Abstract

Chagas disease, leishmaniasis and sleeping sickness affect 20 million people worldwide and lead to more than 50,000 deaths annually1. The diseases are caused by infection with the kinetoplastid parasites Trypanosoma cruzi, Leishmania spp. and Trypanosoma brucei spp., respectively. These parasites have similar biology and genomic sequence, suggesting that all three diseases could be cured with drugs that modulate the activity of a conserved parasite target2. However, no such molecular targets or broad spectrum drugs have been identified to date. Here we describe a selective inhibitor of the kinetoplastid proteasome (GNF6702) with unprecedented in vivo efficacy, which cleared parasites from mice in all three models of infection. GNF6702 inhibits the kinetoplastid proteasome through a non-competitive mechanism, does not inhibit the mammalian proteasome or growth of mammalian cells, and is well-tolerated in mice. Our data provide genetic and chemical validation of the parasite proteasome as a promising therapeutic target for treatment of kinetoplastid infections, and underscore the possibility of developing a single class of drugs for these neglected diseases.

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References

  1. 1.

    Research priorities for Chagas disease, human African trypanosomiasis and leishmaniasis. World Health Organization, WHO Technical Report Series 975, 1–100 (2012)

  2. 2.

    et al. Comparative genomics of trypanosomatid parasitic protozoa. Science 309, 404–409 (2005)

  3. 3.

    Infectious diseases. Overcoming neglect of kinetoplastid diseases. Science 348, 974–976 (2015)

  4. 4.

    & An update on pharmacotherapy for leishmaniasis. Expert Opin. Pharmacother. 16, 237–252 (2015)

  5. 5.

    et al. Recent developments in drug discovery for leishmaniasis and human African trypanosomiasis. Chem. Rev. (2014)

  6. 6.

    Chagas’ Disease. N. Engl. J. Med. 373, 456–466 (2015)

  7. 7.

    Chagas disease drug discovery: toward a new era. J. Biomol. Screen. 20, 22–35 (2015)

  8. 8.

    Clinical features, diagnosis, and treatment of human African trypanosomiasis (sleeping sickness). Lancet Neurol. 12, 186–194 (2013)

  9. 9.

    Control of the leishmaniases. World Health Organization, WHO Technical Report Series 949, 37–39 (2010)

  10. 10.

    & A comparison of the activities of three amphotericin B lipid formulations against experimental visceral and cutaneous leishmaniasis. Int. J. Antimicrob. Agents 13, 243–248 (2000)

  11. 11.

    et al. Pharmacokinetics of miltefosine in Old World cutaneous leishmaniasis patients. Antimicrob. Agents Chemother. 52, 2855–2860 (2008)

  12. 12.

    & Leishmaniasis: clinical syndromes and treatment. QJM 107, 7–14, (2014)

  13. 13.

    Combating the next lethal epidemic. Science 348, 296–297 (2015)

  14. 14.

    & Re-examination of the immunosuppressive mechanisms mediating non-cure of Leishmania infection in mice. Immunol. Rev. 201, 225–238 (2004)

  15. 15.

    , , & Nanodisk-associated amphotericin B clears Leishmania major cutaneous infection in susceptible BALB/c mice. Antimicrob. Agents Chemother. 50, 1238–1244 (2006)

  16. 16.

    et al. Chagas disease: an overview of clinical and epidemiological aspects. J. Am. Coll. Cardiol. 62, 767–776 (2013)

  17. 17.

    & Chagas disease: 100 years after its discovery. A systemic review. Acta Trop. 115, 5–13 (2010)

  18. 18.

    Antitrypanosomal therapy for chronic Chagas’ disease. N. Engl. J. Med. 364, 2527–2534 (2011)

  19. 19.

    et al. Towards a paradigm shift in the treatment of chronic Chagas disease. Antimicrob. Agents Chemother. 58, 635–639 (2014)

  20. 20.

    et al. Randomized trial of posaconazole and benznidazole for chronic Chagas’ disease. N. Engl. J. Med. 370, 1899–1908 (2014)

  21. 21.

    et al. Randomized trial of benznidazole for chronic Chagas’ cardiomyopathy. N. Engl. J. Med. 373, 1295–1306 (2015)

  22. 22.

    et al. Side effects of benznidazole as treatment in chronic Chagas disease: fears and realities. Expert Rev. Anti Infect. Ther. 7, 157–163 (2009)

  23. 23.

    et al. Antitrypanosomal treatment with benznidazole is superior to posaconazole regimens in mouse models of Chagas disease. Antimicrob. Agents Chemother. 59, 6385–6394 (2015)

  24. 24.

