BACKGROUND: Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and leads to ~10,000 deaths each year. Nifurtimox and benznidazole are the only two drugs available but have significant adverse effects and limited efficacy. New chemotherapeutic agents are urgently required. Here we identified inhibitors of the acidic M17 leucyl-aminopeptidase from T. cruzi (LAPTc) that show promise as novel starting points for Chagas disease drug discovery. METHODOLOGY/PRINCIPAL FINDINGS: A RapidFire-MS screen with a protease-focused compound library identified novel LAPTc inhibitors. Twenty-eight hits were progressed to the dose-response studies, from which 12 molecules inhibited LAPTc with IC50 < 34 μM. Of these, compound 4 was the most potent hit and mode of inhibition studies indicate that compound 4 is a competitive LAPTc inhibitor, with Ki 0.27 μM. Compound 4 is selective with respect to human LAP3, showing a selectivity index of >500. Compound 4 exhibited sub-micromolar activity against intracellular T. cruzi amastigotes, and while the selectivity-window against the host cells was narrow, no toxicity was observed for un-infected HepG2 cells. In silico modelling of the LAPTc-compound 4 interaction is consistent with the competitive mode of inhibition. Molecular dynamics simulations reproduce the experimental binding strength (-8.95 kcal/mol), and indicate a binding mode based mainly on hydrophobic interactions with active site residues without metal cation coordination. CONCLUSIONS/SIGNIFICANCE: Our data indicates that these new LAPTc inhibitors should be considered for further development as antiparasitic agents for the treatment of Chagas disease.

Biology Centre Czech Academy of Sciences Institute of Parasitology České Budějovice Czech Republic

Centre for Pharmaceuticals Research and Development La Habana Cuba

Centre for Protein Studies Faculty of Biology University of Havana La Habana Cuba

Department of Biochemistry Faculty of Biology University of Havana La Habana Cuba

Department of Parasitology Faculty of Science Charles University Prague Biocev Vestec Czech Republic

Drug Discovery Unit Wellcome Centre for Anti Infectives Research University of Dundee Dundee United Kingdom

School of Life Sciences University of Dundee Dundee United Kingdom

Wellcome Centre for Anti Infectives Research Division of Biological Chemistry and Drug Discovery School of Life Sciences University of Dundee Dundee United Kingdom

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Mills RM. Chagas disease: Epidemiology and barriers to treatment. Am J Med. 2020. doi: 10.1016/j.amjmed.2020.05.022 PubMed DOI

World Health Organization (WHO). Chagas disease (also known as American tripanosomiasis) fact sheet, 1 April 2021. 2021. Available from: https://www.who.int/news-room/fact-sheets/detail/chagas-disease-(american-trypanosomiasis).

Romano PS, Cueto JA, Casassa AF, Vanrell MC, Gottlieb RA, Colombo MI. Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay. IUBMB Life. 2012;64: 387–396. PubMed PMC

Carod-Artal FJ. American trypanosomiasis. In: Handbook of Clinical Neurology. Elsevier BV; 2013. pp. 103–123. doi: 10.1016/B978-0-444-53490-3.00007–8 PubMed DOI

Malik LH, Singh GD, Amsterdam EA. The epidemiology, clinical manifestations, and management of chagas heart disease. Clin Cardiol. 2015. doi: 10.1002/clc.22421 PubMed DOI PMC

Py MO. Neurologic manifestations of chagas disease. Curr Neurol Neurosci Rep. 2011;11: 536–542. doi: 10.1007/s11910-011-0225-8 PubMed DOI

Echavarría NG, Echeverría LE, Stewart M, Gallego C, Saldarriaga C. Chagas disease: Chronic Chagas cardiomyopathy. Curr Probl Cardiol. 2021. doi: 10.1016/j.cpcardiol.2019.100507 PubMed DOI

Mansoldo FRP, Carta F, Angeli A, Cardoso VdaS, Supuran CT, Vermelho AB. Chagas disease: Perspectives on the past and present and challenges in drug discovery. Molecules. 2020. doi: 10.3390/molecules25225483 PubMed DOI PMC

