top until cool to the touch. At The UIUC Microanalysis

Lab, we measured the weight of each vial. Care was taken to ensure that there were no finger marks on the glass vials and a static gun was applied before each measurement.

Three weight measurements were obtained for each vial and the average was determined for each vial ± 10%.

Second, the desired compounds of interest (more than 1 mg per sample per vial) were dissolved in deuterated DMSO-d6, added to each vial, and frozen. Following, the samples were lyophilized overnight until all the solvent was removed. Next, the weight of

40 each vial was determined as previously described, and the average of three measurements was collected for each vial.

Following, we prepared a 1 mM stock solution in DMSO-d6 for each compound of interest and then generated 20 μL aliquots for each of the following dilutions: 800, 500,

300, 100, and 50 μM. Afterwards, we took each of the aliquots and mixed 5 μL with 500

μL of MeOH in triplicate for each concentration. Then on the UV-VIS spectrophotometer, we obtained spectra from 300 to 500 nm. We graphed the relative absorbance (at λmax) and used the slope to determine the extinction coefficient for both small molecules

2 (Figure 20), where the R > 0.96 for both Can. and Nyst. For Can., we found that the λmax

-1 -1 = 377.5 nm and the extinction coefficient is 33633 M cm . For Nyst., the λmax = 304 nm and the extinction coefficient is 51207 M-1 cm-1.

Figure 20. Determination of the Extinction Coefficients for Small Molecule Ion Channels Candicidin and Nystatin. The extinction coefficients were obtained for both A. Can. as 33633 M-1 cm-1 and B. Nyst. as 51207 M-1 cm-1.

References

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19 Cybulska B. et al. (1995). Identification of the structural elements of amphotericin B and other polyene macrolide antibiotics of the heptaene group influencing the ionic selectivity of the permeability pathways formed in the red cell membrane. Biochimica et Biophysica Acta (BBA)- Biomembranes. 1240(2): 167-178. 20 Cioffi AG, et al. (2015). Restored physiology in protein-deficient yeast by a small molecule channel. J. Am. Chem. Soc. 137: 10096-10099. 21 Khodakhah K, Melishchuk A, Armstrong CM. (1997). Killing K channels with TEA+. Proc. Natl. Acad. Sci. USA. 94: 13335-13338. 22 Cholo MC, van Rensburg EJ, Osman AG, Anderson R. (2015). Expression of the genes encoding the Trk and Kdp potassium transport systems of Mycobacterium tuberculosis during growth in vitro. Biomed. Res. Int. 2015(608682): 1-11. 23 Kotler-Brajtburg J, et al. (1979). Classification of polyene antibiotics according to chemical structure and biological effects. Antimicrobial agents and chemotherapy. 15(5):716-722. 24 Mimouni M, Lyazghi R, Julliard J. (1998). Formation and structure of alkali metal, thallium, silver, and alkaline-earth cation complexes with the Ionophore lasalocid free acid form in methanol from NMR experiments. New. J. Chem. 22: 367-372. 25 Rutkowski J and Brzezinski B. (2013). Structures and properties of naturally occurring polyether antibiotics. Biomed. Res. Int. 2013(162513): 1-31. 26 Lauger P. (1972). Carrier-mediated ion transport. Science. 178(4056): 24-30. 27 Lowicki D, Huczynski A (2012). Structure and antimicrobial properties of monensin A and its derivatives: summary of the achievements. BioMed. Research International. 742149. 28 Nakamura T, Hsu C, Rosen BP. (1986). Cation/Proton antiport systems in Escherichia coli. The Journal of Biological Chemistry. 261(2): 678-683. 29 Muraglia KA et al. CFTR-independent rescue of cystic fibrosis with a small molecule bicarbonate channel. Manuscript in preparation. 30 Jim JB. (2014). Channelopathies. Korean J. Pediatr. 57(1): 1-18. 31 Amberger JS. et al. (2015). OMIM.org: Online Mendelian Inheritance in Man (OMIM), an online catalog of human genes and genetic disorders. Nucleic Acids Res. 43: D789- D798. 32 Brady RO, Kanfer JN, Shapiro D. (1965). Metabolism of glucocerebrosides II. Evidence of an enzymatic deficiency in Gaucher’s disease. Biochemical and Biophysical Research Communications. 18(2): 221-225. 33 Pinnell SR (1982). Molecular defects in the Ehlers-Danlos syndrome. Journal of Investigative Dermatology. 79(1): 90-92. 34 White PC and Speiser PW. (2000). Congenital adrenal hyperplasia due to 21- hydroxylase deficiency. Endocrine Reviews. 21(3): 245-291. 35 Ozen H. (2007). Glycogen storage diseases: new perspectives. World J. Gastroenterol. 13(8): 2541-2553. 36 Kaufman S. et al. (1975). Phenylketonuria due to a deficiency of dihydropteridine reductase. N. Engl. J. Med. 293: 785-790. 37 Wikler MA. et al. (2006). Performance standards for antimicrobial disk susceptibility tests; approved standard- ninth edition. Clinical and Laboratory Standards Institute. 26(1): M2-A9.

38 Sigel A, Sigel H, Sigel RKO. (2016). The Alkali Metal Ions: Their Role for Life. Springer. 628p. 39 Sasaki S. et al. (2002). Comparison of two molecular design strategies for the development of an ammonium Ionophore more highly selective than nonactin. Anal. Chem. 74: 4845-4848. 40 Pfeiffer DR, Taylor RW, Lardy HA. (1978). Ionophore A23187: cation binding and transport properties. Annals of The New York Academy of Sciences. 307: 402-423. 41 CLSI. Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts: Approved Standard – Third Edition. CLSI document M27-A3. Wayne, PA: Clinical and Laboratory Standards Institute: 2008. 42 Mulet JM, Serrano R. (2002). Simultaneous determination of potassium and rubidium content in yeast. Yeast. 19(15): 1295-1298. 43 Palacios DS, et al. (2011). Synthesis-enabled functional group deletions reveal key underpinnings of amphotericin B ion channels and antifungal activities. Proc. Natl. Acad. Sci. USA. 108(17): 6733-8.

CHAPTER 3: SMALL MOLECULE ION CHANNELS RESTORE PHYSIOLOGICAL ION CONTENT AND THEREBY CELL GROWTH

Introduction

Ion homeostasis is a critical cellular process across all kingdoms of life.1,2,3 The dysregulation of ion flow and storage can result in devastating consequences for living systems.4,5 Preliminary studies demonstrate that an imperfect small molecule ion channel mimic could provide enough ion transport function to restore physiology in both yeast6 and in human cells.7 With this discovery in hand, we sought to better understand mechanistically how passive and unrectified small molecule ion channel mimics could rescue physiology in potassium (K+) transporter deficient (trk1Δtrk2Δ) yeast.

K+ is important for several aspects of cellular physiology8 including: promoting enzyme activity, retaining intracellular pH, and sustaining plasma membrane potential.9,10,11,12,13 Yeast possess two primary K+ transporters, Trk1 and Trk2 which are localized to the plasma membrane.14,15,16 Trk1 and Trk2 transporters require an electrochemical gradient to drive K+ into cells.2 This electrochemical gradient is generated through the activity of H+-ATPases Pma1 and V-ATPase, which hydrolyze ATP to drive protons (H+) out of the cell or into vacuoles respectively, thereby producing favorable electrochemical gradients.13 Pma1 and V-ATPase are able to create electrochemical gradients which promote K+ flow through Trk1 and Trk2 transporters into cells to attain cytosolic concentrations around 200-300 mM2 and cation storage into vacuoles.13

Previous reports demonstrated that when trk1Δtrk2Δ cells were grown under ample K+ conditions (50 mM KCl), they showed total cellular K+ content near 500 nmol

K+/mg cells, which was comparable to that observed in WT cells.17 However, when

45 trk1Δtrk2Δ cells were cultured under conditions of K+ limitation (5 mM KCl), after 10 hours their total cellular K+ content decreased dramatically to around 300 nmol K+/mg cells.17

Loss of function of the integral Trk1 and Trk2 K+ transporters18 caused yeast cells to not be able to growth under standard lab conditions of 10 mM K+.19

We propose the following mechanistic framework for how imperfect small molecule ion channel mimics restore physiology in protein-deficient yeast (Figure 21). First, it is well accepted that endogenous H+ pumps (drivers) generate favorable electrochemical gradients across cellular membranes. Furthermore, several groups have demonstrated that these electrochemical gradients persist in trk1Δtrk2Δ yeast, resulting in the hyperpolarization of these cells.2 Next, we hypothesize that passive and unselective small molecule ion channel mimics can harness these gradients to promote ion movement back to physiologically relevant levels, to restore physiology. These studies will provide insights into how small molecule surrogates act in concert with cellular networks of protein ion pumps to restore physiology, and may be applicable to a broad range of protein-deficient systems.

Figure 21. Schematic for the Proposed Pathway for Imperfect Small Molecule Mediated Rescue of Physiology. Yeast lacking K+ transporters are hyperpolarized and have functional H+-ATPases Pma1 and V-ATPase. These H+ pumps generate favorable electrochemical gradients by pumping H+ out of cells and into vacuoles respectively. These gradients are hypothesized to power ions (including K+) though imperfect small molecule ion channels. Small molecule mediated movement of K+ is predicted to increase intracellular K+ levels back to physiologically relevant values and thereby lead to the restoration of cell growth. Furthermore, Tok1 (a voltage-gated K+ efflux channel), Nha1 (an antiporter), and Ena1-5 (Na+ efflux pumps) participate in ion regulation through releasing excess ions.

Results

Under conditions of K+ limitation, trk1Δtrk2Δ yeast are hyperpolarized when compared to WT.2,3 Our findings of hyperpolarization in trk1Δtrk2Δ yeast when grown under K+ limiting conditions (Figure 22) are consistent with prior reports. Additionally, we found that the cells remained hyperpolarized across all time points tested up to 12 hours.

Encouraged by these results, we predicted that these inherent electrical gradients across cellular membranes (Figure 22) could be harnessed by cells, to drive ion movement through passive small molecule ion channels. Specifically, we hypothesized that ion channel forming small molecules would promote increased intracellular K+ content in trk1Δtrk2Δ yeast, thereby restoring cell growth and physiology. To test this hypothesis, we treated WT and trk1Δtrk2Δ yeast with small molecule ion channels Amphotericin B

(AmB) and Nystatin A1 (Nyst.),20 and quantified the total cellular K+ content.

Figure 22. K+ Transporter Deficient Yeast are Hyperpolarized. After transferring the yeast from 50 mM KCl to 15 mM KCl Yeast Nitrogen Base (YNB) medium and incubating with membrane potential dye 3,3-dipropylthiacarbocyanine iodide (DiSC3(3)), trk1Δtrk2Δ cells showed hyperpolarization (greater I580/I560), over the course of 12 hours tested. N > 6 samples for 0 hours, N > 11 samples for 6 hours, N = 12 samples for 9 hours, and N > 11 samples for 12 hours, over N ≥ 3 independent experiments. *P ≤ 0.05, ****P ≤ 0.0001.

First as a negative control, we employed Natamycin (Nat.) which is a small molecule polyene macrolide that does not permeabilize cells nor rescue trk1Δtrk2Δ cell growth. Consistent with previous results, we observed no restoration of cell growth in trk1Δtrk2Δ when tested up to 1 μM (Figure 23A). In the DMSO control, we observed a

48 significant decrease in the total cellular K+ content at 24 hours in trk1Δtrk2Δ yeast (around

200 nmol K+/ mg cells, via inductively coupled plasma-mass spectrometry (ICP-MS) compared to that of WT (around 400 nmol K+/ mg cells) when tested under conditions of

K+ limitation (15 mM KCl YNB pH 5.8) (Figure 23D). These results highlight the major role that Trk1 and Trk2 play in enabling cells to import K+. In contrast, AmB small molecule ion channels dose-dependently rescued cell growth in trk1Δtrk2Δ yeast, with optimal rescue observed at 100 nM (Figure 23B). Furthermore, increases in restored cell growth correlated with a significant and dose-dependent rise in K+ levels for AmB rescued trk1Δtrk2Δ cells (Figure 23E). Similar results were observed for both Nyst. rescue of cell growth (Figure 23C) and K+ content (Figure 23F). Importantly across all concentrations tested, Nat., AmB, and Nyst. did not perturb the K+ levels in WT cells.

