Novel broad-spectrum activity-based probes to profile malarial cysteine proteases

Autoři: Michele S. Y. Tan aff001;  Dara Davison aff001;  Mateo I. Sanchez aff002;  Bethany M. Anderson aff003;  Stephen Howell aff001;  Ambrosius Snijders aff001;  Laura E. Edgington-Mitchell aff003;  Edgar Deu aff001
Působiště autorů: The Francis Crick Institute, London, United Kingdom aff001;  Department of Genetics, Stanford School of Medicine, Stanford, California, United States of America aff002;  Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville Victoria, Australia aff003;  Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia aff004;  Department of Maxillofacial Surgery, College of Dentistry, New York University, New York, New York, United States of America aff005
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227341


Clan CA cysteine proteases, also known as papain-like proteases, play important roles throughout the malaria parasite life cycle and are therefore potential drug targets to treat this disease and prevent its transmission. In order to study the biological function of these proteases and to chemically validate some of them as viable drug targets, highly specific inhibitors need to be developed. This is especially challenging given the large number of clan CA proteases present in Plasmodium species (ten in Plasmodium falciparum), and the difficulty of designing selective inhibitors that do not cross-react with other members of the same family. Additionally, any efforts to develop antimalarial drugs targeting these proteases will also have to take into account potential off-target effects against the 11 human cysteine cathepsins. Activity-based protein profiling has been a very useful tool to determine the specificity of inhibitors against all members of an enzyme family. However, current clan CA proteases broad-spectrum activity-based probes either target endopeptidases or dipeptidyl aminopeptidases, but not both subfamilies efficiently. In this study, we present a new series of dipeptydic vinyl sulfone probes containing a free N-terminal tryptophan and a fluorophore at the P1 position that are able to label both subfamilies efficiently, both in Plasmodium falciparum and in mammalian cells, thus making them better broad-spectrum activity-based probes. We also show that some of these probes are cell permeable and can therefore be used to determine the specificity of inhibitors in living cells. Interestingly, we show that the choice of fluorophore greatly influences the specificity of the probes as well as their cell permeability.

Klíčová slova:

Cysteine – Cysteine proteases – Malaria – Malarial parasites – Parasitic diseases – Plasmodium – Proteases – Merozoites


1. World Health Organization. World Malaria Report 2016. 2017: 1–186.

2. Bhatt S, Weiss DJ, Cameron E, Bisanzio D, Mappin B, Dalrymple U, et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015;526: 207–211. doi: 10.1038/nature15535 26375008

3. Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014;371: 411–423. doi: 10.1056/NEJMoa1314981 25075834

4. Ranson H, Lissenden N. Insecticide resistance in African Anopheles mosquitoes: a worsening situation that needs urgent action to maintain malaria control. Trends Parasitol. 2016;32: 187–196. doi: 10.1016/ 26826784

5. Wells TNC, Hooft van Huijsduijnen R, Van Voorhis WC. Malaria medicines: a glass half full? Nat Rev Drug Discov. 2015;14: 424–442. doi: 10.1038/nrd4573 26000721

6. Drag M, Salvesen GS. Emerging principles in protease-based drug discovery. Nat Rev Drug Discov. 2010;9: 690–701. doi: 10.1038/nrd3053 20811381

7. Olson OC, Joyce JA. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat Rev Cancer. 2015;15: 712–729. doi: 10.1038/nrc4027 26597527

8. Kramer L, Turk D, Turk B. The Future of cysteine cathepsins in disease management. Trends Pharmacol Sci. 2017;38: 873–898. doi: 10.1016/ 28668224

9. Drake MT, Clarke BL, Oursler MJ, Khosla S. Cathepsin K inhibitors for osteoporosis: biology, potential clinical utility, and lessons learned. Endocr Rev. 2017;38: 325–350. doi: 10.1210/er.2015-1114 28651365

