Kizhake, S. | Search bioRxiv

bioRxiv2026

Beyond Dmax and DC50: A simple tool to evaluate the complex PROTAC-mediated kinetic degradation dynamics

Protein degradation mediated by PROteolysis TArgeting Chimeras (PROTACs) is a complex, dynamic, and multi-step process. The structural diversity of PROTACs, coupled with cellular heterogeneity, introduces considerable variability at each stage. Therefore, a straightforward yet comprehensive approach is essential to effectively dissect these mechanisms. The existing methods for evaluating PROTAC degradation are often time-consuming, labor-intensive, or expensive, and many capture only a partial view of the entire degradation process. Consequently, multiple complementary assays are typically required to obtain the complete picture. Here, we use commercially available Cyclin dependent Kinase 6 (CDK6) targeting PROTACs to demonstrate that a simple, single-step, no-wash, additive-free, GFP-based live-cell imaging approach can rapidly generate dynamic degradation metrics (Dmax, Rmax, TDmax, CDmax, DC50, Km, Tmax, Kdeg, Vmax, DT50, {tau} and TRmax) for comparing and ranking PROTACs within a controlled reporter system. The rapid assay turnaround time, excellent assay qualities and compatibility with high-throughput (HT) formats will facilitate quick hit identification and serve as a guiding assay for hit-to-lead optimization campaigns.

molecular biology↗

bioRxiv2024

Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14{Delta}) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14{Delta} using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14{Delta}-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

cancer biology↗