Figure 23. Imperfect Small Molecule Ion Channels Dose-Dependently Rescued Cell Growth and Restored Cellular K+ Content in Protein-Deficient Yeast. A. In the absence of any small molecule added, trk1Δtrk2Δ cells showed limited growth compared to that of WT at 24 hours, under conditions of K+ limitation (15 mM KCl Final YNB, pH 5.8). Nat. did not promote cell growth across all concentrations tested (up to 1 μM). N =

Figure 23. (continued) 3 independent experiments at 24 hours. B. In contrast, AmB dose-dependently promoted cell growth in trk1Δtrk2Δ with optimal rescue occurring at 100 nM. N = 3 - 8 independent experiments at 24 hours. C. Nyst., a second example of an ion channel forming small molecule also restored growth (optimally at 1 μM), and reached values similar to that observed in WT cells. N = 3 independent experiments at 24 hours. D. Consistent with its inability to form ion channels, Nat. did not promote increased total cellular K+ content, even when tested at the highest dose of 1 μM. E. In contrast, AmB dose-dependently and significantly elevated total cellular K+ content. F. Similar results were observed for Nyst., wherein K+ levels were found to be similar to those of WT. Importantly for both AmB and for Nyst. treated WT cells, the presence of either of these small molecules did not perturb the total cellular K+ content and levels remained consistent around 400 – 500 nmol K+/ mg of cells. NS = not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Next, we asked whether small molecule mediated restoration of ion content was sustained over time. To interrogate this question, we selected the optimal rescuing concentration of each small molecule ion channel and evaluated the growth and K+ content as a function of time. Again, we employed Nat. as a negative control. We observed no improvements in trk1Δtrk2Δ cell growth upon 100 nM Nat. treatment across all time points evaluated (Figure 24A). Through ICP-MS analysis, we noted that WT cells accumulated more K+ over time (Figure 24D). Trk1Δtrk2Δ showed minor increases in K+ content by 24 hours. The addition of 100 nM Nat. to trk1Δtrk2Δ cells did not raise the total cellular K+. Even when Nat. was tested at a higher dose of 1 μM final, we did not observe improvements in trk1Δtrk2Δ cell growth nor total cellular K+ content (data not shown).

Second, we investigated whether AmB small molecule ion channels would promote increased K+ content over time. We observed that 100 nM AmB rescued trk1Δtrk2Δ cell growth back to that of WT levels by 12 hours (Figure 24B). Consistent with our hypothesis, we observed that AmB small molecule ion channels led to significant increases in the total cellular K+ content over time (Figure 24E), reaching levels close to that observed in WT. Similar results were observed for Nyst. mediated rescue of cell

50 growth (Figure 24C); and we found concurrent increases in total cellular K+ content

(Figure 24F) in trk1Δtrk2Δ cells that was sustained over the course of 24 hours.

Figure 24. Small Molecule Ion Channel Mimics Sustain Ion Transport and Restored Growth over Time in Protein-Deficient Yeast. A. Negative control Nat. (100 nM) did not re-establish cell growth over the course of 24 hours in trk1Δtrk2Δ cells compared to that of WT (N = 3-4 independent experiments). B. AmB (100 nM) promoted trk1Δtrk2Δ growth back to WT levels over time (N = 5 independent experiments). C. Nyst. (1 μM) also restored growth in trk1Δtrk2Δ cells by 12 to 24 hours (N = 3 independent experiments). D. ICP-MS analysis showed that WT cells increased total cellular K+ content over time commensurate with cell growth, reaching values near 400-500 nmol K+/mg cells. Nat. did not improve K+ levels in trk1Δtrk2Δ cells compared to that of the DMSO control with values near 200 nmol K+/mg cells. E. In contrast, 100 nM AmB treatment promoted increased total cellular K+ content in trk1Δtrk2Δ cells back to WT levels, over time. F. Similar results were found for 1 μM Nyst., where small molecule rescued trk1Δtrk2Δ cells exhibited elevated and sustained total cellular K+ content. NS not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Since endogenous H+ pumps V-ATPase and Pma1 are important for generating favorable electrochemical gradients, we predicted that chemical inhibition of these pumps would disrupt these gradients, reduce the driving force for small molecule mediated transport of ions, and thereby diminish overall cellular K+ content. To test this hypothesis,

51 we employed chemical inhibitors Bafilomycin B1 and Ebselen, a V-ATPase blocker21 and

Pma1 inhibitor22 respectively, and monitored their effect on small molecule rescued cells.

We observed that AmB rescued trk1Δtrk2Δ cells showed increased sensitivity to

Bafilomycin inhibition compared to that of AmB treated WT cells (Figure 25A).

Furthermore, we saw a dose-dependent decrease in the total cellular K+ content for both treatment groups at 12 hours (Figure 25C). The cells did not recover over the course of the experiment, and inhibition of both cell growth and K+ content was sustained even up to 24 hours (data not shown).

Figure 25. Chemical Inhibition of Ion Pumps is Detrimental to Cell Growth and Results in Diminished K+ Content. A. Bafilomycin and B. Ebselen dose-dependently blocked cell growth in both AmB treated WT and AmB rescued trk1Δtrk2Δ cells at 12 hours. These inhibitors, C. Bafilomycin and D. Ebselen also reduced the total cellular K+

Figure 25. (continued) content in AmB treated WT and AmB rescued trk1Δtrk2Δ cells, at 12 hours. N = 3 - 5 independent experiments for Bafilomycin B1. N = 3 - 4 independent experiments for Ebselen. NS = not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Ebselen inhibited cell growth (Figure 25B) and decreased the total cellular K+ content (Figure 25D) in both treatment groups. Current efforts are geared towards utilizing a more selective Pma1 inhibitor, to determine whether differences in sensitivity to this inhibitor, could be detected among treatment groups.

Discussion

We found that imperfect small molecule ion channels can functionally interface with the cells’ endogenous network of protein ion pumps, to restore ion content and physiology. Specifically, our studies further support that H+-ATPases V-ATPase and

Pma1 are important for generating favorable electrochemical gradients. Our data suggest that cells can harness these gradients to drive K+ ions through imperfect small molecule ion channels, back to physiologically meaningful levels for cell growth. Furthermore, blocking either V-ATPase or Pma1 diminished small molecule mediated K+ uptake and thereby cell growth.

Intriguingly, the total cellular K+ content in small molecule rescued trk1Δtrk2Δ cells was found to be similar to that in WT. These observations suggest that mechanisms of ion homeostasis remain active in small molecule rescued trk1Δtrk2Δ cells, albeit these imperfect small molecule ion channels are poorly selective and not regulated. Future studies quantifying the total cellular ion content of other ions23 that polyene macrolides are known to transport, including H+, Na+, and Cl- would provide insights into the dynamic

53 responses of cells to these imperfect ion channels and cellular regulation of ion movement.

Furthermore, could the inherent protein framework of electrogenic ion pumps that generate favorable gradients for small molecule ion channels, be broadly applicable across many different vertebrate systems? These small molecule ion channel mimics could serve as invaluable probes to investigate these ion gradients and protein drivers, in other cellular systems. In addition, it is fascinating to consider whether this approach of using imperfect small molecule ion channels could expand beyond that of ion channels and carriers. Future studies with diverse small molecule mimics that perform different functions (enzyme catalysis, protein scaffolding, etc.) would provide further evidence for the generality of this small molecule approach.

Author Contributions

For scientific contributions, Page N. Daniels developed the membrane potential assay, based on literature precedent. Jenn Hou performed the membrane potential assay

(Figure 22), and developed and conducted the dose- and time-dependent studies

(Figure 23 and Figure 24) for total cellular K+ content and for the chemical inhibition studies (Figure 25). Dr. Kiran Subedi contributed to the inductively coupled plasma-mass spectrometry (ICP-MS) experimental design and with Crislyn Lu performed the ICP-MS analysis for all reported data.

Methods and Materials

Membrane Potential Determination and Statistical Analysis

We measured the membrane potential as previously described.2,24 Briefly, both

WT and trk1∆trk2∆ yeast were cultured overnight in 50 mM KCl supplemented YNB, pH

5.8. Following, the cells were harvested, washed twice with sterile Milli-Q H2O, and resuspended in 15 mM KCl supplemented YNB, pH 5.8. Under a sterile field, the cells were diluted to an OD600 of 0.2 and incubated for the indicated times. We prepared multiple sample tubes, per designated time point. At each time point, we checked the OD600 values of the samples and diluted the cells to an OD600 of 0.2 if necessary. An aliquot of each sample was pipetted into a 96-well black microplate. 3,3-dipropylthiacarbocyanine iodide

(DiSC3(3)) dye was added to the wells to a final concentration of 0.2 µM. The samples were incubated for 30 minutes (at 200 X RPM, 30°C). A fluorescence spectrum with excitation at 531 nm and emission from 549-589 nm, was taken on a BioTek Synergy H1 microplate reader. The ratio of 580 nm/560 nm was recorded for each sample. Results show the average of at least N = 6 biological replicates per time point, for ≥ 3 independent experiments total. For statistical analysis, we compared the two yeast strains at each time point, with the unpaired t-test.

Large Scale Rescue Assays and Statistical Analysis

Dose-Response

5 single colonies of isogenic WT and trk1Δtrk2Δ yeast were cultured overnight in

100 mL each of 100 mM KCl YNB medium pH 5.8, 200 X RPM, 30°C. During the day of the experiment, AmB was freshly prepared. A clean pasteur pipette was used to add a small amount of AmB to 2 mL of DMSO. The solution was briefly vortexed and sonicated.

Then a 1 mL gastight syringe was utilized to add a total of 1.998 mL of methanol (MeOH) to three separate glass vials. Following, we added 2 μL of the AmB solution to dilute the

55 sample 1:1000 fold. Next, we blanked the UV-VIS instrument with MeOH across wavelengths from 300-450 nm and determined the absorbance at 406 nm. Care was taken to ensure that the absorbance values were within 5-10% of the average and more samples were prepared and measured if needed. The Beer-Lambert Law A = εcl was employed to determine the concentration of the solution with ε = 164,000 M-1cm-1 for AmB, l = 1 cm; and we accounted for the 1,000-fold dilution factor. Once the concentration of

AmB was determined, we prepared a 6 mL solution of 80 μM. Then the following dilutions were prepared as indicated in Table 1.

Table 1. AmB Dilution

Initial Concentration (μM) 0 0.4 2 4 Final Concentration (μM) 0 0.01 0.05 0.1 Amount of 80 μM AmB to Add 0 0.03 mL 0.15 mL 0.3 mL (2.5 mL Amount of DMSO to Add per flask) 5.97 mL 5.85 mL 5.7 mL

The same protocol was followed for Nystatin A1, except the UV-VIS instrument was blanked with MeOH for wavelengths from 250-450 nm. The absorbance values were determined at λmax of 304 nm, and the concentration was determined where the ε = 51,207

M-1cm-1. Following, we prepared a 120 μM Nystatin A1 solution and made the following dilutions in DMSO as indicated in Table 2.

Table 2. Nystatin A1 Dilution

Initial Concentration (μM) 0 4 10 20 40 Final Concentration (μM) 0 0.1 0.25 0.5 1 Amount of 120 μM Nystatin A1 to Add 0 0.2 mL 0.5 mL 1 mL 2 mL (2.5 mL Amount of DMSO to Add per flask) 5.8 mL 5.5 mL 5 mL 4 mL

For Natamycin, the UV-VIS instrument was blanked with MeOH for wavelengths from 250-450 nm. The absorbance values were determined at λmax of 317 nm, and the concentration was determined where the ε = 76,000 M-1cm-1. Following, we prepared a

120 μM Natamycin solution and made the following dilutions in DMSO as indicated in

Table 3.

Table 3. Natamycin Dilution

Initial Concentration (μM) 0 0.4 4 20 40 Final Concentration (μM) 0 0.01 0.1 0.5 1 Amount of 120 μM Natamycin to Add 0 0.02 mL 0.2 mL 1 mL 2 mL (2.5 mL Amount of DMSO to Add per flask) 5.98 mL 5.8 mL 5 mL 4 mL

Then between 18-24 hours when both cell cultures were saturated, we harvested

90 mL for both WT and trk1Δtrk2Δ cultures by centrifuging at 1000 X G, 5 minutes, 23°C.

We removed the supernatant, resuspended the cells in equal volumes of sterile Milli-Q

H2O and recentrifuged the samples. The wash step was repeated. This time, we resuspended the cells in 5-10 mL of 15 mM KCl YNB pH 5.8 and combined each respective strain into one conical tube. The cell suspensions were briefly vortexed.

Afterwards, we blanked the UV-VIS instrument with an empty cuvette for spectra from

590-610 nm. Under the sterile field of the Bunsen burner, the cell suspensions were diluted to an OD600 value between 0.090 – 0.110 (after allowing the samples to rest for 2-

3 minutes). We prepared at least 0.7 L of cells suspension at these absorbance values for both strains.

