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The identification of bioactive small molecules is at the heart of chemical biology and medicinal research. The screening for modulators of disease-relevant targets and phenotypes is the first step on the way to new drugs. Therefore, large compound libraries have been synthesized and employed by academia and, particularly, pharmaceutical companies to meet the need for chemical entities that are as diverse as possible. Extensive screening of these compound libraries revealed a portion of small molecules that is inactive in more than 100 different assays and was therefore termed dark chemical matter (DCM). Deorphanization of DCM promises to yield very selective compounds as they, by definition, should have less off-target effects. We employed morphological profiling using the Cell painting assay (CPA) to detect bioactive DCM compounds. CPA is not biased to a given target or phenotype and can detect various unrelated mechanisms and modes of action. Within the DCM collection, we identified bioactive compounds and confirmed several modulators of microtubules, DNA synthesis and pyrimidine biosynthesis. Profiling approaches are therefore powerful tools to probe compound collections for bioactivity in an unbiased manner and particularly suitable for deorphanization of DCM.

Mutations within Ras proteins represent major drivers in human cancer. In this study, we report the structure-based design, synthesis, as well as biochemical and cellular evaluation of nucleotide-based covalent inhibitors for KRasG13C, an important oncogenic mutant of Ras that has not been successfully addressed in the past. Mass spectrometry experiments and kinetic studies reveal promising molecular properties of these covalent inhibitors, and X-ray crystallographic analysis has yielded the first reported crystal structures of KRasG13C covalently locked with these GDP analogues. Importantly, KRasG13C covalently modified with these inhibitors can no longer undergo SOS-catalysed nucleotide exchange. As a final proof-of-concept, we show that in contrast to KRasG13C, the covalently locked protein is unable to induce oncogenic signalling in cells, further highlighting the possibility of using nucleotide-based inhibitors with covalent warheads in KRasG13C-driven cancer., eLife;Vol. 12 2023, Artikel-ID: e82184