Rui Sun, PhD
Icahn School of Medicine at Mount Sinai
Bump-Hole PROTACs: a Novel Chemical Genetic Strategy to Dissect Kinase Functions
Genetic sensitization to bulky adenine analog inhibitors helped define functions of cyclin-dependent kinases (CDKs) in cell division and RNA polymerase II (RNAPII) transcription. Here we develop a chemical-genetic tool by converting bulky adenine analogs into proteolysis targeting chimeras (PROTACs). We designed and optimized bump-hole (BH) PROTACs based on their ability to induce degradation of analog-sensitive (AS) mutant CDK7 in CDK7as/as HCT116 colon cancer cells. Proteomics revealed selective degradation of CDK7 and its partners, cyclin H and MAT1, in CDK7as/as cells. CDK7 has dual functions, as a CDK-activating kinase (CAK) that phosphorylates T loops of downstream CDKs, and as part of transcription factor TFIIH. Both 3-MB-PP1 and 3-MB-C7 efficiently blocked CDK T-loop phosphorylation in CDK7as/as cells. In RNA-seq analysis, there was extensive overlap between genes repressed by 3-MB-PP1 and BH-PROTAC in CDK7as/as cells, but the inhibitor affected more genes than did the degrader. We found that the incomplete repression of transcription by CDK7 degrader was due to partial degradation of CDK7 even though cellular CDK7 was depleted by >90%. Like 3-MB-PP1, 3-MB-C7 specifically impaired cell-cycle progression and viability in CDK7as/as cells. The BH-PROTACs also induced CDK2 degradation in CDK2as/as cells. Therefore, BH-PROTACs are generally applicable for dissection of kinase functions, expanding the chemical-genetic toolbox and potentially guiding drug development.
Genetic sensitization to bulky adenine analog inhibitors helped define functions of cyclin-dependent kinases (CDKs) in cell division and RNA polymerase II (RNAPII) transcription. Here we develop a chemical-genetic tool by converting bulky adenine analogs into proteolysis targeting chimeras (PROTACs). We designed and optimized bump-hole (BH) PROTACs based on their ability to induce degradation of analog-sensitive (AS) mutant CDK7 in CDK7as/as HCT116 colon cancer cells. Proteomics revealed selective degradation of CDK7 and its partners, cyclin H and MAT1, in CDK7as/as cells. CDK7 has dual functions, as a CDK-activating kinase (CAK) that phosphorylates T loops of downstream CDKs, and as part of transcription factor TFIIH. Both 3-MB-PP1 and 3-MB-C7 efficiently blocked CDK T-loop phosphorylation in CDK7as/as cells. In RNA-seq analysis, there was extensive overlap between genes repressed by 3-MB-PP1 and BH-PROTAC in CDK7as/as cells, but the inhibitor affected more genes than did the degrader. We found that the incomplete repression of transcription by CDK7 degrader was due to partial degradation of CDK7 even though cellular CDK7 was depleted by >90%. Like 3-MB-PP1, 3-MB-C7 specifically impaired cell-cycle progression and viability in CDK7as/as cells. The BH-PROTACs also induced CDK2 degradation in CDK2as/as cells. Therefore, BH-PROTACs are generally applicable for dissection of kinase functions, expanding the chemical-genetic toolbox and potentially guiding drug development.