Next, we carefully poured about 0.25 L of each cell suspension into sterile graduated cylinders to coat the inner surface, working with one strain at a time. Then we poured this cell suspension back into the original 1 L bottle. Next, we measured out 0.1 L

57 total of cell suspension and transferred this to a clean and labeled 0.25 L Erlenmeyer flask. This step was repeated for the remaining flasks and the bottle was inverted between each aliquot to ensure that the cells did not settle to the bottom. The same was done for the other stain.

Next, we swirled each flask and removed 2.5 mL of cell suspension from each flask using clean serological pipettes. Following, 2.5 mL of DMSO or small molecule solution was added to each flask. The order of the strains poured and small molecule treatment doses was alternated for different experimental runs to avoid introducing bias. The flasks were brought to the incubating shaker (200 X RPM, 30°C) and incubated for 24 hours.

Time Course

We performed the same overnight culture set-up as described for the dose- response experiments. To prepare the Nat. stock solution, the concentration was determined as previously described. This time, we prepared 14 mL of a 4 μM solution

(with a final concentration of 0.1 μM). We also prepared 14 mL of a 40 μM solution (with a final concentration of 1 μM) for the higher-dose experiment. For AmB, we generated 14 mL of a 4 μM solution (with a final concentration of 0.1 μM). Last, for the Nystatin A1 stock solution, we made 14 mL of a 40 μM solution (with a final concentration of 1 μM).

When the cells were saturated between 18-24 hours, we harvested 90 mL of each strain at 1000 X G, 5 minutes, 23°C. Then the supernatant was poured off and the cells were resuspended in equal volumes of sterile Milli-Q H2O. The cells were briefly vortexed to fully resuspend the cell pellets. Afterwards, we recentrifuged the samples. We repeated the wash step. This time, the cell pellets were resuspended in 15 mM KCl YNB pH 5.8 and vortexed to fully resuspend.

We blanked the UV-VIS instrument with an empty cuvette for wavelengths from

590- 610 nm. Then under the sterile field of a Bunsen burner, we prepared at least 0.6 L of WT cell suspension and 1.1 L of trk1Δtrk2Δ cell suspension with OD600 values between

0.090-0.110. Next, 0.5 L of cell suspension was poured into a 0.5 L graduated cylinder to coat the inner surface. Then this cell suspension was poured back into the original bottle.

Following, 0.5 L of cell suspension was measured and then transferred into sterile 0.5 L

Erlenmeyer flasks for the WT and for the trk1Δtrk2Δ strains (two separate flasks).

Serological pipettes were used to remove 12.5 mL of cell suspension from each flask.

Afterwards, 12.5 mL of DMSO or small molecule solution was added to each Erlenmeyer flask, and we made sure to pipette up and down several times. We measured out 0.1 L for each treatment group (WT + DMSO, trk1Δtrk2Δ + DMSO, and trk1Δtrk2Δ + small molecule) and poured these cell suspensions into labeled 0.25 L Erlenmeyer flasks for the 6, 12, and 24 hour time points. The order by which the cell strains and flasks designated for each time point were measured out and treated with small molecule, was varied from experiment to experiment to avoid bias.

The remaining 200 mL of cell suspension (for the 0 hour timepoint) was rapidly filtered onto 0.8 μm filters, and washed with 100 mL each of ice cold 20 mM MgCl2 before transferring the 0.8 μm filters to clean, pre-labeled 6 well plates for ICP-MS analysis.

Statistical Analysis

For the dose-response studies, we utilized the unpaired t-test for the DMSO controls and one-way ANOVA for the trk1Δtrk2Δ treatment groups. For the time-course studies, we employed one-way ANOVA for each time point.

Chemical Inhibition and Statistical Analysis

We followed the same protocol as described previously for the overnight culturing conditions. For the chemical inhibition studies, at least 25 mL of a 5 μM AmB solution was prepared. The ratio of AmB to cell suspension was 1:50 (where 2 mL of AmB solution was added per 100 mL of cell suspension) and the ratio of inhibitor to cell suspension was 1:200 (where 0.5 mL of inhibitor solution was added per 100 mL of cell suspension).

For Bafilomycin solutions, 1 mg of Bafilomycin B1 (Santa Cruz Biotechnology, sc-202072) was dissolved in 0.5 mL of DMSO to obtain a concentration of 2450.98 μM. Then 3 mL of a 100 μM stock solution was prepared in DMSO. Next, the following dilutions were prepared in DMSO:

Table 4: Bafilomycin B1 Dilution Initial Concentration (μM) 0 12.5 25 50 100 Final Concentration (μM) 0 0.0625 0.125 0.25 0.5 1.5 mL 1.5 mL 1.5 mL of 25 of 50 of 100 μM μM μM (use Serially Dilute Bafilomycin B1 0 stock stock stock direct) (0.5 mL Amount of DMSO to Add per flask) 1.5 mL 1.5 mL 1.5 mL 0

For Ebselen, 5 mg of Ebselen (Cayman Chemical, 70530) were dissolved into 0.5 mL of DMSO. The resulting 36.46 mM Ebselen solution was further diluted in DMSO to generate 3.5 mL of a 5000 μM solution. Then the following solutions were prepared starting with the 5000 μM stock solution:

Table 5: Ebselen Dilution

Initial Concentration (μM) 0 312.5 625 1250 2500 Final Concentration (μM) 0 1.56 3.125 6.25 12.5 1.5 mL 1.5 mL 1.5 mL 1.5 mL of 625 of 1250 of 2500 of 5000 Serially Dilute Ebselen 0 μM μM μM μM (0.5 mL Amount of DMSO to Add per flask) 1.5 mL 1.5 mL 1.5 mL 1.5 mL

The cells were harvested, washed, and resuspended in 15 mM KCl YNB, pH 5.8 medium as previously described. This time, we obtained 0.7 L of WT and 0.9 L of trk1Δtrk2Δ cell suspensions with OD600 values between 0.090-0.110. Next, we measured out 0.5 L of WT cell suspension using a graduated cylinder to coat the inner surface. This cell suspension was poured back into the original 1 L bottle. Then 0.5 L of WT cell suspension was remeasured and then transferred to a sterile 0.5 L Erlenmeyer flask.

Additionally, 0.1 L of WT cell suspension was obtained and transferred to a 0.25 L

Erlenmeyer flask (this sample was designated for the WT + DMSO control). The same was done for the trk1Δtrk2Δ cells. We also included two additional controls for the 24 hour time points: trk1Δtrk2Δ + DMSO and trk1Δtrk2Δ + 100 nM AmB.

Then 2.5 mL of cell suspension was removed from each flask designated for

DMSO, 2 mL of cell suspension was removed from the 0.1 L flasks designated for AmB treatment, and 10 mL of cell suspension was removed from the 0.5 L flasks designated for AmB treatment. Next, 2.5 mL of DMSO was added to flasks designated for DMSO treatment. 2 mL of AmB was added to the 0.1 L flasks designated for AmB treatment. 10 mL of 5 μM AmB was added to the 0.5 L flasks meant for AmB treatment.

Following, we transferred the AmB treated cell suspensions to clean 1 L bottles and gently swirled to ensure homogenous mixing of the AmB solution. We measured out

0.1 L of both AmB treated WT and trk1Δtrk2Δ cell suspensions and placed them into designated 0.25 L Erlenmeyer flasks. Then 0.5 mL of cell suspension was removed from each flask using serological pipettes. Afterwards, 0.5 mL of DMSO or chemical inhibitor was added to each flask in the indicated doses. The cell suspension was pipetted up and down several times to ensure full dispensing of the indicated chemical inhibitor solution.

The flasks were cultured at 200 X RPM, 30°C, for 12 hours. We also had prepared trk1Δtrk2Δ + DMSO, and trk1Δtrk2Δ + AmB samples for the 24 hour controls.

Table 6: Chemical Inhibition Treatment Groups. Flask Number Treatment Group Time Point (Hours) 1 WT + DMSO 12 2 WT + 100 nM AmB 12 3 WT + 100 nM AmB + Dose 1 12 4 WT + 100 nM AmB + Dose 2 12 5 WT + 100 nM AmB + Dose 3 12 6 WT + 100 nM AmB + Dose 4 12 7 trk1Δtrk2Δ + DMSO 12 8 trk1Δtrk2Δ + 100 nM AmB 12 9 trk1Δtrk2Δ + 100 nM AmB + Dose 1 12 10 trk1Δtrk2Δ + 100 nM AmB + Dose 2 12 11 trk1Δtrk2Δ + 100 nM AmB + Dose 3 12 12 trk1Δtrk2Δ + 100 nM AmB + Dose 4 12 13 trk1Δtrk2Δ + DMSO 24 14 trk1Δtrk2Δ + 100 nM AmB 24

Statistical Analysis

For chemical inhibition studies, we used the unpaired t-test to compare treatment groups.

Determination of Ion Content by Inductively Coupled Plasma- Mass Spectrometry

For both the dose-response and time-course studies, a 1 mL aliquot of sample was taken and reserved for absorbance (OD600) measurements by UV-VIS spectroscopy.

Following, each 100 mL sample was rapidly filtered through a 0.8 μm membrane filter

(EMD Millipore, catalog number AAWP04700) and washed with 50 mL of ice cold 20 mM

MgCl2. Then the sample filter membranes were carefully transferred into clean 6 well plates, where each filter membrane was resuspended in 5 mL of ice cold 20 mM MgCl2 to collect the cell suspension. Each of these cell suspensions were then transferred into clean and labeled 15 mL conical tubes. Afterwards, the samples were flash frozen in liquid nitrogen and lyophilized for at least 72 hours. Importantly, after 48 hours of lyophilization, the samples were gently agitated to ensure full removal of water from the samples and re-lyophilized for the remaining 24 hours. These samples were highly hygroscopic and care was taken to avoid exposure to water.

For best practice, the lyophilized samples were transferred into an argon glovebox.

Five cycles of purging the antechamber with argon and then applying vacuum for 5 min. each were performed. Next, each sample was packed into aluminum capsules (provided by The UIUC Microanalysis Lab, with 1 – 10 mg of sample per capsule) and crimped shut with tweezers. The remaining sample was transferred into labeled 7 mL glass vials. The capsules were submitted for total cellular K+ content and the unused samples were stored in desiccators.

References

1 Navarrete C, Petrezselyova S, Barreto L, Marinez JL, Zahradka J, Arino J, Sychrova H, Ramos J. (2010). Lack of main K+ uptake systems in Saccharomyces cerevisiae affects yeast performance in both potassium-sufficient and potassium-limiting conditions. FEMS Yeast Res. 10(5): 508-517. 2 Zahradka J, Sychrova H. (2012). Plasma-membrane hyperpolarization diminishes the cation efflux via Nha1 antiporter and Ena ATPase under potassium-limiting conditions. FEMS Yeast Res. 12(4): 439-446. 3 Gouaux E, MacKinnon R. (2005). Principles of selective ion transport. Science. 310(5753): 1461-1465. 4 Yu JT, Chang RCC, Tan L. (2009). Calcium dysregulation in Alzheimer’s disease: from mechanisms to therapeutic opportunities. Progress in Neurobiology. 89(3): 240-255. 5 Mandel S, Amit T, Bar-Am O, Youdim MBH. (2007). Iron dysregulation in Alzheimer’s disease: multimodal brain permeable iron chelating drugs, possessing neuroprotective- neurorescue and amyloid precursor protein-processing regulatory activities as therapeutic agents. Progress in Neurobiology. 82(6): 348-360. 6 Cioffi AG, Hou J, Grillo AS, Diaz KA, Burke MD (2015) Restored physiology in protein- deficient yeast by a small molecule channel. J. Am. Chem. Soc. 137(22): 10096-10099. 7 Muraglia K. et al. (2017). CFTR-independent rescue of cystic fibrosis with a small molecule bicarbonate channel. Manuscript in preparation. 8 Herrera R, Alavarez MC, Gelis S, Kodedova M, Sychrova H, Kschischo M, Ramos J. (2014). Role of Saccharomyces cerevisiae Trk1 in stabilization of intracellular potassium content upon changes in external potassium levels. Biochim. Biophys. Acta. 1838(1 Pt B): 127-133. 9 Arino J, Ramos J, Sychrova H. (2010). Alkali metal cation transport and homeostasis in yeast. Microbiol. Mol. Biol. Rev. 74(1): 95-119. 10 Herrera R, Alvarez MC, Gelis S, Ramos J. (2013). Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants. Biochem. J. 454(3): 525-532. 11 Barreto L, Canadell D, Valverde-Saubi D, Casamayor A, Arino J. (2012). The short- term response of yeast to potassium starvation. Environ. Microbiol. 14(11): 3026-3042. 12 Kahm M, Navarrete C, Llopis-Torregrosa V, Herrera R, Barreto L, Yenush L, Arino J, Ramos J, Kschischo M. (2012). Potassium starvation in yeast: mechanisms of homeostasis revealed by mathematical modeling. PLoS Comput. Biol. 8(6): e1002548. 13 Cyert MS and Philpott CC. (2013). Regulation of cation balance in Saccharomyces cerevisiae. Genetics. 193(3): 677-713. 14 Gaber RF, Styles CA, Fink GR. (1988). TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol. Cell. Biol. 8(7): 2848-2859. 15 Ko CH, Buckley AM, Gaber RF. (1990). TRK2 is required for low affinity K+ transport in Saccharomyces cerevisiae. Genetics 125(2): 305-312. 16 Ko CH, Gaber RF. (1991). TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 11(8): 4266-4273.

