Scientific paper - Ressources

Structure based design of small macrocyclic CDK9 degraders as chemical biology tools and beyond

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Nerina Dodic | Nicolas Ancellin | Morgane Bordessoules | Anne Bouillot | Nathan Butin | Cédric Charrier | Marie-Hélène Fouchet | Alain Laroze | Anne-Pascale Luzy | Alexandre Moquette | Julia Pilot | Guillaume Serin | Jean-François Mirjolet | Fabrice Viviani | Christophe Parsy


Small molecule macrocyclic kinase inhibitors have attracted significant attention in drug discovery over the past years with drugs approval such as lorlatinib demonstrating the clinical relevance of this approach. We developed our expertise to further optimize those cyclic molecules. Having low molecular weight, they favorably alter the biological and physiochemical properties as well as selectivity, as compared to their linear parent, yielding high-quality drug candidates. While focused on kinase inhibitors, macrocyclic derivatives could be potentially turned into bifunctional protein degrader molecules useful for selective cellular knockdown of targeted proteins and investigation of the pharmacological effects. With macrocyclic “probes” from our proprietary library in nanomolar range IC50, we turned our attention to CDK9 inhibitors with good selectivity profile against CDK1/2/5/7.

Previous CDK9 degraders based on acyclic inhibitors have used thalidomide as recruiter of the CRL4CRBN, resulting in successful ubiquitination and proteasomal degradation of the protein of interest. Applying a three-step approach, we first defined the vector of substitution on our macrocyclic “probes” by homology modeling with known acyclic ligands/CDK9 co-crystal structures. Having defined 2 substitution patterns, we synthesized and tested in a biochemical assay the novels “probes” vectorized with the pro-linker moieties, without loss of activity. ODS’7152 was further derived into fully bifunctional molecules with variation on the linker’s nature and length, keeping thalidomide as the E3 ligase recruiter. Biophysical and biological properties were further evaluated in an MTS assay against several cell lines (HCT116, Jurkat E6.2, MOLT-4 and MV4-11) to assess cell proliferation and viability. The E3 Ligase engagement along with cell penetration was measured in a NanoBRET™ assay and compared to MDR_MDCK permeability. Finally, a selection of 4 PROTACs were evaluated in a dose response assay on MV4-11 for protein degradation phosphorylation of CTD-pol II and selectivity among several CDKs. Our findings support the rapid derivatization of macrocyclic “probes” inhibitors into macrocyclic-PROTAC as a strategy to generate early chemical biology tools with maintained potency and selectivity between homologous targets. Optimization of the physico-chemical and ADME properties of molecules can lead to drug candidates with distinct pharmacological effects as compared to the parent linear kinase inhibitors.


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