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Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness

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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Figure 1: Chemical evolution of GNF6702 from the phenotypic hit GNF5343.
Figure 2: GNF6702 clears parasites in mouse models of kinetoplastid infections.
Figure 3: F24L mutation in proteasome β4 subunit confers selective resistance to GNF6702.
Figure 4: Compounds from GNF6702 series inhibit growth of kinetoplastid parasites by inhibiting parasite proteasome chymotrypsin-like activity.

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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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Frantisek Supek.

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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.

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

Extended data figures and tables

Extended Data Figure 1 Pharmacokinetic profile of GNF6702 in mouse.

a, Time profiles of mean free plasma concentration of GNF6702 in mouse model of visceral leishmaniasis; free GNF6702 concentration values were predicted from measured total plasma concentration values collected on day 1 and day 8 of treatment. Dashed blue lines correspond to intra-macrophage L. donovani EC50 of 18 ± 1.8 nM and EC99 of 42 ± 5.6 nM. Circles, means ± s.d.; n = 3 mice for treatment day 1; n = 5 mice for treatment day 8; fraction unbound in mouse plasma = 0.063. For data points lacking error bars, standard deviations are smaller than circles representing means. b, Time course of total GNF6702 concentration in mouse plasma and brain after single oral dose (20 mg kg−1); n = 2 mice per time point; circles, measured values; rectangles, means.

Extended Data Figure 2 GNF6702 clears parasites from mice infected with T. brucei.

a, In vivo quantification of bioluminescent T. brucei in infected mice before and after treatment. i.p., intraperitoneal; day 21, start of treatment; day 28, 24 h after last GNF6702 dose; day 42, evaluation of early parasite recrudescence in mice treated with diminazene aceturate (n = 3); day 42 and 92, absence of parasite recrudescence in mice treated with GNF6702 (n = 6). Images from uninfected mice (3 mice of 4 are shown) aged-matched for day 0 were collected independently using the same acquisition settings. Parasitemia (blue font) and whole mouse total flux (black font) values of each animal are shown above the image; N.D., not detectable. Within each group the mouse numbers in yellow (top left in each image) refer to the same mouse imaged throughout. Complete sets of parasitemia and whole mouse total flux values collected on individual mice throughout the experiment are listed in Supplementary Tables 4 and 5. b, Brains from mice shown in a were soaked in luciferin and imaged for presence of bioluminescent T. brucei at the indicated time points. For three diminazene-treated mice, two images of each brain are shown, one at a lower sensitivity (left) and the other at a high signal intensity scale.

Extended Data Figure 3 Structures and profiles of GNF3943 and GNF8000 used for selection of resistant T. cruzi lines.

Leishmania donovani, amastigotes proliferating within primary mouse macrophages; T. brucei, the bloodstream form trypomastigotes; T. cruzi, amastigotes proliferating in 3T3 fibroblast cells; macrophage, mouse primary peritoneal macrophages; EC50 and CC50, half-maximum growth-inhibition concentration; F, oral bioavailability in mouse after administering single compound dose (20 mg kg−1) as a suspension; CL, plasma clearance in mouse after single i.v. bolus dose (5 mg kg−1); all EC50 and CC50 values correspond to means ± s.e.m. (n = 4 technical replicates).

Extended Data Figure 4 Mutations in proteasome β4 subunit confer resistance to GNF6702 in T. cruzi and T. brucei.

a, Growth curves of wild-type, GNF3943-resistant and GNF8000-resistant T. cruzi epimastigote strains in the presence of increasing concentrations of GNF6702, nifurtimox, bortezomib and MG132; RU (relative units) corresponds to parasite growth relative to the DMSO control (%); for data points lacking error bars, standard errors are smaller than circles representing means; owing to limited aqueous solubility, the highest tested GNF6702 concentration was 10 μM. b, Growth-inhibition EC50 values of GNF6702, bortezomib, MG132 and nifurtimox on indicated T. cruzi strains. c, Growth-inhibition EC50 values of GNF6702 and bortezomib on T. cruzi epimastigotes and T. brucei bloodstream form trypomastigotes overexpressing wild-type PSMB4 or PSMB4F24L. Data shown in panels a, b and c correspond to means ± s.e.m. (n = 3 technical replicates).

