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Preclinical Candidate for the Treatment of Visceral Leishmaniasis That Acts Through Proteasome Inhibition

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Preclinical Candidate for the Treatment of Visceral Leishmaniasis That Acts Through Proteasome Inhibition Preclinical candidate for the treatment of visceral leishmaniasis that acts through proteasome inhibition Susan Wylliea,1, Stephen Branda,1, Michael Thomasa, Manu De Ryckera, Chun-wa Chungb, Imanol Penac, Ryan P. Binghamb, Juan A. Bueren-Calabuiga, Juan Cantizanic, David Cebrianc, Peter D. Craggsb, Liam Fergusona, Panchali Goswamib, Judith Hobratha, Jonathan Howed, Laura Jeacocka, Eun-Jung Koa, Justyna Korczynskab, Lorna MacLeana, Sujatha Manthria, Maria S. Martinezc, Lydia Mata-Canteroc, Sonia Moniza, Andrea Nühsa, Maria Osuna-Cabelloa, Erika Pintoa, Jennifer Rileya, Sharon Robinsond, Paul Rowlandb, Frederick R. C. Simeonsa, Yoko Shishikuraa, Daniel Spinksa, Laste Stojanovskia, John Thomasa, Stephen Thompsona, Elisabet Viayna Gazaa, Richard J. Walla, Fabio Zuccottoa, David Horna, Michael A. J. Fergusona, Alan H. Fairlamba, Jose M. Fiandorc, Julio Martinc, David W. Graya, Timothy J. Milesc, Ian H. Gilberta, Kevin D. Reada,2, Maria Marcoc,2, and Paul G. Wyatta,2 aDrug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom; bMedicines Research Centre, Stevenage, Hertfordshire SG1 2NY, United Kingdom; cGlobal Health R&D, GlaxoSmithKline, Tres Cantos, 28760, Spain; and dDavid Jack Centre for R&D, GlaxoSmithKline, Ware SG12 0DP, United Kingdom Edited by Carl F. Nathan, Weill Medical College of Cornell University, New York, NY, and approved March 5, 2019 (received for review November 28, 2018) Visceral leishmaniasis (VL), caused by the protozoan parasites the only oral treatment available. These treatments have seri- Leishmania donovani and Leishmania infantum, is one of the ma- ous drawbacks, including prolonged treatment duration (20–30 jor parasitic diseases worldwide. There is an urgent need for new d for antimonials and 28 d for miltefosine and amphotericin B drugs to treat VL, because current therapies are unfit for purpose deoxycholate), parenteral administration (amphotericin B, anti- in a resource-poor setting. Here, we describe the development of a monials, and paromomycin), low tolerability (antimonials and preclinical drug candidate, GSK3494245/DDD01305143/compound amphotericin B deoxycholate), teratogenicity (miltefosine), 8, with potential to treat this neglected tropical disease. The com- SCIENCES pound series was discovered by repurposing hits from a screen Significance Trypanosoma cruzi against the related parasite . Subsequent opti- APPLIED BIOLOGICAL mization of the chemical series resulted in the development of a Safer and more effective oral drugs are urgently required to potent cidal compound with activity against a range of clinically treat visceral leishmaniasis (VL), a neglected parasitic disease L. donovani L. infantum relevant and isolates. Compound 8 dem- that kills 20,000–40,000 people each year in parts of Asia, onstrates promising pharmacokinetic properties and impressive Africa, and Latin America. Here, we describe the development in vivo efficacy in our mouse model of infection comparable with of GSK3494245/DDD01305143/compound 8, a small molecule those of the current oral antileishmanial miltefosine. Detailed that demonstrates clinical-level efficacy in a mouse model of mode of action studies confirm that this compound acts principally VL. Compound 8 exhibits attractive biological and biosafety β by inhibition of the chymotrypsin-like activity catalyzed by the 5 properties, resulting in its selection as a preclinical candidate. L. donovani subunit of the proteasome. High-resolution cryo-EM Target deconvolution and cryo-EM studies reveal that com- Leishmania tarentolae structures of apo and compound 8-bound pound 8 is a potent and selective inhibitor of the chymotrypsin- 20S proteasome reveal a previously undiscovered inhibitor site like activity of the parasite proteasome binding in a site β β that lies between the 4 and 5 proteasome subunits. This induced sandwiched between the β4 and β5 subunits. Compound 8 is β pocket exploits 4 residues that are divergent between humans progressing toward human clinical trials, raising hopes of im- and kinetoplastid parasites and is consistent with all of our exper- proved therapeutics for this disease. imental and mutagenesis data. As a result of these comprehensive studies and due to a favorable developability and safety profile, Author contributions: S.W., S.B., M.T., M.D.R., C.-w.C., I.P., D.C., J. Howe, E.-J.K., M.S.M., compound 8 is being advanced toward human clinical trials. L.M.-C., S.R., D.S., S.T., E.V.G., F.Z., D.H., M.A.J.F., A.H.F., J.M.F., J.M., D.W.G., T.J.M., I.H.G., K.D.R., M.M., and P.G.W. designed research; S.W., S.B., I.P., R.P.B., J.A.B.