Kampen S, Duy Vo D, Zhang X, Panel N, Yang Y, Jaiteh M, et al.. Structure-guided design of G-protein-coupled receptor polypharmacology. Angew Chem Int Ed Engl. 2021;60: 18022–18030. doi: 10.1002/anie.202101478 PubMed DOI PMC

Docherty AJ, Crabbe T, O’Connell JP, Groom CR. Proteases as drug targets. Biochem Soc Symp. 2003;70: 147–161. doi: 10.1042/bss0700147 PubMed DOI

Verhelst SHL. Intramembrane proteases as drug targets. FEBS J. 2017. doi: 10.1111/febs.13979 PubMed DOI

Sajid M, Robertson SA, Brinen LS, McKerrow JH. Cruzain: The path from target validation to the clinic. Adv Exp Med Biol. 2011;712: 100–115. doi: 10.1007/978-1-4419-8414-2_7 PubMed DOI

Álvarez VE, Niemirowicz GT, Cazzulo JJ. The peptidases of Trypanosoma cruzi: Digestive enzymes, virulence factors, and mediators of autophagy and programmed cell death. Biochim Biophys Acta—Proteins Proteomics. 2012. doi: 10.1016/j.bbapap.2011.05.011 PubMed DOI

Cadavid-Restrepo G, Gastardelo TS, Faudry E, De Almeida H, Bastos IM, Negreiros RS, et al.. The major leucyl aminopeptidase of Trypanosoma cruzi (LAPTc) assembles into a homohexamer and belongs to the M17 family of metallopeptidases. BMC Biochem. 2011;12. doi: 10.1186/1471-2091-12-46 PubMed DOI PMC

Timm J, Valente M, García-Caballero D, Wilson KS, González-Pacanowska D. Structural characterization of acidic M17 leucine aminopeptidases from the TriTryps and evaluation of their role in nutrient starvation in Trypanosoma brucei. mSphere. 2017;2: e00226–17. doi: 10.1128/mSphere.00226-17 PubMed DOI PMC

Izquierdo M, Lin D, O’Neill S, Zoltner M, Webster L, Hope A, et al.. Development of a high-throughput screening assay to identify inhibitors of the major M17-leucyl aminopeptidase from Trypanosoma cruzi using RapidFire mass spectrometry. SLAS Discov. 2020. doi: 10.1177/2472555220923367 PubMed DOI

Knowles G. The effects of arphamenine-A, an inhibitor of aminopeptidases, on in-vitro growth of Trypanosoma brucei. J Antimicrob Chemother. 1993;32: 172–174. PubMed

Peña-Díaz P, Vancová M, Resl C, Field MC, Lukeš J. A leucine aminopeptidase is involved in kinetoplast DNA segregation in Trypanosoma brucei. PLoS Pathog. 2017;13: e1006310. doi: 10.1371/journal.ppat.1006310 PubMed DOI PMC

Umezawa H, Aoyagi T, Suda H, Hamada M, Takeuchi T. Bestatin, an inhibitor of aminopeptidase B, produced by actinomycetes. J Antibiot (Tokyo). 1976. doi: 10.7164/antibiotics.29.97 PubMed DOI

Trochine A, Creek DJ, Faral-Tello P, Barrett MP, Robello C. Bestatin induces specific changes in Trypanosoma cruzi dipeptide pool. Antimicrob Agents Chemother. 2015;59: 2921–2925. PubMed PMC

Stack CM, Lowther J, Cunningham E, Donnelly S, Gardiner DL, Trenholme KR, et al.. Characterization of the Plasmodium falciparum M17 leucyl aminopeptidase. A protease involved in amino acid regulation with potential for antimalarial drug development. J Biol Chem. 2007;282: 2069–2080. PubMed