17 Gelis S, Gonzalez-Fernandez R, Herrera R, Jorrin J, Ramos J (2015) A physiological, biochemical and proteomic characterization of Saccharomyces cerevisiae trk1,2 transport mutants grown under limiting potassium conditions. Microbiology 161: 1260-1270. 18 Perez-Valle J, Jenkins H, Merchan S, Montiel V, Ramos J, Sharma S, Serrano R, Yenush L. (2007). Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters. Mol. Cell. Biol. 27(16): 5725-5736. 19 Gelis S, Curto M, Valledor L, Gonzales A, Arino J, Jorrin J, Ramos J. (2012). Adaptation to potassium starvation of wild-type and K+-transport mutant (trk1,2) of Saccharomyces cerevisiae: 2-dimensional gel electrophoresis-based proteomic approach. Microbiologyopen. 1(2): 182-193. 20 Ermishkin LN, Kasumov KM, and Potzeluyev VM (1976) Single ionic channels induced in lipid bilayers by polyene antibiotics amphotericin B and nystatine. Nature. 262(5570): 698-690. 21 Drose S and Altendorf K (1997) Bafilomycins and concanamycins as inhibitors of V- ATPase and P-ATPases. J. Exp. Biol. 200: 1-8. 22 Chan G, Hardej D, Santoro M, Lau-Cam C, Billack B (2007) Evaluation of the antimicrobial activity of ebselen: role of the yeast plasma membrane H+-ATPase. J. Biochem. Mol. Toxicol. 21(5): 252-264. 23 Hartsel SC, Benz SK, Ayenew W, Bolard J. (1994). Na+, K+, Cl- selectivity of the permeability pathways induced through sterol-containing membrane vesicles by amphotericin B and other polyene antibiotics. Eur. Biophys. J. 23(2): 125-132. 24 te Winkel JD, Gray DA, Seistrup KH, Hameon LW, Strahl H. (2016). Analysis of antimicrobial-triggered membrane depolarization using voltage sensitive dyes. Frontiers in Cell and Developmental Biology. 4(29): 1-10.

CHAPTER 4: SMALL MOLECULE ION CHANNELS FUNCTIONALLY INTERFACE WITH ENDOGENOUS PROTEIN ION PUMPS AND TRANSPORTERS

Introduction

Across all kingdoms of life, organisms possess robust mechanisms1,2,3,4,5 that enable them to respond and adapt to genomic and environmental perturbations.6 For example, a recent study found that humans on average harbor 20 genes that are completely inactivated; yet, these individuals show no phenotypic consequence.7 The specific molecular mechanisms of robustness include functional redundancy,8,9,10,11 feedback control,12 parallel signaling,13 and modularity.8

Briefly, functional redundancy occurs through gene duplication (more than one copy of a gene carries out the same biological process) and degeneracy (unrelated genes perform similar functions).14,15 In feedback control, positive or negative feedback signals can enhance sensitivity or reduce noise respectively.12 Parallel signaling allows for multiple pathways to converge onto a similar output.16 For example, in many metabolic pathways, intermediates can quickly be diverted and funneled into parallel reactions, to achieve the desired products. Last, modularity or compartmentalization, stops potential defects from infiltrating other parts of the cell or organism.8

However, when an organism is missing a protein that is critical for carrying out a vital cellular process, that organism’s functional capacity dips below the threshold required for normal physiology and results in a disease phenotype such as the lack of cell growth or disease (Figure 26, middle bar). Yet, we hypothesized that a residual level of functional capacity remains. We predicted that an imperfect small molecule mimic of the missing protein could functionally interface with the organism’s own robustness

66 mechanisms, and thereby provide enough functional capacity to enable the organism to regain normal physiology (Figure 26, right bar).

Figure 26. Model of Small Molecule Mediated Functional Complementation. Wild type organisms (left bar) have many mechanisms of robustness such as functional redundancy, parallel signaling, feedback control, and modularity that enable them to survive and propagate despite environmental and genetic challenges. However, when the organism is missing a key protein (middle bar), the organism’s functional capacity drops below the threshold needed for normal physiology. Several robust mechanisms remain (middle bar). Functionally interfacing with the organism’s own robustness networks, a small molecule is hypothesized to provide the functional capacity necessary to enable the organism to regain normal physiology (right bar).

Excitingly, we discovered that imperfect small molecule mimics are sufficient to restore physiology in cellular systems and organisms ranging from yeast, to human lung epithelia, to vertebrates. For example, Hinokitiol (Hino.), a cedar tree derived tropolone natural product, binds to iron in a 3:1 ratio of Hino. to iron.17 We found that this small molecule carrier binds and transports iron across cellular membranes, by leveraging built- up electrochemical gradients to restore physiology.17 Interestingly, Hino. was also found

67 to functionally interface with the cells’ inherent network of proteins involved in iron homeostasis including ferritin (sequesters iron) and ferroportin (releases excess iron).17

In a completely different system, we discovered that an imperfect small molecule ion channel Amphotericin B (AmB) could restore physiology in a potassium (K+) transporter deficient (trk1Δtrk2Δ) yeast.18 We hypothesized that the interface between endogenous protein networks and small molecules could be broadly applicable. We sought to understand the endogenous proteins involved in promoting small molecule ion channel mediated rescue and utilized yeast as an outstanding eukaryotic model organism.

Ion homeostasis is vital for normal physiology.19,20,21 As in human cells, exquisitely regulated networks of protein ion pumps and cotransporters control the passage of ions across yeast membranes.22 Yeast express ATP driven H+ pumps V-ATPase and Pma1, which localize to the vacuolar and plasma membranes respectively (Figure 27). V-

ATPase and Pma1 hydrolyze ATP to force H+ into vacuoles and out of the cell respectively, leading to the acidification of vacuoles,23 establishment of intracellular pH,24,25,26 and production of favorable electrochemical gradients.23,27,28 These gradients power K+ flux through two K+ transporters, Trk1 and Trk223,27,29,30 and into vacuoles.23

Yeast rigorously regulate intracellular K+ and sodium (Na+) concentrations to enable survival amidst varying extracellular K+ concentrations (2 μM to 2 M)31 and to avoid Na+ toxicity.23,32 Healthy cytoplasmic concentrations range from 200-300 mM19 for K+ and 10-

20 mM for Na+.33 Yeast do not express a selective Na+ transporter,23 and intracellular Na+ increases when cells are exposed to high extracellular Na+.23,27

Figure 27. Yeast Possess Highly Regulated and Integrated Networks of Protein Ion Pumps and Transporters. H+ ATPases Pma1 (found on the plasma membrane) and V- ATPase (localized to the vacuolar membrane) utilize the energy from ATP hydrolysis to establish electrochemical gradients across these membranes. Other protein transporters including Trk K+ transporters harness these gradients and import K+ through secondary active transport. Yeast also express K+ and Na+ exporters including voltage gated K+ efflux pump Tok1, Na+/H+ antiporter Nha1, and Na+-ATPase Ena1-5 that release excess ions.

V-ATPase and Pma1 are widely accepted as the primary H+-ATPases in charge of producing electrochemical gradients. V-ATPase is a hetero-multimeric protein with two

+ distinct structural components V0 (H passageway) and V1 (active site for ATP

26,34 hydrolysis). V-ATPase is regulated by low glucose, where domains Vo and V1

26 + 35 reversibly separate. K starvation triggers P2-type ATPase Pma1 to ramp up its activity, but not its protein expression.36 Trk1Δtrk2Δ cells deprived of K+ significantly increase Pma1 activity relative to that in WT.36 Furthermore, truncation of Pma1 prohibits cell growth under low K+ conditions (1 mM K+).36

Yeast also express plasma membrane localized voltage gated K+ efflux channel

Tok1,37,38 Na+(K+)/(H+) anti-porter Nha1,39,40,41 and Na+-ATPase Ena1-5,42 all of which play an important role in releasing Na+ and/or K+ to maintain ion homeostasis43 (Figure

27). For its regulation, Tok1 is an eight-transmembrane domain channel that responds to depolarization of the plasma membrane.27 Nha1 possesses 12 transmembrane domains, is constitutively expressed, and functions under acidic conditions.27 Last, cells express low levels of Ena1-5 where each protein contains 10 transmembrane domains.44

However, cells show increased expression of Ena1-5 upon exposure to basic pH, perturbations in osmotic pressure, salt stress, and lack of glucose.27

Since AmB small molecule ion channels conduct both K+ and Na+, we hypothesized that Tok1, Nha1, and/or Ena1-5 are critical for AmB mediated ion channel rescue, by expelling excess Na+ and/or K+ ions (Figure 28B). Through both pharmacological and genetic approaches, we tested the hypothesis that protein drivers

(V-ATPase and Pma1, Figure 28A) and protein correctors (Tok1, Nha1, and Ena1-5,

Figure 28B) are critical for small molecule mediated rescue of trk1Δtrk2Δ growth.

Furthermore, we expanded this study to investigate other potential proteins that functionally interface with small molecule mimics through performing DNA microarray analysis.

Figure 28. Protein Drivers and Correctors Functionally Interface with Small Molecule Mimics to Restore Physiology. A. Protein drivers Pma1 and V-ATPase

Figure 28. (continued) generate favorable electrochemical gradients to mediate cation influx through imperfect small molecule ion channels. B. Protein correctors Tok1, Nha1, and Ena1-5 are predicted to address the lack of ion selectivity of small molecule ion channels, through expelling excess Na+ and/or K+ ions.

Results

First, we questioned whether Pma1 and V-ATPase enable imperfect small molecule ion channel mimics to rescue cell growth in trk1Δtrk2Δ yeast. We hypothesized that chemical inhibition of Pma1 and V-ATPase activity would impair small molecule mediated restoration of cell growth, i.e. small molecule ion channel rescued cells would show increased sensitivity to inhibition of Pma1 and V-ATPase compared to that of WT cells. To test this hypothesis, we employed three inhibitors (Figure 29): Nocodazole

(impedes microtubule dynamics, negative control),45,46 Ebselen (inhibits Pma1),47 and

Bafilomycin (blocks V-ATPase).48 We tested these inhibitors against WT and trk1Δtrk2Δ cells treated with small molecule ion channels and grown under standard K+ conditions

(10 mM KCl).

Figure 29. Chemical Inhibition of Protein Drivers. Schematic showing the three targets of the chemical inhibition studies. Nocodazole inhibits microtubule dynamics and served as an off-target pathway inhibitor; microtubule dynamics are not predicted to be directly involved in small molecule mediated rescue. Ebselen and Bafilomycin inhibit Pma1 and V-ATPase respectively.

Nocodazole impairs microtubule dynamics, a process unrelated to K+ transport and thus served as a negative control. We observed no statistically significant difference in the half maximal effective concentration (EC50) between chemical inhibition of WT versus trk1Δtrk2Δ + AmB (Figure 30A and Figure 30D; EC50 ± SEm: 24.8 μM ± 0.2 and 24.5 μM

± 0.3, respectively). Similarly, Nocodazole inhibition of Nystatin (Nyst.)-, Candicidin

(Can.)-, and Mepartricin (Mep.)-treated WT and rescued trk1Δtrk2Δ cells showed little to no statistically significant difference in sensitivity (Figure 30D). In contrast, we observed that AmB-rescued trk1Δtrk2Δ cells showed increased sensitivity to Ebselen compared to that of WT cells (Figure 30B and Figure 30E; EC50: 7.3 μM ± 0.2 and 13.2 μM ± 0.1

72 respectively, ****P). Similar results were obtained in Nyst.-, Can.-, and Mep.-rescued trk1Δtrk2Δ cells (Figure 30E). In addition, we noted exceptional sensitivity of AmB- rescued trk1Δtrk2Δ cells compared to that of WT cells to Bafilomycin inhibition in Figure

30C and Figure 30F (EC50: 0.05 μM ± 0.03 and 0.6 μM ± 0.01 respectively, ****P). Nyst.-

, Can.-, and Mep.-rescued trk1Δtrk2Δ cells also showed high sensitivity towards

Bafilomycin compared to that of WT cells (Figure 30F). These results provide evidence that Pma1 and V-ATPase promote small molecule mediated rescue of trk1Δtrk2Δ growth.