Extended Data Figure 5 Correlation between inhibition of parasite proteasome chymotrypsin-like activity and parasite growth inhibition by the GNF6702 compound series.

IC50, half-maximum inhibition of indicated parasite proteasome; T. brucei EC50, half-maximum growth inhibition on T. brucei bloodstream form trypomastigotes; T. cruzi EC50, half-maximum growth inhibition on T. cruzi amastigotes proliferating inside 3T3 cells; data points correspond to means of 2 technical replicates; red circles, IC50 > 20 μM; yellow circles, IC50 > 20 μM and EC50 > 25 μM; data for 317 analogues are shown.

Extended Data Figure 6 Hypothetical model of GNF6702 binding to T. cruzi proteasome β4 subunit.

a, Alignment of amino acid sequences of proteasome β4 subunits (PSMB4) from L. donovani, T. cruzi, T. brucei and Homo sapiens. Green, amino acid residues conserved between human and kinetoplastid PSMB4 proteins; blue, amino acid residues conserved only among kinetoplastid PSMB4 proteins; black, amino acids mutated in T. cruzi mutants resistant to analogues from the GNF6702 series. b, Surface representation of the modelled T. cruzi 20S proteasome structure showing relative positions of the β5 and β4 subunits. β4 amino acid residues F24 and I29 (coloured yellow) are located at the interface of the two β subunits. GNF6702 is depicted in a sphere representation bound into a predicted pocket on the β4 subunit surface with carbon, nitrogen, oxygen and hydrogen atoms coloured magenta, blue, red and grey, respectively. The other T. cruzi 20S proteasome subunits are coloured grey. c, Close-up of the β5 and β4 subunits. The β5 subunit active site (pocket 1, chymotrypsin-like activity) is coloured pale green. The predicted β4 pocket (pocket 2) with bound GNF6702 is coloured blue. The inhibitor is shown in a stick representation with atoms coloured as described in caption for b. β4 residues F24 and I29 are coloured yellow. The proteasome model shown in b and c was produced in the PyMol Molecule Graphics System, Version 1.8, Schrodinger, LLC.

Extended Data Figure 7 Effect of GNF6702 on accumulation of ubiquitylated proteins by T. cruzi epimastigotes and 3T3 cells.

a, Western blot analysis of T. cruzi whole-cell extracts with anti-ubiquitin antibody after treatment with GNF6702 and bortezomib. b, Western blot analysis of 3T3 whole cell extracts with anti-ubiquitin antibody after treatment with GNF6702 and borteomib. c, Concentrations of GNF6702 and bortezomib effecting half-maximum accumulation of ubiquitylated proteins in T. cruzi and 3T3 cells (means ± s.e.m.; n = 3 technical replicates); total ubiquitin signal values in individual blot lanes shown in a and b were quantified and used for calculation of the listed EC50 values. In a and b, numbers above the blot lanes indicate compound concentrations and D indicates control, DMSO-treated cells. For western blot source data, see Supplementary Fig. 1.

Extended Data Table 1 Point mutations identified by whole-genome sequencing in GNF3943- and GNF8000-resistant T. cruzi epimastigotes
Extended Data Table 2 Enzyme inhibition IC50 values of bortezomib and GNF6702 on three proteolytic activities of wild-type T. cruzi, PSMB4I29M T. cruzi and H. sapiens proteasomes
Extended Data Table 3 Inhibition kinetics parameters of GNF6702 on L. donovani and T. cruzi proteasomes

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Khare, S., Nagle, A., Biggart, A. et al. Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness. Nature 537, 229–233 (2016). https://doi.org/10.1038/nature19339

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