Harbut MB, Velmourougane G, Dalal S, Reiss G, Whisstock JC, Onder O, et al.. Bestatin-based chemical biology strategy reveals distinct roles for malaria M1- and M17-family aminopeptidases. Proc Natl Acad Sci USA. 2011;108: 526–534. doi: 10.1073/pnas.1105601108 PubMed DOI PMC

Lee Y-R, Na B-K, Moon E-K, Song S-M, Joo S-Y, Kong H-H, et al.. Essential role for an M17 leucine aminopeptidase in encystation of Acanthamoeba castellanii. PLoS ONE. 2015;10: e0129884. doi: 10.1371/journal.pone.0129884 PubMed DOI PMC

Aboge GO, Cao S, Terkawi MA, Masatani T, Goo Y, AbouLaila M, et al.. Molecular characterization of Babesia bovis M17 leucine aminopeptidase and inhibition of Babesia growth by bestatin. J Parasitol. 2015;101: 536–541. PubMed

Rinaldi G, Morales ME, Alrefaei YN, Cancela M, Castillo E, Dalton JP, et al.. RNA interference targeting leucine aminopeptidase blocks hatching of Schistosoma mansoni eggs. Mol Biochem Parasitol. 2009;167: 118–126. PubMed PMC

Zheng J, Jia HL, Zheng YH. Knockout of leucine aminopeptidase in Toxoplasma gondii using CRISPR/Cas9. Int J Parasitol. 2015;45: 141–148. PubMed

González-Bacerio J, Arocha I, Aguado ME, Méndez Y, Marsiccobetre S, Izquierdo M, et al.. KBE009: A bestatin-like inhibitor of the Trypanosoma cruzi acidic M17 aminopeptidase with in vitro anti-trypanosomal activity. Life. 2021;11: 1037. doi: 10.3390/life11101037 PubMed DOI PMC

Skinner-Adams TS, Lowther J, Teuscher F, Stack CM, Grembecka J, Mucha A, et al.. Identification of phosphinate dipeptide analog inhibitors directed against the Plasmodium falciparum M17 leucine aminopeptidase as lead antimalarial compounds. J Med Chem. 2007;50: 6024–6031. PubMed

Mistry SN, Drinkwater N, Ruggeri C, Sivaraman KK, Loganathan S, Fletcher S, et al.. Two-pronged attack: Dual inhibition of Plasmodium falciparum M1 and M17 metalloaminopeptidases by a novel series of hydroxamic acid-based inhibitors. J Med Chem. 2014;57: 9168–9183. PubMed

Drinkwater N, Vinh NB, Mistry SN, Bamert RS, Ruggeri C, Holleran JP, et al.. Potent dual inhibitors of Plasmodium falciparum M1 and M17 aminopeptidases through optimization of S1 pocket interactions. Eur J Med Chem. 2016;110: 43–64. PubMed

Vinh NB, Drinkwater N, Malcolm TR, Kassiou M, Lucantoni L, Grin PM, et al.. Hydroxamic acid inhibitors provide cross-species inhibition of Plasmodium M1 and M17 aminopeptidases. J Med Chem. 2019;62: 622–640. PubMed

Izquierdo M, Aguado ME, Zoltner M, González-Bacerio J. High-level expression in Escherichia coli, purification and kinetic characterization of LAPTc, a Trypanosoma cruzi M17-aminopeptidase. Protein J. 2019;38: 167–180. PubMed

MacLean LM, Thomas J, Lewis MD, Cotillo I, Gray DW, De Rycker M. Development of Trypanosoma cruzi in vitro assays to identify compounds suitable for progression in Chagas’ disease drug discovery. PLoS Negl Trop Dis. 2018;12: e0006612. doi: 10.1371/journal.pntd.0006612 PubMed DOI PMC

Silveira FT, Viana Dias MG, Pereira Pardal P, Oliveira Lobão A, Britto Melo G. Nono caso-autóctone de doença de Chagas registrado no estado do Pará, Brasil (Nota prévia). Hiléia Médica. 1979;1: 61–62.

