Chemoproteomic Profiling of C. albicans for Characterization of Anti-fungal Kinase Inhibitors

Shirley, D. J.; Nandakumar, M.; Cabrera, A.; Yiu, B.; Puumala, E.; Liu, Z.; Robbins, N.; Whitesell, L.; Smith, J. L.; Lyons, S. P.; Mordant, A. L.; Herring, L. E.; Graves, L. M.; Counago, R. L. M.; Drewry, D. H.; Cowen, L. E.; Willson, T. M.

doi:10.1101/2025.01.10.632200

bioRxiv · 10.1101/2025.01.10.632200

Chemoproteomic Profiling of C. albicans for Characterization of Anti-fungal Kinase Inhibitors

Shirley, D. J.·Nandakumar, M.·Cabrera, A.·Yiu, B.·Puumala, E.·Liu, Z.·Robbins, N.·Whitesell, L.·Smith, J. L.·Lyons, S. P.·Mordant, A. L.·Herring, L. E.·Graves, L. M.·Counago, R. L. M.·Drewry, D. H.·Cowen, L. E.·Willson, T. M.

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Abstract

Candida albicans is a growing health concern as the leading causal agent of systemic candidiasis, a life-threatening fungal infection with a mortality rate of [~]40% despite best available therapy. Yck2, a fungal casein kinase 1 (CK1) family member, is the cellular target of inhibitors YK-I-02 (YK) and MN-I-157 (MN). Here, multiplexed inhibitor beads paired with mass spectrometry (MIB/MS) employing ATP-competitive kinase inhibitors were used to define the selectivity of these Yck2 inhibitors across the global C. albicans proteome. The MIB matrix captured 89% of the known and predicted C. albicans protein kinases present in cell lysate. In MIB/MS competition assays, YK and MN demonstrated exquisite selectivity across the C. albicans fungal kinome with target engagement of only three CK1 homologs (Yck2, Yck22, and Hrr25) and a homolog of human p38 (Hog1). Additional chemoproteomics using a custom MN-kinobead identified only one additional C. albicans protein, confirming its remarkable fungal proteome-wide selectivity. To identify new Yck2 inhibitors with selectivity over Hog1, thirteen human CK1 kinase inhibitors were profiled for fungal kinase-binding activity using MIB/MS competition assays and in-cell NanoBRET target engagement assays. A new chemotype of family-selective Yck2 inhibitors with antifungal activity was identified. Together, these findings expand the application of MIB/MS proteomic profiling for non-human kinomes and demonstrate its utility in the discovery and development of selective inhibitors of fungal kinases with potential antimicrobial activity. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/632200v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1606f86org.highwire.dtl.DTLVardef@39c92borg.highwire.dtl.DTLVardef@1c666cborg.highwire.dtl.DTLVardef@869b5c_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Shirley, D. J., Nandakumar, M., Cabrera, A., Yiu, B., Puumala, E., Liu, Z., Robbins, N., Whitesell, L., Smith, J. L., Lyons, S. P., Mordant, A. L., Herring, L. E., Graves, L. M., Counago, R. L. M., Drewry, D. H., Cowen, L. E., Willson, T. M.. 2025-01-11. Chemoproteomic Profiling of C. albicans for Characterization of Anti-fungal Kinase Inhibitors. https://doi.org/10.1101/2025.01.10.632200

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