Figure 30. Chemical Inhibition of Small Molecule Mediated Rescue. A. Both WT + AmB and trk1Δtrk2Δ + AmB treatment groups responded similarly to Nocodazole inhibition. B. trk1Δtrk2Δ + AmB cells showed diminished cell growth at lower concentrations of Ebselen (blocks Pma1) than that observed in WT + AmB. C. Bafilomycin (inhibits V-ATPase) reduced cell growth of trk1Δtrk2Δ + AmB cells at lower concentrations compared to that of WT + AmB. D. AmB, Nyst., Can., and Mep. rescued trk1Δtrk2Δ cells were similarly affected by Nocodazole inhibition as AmB, Nyst., Can., and Mep. treated WT cells at 24 hours. E. Ebselen blocking of cell growth was achieved at lower concentrations in small molecule rescued trk1Δtrk2Δ cells compared to that of small molecule treated WT cells at 24 hours. F. Bafilomycin dramatically reduced small molecule rescue of trk1Δtrk2Δ cell growth compared to that of WT cells at 24 hours. WT = black bars, trk1Δtrk2Δ = gray bars. NS not significant, *P ≤ 0.05, ***P ≤ 0.001, ****P ≤ 0.0001.

The ion channel formed by AmB is unrectified, unregulated, and unselective

+ + - - 49,50 (enables passive transport of K , Na , chloride (Cl ), and bicarbonate (HCO3 ) ions).

We next questioned whether protein ion efflux channels and exchangers play a role in enabling tolerance for the lack of ion selectivity of small molecule ion channel mimics.

Voltage gated K+ efflux channel Tok1, plasma membrane localized anti-porter Nha1

(couples H+ influx for Na+/K+ efflux), and Na+-ATPase Ena1-5 play an important role in mediating Na+ and/or K+ efflux to maintain ion homeostasis. We hypothesized that Tok1,

Nha1, and/or Ena1-5 may contribute to imperfect small molecule ion channel rescue through extruding excess Na+ and/or K+ ions. To test this hypothesis, we acquired a series of isogenic yeast strains lacking Trk1 and Trk2, Tok1, Nha1, Ena1-5, or their combination.

Under standard K+ conditions, we observed no differences in growth between WT and nha1Δ strains (Figure 31B). Consistent with previous results, AmB restored cell growth in trk1Δtrk2Δ cells.18 However upon deletion of Nha1, we observed no AmB mediated restoration of cell growth in trk1Δtrk2Δnha1Δ. This result was similarly observed for Nyst. (Figure 31E), Can. (Figure 31H), and Mep. (Figure 31K) treated cells. In contrast, deletion of either Tok1 or Ena1-5 did not affect AmB (Figure 31A and Figure

31C), Nyst. (Figure 31D and Figure 31F), Can. (Figure 31G and Figure 31I), or Mep.

(Figure 31J and Figure 31L) mediated rescue of cell growth in trk1Δtrk2Δtok1Δ and trk1Δtrk2Δena1-5Δ, respectively. Therefore, these results show that Nha1 is important for small molecule mediated rescue of growth in trk1Δtrk2Δ.

Figure 31. Nha1 is Critical for Small Molecule Mediated Rescue. A. Deletion of voltage-gated, K+ efflux pump Tok1 did not impact AmB mediated rescue of growth in trk1Δtrk2Δtok1Δ. Furthermore, we did not observe differences in growth between WT and tok1Δ cells. B. In contrast, deletion of Na+/H+ antiporter, Nha1 impaired AmB mediated restoration of trk1Δtrk2Δnha1Δ growth, while loss of Nha1 alone did not have any

Figure 31. (continued) detrimental effects on cell growth. C. ATP-driven Na+ pumps, Ena1-5 are not required for AmB mediated rescue of trk1Δtrk2Δena1-5Δ. Similar trends were observed for Nyst., in D.–F. WT and trk1Δtrk2Δ values were replotted through graphs A.-F. for clarity. These experiments were performed under conditions of 15 mM KCl Yeast Nitrogen Base (YNB), pH 5.8, 200 X RPM, 30°C and absorbance values (OD600) were obtained at 72 hours. (Graphs represent the average of 2 independent experiments, with 3 biological replicates per experiment). Similar results were observed for Can. where rescue was observed for G. trk1Δtrk2Δtok1Δ, not for H. trk1Δtrk2Δnha1Δ, and growth was noted in I. trk1Δtrk2Δena1-5Δ. We observed the same trends for Mep. with J. trk1Δtrk2Δtok1Δ, K. trk1Δtrk2Δnha1Δ, and L. trk1Δtrk2Δena1-5Δ. Graphs G.-L. were performed in 10 mM KCl YNB, pH 5.8 and absorbance values (OD600) were obtained at 24 hours. The graphs depict the average of 2 independent experiments with 3 biological replicates per experiment (except for N = 5 total biological replicates for graph H.).

We next asked whether trk1Δtrk2Δ cells would undergo global transcriptional changes in response to K+ limitation. Previous reports show that yeast react to changes in extracellular K+ by altering gene expression.28 Trk1Δtrk2Δ cells decreased the expression of 79 genes and elevated transcript levels of 69 genes compared to that of

WT in 50 mM K+ Yeast Nitrogen Base (YNB).28 We hypothesized that we would observe an even greater transcriptional response of trk1Δtrk2Δ cells subjected to an even more stringent K+ limitation of 10 mM KCl YNB and predicted that AmB mediated rescue would mitigate these transcriptional changes. Furthermore, through pursuing a global transcriptional analysis, this could possibly lead to the identification of other key proteins that functionally interface with small molecule mimics.

To probe this question, we investigated the changes in gene expression by performing a DNA microarray analysis. First, we developed a large-scale rescue assay to obtain sufficient quantities of high-quality RNA suitable for DNA microarray analysis.

We treated both WT and trk1Δtrk2Δ cells with and without AmB and collected samples at

0 and 180 minutes. We chose this early time point to capture cells that are in the same

76 cell cycle stage. Importantly, we validated small molecule mediated rescue in these samples by measuring the growth of the trk1Δtrk2Δ + AmB cells at 24 hours (Figure 32).

Figure 32. Large Volume Rescue Assay for DNA Microarray Analysis. The absorbance at 600 nm (OD600) was determined for each sample at 24 hours for experimental trials 23-25. N = 3 independent experiments. NS = not significant and **P ≤ 0.01.

For the DNA microarray results, we generally observed similar expression profiles for WT + DMSO vs. WT + AmB at 0 minutes, WT + DMSO vs. WT + AmB at 180 minutes, and trk1Δtrk2Δ + DMSO vs. trk1Δtrk2Δ + AmB at 0 minutes (Figure 33). In contrast, we found marked differences in gene expression profiles for trk1Δtrk2Δ + DMSO vs. trk1Δtrk2Δ + AmB at 180 minutes. To identify candidate genes, we first generated a weighted gene co-expression network51,52,53 (WGCNA). The WGCNA reveals correlation networks among the genes investigated and yields “biologically relevant modules and

77 corresponding intramodular hubs,”52 wherein one would expect that per module, there would be an over-representation of genes involved in a similar biological process.

Figure 33. DNA Microarray Analysis of AmB Rescued trk1Δtrk2Δ Cells at 0 and 180 Minutes. 3910 genes with an analysis of variance (ANOVA) false discovery rate (FDR), p < 0.05 were analyzed of samples from N = 3-4 independent experiments.

Next, we clustered this network into 24 distinct modules,51 wherein the genes in each of these modules were highly related. We associated these modules with their gene ontology for biological process, molecular function, or cellular component. Afterwards, we performed a KME analysis (Figure 34). The kME indicates how well a specific gene fits

54 within a module’s expression profile. Over 6256 genes were examined wherein the kME threshold was set at 0.8 and the -log(raw p-value) was set to 5 = 0.00001. This analysis yielded 73 genes or open reading frames (ORF) that displayed significantly increased or decreased expression in trk1Δtrk2Δ + AmB vs. trk1Δtrk2Δ + DMSO.

Figure 34. kME Analysis of AmB Mediated Restoration of Physiology in trk1Δtrk2Δ Yeast. 6256 genes were investigated and a cut-off value was set, where -log(raw p-value) of 5 = 0.00001.

The kME analysis revealed both overexpressed Table 7 and under-expressed

Table 8 genes in AmB rescued trk1Δtrk2Δ cells. These genes encode proteins with diverse functions including transport, enzyme activity, cell cycle, spore wall structure, and stress response. Intriguingly, SIT1 (ARN family transporter),55 AQR2 (cell surface transporter),56 and QDR2 (mono- and divalent cation transporter)57 were found to be greatly upregulated with fold changes of 5.2, 4, and 2.1 respectively (Table 7).

Table 7: kME Analysis Revealed Overexpressed Genes for AmB Rescued trk1Δtrk2Δ Cells. Genes involved in various biological processes are highlighted in different colors: transport (green), stress (purple), enzyme function (blue), other (black).58,59

In contrast, YGP1 (secretory glycoprotein),60 ADH2 (alcohol dehydrogenase),61 and PHO84 (inorganic phosphate transporter)62 were all found to be downregulated with a fold change of -10.8, -5.4, and -5.0 respectively (Table 8). These findings warrant further investigation through validation with RT-qPCR, and knock-down and overexpression studies. In addition, investigating how these gene expression levels change over time and in response to reintroducing normal K+ levels may reveal insights into the contributing roles of these proteins.

Table 8: kME Analysis Showed Decreased Gene Expression in AmB Rescued trk1Δtrk2Δ Cells. Genes involved in various biological processes are highlighted in different colors: transport (green), stress (purple), enzyme function (blue), cell cycle (orange), spore wall (red), other (black).58,59

Next, we investigated the change in gene expression of our candidate proteins that we hypothesized would functionally interface with small molecules. DNA microarray analysis and subsequent RT-qPCR validation showed no significant changes in the gene expression of PMA1 (Figure 35A), V-ATPase subunit63 VPH1 (Figure 35B), TOK1

(Figure 35C), NHA1 (Figure 35D), and ENA1 (Figure 35E) among treatment groups at

180 min. These results demonstrate that AmB rescued trk1Δtrk2Δ cells may not transcriptionally regulate these genes at this early time point. The expression of these genes may change at later time points or the cells may instead regulate the activity of the corresponding proteins.

Figure 35. Real Time Quantitative Polymerase Chain Reaction Analysis of Candidate Genes. RT-qPCR analysis was performed to determine A. PMA1, B. VPH1 (subunit of V-ATPase), C. TOK1, D. NHA1, E. ENA1 gene expression levels. Solid bars represent 0 minutes and patterned bars indicate 180 minutes of treatment, N = 3 independent experiments. NS = not significant.

Discussion

We demonstrated through pharmacological and genetic approaches that endogenous protein pumps (drivers, V-ATPase and Pma1) and protein correctors (Nha1) functionally interface with imperfect small molecule mimics to restore physiology. Loss of activity through chemical inhibition and loss of function through genetic deletion abrogates small molecule mediated rescue of cell growth. These studies show that small molecule surrogates can interface with cellular homeostatic mechanisms for ion movement. These findings also suggest the broad utility of small molecule surrogates in diverse eukaryotic systems, through working in concert with highly conserved mechanisms of ion homeostasis.

Intriguingly, loss of Nha1 by gene knockout completely abolished small molecule mediated rescue. Nha1 is known to extrude excess Na+ ions under acidic conditions. We hypothesize that Na+ enters cells through unselective small molecule ion channels. In cells missing Nha1, Na+ ions may accumulate to toxic levels. To elucidate this hypothesis, we intend to quantify the change in total cellular Na+ content in small molecule treated trk1Δtrk2Δnha1Δ cells.

No significant changes in gene expression were observed for PMA1, VPH1, TOK1, nor NHA1. These proteins may either be constitutively expressed or their activity is regulated. We were surprised to observe no change in ENA1-5 expression. Literature reports describe an increase in expression of ENA1-5 upon exposure to salt stress and alterations in osmotic pressure.27 Three hours may potentially be insufficient to induce a change in ENA1-5 gene expression, as the small molecule rescued trk1Δtrk2Δ cells may still exist in lag phase. Investigation of later time points including 6 and 12 hours may

83 reveal greater changes in gene expression as small molecule rescued trk1Δtrk2Δ cells demonstrate entry into the exponential phase.