De Rycker M, Thomas J, Riley J, Brough SJ, Miles TJ, Gray DW. Identification of trypanocidal activity for known clinical compounds using a new Trypanosoma cruzi hit-discovery screening cascade. PLoS Negl Trop Dis. 2016;10: e0004584. doi: 10.1371/journal.pntd.0004584 PubMed DOI PMC

Nühs A, De Rycker M, Manthri S, Comer E, Scherer CA, Schreiber SL, et al.. Development and validation of a novel Leishmania donovani screening cascade for high-throughput screening using a novel axenic assay with high predictivity of leishmanicidal intracellular activity. PLoS Negl Trop Dis. 2015;9: e0004094. doi: 10.1371/journal.pntd.0004094 PubMed DOI PMC

Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR. Avogadro: An advanced semantic chemical editor, visualization, and analysis platform. J Cheminf. 2012;4: 17. doi: 10.1186/1758-2946-4-17 PubMed DOI PMC

Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al.. Autodock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem. 2009;30: 2785–2791. doi: 10.1002/jcc.21256 PubMed DOI PMC

Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al.. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem. 2004;25: 1605–1612. doi: 10.1002/jcc.20084 PubMed DOI

Verma K, Kannan K, Shanthi V, Sethumadhavan R, Karthick V, Ramanathan K. Exploring β-tubulin inhibitors from plant origin using computational approach. Phytochem Anal. 2017;28: 230–241. PubMed

Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J Comput Chem. 2010;31: 455–461. doi: 10.1002/jcc.21334 PubMed DOI PMC

Phillips JC, Braun R, Wang W, Gumbart J, Tajkhorshid E, Villa E, et al.. Scalable molecular dynamics with NAMD. J Comput Chem. 2005;26: 1781–1802. doi: 10.1002/jcc.20289 PubMed DOI PMC

Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S, Shim J, et al.. CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J Comput Chem. 2010;31: 671–690. doi: 10.1002/jcc.21367 PubMed DOI PMC

Huang J, Rauscher S, Nawrocki G, Ran T, Feig M, de Groot BL, et al.. CHARMM36m: An improved force field for folded and intrinsically disordered proteins. Nat Meth. 2017;14: 71–73. doi: 10.1038/nmeth.4067 PubMed DOI PMC

Mayne CG, Saam J, Schulten K, Tajkhorshid E, Gumbart JC. Rapid parameterization of small molecules using the Force Field Toolkit. J Comput Chem. 2013;34: 2757–2770. doi: 10.1002/jcc.23422 PubMed DOI PMC

Humphrey W, Dalke A, Schulten K. VMD: Visual molecular dynamics. J Mol Graph. 1996;14: 33–38. doi: 10.1016/0263-7855(96)00018-5 PubMed DOI

Won Y. Force field for monovalent, divalent, and trivalent cations developed under the solvent boundary potential. J Phys Chem A. 2012;116: 11763–11767. doi: 10.1021/jp309150r PubMed DOI

Davidchack RL, Handel R, Tretyakov MV. Langevin thermostat for rigid body dynamics. J Chem Phys. 2009;130: 234101. doi: 10.1063/1.3149788 PubMed DOI

Sajadi F, Rowley CN. Simulations of lipid bilayers using the CHARMM36 force field with the TIP3P-FB and TIP4P-FB water models. PeerJ. 2018;6: e5472. doi: 10.7717/peerj.5472 PubMed DOI PMC

Feller SE, Zhang Y, Pastor RW. Constant pressure molecular dynamics simulation: The Langevin piston method. J Chem Phys. 1995;103: 4613. doi: 10.1063/1.470648 DOI

Laskowski RA, Swindells MB. LigPlot+: Multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model. 2011;51: 2778–2786. doi: 10.1021/ci200227u PubMed DOI

