Activity-Based Chemoproteomic Profiling Reveals the Active Kinome of Leishmania

Exequiel O. J. Porta^1*Karunakaran Kalesh^2,3*Patrick G. Steel^4*

• ¹University College London, London, United Kingdom
• ²National Horizons Centre, Darlington, United Kingdom
• ³Teesside University School of Health and Life Sciences, Middlesbrough, United Kingdom
• ⁴Durham University Department of Chemistry, Durham, United Kingdom

Background: Leishmania parasites cause neglected tropical diseases such as cutaneous and visceral leishmaniasis, which have limited treatment options and rising drug resistance. Protein kinases are pivotal in Leishmania biology and attractive drug targets, but their functional status in the parasite remains largely unexplored. Methods: We applied activity-based protein profiling (ABPP) with custom in-house cell-permeable ATP-site directed probes to map the ligandable, "active kinome" of Leishmania mexicana. Three related covalent probes featuring an ATP-mimetic scaffold, electrophilic warhead (targeting catalytic lysines or cysteines), and alkyne tag were synthesised to broadly capture active kinases. Live parasites were labelled with probes, followed by click-chemistry tagging, in-gel fluorescence, and tandem mass tag (TMT) proteomics for kinase identification and quantification. Comparative profiling was performed across Leishmania species and life stages. Key findings were validated by competition experiments with ibrutinib and parasite viability assays. Results: We uncovered 16 metabolic kinases and 32 protein kinases spanning all major kinase families (CMGC, AGC, STE, CAMK, CK1, and NEK), including 9 protein kinase enzymes encoded by essential genes and several kinases lacking human orthologs. Notable hits included CRK1, MPK4, CK1.2, and an atypical kinase, underscoring their potential as drug targets. Conclusion: This study provides the first ABPP survey of the Leishmania kinome, revealing multiple ligandable, active kinases that drive parasite survival and virulence. Our chemoproteomic approach highlights both essential protein kinases and unique metabolic kinases as a rich source of potential drug targets. These findings demonstrate that ABPP can unveil the biochemically active kinases in Leishmania, offering a new strategy for prioritizing kinase targets and accelerating kinase inhibitor development against leishmaniasis. The work lays a foundation for next-generation antileishmanial therapies directed at the parasite kinome, particularly those kinases indispensable for the parasite yet sufficiently divergent from the human host.