Mechanistic Insights into a CDK9 Inhibitor Via Orthogonal Proteomics Methods

Cyclin-dependent-kinases
(CDKs) are members of the serine/threonine
kinase family and are highly regulated by cyclins, a family of regulatory
subunits that bind to CDKs. CDK9 represents one of the most studied
examples of these transcriptional CDKs. CDK9 forms a heterodimeric
complex with its regulatory subunit cyclins T1, T2 and K to form the
positive transcription elongation factor b (P-TEFb). This complex
regulates transcription via the phosphorylation of RNA polymerase
II (RNAPolII) on Ser-2, facilitating promoter clearance and transcription
elongation and thus remains an attractive therapeutic target. Herein,
we have utilized classical affinity purification chemical proteomics,
kinobeads assay, compressed CEllular Thermal Shift Assay (CETSA)-MS
and Limited Proteolysis (LiP) to study the selectivity, target engagement
and downstream mechanistic insights of a CDK9 tool compound. The
above experiments highlight the value of quantitative mass spectrometry
approaches to drug discovery, specifically proteome wide target identification
and selectivity profiling. The approaches utilized in this study unanimously
indicated that the CDK family of kinases are the main target of the
compound of interest, with CDK9, showing the highest target affinity
with remarkable consistency across approaches. We aim to provide guidance
to the scientific community on the available chemical biology/proteomic
tools to study advanced lead molecules and to highlight pros and cons
of each technology while describing our findings in the context of
the CDKs biology.