TY - JOUR
T1 - Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity
AU - Vanaerschot, Manu
AU - Murithi, James M.
AU - Pasaje, Charisse Flerida A.
AU - Ghidelli-Disse, Sonja
AU - Dwomoh, Louis
AU - Bird, Megan
AU - Spottiswoode, Natasha
AU - Mittal, Nimisha
AU - Arendse, Lauren B.
AU - Owen, Edward S.
AU - Wicht, Kathryn J.
AU - Siciliano, Giulia
AU - Bösche, Markus
AU - Yeo, Tomas
AU - Kumar, T. R. Santha
AU - Mok, Sachel
AU - Carpenter, Emma F.
AU - Giddins, Marla J.
AU - Sanz, Olalla
AU - Ottilie, Sabine
AU - Alano, Pietro
AU - Chibale, Kelly
AU - Llinás, Manuel
AU - Uhlemann, Anne Catrin
AU - Delves, Michael
AU - Tobin, Andrew B.
AU - Doerig, Christian
AU - Winzeler, Elizabeth A.
AU - Lee, Marcus C.S.
AU - Niles, Jacquin C.
AU - Fidock, David A.
N1 - Funding Information:
This work was supported by the Bill and Melinda Gates Foundation that funds the Malarial Drug Accelerator (MalDA) consortium (OPP1054480 to E.A.W. D.A.F. M.C.S.L. and M.L. and OPP1162467 to J.C.N.), the Medicines for Malaria Venture (08/0015 to D.A.F.), the NIH (R01 AI090141 to E.A.W.) the Italian Ministries of Health and Foreign Affairs (ISARP2018-ISS to P.A.), and the LSHTM/Wellcome ISSF (to M.D.). K.C. gratefully acknowledges the support of the University of Cape Town, the South African Medical Research Council, the South African Research Chairs Initiative of the Department of Science and Innovation administered through the South African National Research Foundation, and the Global Challenges Research Fund (Synchrotron Techniques for African Research and Technology project). S.M. was supported by a Human Frontier Science Program long-term fellowship award. We sincerely thank the team at Cellzome for the chemoproteomic experiments, Drs. Louis Miller and Karthigayan Gunalan at the NIH for providing the CDPK1T145M NF54 mutant line, and Dr. David Baker at the London School of Hygiene & Tropical Medicine, UK for providing the PfPKG expression construct. MMV030084 (TCMDC-140369) and MMV030734 (TCMDC-141019) were kindly provided by MMV, and the other TCMDC analogs (Figure S1A) were kindly provided by GlaxoSmithKline, Tres Cantos, Spain, through the MalDA consortium. M.V. and J.M.M. performed ABS specificity assays and exposure studies. N.M. S.O. and E.A.W. performed liver stage assays. G.S. P.A. S.O. and E.A.W. assessed gametocytocidal activity of compounds. M.D. performed the dual gamete formation assay. E.S.O. and M.L. performed metabolomics experiments. L.D. and A.B.T. performed phosphoproteomic studies. S.G.-D. supervised the chemoproteomic studies that were performed at Cellzome with help from S.O. and B.M. C.F.A.P. and J.C.N. generated and phenotyped the cKD lines. K.J.W. performed PKG docking studies. L.B.A. and K.C. performed recombinant PfPKG kinase inhibition studies. M.V. J.M.M. and K.T.S. performed resistance selections. T.Y. S.M. M.J.G. and A.-C.U. performed whole-genome sequencing of these lines. M.C.S.L. and E.F.C. designed and produced CRISPR-Cas9 plasmids. M.V. J.M.M. N.S. and E.F.C. performed CRISPR-Cas9 editing experiments to validate resistance mediators. M.B. and C.D. performed recombinant TKL3 studies. M.V. compiled all the data and wrote the manuscript with J.M.M. and D.A.F. with input from other authors. All authors approved the final manuscript. The authors declare no competing interests.
Funding Information:
This work was supported by the Bill and Melinda Gates Foundation that funds the Malarial Drug Accelerator (MalDA) consortium ( OPP1054480 to E.A.W., D.A.F., M.C.S.L., and M.L., and OPP1162467 to J.C.N.), the Medicines for Malaria Venture (08/0015 to D.A.F.), the NIH ( R01 AI090141 to E.A.W.) the Italian Ministries of Health and Foreign Affairs ( ISARP2018-ISS to P.A.), and the LSHTM /Wellcome ISSF (to M.D.). K.C. gratefully acknowledges the support of the University of Cape Town, the South African Medical Research Council , the South African Research Chairs Initiative of the Department of Science and Innovation administered through the South African National Research Foundation , and the Global Challenges Research Fund (Synchrotron Techniques for African Research and Technology project). S.M. was supported by a Human Frontier Science Program long-term fellowship award. We sincerely thank the team at Cellzome for the chemoproteomic experiments, Drs. Louis Miller and Karthigayan Gunalan at the NIH for providing the CDPK1 T145M NF54 mutant line, and Dr. David Baker at the London School of Hygiene & Tropical Medicine , UK for providing the PfPKG expression construct. MMV030084 (TCMDC-140369) and MMV030734 (TCMDC-141019) were kindly provided by MMV, and the other TCMDC analogs ( Figure S1 A) were kindly provided by GlaxoSmithKline, Tres Cantos, Spain, through the MalDA consortium.
Publisher Copyright:
© 2020 The Authors
PY - 2020/7/16
Y1 - 2020/7/16
N2 - The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.
AB - The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.
KW - cGMP-dependent protein kinase (PKG)
KW - chemoproteomics
KW - conditional knockdown
KW - kinase
KW - malaria drug discovery
KW - phosphoproteomics
KW - Plasmodium falciparum
KW - resistance
KW - target identification
UR - http://www.scopus.com/inward/record.url?scp=85087768957&partnerID=8YFLogxK
U2 - 10.1016/j.chembiol.2020.04.001
DO - 10.1016/j.chembiol.2020.04.001
M3 - Article
C2 - 32359426
AN - SCOPUS:85087768957
SN - 2451-9456
VL - 27
SP - 806
EP - 816
JO - Cell Chemical Biology
JF - Cell Chemical Biology
IS - 7
ER -