Future studies will focus on the candidate genes found to be increased (Table 7) or decreased (Table 8) in expression. In addition, we are interested in investigating whether other small molecule ion channels also induce similar changes in the expression of these candidate genes.

Author Contributions

Jenn Hou performed the chemical inhibition studies (Figure 30), conducted the liquid broth dilution assays for the isogenic mutant strains (Figure 31), developed and executed the large scale rescue assays (Figure 32), isolated RNA and prepared cDNA samples for DNA microarray analysis, and ran the RT-qPCR assays for the candidate genes (Figure 35). Dr. Jenny Drnevich of The Roy J. Carver Biotechnology Center at

UIUC designed the DNA microarray assay, performed the DNA microarray data analysis

(Figure 33), and conducted the WGCNA and kME analyses. Mary Majewski of The

Edward R. Madigan Lab at UIUC, performed the BioAnalyzer quality analysis for all cDNA samples and ran the DNA microarray.

Methods and Materials

Yeast Strains

Isogenic yeast strains BY4741 (WT), BYT3 (tok1Δ), BYT4 (nha1Δ), BYT5 (ena1-

5Δ), BYT12 (trk1Δtrk2Δ), BYT123 (trk1Δtrk2Δtok1Δ), BYT124 (trk1Δtrk2Δnha1Δ), and

BYT125 (trk1Δtrk2Δena1-5Δ) were kindly gifted by Dr. Joaquín Ariño from The Universitat

Autònoma de Barcelona and Dr. Hana Sychrová from The Academy of Sciences of the

Czech Republic.

Chemical Inhibition Assay

Before conducting the experiment, we suspended 3-5 colonies each of wild-type

Saccharomyces cerevisiae (S. cerevisiae, ATCC9763) and of trk1∆trk2∆ S. cerevisiae

(SGY1528) in 100 mM KCl supplemented YPAD pH 5.0 medium, and cultured overnight at 30°C and speed 200 X RPM for 14 to 18 hours. Prior to harvesting the cells, small molecules (AmB, HPLC purified AKScientific), Nyst. (Riedel-de-Haen), Can. (TOKU-E), or Mep. (Santa Cruz Biotechnology) and chemical inhibitors Nocodazole (Sigma Aldrich),

Ebselen (Cayman Chemical), and Bafilomycin B1 (Santa Cruz Biotechnology) stock solutions were prepared in DMSO. The chemical inhibitor working solutions comprise a range of incremental concentrations.

After 14-18 hours of cell culture, we centrifuged 20 mL of cells at 1000 X G for 5 min. at 23°C. The supernatant was decanted, then the cells were resuspended in 20 ml of sterile milliQ H2O per cell pellet (cell pellet was vortexed until fully dispersed). Next, the cells were centrifuged at 1000 X G for 5 min at 23°C. The wash step was repeated.

Following, we decanted the supernatant and resuspended the cells in YPAD pH

5.0 medium (with no K+ added). Then we diluted both WT and trk1∆trk2∆ samples in

+ YPAD pH 5.0 medium (with no K added) to an OD600 value between 0.095 and 0.105 after allowing the samples to stabilize after 5 min. Afterwards, both WT and trk1∆trk2∆

cells with OD600 values between 0.095 and 0.105 were diluted 10-fold in YPAD, pH 5.0 medium (no K+ added). To these diluted cell suspensions, the appropriate amount of

AmB, Nyst., Can., or Mep. were added to give final rescuing concentration of (125 nM, 1

μM, 8 nM, and 8 nM respectively). A control was prepared by adding the corresponding volume of DMSO vehicle to the trk1∆trk2∆ cell suspension, to check for contamination.

Under a sterile field, 195 μL of WT + small molecule, 195 μL of trk1∆trk2∆ + small molecule, and 195 μL of trk1∆trk2∆ + DMSO were added to the appropriate wells. Next,

5 μL of DMSO or chemical inhibitor was added to each well, with each concentration tested in triplicate for both WT and trk1∆trk2∆ treatment groups. The 96 well plate was gently rotated several times before placing into the 30°C incubator. After 24 hours, all wells were gently resuspended before obtaining a plate reader (BioTek Synergy H1) absorbance measurement at 600 nm.

EC50 Determination and Statistical Analysis of Chemical Inhibition

Utilizing GraphPad PRISM, we fitted the data (nonlinear regression, inhibition dose response, variable slope (four parameters)) to yield IC50 values with SEm. For statistical analysis of the IC50 values, we compared the two treatment groups with the unpaired t- test.

Small Molecule Treatment of Mutant Tok1, Nha1, and Ena1-5 Yeast Strains

The following experiments were performed under sterile conditions. Five single colonies of each yeast strain were resuspended into 1 mL of 100 mM KCl YPD medium and then this 1 mL cell suspension was added to 100 mL of 100 mM KCl YPD medium.

The cultures were grown overnight at 200 X RPM, 30°C for 12 - 24 hours until fully saturated. Next, approx. 25 mL of each cell suspension was centrifuged at 1000 X G for

5 min, at 23°C. The supernatant was removed and the cells were resuspended in 25 mL of sterile Milli-Q H2O. Afterwards, the cells were vortexed to fully re-suspend them and

86 then re-centrifuged. This step was repeated once more. Following, the cells were resuspended in 15 mM KCl YNB pH 5.8 medium (for AmB and Nyst. studies) or in 10 mM

KCl YNB pH 5.8 (for Can. and Mep. studies). Under the sterile field of the Bunsen burner, the cells were diluted in either 10 mM or 15 mM KCl YNB pH 5.8 medium to OD600 values between 0.090-0.110. 195 μL of cell suspension was pipetted into 96 well plates and dosed with 5 μL of DMSO or each corresponding small molecule (AmB, Nyst., Can., and

Mep., as described above) to give the indicated final concentrations, in triplicate. The plates were incubated at 30°C for either 24 hours (for Can. and Mep. studies) or 72 hours

(for AmB and Nyst. studies, at 200 X RPM), protected from light. Absorbance values were determined at 600 nm on a BioTek plate reader.

Large Scale Rescue Assays for Gene Expression Analysis

Isogenic BY4741 (WT) and BYT12 (trk1Δtrk2Δ) yeast were streaked onto 100 mM

KCl supplemented YPD agar plates and incubated for at least 48 hours at 30°C. The plates were kept upside down during this time to avoid dispersing the single colonies, due to condensation. To prepare starter cultures, one single colony was pipetted per culture tube of 4 mL of 100 mM KCl YNB pH 5.8. At least three separate tubes of each strain were prepared.

We checked the optical density (OD600) of the starter cultures by UV-VIS. When the cultures were saturated with an OD600>1, we then transferred 3 mL of cell suspension to 52 mL each of 100 mM KCl supplemented YNB pH 5.8. We incubated the cells at 200

X RPM, 30°C. The OD600 was monitored by UV-VIS and the cells were harvested during exponential phase with OD600 values ranging from 0.6-0.8.

A fresh stock solution of AmB was prepared by dissolving a small amount in

DMSO. A gas tight syringe was used to measure out 1.998 mL of methanol (MeOH) to three separate glass vials. Then 2 μL of AmB was added per vial to dilute 1:1000. Next, we blanked the UV-VIS with MeOH and determined the absorbance at 406 nm. The concentration was determined by applying the Beer-Lambert Law with ε = 164,000M-1cm-

1. Following, 7 mL of a 5 μM stock was prepared in DMSO.

We harvested 50 mL of each cell strain by centrifuging at 1000 X G, 23°C for 5 min. Following, we removed the supernatant and then resuspended the cells in 50 mL each of sterile milli-Q H2O. Afterwards, we vortexed the cells at speed 10, with 5 pulses each. Next, we centrifuged the cells. We performed the wash step once more.

Once the cells were washed, we resuspended the cells in 7.5 mL each of 10 mM

KCl YNB pH 5.8 and gently swirled each conical tube. Then we vortexed the cell suspensions. We utilized the UV-VIS to dilute each cell strain in 10 mM KCl YNB pH 5.8 to achieve an OD600 between 0.090-0.110 with a total volume of 250 mL for each strain.

Afterwards, we measured 125 mL of each cell suspension using clean graduated cylinders and transferred these cell suspensions into labeled 500 mL Erlenmeyer flasks.

Next, we carefully removed 3.125 mL of cell suspension from each flask and added

3.125 mL of 5 μM AmB to all AmB labeled flasks. DMSO was added to the respective

DMSO labeled flasks. We tested the following treatment groups of WT + DMSO, WT +

AmB, trk1Δtrk2Δ + DMSO, and trk1Δtrk2Δ + AmB. Across all four independent experiments, we made sure to vary the order in which each compound was added, and the order in which the strains were harvested to avoid batch effects. We used a clean serological pipette for each addition, and made sure to pipette up and down several times

88 to fully dispense the DMSO or AmB solutions. Then we withdrew 90 mL of treated cell suspension from each flask and transferred this into chilled, labeled conical tubes, with

30 mL of cell suspension per conical tube; the tubes were kept on ice. We centrifuged the samples at 3000 X G, 5 min, 4°C. During this time, we transferred the flasks into the shaking incubator and cultured at 200 X RPM, 30°C.

For the centrifuged samples, the supernatant was carefully removed from the small cell pellets. The respective cell pellets were combined and we filled the conical tubes with cold 1 X PBS (45 mL maximum). We gently inverted each tube 3X (no vortexing) and centrifuged at 3000 X G, 5 min, 4°C. We carefully removed the supernatant by tilting each conical tube to the side and then aspirating off any fluid. We then proceeded to isolate

RNA.

The same procedure described was performed for the 3 hour time point, except only 25 mL of treated cells were harvested, and 20 mL of ice cold 1 X PBS was used to wash.

RNA Isolation

This protocol was performed following the Qiagen’s RNeasy Kit instructions.64 We describe the company’s protocol here. All surfaces, the microcentrifuge, and pipettors were sprayed with RNAse Zap. We carefully added 100 μL of Y1 buffer per pellet (1 mL

Qiagen Y1 buffer with a speck of lyticase, and 1.60 μL of β-mercaptoethanol) and incubated at 30°C, 200 X RPM, for 30 minutes. We prepared buffer RLT ahead of time by adding 10 μL of β-mercaptoethanol per 1 mL of buffer RLT. DNAse I was also prepared by adding 350 μL of buffer RDD per 50 μL of DNAse I stock. After the 30 minute lyticase incubation was complete, we carefully added 350 μL of buffer RLT per conical tube and

89 vortexed at maximum speed for 10 seconds. Then we vortexed each sample for an additional 5 seconds. Next, we added 250 μL of 100% EtOH per sample and gently pipetted up and down 10 times. We transferred this solution to labeled spin columns. We centrifuged the samples at 10,000 X RPM, for 30 seconds, at 23°C. Then we removed the flow through. Afterwards, we added 350 μL of buffer RWI and centrifuged at 10,000

X RPM, 23°C, for 30 seconds. Afterwards, we removed the flow through. Next, we added

80 μL of DNAse I directly to the column and let this sit for 15 minutes at room temperature.

Next, we added 350 μL of buffer RWI and centrifuged at 10,000 X RPM, 23°C, for

30 seconds. We removed the flow through. Then we added 500 μL of buffer RPE and let the tubes sit for 5 minutes. We made sure to coat the inner surfaces. Then we centrifuged at 10,000 X RPM, 23°C, for 30 seconds. We removed the flow through. Following we added 500 μL of buffer RPE per spin column and centrifuged at 10,000 X RPM, 23°C, for

2 minutes. We removed the flow through.

We transferred the spin columns to new collection tubes and centrifuged at 10,000

X RPM, 23°C, for 1 minute. We removed the collection tubes and transferred the spin columns to newly labeled, RNAse free Eppendorf tubes. We added 20 μL of RNase free

H2O and let this sit for 5 minutes. Then we centrifuged at 10000 X RPM, 23°C, for 1 minute. We repeated the RNase free H2O step once more. We kept a small 2.5 μL aliquot for NanoDrop concentration measurement. We stored the rest of the RNA samples in labeled tubes and kept the samples at -80°C. We repeated these steps for the 180 minute samples but modified the following steps including that we only collected 25 mL of cell suspension, and we only used 20 mL of ice cold 1 X PBS to wash. All samples demonstrated 260 nm/280 nm values greater than 2.0.