10. Korkmaz B, Caughey GH, Chapple I, Gauthier F, Hirschfeld J, Jenne DE, et al. Therapeutic targeting of cathepsin C: from pathophysiology to treatment. Pharmacol Ther. 2018;190: 202–236. doi: 10.1016/j.pharmthera.2018.05.011 29842917

11. Miller BE, Mayer RJ, Goyal N, Bal J, Dallow N, Boyce M, et al. Epithelial desquamation observed in a phase I study of an oral cathepsin C inhibitor (GSK2793660). Br J Clin Pharmacol. 2017;83: 2813–2820. doi: 10.1111/bcp.13398 28800383

12. Palmér R, Mäenpää J, Jauhiainen A, Larsson B, Mo J, Russell M, et al. Dipeptidyl peptidase 1 inhibitor AZD7986 induces a sustained, exposure-dependent reduction in neutrophil elastase activity in healthy subjects. Clin Pharmacol Ther. 2018;104: 1155–1164. doi: 10.1002/cpt.1053 29484635

13. Ferreira LG, Andricopulo AD. Targeting cysteine proteases in trypanosomatid disease drug discovery. Pharmacol Ther. 2017;180: 49–61. doi: 10.1016/j.pharmthera.2017.06.004 28579388

14. Deu E. Proteases as antimalarial targets: strategies for genetic, chemical, and therapeutic validation. FEBS J. 2017;284: 2604–2628. doi: 10.1111/febs.14130 28599096

15. Sanman LE, Bogyo M. Activity-based profiling of proteases. Annu Rev Biochem. 2014;83: 249–273. doi: 10.1146/annurev-biochem-060713-035352 24905783

16. Greenbaum D, Medzihradszky KF, Burlingame A, Bogyo M. Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem Biol. 2000;7: 569–581. doi: 10.1016/s1074-5521(00)00014-4 11048948

17. Greenbaum DC, Baruch A, Grainger M, Bozdech Z, Medzihradszky KF, Engel J, et al. A role for the protease falcipain 1 in host cell invasion by the human malaria parasite. Science. 2002;298: 2002–2006. doi: 10.1126/science.1077426 12471262

18. Arastu-Kapur S, Ponder EL, Fonović UP, Yeoh S, Yuan F, Fonović M, et al. Identification of proteases that regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum. Nat Chem Biol. 2008;4: 203–213. doi: 10.1038/nchembio.70 18246061

19. Yuan F, Verhelst SHL, Blum G, Coussens LM, Bogyo M. A selective activity-based probe for the papain family cysteine protease dipeptidyl peptidase I/cathepsin C. J Am Chem Soc. 2006;128: 5616–5617. doi: 10.1021/ja060835v 16637611

20. Miller SK, Good RT, Drew DR, Delorenzi M, Sanders PR, Hodder AN, et al. A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle. J Biol Chem. 2002;277: 47524–47532. doi: 10.1074/jbc.M206974200 12228245

21. Collins CR, Hackett F, Strath M, Penzo M, Withers-Martinez C, Baker DA, et al. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress. PLoS Pathog. 2013;9: e1003344. doi: 10.1371/journal.ppat.1003344 23675297

22. Thomas JA, Tan MSY, Bisson C, Borg A, Umrekar TR, Hackett F, et al. A protease cascade regulates release of the human malaria parasite Plasmodium falciparum from host red blood cells. Nat Microbiol. 2018;3: 447–455. doi: 10.1038/s41564-018-0111-0 29459732

23. Aly ASI, Matuschewski K. A malarial cysteine protease is necessary for Plasmodium sporozoite egress from oocysts. J Exp Med. 2005;202: 225–230. doi: 10.1084/jem.20050545 16027235

24. Shenai BR, Sijwali PS, Singh A, Rosenthal PJ. Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum. J Biol Chem. 2000;275: 29000–29010. doi: 10.1074/jbc.M004459200 10887194