    , & Drug-induced cure drives conversion to a stable and protective CD8+ T central memory response in chronic Chagas disease. Nature Med. 14, 542–550 (2008)

  25. 25.

    et al. In vivo imaging of trypanosome-brain interactions and development of a rapid screening test for drugs against CNS stage trypanosomiasis. PLoS Negl. Trop. Dis. 7, e2384 (2013)

  26. 26.

    et al. Structure- and function-based design of Plasmodium-selective proteasome inhibitors. Nature 530, 233–236 (2016)

  27. 27.

    & Trypanocidal activity of the proteasome inhibitor and anti-cancer drug bortezomib. Parasites & Vectors 2, 29 (2009)

  28. 28.

    et al. The genome of the kinetoplastid parasite, Leishmania major. Science 309, 436–442 (2005)

  29. 29.

    et al. Effect of noncompetitive proteasome inhibition on bortezomib resistance. J. Natl. Cancer Inst. 102, 1069–1082 (2010)

  30. 30.

    et al. Chemical blockage of the proteasome inhibitory function of bortezomib: impact on tumor cell death. J. Biol. Chem. 281, 1107–1118 (2006)

  31. 31.

    , & The isolation and characterization of murine macrophages. Curr. Protoc. Immunol. Chapter 14, Unit1 4.1 (2008)

  32. 32.

    et al. Utilizing chemical genomics to identify cytochrome b as a novel drug target for Chagas disease. PLoS Pathog. 11, e1005058 (2015)

  33. 33.

    , , & Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase. Antimicrob. Agents Chemother. 40, 2592–2597 (1996)

  34. 34.

    et al. GeneDB–an annotation database for pathogens. Nucleic Acids Res. 40, D98–D108 (2012)

  35. 35.

    & pTcINDEX: a stable tetracycline-regulated expression vector for Trypanosoma cruzi. BMC Biotechnol. 6, 32 (2006)

  36. 36.

    , & Stable transformation of Trypanosoma cruzi: inactivation of the PUB12.5 polyubiquitin gene by targeted gene disruption. Mol. Biochem. Parasitol. 57, 15–30 (1993)

  37. 37.

    , , & A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89–101 (1999)

  38. 38.

    & Purification of the eukaryotic 20S proteasome. Curr. Protoc. Protein Sci. Chapter 21 (2001)

  39. 39.

    et al. The structure of the mammalian 20S proteasome at 2.75 A resolution. Structure 10, 609–618 (2002)

  40. 40.

    et al. An efficient rapid system for profiling the cellular activities of molecular libraries. Proc. Natl Acad. Sci. USA 103, 3153–3158 (2006)

  41. 41.

    , , & Validation of a rapid equilibrium dialysis approach for the measurement of plasma protein binding. J. Pharm. Sci. 97, 4586–4595 (2008)

  42. 42.

    & Recent advances in physicochemical and ADMET profiling in drug discovery. Chem. Biodivers. 6, 1887–1899 (2009)

  43. 43.

    , , , & Influence of microsomal concentration on apparent intrinsic clearance: implications for scaling in vitro data. Drug Metab. Dispos. 29, 1332–1336 (2001)

  44. 44.

    et al. A modern in vivo pharmacokinetic paradigm: combining snapshot, rapid and full PK approaches to optimize and expedite early drug discovery. Drug Discov. Today 18, 71–78 (2013)

  45. 45.

