Native mass spectrometry can effectively predict PROTAC efficacy

Protein degraders, also known as proteolysis targeting chimeras (PROTACs), are bifunctional small molecules that promote cellular degradation of a protein of interest (POI). PROTACs act as molecular mediators, bringing an E3 ligase and a POI into proximity, thus promoting ubiquitination and degradation of the targeted POI. Despite their great promise as next-generation pharmaceutical drugs, the development of new PROTACs is challenged by the complexity of the system, which involves binary and ternary interactions between components. Here, we demonstrate the strength of native mass spectrometry (nMS), a label-free technique, to provide novel insight into PROTAC-mediated protein interactions. We show that nMS can monitor the formation of ternary E3-PROTAC-POI complexes and detect various intermediate species in a single experiment. A unique benefit of the method is its ability to reveal preferentially formed E3-PROTAC-POI combinations in competition experiments with multiple substrate proteins, thereby positioning it as an ideal high-throughput screening strategy during the development of new PROTACs

Highly Selective PTK2 Proteolysis Targeting Chimeras to Probe Focal Adhesion Kinase Scaffolding Functions

Focal adhesion tyrosine kinase (PTK2) is often overexpressed in human hepatocellular carcinoma (HCC), and several reports have linked PTK2 depletion and/or pharmacological inhibition to reduced tumorigenicity. However, the clinical relevance of targeting PTK2 still remains to be proven. Here, we present two highly selective and functional PTK2 proteolysis-targeting chimeras utilizing von Hippel–Lindau and cereblon ligands to hijack E3 ligases for PTK2 degradation. BI-3663 (cereblon-based) degrades PTK2 with a median DC₅₀ of 30 nM to >80% across a panel of 11 HCC cell lines. Despite effective PTK2 degradation, these compounds did not phenocopy the reported antiproliferative effects of PTK2 depletion in any of the cell lines tested. By disclosing these compounds, we hope to provide valuable tools for the study of PTK2 degradation across different biological systems

Targeting cancer with small molecule pan-KRAS degraders

Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders

Progress towards a public chemogenomic set for protein kinases and a call for contributions

Protein kinases are highly tractable targets for drug discovery. However, the biological function and therapeutic potential of the majority of the 500+ human protein kinases remains unknown. We have developed physical and virtual collections of small molecule inhibitors, which we call chemogenomic sets, that are designed to inhibit the catalytic function of almost half the human protein kinases. In this manuscript we share our progress towards generation of a comprehensive kinase chemogenomic set (KCGS), release kinome profiling data of a large inhibitor set (Published Kinase Inhibitor Set 2 (PKIS2)), and outline a process through which the community can openly collaborate to create a KCGS that probes the full complement of human protein kinases