Miranda WE, Noskov SY, Valiente PA. Improving the LIE method for binding free energy calculations of protein-ligand complexes. J Chem Inf Model. 2015;55: 1867–1877. doi: 10.1021/acs.jcim.5b00012 PubMed DOI

de Almeida Nogueira NP, Morgado-Díaz JA, Menna-Barreto RFS, Paes MC, da Silva-López RE. Effects of a marine serine protease inhibitor on viability and morphology of Trypanosoma cruzi, the agent of Chagas disease. Acta Trop. 2013;128: 27–35. PubMed

Manchola NC, Rapado LN, Barison MJ, Silber AM. Biochemical characterization of branched chain amino acids uptake in Trypanosoma cruzi. J Eukaryot Microbiol. 2015;63: 299–308. PubMed

Highkin MK, Yates MP, Nemirovskiy OV, Lamarr WA, Munie GE, Rains JW, et al.. High-throughput screening assay for sphingosine kinase inhibitors in whole blood using RapidFire Mass Spectrometry. J Biomol Screen. 2011;16: 272–277. PubMed

Asano W, Takahashi Y, Kawano M, Hantani Y. Identification of an arginase II inhibitor via RapidFire Mass Spectrometry combined with hydrophilic interaction chromatography. SLAS Discov. 2019;24: 457–465. doi: 10.1177/2472555218812663 PubMed DOI

Leveridge M, Collier L, Edge C, Hardwicke P, Leavens B, Ratcliffe S, et al.. A high-throughput screen to identify LRRK2 kinase inhibitors for the treatment of Parkinson’s disease using RapidFire Mass Spectrometry. J Biomol Screen. 2016;21: 145–155. doi: 10.1177/1087057115606707 PubMed DOI

Plant M, Dineen T, Cheng A, Long AM, Chen H, Morgenstern KA. Screening for lysine-specific demethylase-1 inhibitors using a label-free high-throughput mass spectrometry assay. Anal Biochem. 2011;419: 217–227. doi: 10.1016/j.ab.2011.07.002 PubMed DOI

Adachi R, Ishii T, Matsumoto S, Satou T, Sakamoto J, Kawamoto T. Discovery of human intestinal MGAT inhibitors using high-throughput mass spectrometry. SLAS Discov. 2017;22: 360–365. doi: 10.1177/1087057116673181 PubMed DOI

Hutchinson S, Leveridge M, Heathcote M, Francis P, Williams L, Gee M, et al.. Enabling lead discovery for histone lysine demethylases by high-throughput RapidFire Mass Spectrometry. J Biomol Screen. 2012;17: 39–48. doi: 10.1177/1087057111416660 PubMed DOI

Jonas M, LaMarr WA, Özbal C. Mass spectrometry in high throughput screening: A case study on acetyl-coenzyme A carboxylase using RapidFire—Mass Spectrometry (RF-MS). Comb Chem High Through Screen. 2009;12: 752–759. doi: 10.2174/138620709789104924 PubMed DOI

Drinkwater N, Malcolm TR, McGowan S. M17 aminopeptidases diversify function by moderating their macromolecular assemblies and active site environment. Biochimie. 2019;166: 38–51. doi: 10.1016/j.biochi.2019.01.007 PubMed DOI

McGowan S, Oellig CA, Birru WA, Caradoc-Davies TT, Stack CM, Lowther J, et al.. Structure of the Plasmodium falciparum M17 aminopeptidase and significance for the design of drugs targeting the neutral exopeptidases. Proc Natl Acad Sci USA. 2010;107: 2449–2454. PubMed PMC

N’Guessan H, Megnassan E. In silico design of phosphonic arginine and hydroxamic acid inhibitors of Plasmodium falciparum M17 leucyl aminopeptidase with favorable pharmacokinetic profile. J Drug Design Med Chem. 2017;3: 98–125.

González-Bacerio J, Maluf SEC, Méndez Y, Pascual I, Florent I, Melo PMS, et al.. KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library. Bioorg Med Chem. 2017;25: 4628–4636. PubMed

Identification and Validation of Compounds Targeting Leishmania major Leucyl-Aminopeptidase M17