Targeting proteolytic enzymes in the hemoglobin degradation pathway to inhibit Plasmodium falciparum: An in silico approach
Paper Details
Targeting proteolytic enzymes in the hemoglobin degradation pathway to inhibit Plasmodium falciparum: An in silico approach
Abstract
Malaria caused by Plasmodium falciparum continues to be a major health burden, and the parasite depends on hemoglobin digestion for its survival inside human red blood cells. The enzymes operating in this pathway plasmepsins, falcipains, aminopeptidases and serine proteases play specific roles in the stepwise breakdown of hemoglobin, and therefore serve as important points for drug intervention. In this work, twelve proteases with experimentally solved structures were prepared using standard protein-refinement procedures, binding sites were identified, and a library of 326 compounds was docked using Glide-SP. The screened molecules included approved antimalarial drugs, repurposable candidates and selected marine natural products with antimalarial and antiprotozoal activity. Plasmepsin IV produced the strongest binding scores, with WEHI-842 and saquinavir showing tight fitting in the active site. HIV-protease inhibitors and diamidines also showed good interactions with several proteases, which is consistent with earlier reports of their antimalarial effects. Cysteine proteases and metallo-aminopeptidases gave moderate binding for a few compounds like pentamidine, aliskiren and carvedilol. Some large natural molecules also showed notable pocket fitting, suggesting scope for future exploration. The results indicate that structure-based screening can help narrow down promising molecules before lab testing and can support repurposing ideas when the mechanism of binding overlaps with the parasite targets. Since ligand–protein interactions mainly depend on shape, charge and other physical forces, a few drugs originally made for different diseases can also show affinity towards parasite enzymes. This study provides a set of candidates that can be taken forward to biochemical assays and parasite-culture studies to check their actual antimalarial activity.
Abdulhameed Almuqdadi HT, Shakir M, Shoaib R, Alrehaili J, Anwer R, Singh S, Abid M. 2025. Integrative computational and experimental approaches for identifying potent antimalarials by targeting falcipain-2 of Plasmodium falciparum . Journal of Biomolecular Structure and Dynamics, 1–20. https://doi.org/10.1080/07391102.2025.2516143
Achan J, Kakuru A, Ikilezi G, Ruel T, Clark TD, Nsanzabana C, Charlebois E, Aweeka F, Dorsey G, Rosenthal PJ, Havlir D, Kamya MR. 2012. Antiretroviral agents and prevention of malaria in HIV-infected Ugandan children. New England Journal of Medicine 367(22), 2110–2118. https://doi.org/10.1056/NEJMoa1200501
Alemayehu A. 2023. Biology and epidemiology of Plasmodium falciparum and Plasmodium vivax gametocyte carriage: Implication for malaria control and elimination. Parasite Epidemiology and Control 21, e00295. https://doi.org/10.1016/j.parepi.2023.e00295
Ali F, Wali H, Jan S, Zia A, Aslam M, Ahmad I, Afridi SG, Shams S, Khan A. 2021. Analysing the essential proteins set of Plasmodium falciparum PF3D7 for novel drug targets identification against malaria. Malaria Journal 20(1), 335. https://doi.org/10.1186/s12936-021-03865-1
Almuqdadi HTA, Kifayat S, Anwer R, Alrehaili J, Abid M. 2024. Fragment-based virtual screening identifies novel leads against Plasmepsin IX (PlmIX) of Plasmodium falciparum : Homology modeling, molecular docking, and simulation approaches. Frontiers in Pharmacology 15, 1387629. https://doi.org/10.3389/fphar.2024.1387629
Ashburn TT, Thor KB. 2004. Drug repositioning: Identifying and developing new uses for existing drugs. Nature Reviews Drug Discovery 3(8), 673–683. https://doi.org/10.1038/nrd1468
