Slater, M. R. | bioRxiv Science

bioRxiv2025

Cellular Selectivity Analyses Reveal Distinguishing Profiles for Type II Kinase Inhibitors

A pivotal part of kinase chemical probe and drug development is assessment of the selectivity of a putative lead compound. While there is no consensus around the panel size or the type of assay(s) that are most appropriate, there is concurrence that gauging the number of on- and off-targets of a kinase inhibitor is essential. As pharmacology takes place in cells, we have compared profiling results for ten kinase inhibitors generated using the cell-free assays to those obtained when a panel of cellular target engagement NanoBRET assays is used to assess selectivity in intact cells. This is the first systematic comparison of these two approaches across a broad kinase panel. Comparison of the data sets demonstrates divergent results that can influence chemical probe prioritization. We identify unanticipated kinase interactions in cells for type II kinase inhibitors that are not observed in biochemical, cell-free systems. Furthermore, we characterize TPKI-39 as a DDR1, DDR2, and FLT1 chemical probe based on its in-cell selectivity profile. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/681452v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@15abd7borg.highwire.dtl.DTLVardef@4dfbdcorg.highwire.dtl.DTLVardef@1d25bf6org.highwire.dtl.DTLVardef@1939228_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Simultaneous inhibition of DNA-PK and Pol{Theta} improves integration efficiency and precision of genome editing

Genome editing tools, especially CRISPR/Cas9-based strategies, have transformed biomedical research and opened opportunities for developing curative treatments for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of undesired mutations represent major limitations for genome editing applications. Both issues arise from the interplay between the main DNA Double-Strand Break (DSB) repair pathways, Homology-Directed Repair (HDR), Non-Homologous End Joining (NHEJ), and Microhomology-Mediated End Joining (MMEJ). To improve efficiencies of targeted CRISPR-Cas9 genome editing, we screened a large compound library. This led to the discovery of AZD7648, a DNA-dependent protein kinase (DNA-PK) inhibitor and potent enhancer of CRISPR-Cas9-mediated integration. We demonstrated that AZD7648 increased HDR and decreased mutagenic NHEJ repair, thus resulting in improved performance of precise gene editing. Furthermore, we observed additional improvement of integration efficiency by impairing MMEJ repair through DNA polymerase {ominus} (Pol{ominus}) inhibition. Combined treatment with AZD7648 and Pol{ominus} inhibitors (which we named 2iHDR) substantially increased precision of templated insertions, with efficiencies of up to 80%, and nearly no formation of undesired Insertion-Deletions (InDels). Importantly, 2iHDR also decreased Cas9-associated off-target activity, dramatically improving the performance and fidelity of CRISPR-Cas9 gene editing.

molecular biology↗