Farmwald, M. | bioRxiv Science

bioRxiv2025

Molecular Dynamics-Guided Design and Chemoproteomic Profiling of Covalent Kinase Activity Probes

Covalent small molecule activity probes can be powerful tools to interrogate protein function in native cellular environments. The design of family-wide activity probes requires an understanding of the molecular sources of general targeting potential and specificity to enable broad targeting of protein family members. Here, we developed and applied a multifaceted docking and molecular dynamics (MD) simulation pipeline to design and test cell-permeable covalent kinase activity probes from a set of hinge-binding pharmacophores. This computationally-guided approach yielded a new cell-active probe, K60P, which targets around 114 kinases across distinct kinase classes in live cells. Chemoproteomic profiling of this probe and a clinical candidate sharing the same indazole core, KW-2449, identified kinase and non-kinase target profiles that differ from recombinant protein assay profiles, underscoring the utility of native kinase profiling in situ. Biochemical studies with a model target kinase, ABL1, confirmed covalent labeling of the active site lysine across several kinase probes with distinct kinetics, as well as covalent labeling of key tyrosines in trans between ABL1 monomers. Finally, focused proteomics, kinetic modeling, and molecular dynamics simulations revealed that K60P, as well as the comparator probe XO44, preferentially engage with target kinases in their active, DFG-in conformations, which is driven by increasing population of reaction-ready small molecule conformation. These results together establish a computational and kinetic modeling framework for designing covalent activity probes and highlight the balance of target selectivity and kinetic efficiency as a key factor in determining their proteome-wide reactivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=155 SRC="FIGDIR/small/683178v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1ea3e5forg.highwire.dtl.DTLVardef@1a4e999org.highwire.dtl.DTLVardef@1e468c3org.highwire.dtl.DTLVardef@99e04e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Reproducible autosomal gene expression changes with loss of typical X and Y complement across tumor types

Although there are known sex differences in cancer incidence, severity, and treatment, the sex chromosomes are typically excluded from genomic analyses because of the unique technical challenges associated with assessing their copy number, sequence variation, and expression. Here we assess sex chromosome complement in three widely-used human genomics datasets from normal (non-cancerous) tissues, primary tumors, and cancer cell lines and study the effects on genome-wide gene expression. Expected sex chromosome complements based on reported patient sex were observed in non-cancerous tissues, but about half of tumors and cancer cell lines showed loss of typical sex chromosome gene expression across tissue types with three categories: loss of chromosome Y (LOY), loss of chromosome X (LOX) and reactivation of the inactive X chromosome (XaXa). Genes consistently differentially expressed in tumors with loss of chromosome X, loss of chromosome Y, or loss of X chromosome inactivation are associated with the hallmarks of cancer and include both sex-linked and autosomal genes from nearly all chromosomes, druggable genes, and genes with molecular functions relevant to cancer signaling, such as kinase activity. Strikingly, tumors that are X0, including tumors from female patients that have lost an X chromosome and tumors from male patients that have lost a Y chromosome, cluster together by gene expression profile. Patients with tumors that have LOX or LOY had poorer survival outcomes compared to those with tumors that had maintained their sex chromosome complement. Further, LOX and LOY eliminates nearly all of the differential gene expression between tumors from different patient sexes, affecting sex chromosomal and autosomal gene expression. Going forward, considering patient sex as well as the entire genome, including assessment of the sex chromosome complement, will provide additional insights into personalized tumor etiology, progression, treatment, and patient outcome. TeaserLoss of typical sex chromosome complement is present in primary tumors and cancer cell lines across tissue types. We identify consistent autosomal gene expression changes across multiple cancers when the sex chromosome complement is altered, and show that loss of sex chromosomes reduces tumor gene expression differences between patient sexes. We show that loss of sex chromosomes is also associated with poorer patient survival.

cancer biology↗