Fathi, S. | Search bioRxiv

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↗

bioRxiv2021

mTOR controls neurotoxic lysosome exocytosis in inflammatory reactive astrocytes

Astrocytes respond and contribute to neuroinflammation by adopting inflammatory reactive states. Although recent efforts have characterized the gene expression signatures associated with these reactive states, the cell biology underlying inflammatory reactive astrocyte phenotypes remains under-explored. Here, we used CRISPR-based screening in human iPSC-derived astrocytes to identify mTOR activation a driver of cytokine-induced endolysosomal system remodeling, manifesting as alkalinization of endolysosomal compartments, decreased autophagic flux, and increased exocytosis of certain endolysosomal cargos. Through endolysosomal proteomics, we identified and focused on one such cargo - IL-32, a disease-associated pro-inflammatory cytokine not present in rodents, whose secretion mechanism is not well understood. We found that IL-32 was partially secreted in extracellular vesicles likely to be exosomes. Furthermore, we found that IL-32 was involved in the polarization of inflammatory reactive astrocyte states, was upregulated in astrocytes in multiple sclerosis lesions, and preferentially co-localized with astrocytes in hypoxic-ischemic encephalopathy. We believe that our results advance our understanding of cell biological pathways underlying inflammatory reactive astrocyte phenotypes and identify potential therapeutic targets.

cell biology↗