-C., J.C., P.D.C., L.F., P.G., J. Hobrath, L.J., E.-J.K., J.K., L.M., S. Manthri, L.M.-C., S. Moniz, A.N., M.O.-C., E.P., J.R., Leishmania | proteasome | cryo-EM | drug discovery P.R., F.R.C.S., Y.S., D.S., L.S., J.T., S.T., E.V.G., R.J.W., and F.Z. performed research; S.W., S.B., M.T., M.D.R., C.-w.C., I.P., J.A.B.-C., D.C., P.G., J. Hobrath, J. Howe, E.-J.K., M.S.M., L.M.-C., S. isceral leishmaniasis (VL), the most serious form of leish- Moniz, M.O.-C., S.R., D.S., L.S., S.T., E.V.G., F.Z., D.H., M.A.J.F., A.H.F., J.M.F., J.M., D.W.G., T.J.M., I.H.G., K.D.R., M.M., and P.G.W. analyzed data; and S.W., M.T., M.D.R., C.-w.C., I.P., Vmaniasis, is invariably fatal if left untreated (1). This D.C., P.G., J. Howe, J.K., L.M.-C., P.R., F.Z., J.M., I.H.G., K.D.R., M.M., and P.G.W. wrote neglected tropical disease is caused by infection with the pro- the paper. tozoan parasite Leishmania donovani or Leishmania infantum. Conflict of interest statement: The following authors have shares in GlaxoSmithKline: C.- There are over 600 million people at risk from infection, and the w.C., I.P., R.P.B., J.C., D.C., P.D.C., P.G., J. Howe, J.K., M.S.M., L.M-C., S.R., P.R., J.M.F., J.M., annual death toll is 20,000–40,000 (2). The disease is spread D.W.G., T.J.M., K.D.R., M.M., and P.G.W. through the bite of infected sandflies, giving rise to a systemic This article is a PNAS Direct Submission. infection in the human host with diverse symptoms, including This open access article is distributed under Creative Commons Attribution License 4.0 (CC fever, weight loss, anemia, and hepatosplenomegaly. More than BY). 90% of VL cases are reported in India, Sudan, southern Sudan, Data deposition: The EM maps and structures have been deposited in the Protein Data Ethiopia, Kenya, Somalia, and Brazil; however, this disease has a Bank, www.wwpdb.org (liganded structure EMD-4590: PDB ID code 6QM7 and apo struc- ture EMD-4591: PDB ID code 6QM8). Illumina sequencing data reported in this paper have worldwide presence, with cases in Asia, East Africa, South America, been deposited in the European Nucleotide Archive, https://www.ebi.ac.uk/ena (accession and the Mediterranean region (3). HIV/VL coinfections are com- no. PRJEB31738). monplace and can accelerate the progression of both diseases (4). 1S.W. and S.B. contributed equally to this work. Significantly, VL-associated morbidity has a considerable economic 2To whom correspondence may be addressed. Email: [email protected], maria.m. impact, contributing to a perpetual cycle of poverty in some of the [email protected], or [email protected]. poorest regions of the world. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Current available treatments for VL are limited to pentavalent 1073/pnas.1820175116/-/DCSupplemental. antimonials, amphotericin B, paromomycin, and miltefosine, www.pnas.org/cgi/doi/10.1073/pnas.1820175116 PNAS Latest Articles | 1of6 Downloaded by guest on September 30, 2021 treatment failures (5, 6) (paromomycin, miltefosine, and antimo- nials), and requirement for cold storage and high cost (liposomal amphotericin B). These drugs also show significant geographical variation in effectiveness for reasons that are poorly understood. In particular, none of the current VL drugs have high levels of efficacy in East Africa (7, 8). Thus, identification of a low-cost, safe, effec- tive, oral, and short-course drug for VL is urgently needed (9). Here, we report the discovery of a preclinical candidate for the treatment of VL that acts principally by inhibition of the chymo- trypsin-like activity catalyzed by the β5 subunit of the L. donovani proteasome. Results and Discussion Discovery of Compound 8. Identification of new chemical entities capable of treating kinetoplastid infections has proven to be extremely challenging. There are few robustly validated drug targets in the kinetoplastid parasites, making target-based ap- proaches speculative (10), and drug discovery programs are usually reliant on phenotypic screening to find suitable chemical start points. The identification of compounds with activity against these intracellular parasites has also proven difficult, particularly in the case of L. donovani, where the mammalian stage of the parasite, the amastigote, resides within human macrophages. High-throughput screening against the intra- macrophage stage of the parasite is plagued by extremely low hit rates; however, activity for a compound series in this assay usu- ally translates into activity in animal models of disease if phar- macokinetic properties can be optimized (11, 12). The hit compound 1 was identified from phenotypic screening of a 15,659-compound diversity library against the related kinetoplastid parasite Trypanosoma cruzi [EC50 = 0.22 μM; 95% confidence interval (95% CI) = 0.087–0.36 μM; n = 3] (SI Ap- Fig.
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