Janssen, A. P. A. | bioRxiv Science

bioRxiv2024

CellEKT: A robust chemical proteomics workflow to profile cellular target engagement of kinase inhibitors

The human genome encodes 518 protein kinases that are pivotal for drug discovery in various therapeutic areas such as cancer and autoimmune disorders. The majority of kinase inhibitors target the conserved ATP-binding pocket, making it difficult to develop selective inhibitors. To characterize and prioritize kinase-inhibiting drug candidates, efficient methods are desired to determine target engagement across the cellular kinome. In this study, we present CellEKT (Cellular Endogenous Kinase Targeting), an optimized and robust chemical proteomics platform for investigating cellular target engagement of endogenously expressed kinases using the sulfonyl fluoride-based probe XO44 and two new probes ALX005 and ALX011. The optimized workflow enabled the determination of the kinome interaction landscape of covalent and non-covalent drugs across over 300 kinases, expressed as half maximum inhibitory concentration (IC50), which were validated using distinct platforms like phosphoproteomics and NanoBRET. With CellEKT, target engagement profiles were linked to their substrate space. CellEKT has the ability to decrypt drug actions and to guide the discovery and development of drugs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/616061v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@5dd891org.highwire.dtl.DTLVardef@1353379org.highwire.dtl.DTLVardef@1c67382org.highwire.dtl.DTLVardef@1c964eb_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

High-throughput competitive binding assay for targeting RNA with small molecules: discovery of new PreQ1 riboswitch ligands

In the evolving landscape of RNA targeting, there is an indisputable need for new screening methodologies to find small molecules targeting relevant tertiary RNA structures, like viral pseudoknots or bacterial riboswitches. Here, we developed a competitive binding high-throughput screening assay to identify ligands for the bacterial PreQ1-I riboswitch. In this assay, ligands compete with a rationally designed quencherlabeled antisense for binding to the riboswitch. The method is validated for the Fusobacterium nucleatum (Fnu), Thermoanaerobacter tengcongensis (Tte), Bacillus subtilis (Bsu) and Enterococcus faecalis (Efa) PreQ1 riboswitches, using the natural riboswitch ligand PreQ1 and various analogues. A commercial RNA-focused library consisting of [~]15,000 compounds was then screened against the Fnu riboswitch, leading to the identification of 4 hits exhibiting competitive binding activity. These hits were evaluated in in vitro translation assays against several PreQ1 riboswitches. The most promising hit 4494 showed competitive binding activity to the Fnu, Tte, Bsu and Efa riboswitches, and was able to inhibit translation of a Tte riboswitch-regulated reporter gene, making it an interesting starting point for the development of new antibiotics. In essence, this HTS assay has the potential to discover highly sought-after RNA targeting small molecules for complex and clinically relevant RNA structures. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/595132v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@d7af01org.highwire.dtl.DTLVardef@227e93org.highwire.dtl.DTLVardef@a3f24corg.highwire.dtl.DTLVardef@17883e9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