25. Singh N, Sijwali PS, Pandey KC, Rosenthal PJ. Plasmodium falciparum: biochemical characterization of the cysteine protease falcipain-2’. Exp Parasitol. 2006;112: 187–192. doi: 10.1016/j.exppara.2005.10.007 16337629

26. Sijwali PS, Shenai BR, Gut J, Singh A, Rosenthal PJ. Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3. Biochemical Journal. 2001;360: 481–489. doi: 10.1042/0264-6021:3600481 11716777

27. Sijwali PS, Koo J, Singh N, Rosenthal PJ. Gene disruptions demonstrate independent roles for the four falcipain cysteine proteases of Plasmodium falciparum. Mol Biochem Parasitol. 2006;150: 96–106. doi: 10.1016/j.molbiopara.2006.06.013 16890302

28. Sijwali PS, Kato K, Seydel KB, Gut J, Lehman J, Klemba M, et al. Plasmodium falciparum cysteine protease falcipain-1 is not essential in erythrocytic stage malaria parasites. Proc Natl Acad Sci USA. 2004;101: 8721–8726. doi: 10.1073/pnas.0402738101 15166288

29. Hopp CS, Bennett BL, Mishra S, Lehmann C, Hanson KK, Lin J-W, et al. Deletion of the rodent malaria ortholog for falcipain-1 highlights differences between hepatic and blood stage merozoites. PLoS Pathog. 2017;13: e1006586. doi: 10.1371/journal.ppat.1006586 28922424

30. Klemba M, Gluzman I, Goldberg DE. A Plasmodium falciparum dipeptidyl aminopeptidase I participates in vacuolar hemoglobin degradation. J Biol Chem. 2004;279: 43000–43007. doi: 10.1074/jbc.M408123200 15304495

31. Deu E, Leyva MJ, Albrow VE, Rice MJ, Ellman JA, Bogyo M. Functional Studies of Plasmodium falciparum Dipeptidyl Aminopeptidase I Using Small Molecule Inhibitors and Active Site Probes. Chem Biol. 2010;17: 808–819. doi: 10.1016/j.chembiol.2010.06.007 20797610

32. Tanaka TQ, Deu E, Molina-Cruz A, Ashburne MJ, Ali O, Suri A, et al. Plasmodium dipeptidyl aminopeptidases as malaria transmission-blocking drug targets. Antimicrob Agents Chemother. 2013;57: 4645–4652. doi: 10.1128/AAC.02495-12 23836185

33. Suárez-Cortés P, Sharma V, Bertuccini L, Costa G, Bannerman N-L, Sannella AR, et al. Comparative proteomics and functional analysis reveal a role of Plasmodium falciparum osmiophilic bodies in malaria parasite transmission. Mol Cell Proteomics. 2016;15: 3243–3255. doi: 10.1074/mcp.M116.060681 27432909

34. Lehmann C, Tan MSY, de Vries LE, Russo I, Sanchez MI, Goldberg DE, et al. Plasmodium falciparum dipeptidyl aminopeptidase 3 activity is important for efficient erythrocyte invasion by the malaria parasite. PLoS Pathog. 2018;14: e1007031. doi: 10.1371/journal.ppat.1007031 29768491

35. Blackman MJ. Purification of Plasmodium falciparum merozoites for analysis of the processing of merozoite surface protein-1. Methods Cell Biol. 1994;45: 213–220. doi: 10.1016/s0091-679x(08)61853-1 7707987

36. Deu E, de Vries LE, Sanchez MI, Groborz K, Kuppens L, Poreba M, et al. Characterization of P. falciparum dipeptidyl aminopeptidase 3 specificity identifies differences in amino acid preferences between peptide-based substrates and inhibitors. Preprint. Available from bioRxiv. 2018;: 1–32.

37. Edgington-Mitchell LE, Bogyo M, Verdoes M. Live cell imaging and profiling of cysteine cathepsin activity using a quenched activity-based probe. Methods Mol Biol. 2017;1491: 145–159. doi: 10.1007/978-1-4939-6439-0_11 27778287

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2020 Číslo 1