Chugh M, Sundararaman V, Kumar S, Reddy VS, Siddiqui WA, Stuart KD, Malhotra P. 2013. Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum . Proceedings of the National Academy of Sciences 110(14), 5392–5397. https://doi.org/10.1073/pnas.1218412110
Coronado LM, Nadovich CT, Spadafora C. 2014. Malarial hemozoin: From target to tool. Biochimica et Biophysica Acta – General Subjects 1840(6), 2032–2041. https://doi.org/10.1016/j.bbagen.2014.02.009
Crutcher JM, Hoffman SL. 1996. Malaria. In: Baron S (ed.). Medical Microbiology, 4th ed. University of Texas Medical Branch at Galveston. http://www.ncbi.nlm.nih.gov/books/NBK8584/
Darmin WOYPN, Arfan A, Ruslin R, Wu C, Arba M. 2024. Docking-based virtual screening to identify the cysteine protease falcipain-2 inhibitors of Plasmodium falciparum. Molekul 19(3), 406. https://doi.org/10.20884/1.jm.2024.19.3.7376
Deu E. 2017. Proteases as antimalarial targets: Strategies for genetic, chemical, and therapeutic validation. The FEBS Journal 284(16), 2604–2628. https://doi.org/10.1111/febs.14130
Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, Shaw DE, Francis P, Shenkin PS. 2004. Glide: A new approach for rapid, accurate docking and scoring. 1. Method and assessment of docking accuracy. Journal of Medicinal Chemistry 47(7), 1739–1749. https://doi.org/10.1021/jm0306430
Gluzman IY, Francis SE, Oksman A, Smith CE, Duffin KL, Goldberg DE. 1994. Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway. Journal of Clinical Investigation 93(4), 1602–1608. https://doi.org/10.1172/JCI117140
Goldberg DE, Slater AF, Beavis R, Chait B, Cerami A, Henderson GB. 1991. Hemoglobin degradation in the human malaria pathogen Plasmodium falciparum : A catabolic pathway initiated by a specific aspartic protease. The Journal of Experimental Medicine 173(4), 961–969. https://doi.org/10.1084/jem.173.4.961
Gu WG, Zhang X, Yuan JF. 2014. Anti-HIV drug development through computational methods. The AAPS Journal 16(4), 674–680. https://doi.org/10.1208/s12248-014-9604-9
Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, Banks JL. 2004. Glide: A new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. Journal of Medicinal Chemistry 47(7), 1750–1759. https://doi.org/10.1021/jm030644s
Hodder AN, Sleebs BE, Czabotar PE, Gazdik M, Xu Y, O’Neill MT, Lopaticki S, Nebl T, Triglia T, Smith BJ, Lowes K, Boddey JA, Cowman AF. 2015. Structural basis for plasmepsin V inhibition that blocks export of malaria proteins to human erythrocytes. Nature Structural and Molecular Biology 22(8), 590–596. https://doi.org/10.1038/nsmb.3061
Jain S, Asad M, Caldelari R, Datta G, Singh S, Rathore S, Stanway RR, Heussler VT, Mohmmed A. 2022. Small-molecule inhibitors targeting prokaryotic ClpQ protease in Plasmodium display anti-malarial efficacies against blood and liver stages. Microbiology. https://doi.org/10.1101/2022.05.12.491652
Jeninga MD, Tang J, Selvarajah SA, Maier AG, Duffy MF, Petter M. 2023. Plasmodium falciparum gametocytes display global chromatin remodelling during sexual differentiation. BMC Biology 21(1), 65. https://doi.org/10.1186/s12915-023-01568-4
Ji X, Wang Z, Chen Q, Li J, Wang H, Wang Z, Yang L. 2022. In silico and in vitro antimalarial screening and validation targeting Plasmodium falciparum plasmepsin V. Molecules 27(9), 2670. https://doi.org/10.3390/molecules27092670
Jimoh Y, Abdullah IY, Hamza AN, Abdullahi M, Ahmadu J, Hassan LA, Yakubu MS, Salami Z. 2024. In silico evaluation of novel 2-pyrazoline carboxamide derivatives as potential protease inhibitors against Plasmodium parasites. ECSOC 2024, 57. https://doi.org/10.3390/ecsoc-28-20224
Johnson BC, Métifiot M, Pommier Y, Hughes SH. 2012. Molecular dynamics approaches estimate the binding energy of HIV-1 integrase inhibitors and correlate with in vitro activity. Antimicrobial Agents and Chemotherapy 56(1), 411–419. https://doi.org/10.1128/AAC.05292-11
Koussis K, Withers-Martinez C, Yeoh S, Child M, Hackett F, Knuepfer E, Juliano L, Woehlbier U, Bujard H, Blackman MJ. 2009. A multifunctional serine protease primes the malaria parasite for red blood cell invasion. The EMBO Journal 28(6), 725–735. https://doi.org/10.1038/emboj.2009.22
