Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors
Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors
ABSTRACT The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei the proteins that make up the kinetochore are highly divergent, with the inner kinetochore comprising at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2 and KKT3. We performed a phenotypic screen of T. brucei bloodstream forms with a Novartis kinase-focused inhibitor library, which identified a number of selective inhibitors with potent pan-kinetoplastid activity. Deconvolution of an amidobenzimidazole series using a selection of 37 T. brucei mutants that over-express known essential protein kinases identified CLK1 as the primary target. Biochemical studies show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with C215 in the ATP binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. We show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 to be essential for KKT2 function and for kinetochore assembly. We propose that CLK1 is part of a novel signalling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. This work highlights a novel drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.
Jiricek Jan、Mottram Jeremy C.、Saldivia Manuel、Fang Eric、Myburgh Elmarie、Ritchie Ryan、Patra Debjani、Paape Daniel、Bower-Lepts Christopher、McCulloch Richard、Kaiser Marcel、Lakhsminarayana Suresh B、Chen Yen Liang、Koh Hazel X. Y.、Supek Frantisek、Rao Srinivasa P.S.、Leake Mark C.、Barrett Michael P.、Diagana Thierry T.、Brown Elaine、Williams Sarah、Wollman Adam J. M.
Novartis Institute for Tropical DiseasesYork Biomedical Research Institute, Department of Biology, University of YorkYork Biomedical Research Institute, Department of Biology, University of YorkYork Biomedical Research Institute, Hull York Medical School, University of YorkInstitute of Infection, Immunity and Inflammation, University of GlasgowNovartis Institute for Tropical DiseasesInstitute of Infection, Immunity and Inflammation, University of GlasgowYork Biomedical Research Institute, Department of Biology, University of YorkInstitute of Infection, Immunity and Inflammation, University of GlasgowSwiss Tropical and Public Health Institute||University of BaselNovartis Institute for Tropical DiseasesNovartis Institute for Tropical DiseasesNovartis Institute for Tropical DiseasesNovartis Institute for Tropical DiseasesNovartis Institute for Tropical DiseasesYork Biomedical Research Institute, Department of Biology, University of York||Department of Physics, University of YorkInstitute of Infection, Immunity and Inflammation, University of GlasgowNovartis Institute for Tropical DiseasesYork Biomedical Research Institute, Department of Biology, University of YorkNovartis Institute for Tropical DiseasesYork Biomedical Research Institute, Department of Biology, University of York||Department of Physics, University of York
基础医学分子生物学微生物学
Jiricek Jan,Mottram Jeremy C.,Saldivia Manuel,Fang Eric,Myburgh Elmarie,Ritchie Ryan,Patra Debjani,Paape Daniel,Bower-Lepts Christopher,McCulloch Richard,Kaiser Marcel,Lakhsminarayana Suresh B,Chen Yen Liang,Koh Hazel X. Y.,Supek Frantisek,Rao Srinivasa P.S.,Leake Mark C.,Barrett Michael P.,Diagana Thierry T.,Brown Elaine,Williams Sarah,Wollman Adam J. M..Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors[EB/OL].(2025-03-28)[2025-05-07].https://www.biorxiv.org/content/10.1101/616417.点此复制
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