    & Animal models for the analysis of immune responses to leishmaniasis. Curr. Protoc. Immunol. Chapter 19, Unit 19.12 (2001)

  46. 46.

    et al. Highly sensitive in vivo imaging of Trypanosoma brucei expressing “red-shifted” luciferase. PLoS Negl. Trop. Dis. 7, e2571 (2013)

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Acknowledgements

This work was supported in part by grants from the Wellcome Trust (091038/Z/09/Z to R.J.G. and F.S., and 104976/Z/14/Z, 104111/15/Z to J.C.M. and E.M.) and NIH (AI106850 to F.S.B.). We thank S. Croft, R. Don, L. Gredsted, A. Hudson and J. Mendlein for discussions, R. Tarleton for T. cruzi CL strain, and G. Cross for T. brucei Lister 427 strain. We thank A. Kreusch for help with proteasome purification, and F. Luna for help with T. cruzi whole-genome sequencing. We acknowledge technical assistance of O. Faghih in generating the plasmids for ectopic expression of PSMB4 in T. cruzi, R. Ritchie for IVIS in vivo imaging, and A. Mak, J. Matzen and P. Anderson for execution of high-throughput screens. We thank J. Isbell and T. Hollenbeck for profiling GNF6702 in ADME assays.

Author information

Author notes

    • Shilpi Khare
    •  & Advait S. Nagle

    These authors contributed equally to this work.

Affiliations

  1. Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA

    • Shilpi Khare
    • , Advait S. Nagle
    • , Agnes Biggart
    • , Yin H. Lai
    • , Fang Liang
    • , Lauren C. Davis
    • , S. Whitney Barnes
    • , Casey J. N. Mathison
    • , Mu-Yun Gao
    • , Xianzhong Liu
    • , Jocelyn L. Tan
    • , Monique Stinson
    • , Ianne C. Rivera
    • , Jaime Ballard
    • , Vince Yeh
    • , Todd Groessl
    • , Glenn Federe
    • , John D. Venable
    • , Badry Bursulaya
    • , Michael Shapiro
    • , Pranab K. Mishra
    • , Glen Spraggon
    • , Ansgar Brock
    • , Ben G. Wen
    • , John R. Walker
    • , Tove Tuntland
    • , Valentina Molteni
    • , Richard J. Glynne
    •  & Frantisek Supek
  2. Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8TA, UK

    • Elmarie Myburgh
    •  & Jeremy C. Mottram
  3. Centre for Immunology and Infection, Department of Biology, University of York, Wentworth Way, Heslington, York YO10 5DD, UK

    • Elmarie Myburgh
    •  & Jeremy C. Mottram
  4. Department of Medicine, University of Washington, Seattle, Washington 98109, USA

    • J. Robert Gillespie
    •  & Frederick S. Buckner
  5. Novartis Institute for Tropical Diseases, 10 Biopolis Road, Singapore 138670.

    • Hazel X. Y. Koh
    •  & Srinivasa P. S. Rao

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Contributions

A.B., F.L., C.J.N.M., P.K.M., A.S.N., J.L.T. and V.Y. designed chemical analogues, and performed chemical synthesis and purification of synthesized analogues. F.S.B., J.B., J.R.G., S.K., H.X.Y.K., Y.H.L., S.P.S.R., F.S. and X.L. conducted and analysed data from in vitro growth-inhibition assays. L.C.D., X.L., J.C.M., E.M., I.C.R., S.P.S.R., M.S., F.S. and B.G.W. conducted and analysed data from in vivo efficacy assays. J.B., M.-Y.G., S.K. and F.S. conducted proteasome purification, proteasome inhibition assays and biochemical data analysis. S.W.B., G.F., S.K., F.S. and J.R.W. designed, conducted and analysed experiments resulting in identification of proteasome resistance mutations. G.S. and B.B. built the homology model of T. cruzi proteasome structure and performed GNF6702 docking. A.B. and J.D.V. analysed T. cruzi proteasome by mass spectrometry. A.N., T.G., M.S., F.S. and T.T. designed, conducted, and analysed PK data. A.N. and V.M. led the chemistry team. F.S. led the biology team. R.J.G. and F.S. supervised and led the overall project, and led the writing of the manuscript. All authors contributed to writing of the manuscript.

Competing interests

Patents related to this work have been filed (WO 2015/095477 A1, WO 2014/151784 A1, WO 2014/151729). Several authors own shares of Novartis.

Corresponding author

Correspondence to Frantisek Supek.

Reviewer Information

Nature thanks M. PhilIips, S. Schreiber and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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https://doi.org/10.1038/nature19339

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