Li H, Child MA, Bogyo M. 2012. Proteases as regulators of pathogenesis: Examples from the Apicomplexa. Biochimica et Biophysica Acta – Proteins and Proteomics 1824(1), 177–185. https://doi.org/10.1016/j.bbapap.2011.06.002
McGillewie L., Soliman ME. 2016. The binding landscape of plasmepsin V and the implications for flap dynamics. Molecular BioSystems 12(5), 1457–1467. https://doi.org/10.1039/C6MB00077K
Mahanta PJ, Lhouvum K. 2024. Plasmodium falciparum proteases as new drug targets with special focus on metalloproteases. Molecular and Biochemical Parasitology 258, 111617. https://doi.org/10.1016/j.molbiopara.2024.111617
Meyers JM, Goldberg DE. 2012. Recent advances in plasmepsin medicinal chemistry and implications for future antimalarial drug discovery efforts. Current Topics in Medicinal Chemistry 12(5), 445–455. https://doi.org/10.2174/156802612799362959
Mishra M, Singh V, Singh S. 2019. Structural insights into key Plasmodium proteases as therapeutic drug targets. Frontiers in Microbiology 10, 394. https://doi.org/10.3389/fmicb.2019.00394
Nasamu AS, Polino AJ, Istvan ES, Goldberg DE. 2020. Malaria parasite plasmepsins: More than just plain old degradative pepsins. Journal of Biological Chemistry 295(25), 8425–8441. https://doi.org/10.1074/jbc.REV120.009309
Pinzi L, Bisi N, Rastelli G. 2024. How drug repurposing can advance drug discovery: Challenges and opportunities. Frontiers in Drug Discovery 4, 1460100. https://doi.org/10.3389/fddsv.2024.1460100
Porter KA, Cole SR, Eron JJ, Zheng Y, Hughes MD, Lockman S, Poole C, Skinner-Adams TS, Hosseinipour M, Shaffer D, D’Amico R, Sawe FK, Siika A, Stringer E, Currier JS, Chipato T, Salata R, McCarthy JS, Meshnick SR. 2012. HIV-1 protease inhibitors and clinical malaria: A secondary analysis of the AIDS clinical trials group A5208 study. Antimicrobial Agents and Chemotherapy 56(2), 995–1000. https://doi.org/10.1128/AAC.05322-11
Pushpakom S, Iorio F, Eyers PA, Escott KJ, Hopper S, Wells A, Doig A, Guilliams T, Latimer J, McNamee C, Norris A, Sanseau P, Cavalla D, Pirmohamed M. 2019. Drug repurposing: Progress, challenges and recommendations. Nature Reviews Drug Discovery 18(1), 41–58. https://doi.org/10.1038/nrd.2018.168
Qidwai T. 2015. Hemoglobin degrading proteases of Plasmodium falciparum as antimalarial drug targets. Current Drug Targets 16(10), 1133–1141. https://doi.org/10.2174/1389450116666150304104123
Rivero CV, Martínez SJ, Novick P, Cueto JA, Salassa BN, Vanrell MC, Li X, Labriola CA, Polo LM, Engman DM, Clos J, Romano PS. 2021. Repurposing carvedilol as a novel inhibitor of the Trypanosoma cruzi autophagy flux that affects parasite replication and survival. Frontiers in Cellular and Infection Microbiology 11, 657257. https://doi.org/10.3389/fcimb.2021.657257
Roos K, Wu C, Damm W, Reboul M, Stevenson JM, Lu C, Dahlgren MK, Mondal S, Chen W, Wang L, Abel R, Friesner RA, Harder ED. 2019. OPLS3e: Extending force field coverage for drug-like small molecules. Journal of Chemical Theory and Computation 15(3), 1863–1874. https://doi.org/10.1021/acs.jctc.8b01026
Sabnis YA, Desai PV, Rosenthal PJ, Avery MA. 2003. Probing the structure of falcipain-3, a cysteine protease from Plasmodium falciparum : Comparative protein modeling and docking studies. Protein Science 12(3), 501–509. https://doi.org/10.1110/ps.0228103
Salam SS, Chetia P, Kardong D. 2020. In silico docking, ADMET and QSAR study of few antimalarial phytoconstituents as inhibitors of plasmepsin II of P. falciparum against malaria. Current Drug Therapy 15(3), 264–273. https://doi.org/10.2174/1574885514666190923112738
Segireddy RR, Belda H, Yang ASP, Dundas K, Knoeckel J, Galaway F, Wood L, Quinkert D, Knuepfer E, Treeck M, Wright GJ, Douglas AD. 2024. A screen for Plasmodium falciparum sporozoite surface protein binding to human hepatocyte surface receptors identifies novel host–pathogen interactions. Malaria Journal 23(1), 151. https://doi.org/10.1186/s12936-024-04913-2