Bioanalyzer Analysis and DNA Microarray Sample Submission

We first determined the concentration of the RNA samples by NanoDrop. Then we diluted the samples in DEPC treated H2O to obtain concentrations around 250 ng/μL. We aliquoted 3 μL of each treatment group for the Bioanalyzer analysis and 4 μL of each treatment group for the DNA Microarray analysis. The samples were kept at -80°C until they were ready to submit to the Roy J. Carver Biotechnology Center. Bioanalyzer analysis showed that all samples had an RNA integrity number (RIN) > 9.0.

cDNA Synthesis

We followed the manufacturer’s instructions for the Superscipt VILO.65 All surfaces were cleaned with RNAse Zap. First, VILO reaction mix and 10X superscript enzyme were added as instructed. Then 2500 ng of RNA per sample was added per respective tube.

Following, the reaction mixtures were added to RNAse free strip tubes and incubated at

25°C for 10 minutes, 42°C for 90 minutes and 85°C for 5 minutes. After the reactions were completed, we transferred the cDNA to labeled tubes and stored them at -80°C.

Real Time Quantitative Polymerase Chain Reaction (RT-qPCR)

The following protocol was adapted from Taqman Gene Expression Assays.66 We utilized RNAse free tubes and RT-qPCR Grade H2O. Per tube, we added 25 μL of nuclease free H2O, 20 μL of cDNA template, and 55 μL of probe master mix. For the master mix, we added per probe 47 μL of gene expression assay and 470 μL of Taqman

Gene Expression mix. Then we carefully pipetted 20 μL per well. We also prepared no template controls by adding 20 μL of nuclease free H2O instead of cDNA template. We gently covered the plate with a PCR adhesive film, vortexed the plate briefly, and then

91 centrifuged at 1000 X RPM for 1 minute, at 23°C. Then we setup the RT-qPCR instrument to run at 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. To assess for statistical significance, we analyzed the data via unpaired t-test.

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39 Prior C, Potier S, Souciet JL, Sychrova H. (1996). Characterization of the NHA1 gene encoding a Na+/H+-antiporter of the yeast Saccharomyces cerevisiae. FEBS Letters 387(1): 89-93. 40 Ruiz A, Arino J. (2007). Function and regulation of the Saccharomyces cerevisiae ENA sodium ATPase system. Eukaryot. Cell. 6(12): 2175-2183. 41 Ke R, Ingram PJ, Haynes K. (2013). An integrative model of ion regulation in yeast. PLoS Comput. Biol. 9(1): e1002879. 42 Garciadeblas B, Rubio F, Quintero FJ, Banuelos MA, Haro R, Rodriguez-Navarro A. (1993). Differential expression of two genes encoding isoforms of the ATPase involved in sodium efflux in Saccharomyces cerevisiae. Mol. Gen. Genet. 236(2-3): 363-368. 43 Banuelos MA, Sychrova H, Bleykasten-Grosshans C, Souciet JL, Potier S. (1998). The Nha1 antiporter of Saccharomyces cerevisiae mediates sodium and potassium efflux. Microbiology. 144(Pt 10): 2749-2758. 44 Ruiz A, Arino J. (2007). Function and regulation of the Saccharomyces cerevisiae ENA sodium ATPase system. Eukaryot. Cell. 6(12): 2175-2183. 45 Kunkel W (1979) Effects of the antimicrotubular cancerostatic drug nocodazole on the yeast Saccharomyces cerevisiae. Zeitschrift fur Allgemeine Mikrobiologie 20(5): 315-324. 46 Endo K, Mizuguchi M, Harata A, Itoh G, Tanaka K (2010) Nocodazole induces mitotic cell death with apoptotic like features in Saccharomyces cerevisiae. FEBS Letters 584: 2387-2392. 47 Chan G, Hardej D, Santoro M, Lau-Cam C, Billack B (2007) Evaluation of the antimicrobial activity of ebselen: role of the yeast plasma membrane H+-ATPase. J. Biochem. Mol. Toxicol. 21(5): 252-264. 48 Drose S and Altendorf K (1997) Bafilomycins and concanamycins as inhibitors of V- ATPase and P-ATPases. J. Exp. Biol. 200: 1-8. 49 Ermishkin LN, Kasumov KM, Potzeluyev VM (1976) Single ionic channels induced in lipid bilayers by polyene antibiotics amphotericin B and nystatine. Nature 262: 698-699. 50 Ermishkin LN, Kasumov KM, Potseluyev VM (1977) Properties of amphotericin B channels in a lipid bilayer. Biochim. Biophys. Acta 470: 357-367. 51 Langfelder P and Horvath S. 2008. WGCNA: An R package for weighted correlation network analysis. BMC Bioinformatics. 9: 559. 52 Langfelder P, Mischel PS, Horvath S. 2013. When is hub gene selection better than standard meta-analysis. PLos ONE. 8(4): e61505. 53 This work was performed by Dr. Jenny Drnevich. 54 Coppola G. (2014). The OMICS: applications in neuroscience. England: Oxford University Press. 374p. 55 Anderson GJ, and McLaren GD. (2012). Iron Physiology and Pathophysiology in Humans. Springer Science and Business Media. 704p. 56 Gbelska Y, Krijger JJ, Breunig KD. (2006). Evolution of gene families: the multidrug resistance transporter genes in five related yeast species. FEMS Yeast Research. 6(3): 345-355. 57 Rios G, et al. (2012). Role of the yeast multidrug transporter Qdr2 in cation homeostasis and the oxidative stress response. FEMS. 13: 97-106. 58 Gene Ontology Consortium. 2004. The gene ontology (GO) database and informatics resource. Nucleic Acids Research. 32: 1-4.

59 Cherry JM et al. 2012. Saccharomyces Genome Database: the genomics resource of budding yeast. Nucleic Acids Res. Jan;40(Database issue): D700-5. 60 Destruelle M, Holzer H, Klionsky DJ. (1994). Identification and characterization of a novel yeast gene: the YGP1 gene product is a highly glycosylated secreted protein that is synthesized in response to nutrient limitation. Mol. Cell. Biol. 14(4): 2740-2754. 61 Lutstorf U and Megnet R. (1968). Multiple forms of alcohol dehydrogenase in Saccharomyces cerevisiae: I. physiological control of ADH-2 and properties of ADH-2 and ADH-4. Archives of Biochemistry and Biophysics. 126(3): 933-944. 62 Bun-Ya M, Nishimura M, Harashima S, Oshima Y. (1991). The PHO84 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol. Cell. Biol. 11(6): 3229-3338. 63 Manolson MF, et al. (1992). The VPH1 gene encodes a 95-kDA integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase. J. Biol. Chem. 267(20): 14294-303. 64 Qiagen, June 2012. RNeasy Mini Handbook. < https://www.qiagen.com/us/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc- bf9f6fa33e24&lang=en>. November 18, 2017. 65 ThermoFisher Scientific, 2015. SuperScript VILO cDNA Synthesis Kit. < https://www.thermofisher.com/order/catalog/product/11754050>. November 18, 2017. 66 Applied Biosystems, November 2010. TaqMan Gene Expression Assays. . November 18, 2017.

CHAPTER 5: IDENTIFICATION OF NOVEL SMALL MOLECULE MIMICS

Introduction

Polyene macrolides represent a diverse and chemically complex group of natural products,1 with several universal structural features including a core macrocyclic ring, polyene region of varying lengths, and mycosamine sugar appendage.2 Since the initial isolation of Nystatin (Nyst., 1950),3 Rimocidin (1951),4 and Trichomycin (1952),5 the field of polyene natural products has continued to capture immense attention. The study of polyene macrolides has not only unveiled insights into the polyketide synthase (PKS) machinery,6,7 but has also demonstrated their utility as broad spectrum antifungal agents.8

For example, for more than 60 years Amphotericin B (AmB) has served as a potent antifungal agent for the treatment of systemic and life-threatening infections without the emergence of resistance.9,10 However, one major limitation of AmB is its dose-limiting toxicity,11 which precludes its use at high concentrations.

Previously, The Burke Group found that AmB primarily exerts its antifungal activity via sterol binding and extraction, contrary to the widely accepted mechanism of ion channel formation.12 With this key finding in hand, current synthetic efforts are heavily geared towards the generation of less toxic AmB derivatives that preferentially bind ergosterol (primary sterol in yeast) over cholesterol (primary sterol in mammals),13 while retaining efficacy against a broad spectrum of pathogenic yeast and avoiding the development of resistance.

This mechanistic discovery enabled the distinction of AmB’s ion channel activity from its cell killing activity; wherein use of this small molecule at low concentrations favors ion channel formation.12 Through repurposing AmB as an imperfect small molecule ion

97 channel mimic, we found that this small molecule surrogate was sufficient to restore physiology in protein ion channel deficient yeast14 and in cystic fibrosis transmembrane conductance regulator (CFTR) deficient human lung epithelia.15

In this vein, we are invested in identifying novel polyene macrolides for their potential use as antifungal agents or as therapies for protein ion channel deficiencies.

Structural comparison of at least 30 reported polyene macrolides2 and their variants demonstrates a high degree of conservation of the hemiketal region and mycosamine appendage (Figure 36,16 shaded region). We hypothesized that an in-depth biosynthetic gene cluster analysis of several highly conserved domains and/or enzymes, taken in the context of neighboring genomic regions, in Streptomyces and related genus members could yield potentially uncharacterized producing organisms of polyene macrolides.

To probe this hypothesis, we teamed up with Professor Doug Mitchell and his group at UIUC. They are leaders in the field of biosynthetic gene cluster (BCG) analysis and natural product discovery. The Mitchell Group created a powerful and user-friendly biosynthetic gene cluster analysis program called RODEO (Rapid ORF Description and

Evaluation Online).17 RODEO was highly successful in identifying previously uncharacterized lasso peptides.17 We predicted that RODEO analysis of mycopolyene

BCGs from Streptomyces18 and related species, taken together with Prediction

Informatics for Secondary Metabolomes (PRISM) analysis would reveal previously uncharacterized polyene macrolides. With these potential targets and our established disc diffusion assay, we could thus effectively screen bacterial extracts predicted to contain uncharacterized polyene macrolides, and evaluate them for their antifungal and/or ion channel activity.

Figure 36. Conserved Structural Region of Polyene Macrolide Natural Products.16 Like protein sequences, we analyzed the structural sequences of at least 30 polyene

Figure 36. (continued) macrolides. The hemiketal and mycosamine appendage were found to be highly conserved. [This analysis was performed by and this figure was reproduced from Dailey, I. (June 6, 2012). Synthesis and function of the conserved motif of mycosamine- containing polyene macrolides. Retrieved from Graduate Dissertations and Theses at Illinois (http://hdl.handle.net/2142/95658)].

RODEO and PRISM analyses and the identification of novel polyene macrolides will also enable the characterization of the relationship between polyene length and ion channel activity.19 Literature reports,20 as well as our own studies suggest that the length of the polyene region influences the capacity of these small molecules to form ion channels. For example, Natamycin (Nat., a tetraene) effectively binds sterols but does not permeabilize cells.14 In contrast, AmB (a heptaene) acts both as an antifungal agent as well as a small molecule ion channel mimic.14 Based on these findings, we sought to characterize the minimum length or number of double bonds in the polyene region required for ion channel formation. Identification of a pentaene or hexaene polyene macrolide (Figure 37), would enable a systematic investigation of the impact of polyene length on ion channel activity.

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Figure 37. Predicted Chemical Structures of Polyene Macrolides with Varying Polyene Lengths.21 Based on biosynthetic gene cluster analysis and Prediction Informatics for Secondary Metabolomes (PRISM), we show the following chemical structures for known or predicted polyene macrolides and their producing organisms A. Lucensomycin (tetraene), B. small molecule produced by Lentzea albida (tetraene), C. Eurocidin D (pentaene), D. small molecule produced by Amycolatopsis saalfeldensis (hexaene), E. small molecule produced by Lentzea waywayandensis (heptaene), F. Levorin a.k.a. Candicidin (heptaene), and G. Partricin A a.k.a. Mepartricin (heptaene).

In addition, our preliminary results suggest that p-amino benzoic acid (PABA) containing polyene macrolides show at least a 10-fold reduction in concentration required for ion channel formation. Non-PABA containing polyene macrolides AmB and Nyst. restored cell growth in potassium (K+) transporter deficient yeast at 125 nM and 1 μM respectively. In contrast, PABA containing polyene macrolides Candicidin (Can.) and

Mepartricin (Mep.), showed potent rescue of cell growth at 8 nM. Mechanistically, we predict that the PABA motif may anchor the polyene macrolide to lipid membranes to increase the probability of ion channel formation. To further understand this phenomenon, we are interested in identifying new PABA containing polyene macrolides and studying

101 their ability to bind to sterols and/or to form ion channels. We intend to characterize the binding interactions of these polyene macrolides to sterols through solid state nuclear magnetic resonance (ssNMR) and ultraviolet-visible (UV-VIS) spectrometry studies.

