Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target

Alam, M. M. et al. (2019) Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target. Science, 365(6456), eaau1682. (doi:10.1126/science.aau1682)

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Abstract

Introduction: Despite the positive effects of intervention strategies that include insecticide-impregnated bed nets and artemisinin-based drug therapies, malaria still kills nearly 500,000 people per year and infects more than 200 million individuals globally. This, together with the emerging resistance of the parasite to frontline antimalarials, means that there is an urgent need for novel treatments that not only offer a cure for malaria but also prevent transmission. We show that by inhibiting an essential protein kinase that is a key regulator of RNA processing, we are able to kill the parasite in the blood and liver stages as well as prevent the development of the sexual-stage gametocytes, thereby blocking transmission to the mosquito. Rationale: Our group has previously published a list of 36 protein kinases that are essential for blood-stage survival of the most virulent form of the human malaria parasite, Plasmodium falciparum. Here, we focused on one of these protein kinases from the P. falciparum CLK (cyclin-dependent–like kinase) family, PfCLK3, and reasoned that inhibition of this protein kinase by a small drug-like molecule would be effective at killing blood-stage parasites. We further hypothesized that because PfCLK3 plays a key role in RNA splicing, inhibition of this kinase would be effective at killing the parasite at all stages of the life cycle where RNA splicing is required. This would include blood, liver, and sexual stages. Results: By screening a focused library of nearly 30,000 compounds, we identified a probe molecule that selectively inhibited PfCLK3 and killed blood-stage P. falciparum. Using a combination of evolved resistance and chemogenetics, we established that our probe molecule had parasiticidal activity by inhibition of PfCLK3. We further showed that inhibition of PfCLK3 in parasites resulted in a reduction in more than 400 gene transcripts known to be essential for parasite survival. The finding that the vast majority of the genes down-regulated by PfCLK3 inhibition contained introns supported the notion that inhibition of PfCLK3 killed the malaria parasite by preventing the splicing of essential parasite genes. Because there is a high degree of homology between orthologs of CLK3 in other Plasmodium species, it might be expected that our probe molecule would both inhibit CLK3 contained in other malaria parasite species and have effective parasiticidal activity in these parasites. This was indeed found to be the case, with our molecule showing potent inhibition of CLK3 from P. vivax and P. berghei as well as killing the blood stages of P. berghei and P. knowlesi. Furthermore, we demonstrated that CLK3 inhibition also kills liver-stage P. berghei parasites and prevents P. berghei infection in mice. Finally, we showed that inhibition of PfCLK3 prevents the development of P. falciparum gametocytes, thereby blocking the infection of mosquitoes. Conclusion: We found that inhibition of the essential malaria protein kinase CLK3 can kill multiple species of malaria parasites at the blood stage as well as killing liver-stage parasites and blocking transmission of the parasite to mosquitoes by preventing gametocyte development. In this way, we validate Plasmodium spp. CLK3 as a target that can offer prophylactic, curative, and transmission-blocking potential.

Item Type:Articles
Additional Information:Supported by an MRC Toxicology Unit program grant (A.B.T., M.M.A.), MRC Developmental Gap Fund (A.S.-A.), Lord Kelvin Adam Smith Fellowship (M.M.A.), GSK Open Lab Foundation Award (A.S.-A.), joint MRC Toxicology Unit and MRC Unit the Gambia PhD program (O.J.), and Daphne Jackson Fellowship (D.M.). A.P.W., M.M., M.M.A., K.C., N.V.S., and S.B.M. are supported by Wellcome Centre for Integrative Parasitology Core support award WT104111AIA. E.A.W. is supported by grants from the NIH (5R01AI090141 and R01AI103058) and by grants from the Bill & Melinda Gates Foundation (OPP1086217, OPP1141300) as well as by Medicines for Malaria Venture (MMV). Drug WR99210 for selection of transgenic parasites was a gift from Jacobus Pharmaceuticals. M.M. is supported through WT award 172862-01 and a Wolfson Merit award from the Royal Society. The Structural Genomics Consortium (SGC) is a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, the Canada Foundation for Innovation, the Eshelman Institute for Innovation, Genome Canada, the Innovative Medicines Initiative (European Union [EU]/European Federation of Pharmaceutical Industries and Associations [EFPIA]) (ULTRA-DD grant no. 115766), Janssen, Merck & Company, Merck KGaA, Novartis Pharma AG, the Ontario Ministry of Economic Development and Innovation, Pfizer, the São Paulo Research Foundation (FAPESP number 2013/50724-5), Takeda, and the Wellcome Trust (106169/ZZ14/Z). E.F.A. was supported by the Tres Cantos Lab Foundation (grant TC125). A.B.C. was supported by a Scottish Funding Council Global Challenges Research Fund award to L.C.R.-C
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Janha, Dr Omar and Millar, Mr Scott and Charpenay, Miss Aude and Crouch, Dr Kathryn and Mahindra, Dr Amit and Mapesa, Mr Kopano and Ranford-Cartwright, Dr Lisa and Simwela, Nelson and Waters, Professor Andy and Jamieson, Dr Andrew and Tobin, Andrew and Marti, Professor Matthias and Sanchez Azqueta, Dr Rer Nat Ana and Alam, Dr Mahmood and Brancucci, Dr Nicolas
Authors: Alam, M. M., Sanchez-Azqueta, A., Janha, O., Flannery, E. L., Mahindra, A., Mapesa, K., Charpenay, A., Sriranganadane, D., Brancucci, N. M.B., Antonova-Koch, Y., Crouch, K., Simwela, N. V., Millar, S., Akinwale, J., Mitcheson, D., Solyakov, L., Dudek, K., Jones, C., Zapatero, C., Doerig, C., Nwakanma, D. C., Vázquez, M. J., Colmenarejo, G., Lafuente-Monasterio, M. J., Leon, M. L., Godoi, P. H. C., Elkins, J. M., Waters, A. P., Jamieson, A. G., Álvaro, E. F., Ranford-Cartwright, L., Marti, M., Winzeler, E. A., Gamo, F. J., and Tobin, A. B.
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
College of Medical Veterinary and Life Sciences > Institute of Infection Immunity and Inflammation
College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
College of Science and Engineering > School of Chemistry
Journal Name:Science
Publisher:American Association for the Advancement of Science
ISSN:0036-8075
ISSN (Online):1095-9203
Copyright Holders:Copyright © 2019 The Authors
First Published:First published in Science 365(6456):eaau1682
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
371799The Wellcome Centre for Molecular Parasitology ( Core Support )Andrew WatersWellcome Trust (WELLCOTR)104111/Z/14/Z & AIII - PARASITOLOGY