Sharma PP, Kumar S, Kaushik K, Singh A, Singh IK, Grishina M, Pandey KC, Singh P, Potemkin V, Poonam, Singh G, Rathi B. 2022. In silico validation of novel inhibitors of malarial aspartyl protease, plasmepsin V, and antimalarial efficacy prediction. Journal of Biomolecular Structure and Dynamics 40(18), 8352–8364. https://doi.org/10.1080/07391102.2021.1911855
Shenai BR, Lee BJ, Alvarez-Hernandez A, Chong PY, Emal CD, Neitz RJ, Roush WR, Rosenthal PJ. 2003. Structure–activity relationships for inhibition of cysteine protease activity and development of Plasmodium falciparum by peptidyl vinyl sulfones. Antimicrobial Agents and Chemotherapy 47(1), 154–160. https://doi.org/10.1128/AAC.47.1.154-160.2003
Silva AM, Lee AY, Gulnik SV, Maier P, Collins J, Bhat TN, Collins PJ, Cachau RE, Luker KE, Gluzman IY, Francis SE, Oksman A, Goldberg DE, Erickson JW. 1996. Structure and inhibition of plasmepsin II, a hemoglobin-degrading enzyme from Plasmodium falciparum . Proceedings of the National Academy of Sciences 93(19), 10034–10039. https://doi.org/10.1073/pnas.93.19.10034
Soh BY, Song HO, Lee Y, Lee J, Kaewintajuk K, Lee B, Choi YY, Cho JH, Choi S, Park H. 2013. Identification of active Plasmodium falciparum calpain to establish screening system for Pf-calpain-based drug development. Malaria Journal 12(1), 47. https://doi.org/10.1186/1475-2875-12-47
Stead AMW, Bray PG, Edwards IG, DeKoning HP, Elford BC, Stocks PA, Ward SA. 2001. Diamidine compounds: Selective uptake and targeting in Plasmodium falciparum. Molecular Pharmacology 59(5), 1298–1306. https://doi.org/10.1124/mol.59.5.1298
Subramanian S, Hardt M, Choe Y, Niles RK, Johansen EB, Legac J, Gut J, Kerr ID, Craik CS, Rosenthal PJ. 2009. Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3. PLoS ONE 4(4), e5156. https://doi.org/10.1371/journal.pone.0005156
Summa V, Petrocchi A, Bonelli F, Crescenzi B, Donghi M, Ferrara M, Fiore F, Gardelli C, Gonzalez Paz O, Hazuda DJ, Jones P, Kinzel O, Laufer R, Monteagudo E, Muraglia E, Nizi E, Orvieto F, Pace P, Pescatore G, Rowley M. 2008. Discovery of raltegravir, a potent, selective orally bioavailable HIV-integrase inhibitor for the treatment of HIV-AIDS infection. Journal of Medicinal Chemistry 51(18), 5843–5855. https://doi.org/10.1021/jm800245z
Tehlan A, Saha A, Dhar SK. 2023. Targeting proteases and proteolytic processing of unusual N-terminal extensions of Plasmodium proteins: Parasite peculiarity. Frontiers in Drug Discovery 3, 1223140. https://doi.org/10.3389/fddsv.2023.1223140
Voß Y, Klaus S, Guizetti J, Ganter M. 2023. Plasmodium schizogony: A chronology of the parasite’s cell cycle in the blood stage. PLOS Pathogens 19(3), e1011157. https://doi.org/10.1371/journal.ppat.1011157
Wiser MF. 2024. The digestive vacuole of the malaria parasite: A specialized lysosome. Pathogens 13(3), 182. https://doi.org/10.3390/pathogens13030182
Xie SC, Metcalfe RD, Mizutani H, Puhalovich T, Hanssen E, Morton CJ, Du Y, Dogovski C, Huang SC, Ciavarri J, Hales P, Griffin RJ, Cohen LH, Chuang BC, Wittlin S, Deni I, Yeo T, Ward KE, Barry DC, Tilley L. 2021. Design of proteasome inhibitors with oral efficacy in vivo against Plasmodium falciparum and selectivity over the human proteasome. Proceedings of the National Academy of Sciences 118(39), e2107213118. https://doi.org/10.1073/pnas.2107213118
Sethupathi Virumandi, Elumalai Balamurugan, Aakash Ganesan, Sowmiya Ganesan, Srinidhi Raveenthiran, 2025. Targeting proteolytic enzymes in the hemoglobin degradation pathway to inhibit Plasmodium falciparum: An in silico approach. Int. J. Biosci., 27(5), 182-197.
Copyright © 2025 by the Authors. This article is an open access article and distributed under the terms and conditions of the Creative Commons Attribution 4.0 (CC BY 4.0) license.