Results

We first sought to identify novel polyene macrolides from producing organisms, the majority of which are anticipated to come from the Streptomyces genus and related members. We hypothesized that analysis of the biosynthetic gene clusters from these known natural product producers would yield closely related species that potentially biosynthesize previously uncharacterized polyene macrolides. To probe our hypothesis, we input the genomes of known polyene macrolide producing strains through RODEO analysis, identified strains which potentially produce uncharacterized polyene macrolides, and attempted to express extracts from them (Figure 38).

Figure 38. Identification of Potential Polyene Macrolide Producing Organisms. The general workflow for natural product discovery includes the input of biosynthetic gene clusters from known polyene macrolide producing organisms into RODEO, to identify related and uncharacterized species. For demonstration purposes, a known producer of Trichomycin, S. hachijoensis is shown here. Next, through PRISM analysis, structural predictions can be made of the predicted, expressed small molecules. Next, candidate producing organisms are cultured under various media conditions to enhance the probability of natural product expression. Last, extracts are isolated and characterized via a suite of analytical and functional assays.

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RODEO analysis revealed four main groups (Figure 39) including producing organisms of Nat. like compounds, Nyst./AmB like compounds, PABA containing compounds, and other uncatergorized compounds.

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Figure 39. RODEO Analysis of Mycopolyene Biosynthetic Gene Clusters. Biosynthetic gene cluster analysis of known polyene macrolide producing organisms

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Figure 39. (continued) revealed organisms that produce Natamycin like compounds (highlighted in purple), Nystatin/Amphotericin like compounds (shown in green), PABA containing compounds (depicted in orange), and potential uncharacterized compounds (shown in blue).21

With our candidate producing strains in hand, we cultured each bacterial strain under a suite of different media conditions including glycerol-sucrose-beef extract- casamino acids (GuBC), International Streptomyces Project 2 (ISP2), ISP4, mannitol soy

(MS), nutrient broth (NB), Reasoner’s 2A (R2A), and/or Staibs. We tested a variety of media conditions to increase the probability of small molecule expression because different bacterial strains have specific nutritional requirements. Next, we extracted with methanol (MeOH), as most polyene macrolides are soluble in this organic solvent. We analyzed the extracts via tetrazine labeling which specifically detects the presence of electron-rich alkenes in the polyene region. To check for biological activity, we applied our disc diffusion assay to assess for yeast cell killing, and for cell rescue via ion channel activity.

First, to determine whether each of our bacterial extracts possesses antifungal activity, wild-type (WT) yeast were streaked onto agar plates. We then added either 10

μL of DMSO control or 10 μL of each bacterial extract and incubated the plates for 24 hours. Extracts with antifungal activity showed prominent zones of cell inhibition around the extract containing discs (Figure 40), while the DMSO controls did not.

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Figure 40. Representative Disc Diffusion Assay Images for Yeast Cell Killing. To assess for antifungal activity of these bacterial extracts, we performed disc diffusion assays. On agar plates coated with wild-type (WT) yeast, we placed sterile paper discs and then applied 10 μL of DMSO and 10 μL of each bacterial extract. After 24 hours of incubation, plates were examined for fungal cell killing i.e. the presence of a zone of inhibition. Shown are the bacterial extracts tested from strain B3253 grown under an array of media conditions, A. GuBC, B. ISP2, C. ISP4, D. MS, E. R2A, F. Staibs. Each bacterial extract showed antifungal activity compared to the DMSO control.22

We observed robust antifungal activity of extracts (Table 9) from AmB producer S. nodosus (positive control), a Eurocidin and congeners producer (ISP5604), Levorin

106

(a.k.a. Can.) and congeners producers (strains B3253, S37, B16016, and B12325), a

Partricin (a.k.a. Mep.) producer (B1868), and uncharacterized producers (WC3896 and

B3698).

Table 9. Yeast Cell Killing Results from Potential Polyene Macrolide Producing Organisms. Extracts were isolated from the indicated bacterial strains grown under a suite of media conditions including GuBC, ISP2, ISP4, MS, NB, R2A, and Staibs. Extracts with antifungal activity after 24 hours are indicated, ND = not determined.

We then evaluated extracts for ion channel activity. To do so, we employed our disc diffusion assay, but this time streaked potassium (K+) transporter deficient trk1Δtrk2Δ yeast that are unable to grow under standard laboratory conditions (10 mM KCl YPAD).

Extracts containing small molecules with ion channel activity would promote restoration of cell growth in trk1Δtrk2Δ yeast. We observed that in contrast to the DMSO controls,

107 extracts containing potential polyene macrolides with the capacity for forming ion channels, promoted vigorous rescue of trk1Δtrk2Δ cell growth (Figure 41) by 24 hours.

Figure 41. Representative Disc Diffusion Assay Images for Yeast Cell Rescue. To assess for ion channel activity of these bacterial extracts, we performed disc diffusion assays. Trk1Δtrk2Δ yeast were streaked onto agar plates. After, we applied sterile paper discs and then pipetted onto each disc either 10 μL of DMSO or 10 μL of each bacterial extract. After 24 hours of incubation, plates were examined for rescue of cell growth i.e. zone of functional complementation. Shown are the bacterial extracts tested from strain B3253 grown under an array of media conditions, A. GuBC, B. ISP2, C. ISP4, D. MS, E. R2A, F. Staibs. Each bacterial extract showed ion channel activity compared to the DMSO control.22

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We observed ion channel activity of extracts (Table 10) from AmB producer S. nodosus (positive control), Can. and congeners producers (B3253, S37, B16016,

B12325), a Mep. and congeners producer (B1868), and uncharacterized strain B3698.

Eurocidin D (pentaene) did not promote rescue of cell growth.

Table 10. Results of Yeast Cell Rescue Assay for Tested Extracts from Potential Producing Organisms. Extracts were isolated from the indicated bacterial strains, grown under a suite of media conditions including GuBC, ISP2, ISP4, MS, NB, R2A, and Staibs. The outcomes of the rescue assays after 24 hours are shown, ND = not determined.

These findings suggest that a pentaene polyene core (e.g. as in Eurocidin) is insufficient in length for ion channel activity. To further determine the length of the polyene core necessary for ion channel activity, we are immensely interested in identifying a producer of a hexane polyene macrolide. Utilization of this hexane small molecule as a

109 chemical probe will show the minimum polyene length required for ion channel activity.

Current efforts are geared towards expressing a predicted hexane polyene macrolide from producing organisms including A. jejuensis and A. saalfeldensis, based on RODEO and PRISM analyses (Table 11). Due to the lack of detectable tetrazine labeling, we believe that there was no expression of these natural products, from most of these strains.

We are actively pursuing various methods to try to improve expression, including culturing cells under an even more extensive set of media conditions, and employing different expression vector systems.

Table 11. Potential Producing Organisms of Polyene Macrolides of Varying Length. RODEO and PRISM analyses yielded several, uncharacterized producing organisms predicted to generate polyene macrolides of different lengths including a tetraene, pentaenes, hexaenes, heptaenes, and an octaene. Tetrazine labeling was only detected in the extracts from S. eurocidicus.

To rule out the possibility of unintentionally adding exogenous K+ in the trk1Δtrk2Δ rescue of cell growth assays (i.e. false positive), we quantified the total K+ content of the bacterial extracts. Potentially, we are observing restored cell growth in the trk1Δtrk2Δ yeast due to high K+ supplementation (from the extracts). We determined the total K+ content through performing inductively coupled plasma-mass spectrometry (ICP-MS).23

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ICP-MS analysis (Figure 42) revealed raw K+ values under 40 parts per million (ppm).

Taking into account the dilution factor (5 μL of sample per 245 μL of solvent), the final K+ content is below 2000 ppm per sample and is low. Thus, these results further support that trk1Δtrk2Δ rescue of cell growth is due to the ion channel activity of the extracts rather than direct K+ supplementation.

Figure 42. ICP-MS Quantification of K+ Content in Extracts. The raw K+ content was analyzed for the shown sample extracts.

Discussion

Through RODEO and PRISM analyses, we uncovered previously uncharacterized polyene macrolide natural products that may serve as promising novel antifungal agents and small molecule mimics. Members of the Can. genus (B3253, S37, B16016, B12325) produced natural products with both antifungal and ion channel activity. Future efforts will be geared towards the purification (HPLC) and in-depth structural characterization

(ssNMR, UV-VIS, etc.) of these extracts. In addition, we are interested in determining the relative ion selectivities of each predicted small molecule ion channel. Do these small

111 molecule ion channels show a difference in selectivity for cations (K+, H+, and/or Na+) vs.

- - anions (Cl and/or HCO3 )? Customizing small molecule ion channels that have different ion selectivities, for different protein ion channel deficiencies could improve therapeutic outcomes, by minimizing the dosage required for effective transport of specific ions.

Furthermore, use of more selective ion channels could also minimize unwanted side effects by reducing the undesired transport of other ions.

We found that the pentaene Eurocidin effectively kills yeast but does not promote ion channel formation. This key finding provides further clues into the structure activity relationship between polyene length and ion channel formation; wherein shorter polyene macrolides still bind sterols but do not promote ion channel formation. We are heavily interested in identifying a hexaene polyene macrolide to further probe the minimum polyene length required for ion channel activity.

Furthermore, our group is interested in understanding from an evolutionary perspective how the core scaffold of polyene macrolides has changed throughout time.

We hypothesize that polyene macrolide producing organisms share a common ancestor that expressed a tetraene like polyene macrolide. Then through gene transfer and/or duplication events, the length of the polyene region increased to eventually give rise to heptaenes. Possibly, octaene polyene macrolides exist but to our knowledge this class of polyene macrolides has not been identified. Moreover, we predict that organisms producing PABA containing small molecules appeared later, as these more potent antifungal agents could provide some evolutionary benefit for survival. Potentially, organisms that produced PABA containing small molecules, could produce less of these small molecules needed for effective protection against invading yeast cells, thereby

112 enabling them to conserve energy and nutrients for other biological processes.

Alternatively, the opposite sequence of events may have occurred wherein through loss of function or gene deletion events, polyene macrolides became less functionalized and shorter. Further study into the lineage of polyene macrolide producing organisms is warranted.

Author Contributions

For author contributions to this project, Xiao Rui Guo performed the RODEO and

PRISM analyses, identification of novel strains, culture and expression of natural product extracts from these strains, HPLC purification, and characterization of extracts through tetrazine labeling and liquid chromatography-mass spectrometry (LC-MS). Page N.

Daniels, Jordan T. Holler, and Jenn Hou conducted the yeast disc-diffusion assays.

Methods and Materials

Mid-Throughput Yeast Screening Assay

This protocol was modified from The National Committee of Clinical Laboratory

Standards document M2-A9.24 We cultivated 3-5 colonies of nonisogenic WT and nonisogenic trk1Δtrk2Δ cells in 50-100 mL each of 100 mM KCl YPAD pH 5.0 medium.

At 24 hours, we harvested about 30 mL of saturated cell cultures by centrifuging at 500

X G for 5 min, 23°C. We poured off the supernatant. Then we resuspended the cells in

35 mL each of sterile Milli-Q H2O. Next, we vortexed the cells and recentrifuged. We repeated the wash step once more. This time, we removed the supernatant and resuspended the cells in 5 mL each of no K+ added YPAD pH 5.0 medium. We briefly

113 vortexed the cells and brought them over to the UV-VIS. Under the sterile field, we diluted the WT and trk1Δtrk2Δ cells to OD600 values between 0.090 to 0.110.

Next, we streaked the trk1Δtrk2Δ cells onto no K+ added YPAD pH 5.0 agar plates

(large pie size plates). To do so, we used sterile cotton applicators and dipped the cotton tip into the trk1Δtrk2Δ cell suspension. We streaked 1/4 of the plate. Then we got a new cotton tip and dipped it into the trk1Δtrk2Δ cell suspension and streaked the next quarter of the plate. We did this again for the remaining half of the plate. We rotated the plate 60° and repeated this process. We rotated the plate an additional 60° and repeated this step.

We did these steps three more times, for a total of 6 rotations. Then we used the cotton applicator to trace the inner rim to ensure an even coating of cells. We let the trk1Δtrk2Δ plates settle for 15-20 minutes upright. Then we placed sterile paper discs onto each plate. We did the same for the WT cells. We left the plates undisturbed for 10 minutes.

Afterwards, either 10 μL of DMSO, S. nodosus extract, purified AmB (4 mg/mL,

Ampho99 from Gold Biotechnology), purified Nat. (4 mg/mL) controls, or 10 μL of extracts were pipetted onto each respective disc. The plates rested upright for 20 minutes. Then we incubated the plates (upside down) in the 30°C incubator for 24 hours before imaging.

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