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[Preprint]. 2025 Jan 10:2025.01.10.632200.
doi: 10.1101/2025.01.10.632200.

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

David J Shirley  1 , Meganathan Nandakumar  1 , Aurora Cabrera  2   3 , Bonnie Yiu  4 , Emily Puumala  4 , Zhongle Liu  4 , Nicole Robbins  4 , Luke Whitesell  4 , Jeffrey L Smith  1 , Scott P Lyons  2   3 , Angie L Mordant  2   3 , Laura E Herring  2 , Lee M Graves  2   3 , Rafael M Couñago  1   5 , David H Drewry  1   3 , Leah E Cowen  4 , Timothy M Willson  1
Affiliations

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

David J Shirley et al. bioRxiv. .

Update in

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 p38α (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.

Keywords: antifungal; chemoproteomics; in-cell target engagement assay; kinase inhibitor; multiplexed inhibitor beads (MIBS); multiplexed inhibitor beads analyzed by tandem mass-spectrometry (MIB/MS).

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Figures

Figure 1.
Figure 1.. MIB/MS captures the majority of protein kinases in C. albicans.
A. 6,039 proteins are predicted by UniProtKB to be encoded in the genome of C.albicans. 3,928 proteins were detected by global proteomic analysis. 1,211 of these proteins were captured by the MIB matrix of which 152 were annotated a potential kinase. 89 of these potential kinases were identified by Goswami et al., while 83 were captured by MIB/MS. B. Venn diagram displaying overlap between the Goswami annotation, MIB/MS experiments, and global proteomic analysis. C. Functional classification of the 1,211 fungal proteins captured by MIB/MS. D. The fungal protein kinome displayed by family. Kinases captured by MIB are shown by green dots on the sunburst plot. Open circles indicate kinases not captured by MIB.
Figure 2.
Figure 2.. GW, YK, and MN demonstrate high selectivity across the fungal kinome.
Fold change of fungal protein kinase targets captured in MIB/MS competition assays for each compound compared to DMSO. Assays were performed in triplicate.
Figure 3.
Figure 3.. MN-kinobead pulldown experiment.
A. Synthetic scheme for the MN-kinobead. B. Western blot for p38α following pull down from HEK293 cell lysate. C. C. albicans proteins competed by MN (10 μM) following MN-kinobead pull down from C. albicans cell lysate.
Figure 4.
Figure 4.. YK and MN selectivity across the human kinome.
A. Number of human kinases assayed by MIB/MS in blue (269) and NanoBRET (192) in green. A total of 355 human kinases are assayed by the two techniques combined. B. Human kinases showing significant dose dependent competition by MIB/MS for either YK or MN. C. Target engagement of human kinases expressed as percent occupancy using NanoBRET by 1.0 μM of YK or MN.
Figure 5.
Figure 5.. Repurposing of CK1 as potential Yck2 inhibitors.
A. Flowchart of compound triage for candidate Yck2 inhibitors. B. Source of CK1 inhibitor screening data. C. CK1 inhibitor chemotypes
Figure 6.
Figure 6.. Yck2 NanoBRET assay.
A. Synthesis of the MN-Bodipy tracer. B. Tracer titration of MN-Bodipy on NLuc-Yck2.
Figure 7.
Figure 7.. MIB/MS competition assay with LY and GSK.
A. MIB/MS profile of LY. B. MIB/MS profile of GSK.
Figure 8.
Figure 8.. C. Albicans antifungal activity of LY. A.
Wild-type CaSS1. B. tetO-YCK2/yck2Δ. C. cdr1Δ/Δ, cdr2Δ/Δ, mdr1Δ/Δ, flu1Δ/Δ. D. tetO-ERG6/erg6Δ. DOX = doxycyclin (5 or 10 μg/mL).
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