YKL-5-124

The growth rate assay comparing WT cells to CDK7-C312S expressing cells is quite convincing at 100 nM. This data increases confidence in on-target selectivity towards CDK7 and usage at this concentration in cells. One note is that, because of this, cells do need to have the WT C312 for reliable evaluation.

This covalent inhibitor exhibits low nanomolar potency with a kinact/KI = 1.03*10^5 M-1s-1. Selectivity against the related CDKs 2/9/12/13 was demonstrated through kinase assays. A chemoproteomic Kinativ screen (~180 kinases covered) conducted at a 1 µM concentration suggests high selectivity in the kinome, but no larger in vitro kinase panel screen or chemoproteomic analysis in live cells was performed in the original publication. Covalent target engagement and labeling site were confirmed by mass spectrometry and C312S mutation. Dependence of cellular effects on CDK7 was confirmed with C312S mutant cell lines. Cellular target engagement below 100 nM was demonstrated by pulldown assays. Quantification of target engagement in intact cells at the suggested concentration of up to 100 nM (e.g., by a NanoBRET assay) would have been desirable. As a covalent inhibitor, potency of this compound increases over time. Off-target activity may also increase over time. It is recommended to use the compound in conjunction with the non-reactive close analog YKL-5-167 as a negative control. Combined use with an orthogonal probe is recommended.

YKL-5-124 is 9.7 nM biochemical inhibitor of CDK7 (in the CAK complex) that labels C312 covalently, derived from a combination of PAK inhibitor scaffold (PF-3758309) and a covalent compound, THZ1. Selectivity vs isolated kinase compared to CDK12 or CDK13 was 100x. NanoBRET data could have been run to add more information about selectivity in a cellular context. Mutagenesis studies in cells showed that the CDK7 C213S mutant did not respond to YKL-5-124, indicating that covalent reactivity is critical for inhibition. Kinome profiling against a set of ~200 kinases showed CDK7 as the strongest engaged target, with several other kinases also being flagged. An orthogonal larger kinome panel would add additional information about broader kinome selectivity. Given the presence of the reactive acrylamide Michael acceptor, global proteomics studies could have been carried out to determine other targets that YKL-5-124 may modify. Cysteine or glutathione stability assays are another way to measure general reactivity, especially given the aniline nature of the acrylamide, which are known to have increased reactivity compared to their alkyl variants.