Shirley, D. J. | Search bioRxiv

bioRxiv2025

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

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

microbiology↗

bioRxiv2024

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

microbiology↗