The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study

Abstract

Proteolysis targeting chimera (PROTAC) technology has emerged over the last two decades as a powerful tool for targeted degradation of endogenous proteins. Herein we describe the development of PROTACs for receptor tyrosine kinases, a protein family yet to be targeted for induced protein degradation. The use of VHL-recruiting PROTACs against this protein family reveals several advantages of degradation over inhibition alone: direct comparisons of fully functional, target-degrading PROTACs with target-inhibiting variants that contain an inactivated E3 ligase-recruiting ligand show that degradation leads to more potent inhibition of cell proliferation and a more durable and sustained downstream signaling response, and thus addresses the kinome rewiring challenge seen with many receptor tyrosine kinase inhibitors. Combined, these findings demonstrate the ability to target receptor tyrosine kinases for degradation using the PROTAC technology and outline the advantages of this degradation-based approach.

Keywords: EGFR; HER2; PROTAC; VHL; c-Met; receptor tyrosine kinases; targeted degradation.

Figure 1. Small molecule induced degradation of EGFR and mutants

A–F – Immunoblots of cells expressing different EGFR variants treated with increasing doses of the indicated compound for 24 hours. A – OVCAR8 cells treated with lapatinib-based PROTAC 1. B – OVCAR8 cells treated with compound 2, an inactive diastereomer of lapatinib-based PROTAC 1. C – HeLa cells expressing FLAG-tagged exon 20 ins (ASV duplication) EGFR treated with lapatinib-based PROTAC 1. D – HCC827 cells expressing exon 19 del EGFR treated with gefitinib-based PROTAC 2. E – H3255 cells expressing L858R EGFR treated with gefitinib-based PROTAC 3. F – H1975 cells expressing double mutant (L858R/T790M) EGFR treated with afatinib-based PROTAC 4. G – Summary table of DC₅₀ (the concentration at which half-maximal degradation is achieved) and D_max (the maximum percentage of degradation) values. See also Figure S1 and S2.

Figure 2. Selective PROTAC-mediated degradation of HER2 and implications for kinome re-wiring

A – Employing different linker lengths imparts PROTAC selectivity for EGFR over HER2. OVCAR8 cells were treated with PROTAC 1 or 5 for 24 hours before being lysed and probed for EGFR, HER2 and tubulin (as a loading control). B – Cell proliferation assay in SKBr3 cells after 72 hours of treatment with the indicated compound concentrations. C - Treatment of SKBr3 cells with sub-lethal concentrations (500 nM) of PROTAC 1 or diastereomer 2 over 48 hours shows a gradual increase in downstream signalling consistent with kinome re-wiring, previously observed in SKBr3 cells, with diastereomer but not with PROTAC. D – Immunoblotting analysis of c-Met phosphorylation after 48 hours with 500 nM PROTAC 1 or diastereomer 2. See also Figure S2.

Figure 3. PROTAC mediated degradation of c-Met

A–B – MDA-MB-231 cells treated for 24 hours with increasing concentrations of foretinib-based PROTAC 7 (A) or diastereomer 8 (B). C – Cell proliferation assay in GTL16 cells (PROTAC 7 IC₅₀ = 66.7 nM, diastereomer 8 IC₅₀ = 156 nM) D – Time course of c-Met degradation by foretinib-based PROTAC (500 nM) 7. E – PROTAC effects are longer lasting in cell culture. Cells were treated for 24 hours with 500 nM PROTAC 7 or diastereomer 8 before re-plating on new plastic, in fresh media for 24 or 48 hours. Excess VHL (25 μM) ligand was added to the indicated wells. See also Figure S3 and S6.

Figure 4. PROTAC mediated internalization

A/B – Cell surface proteins were labelled with a cell membrane impermeant biotin reagent prior to treatment with 500 nM foretinib-based PROTAC 7 (A) or 100 ng/ml HGF (B) for the indicated times and lysed. Biotinylated proteins were enriched by streptavidin pulldown and immunoblotted for c-Met. Biotinylated proteins represent the cell surface fraction. Corresponding whole cell lysates are also shown C - Representative confocal microscopy images of c-Met (green) internalization in response to PROTAC 7 (500 nM) treatment for the indicated times (DAPI nuclear stain - blue). See also Figure S5.

Figure 5. Exon 14-deleted c-Met has increased stability and resistance to HGF-mediated degradation that can be combated by foretinib-based PROTAC 7

A – Quantitation of WT c-Met or exon 14-deleted c-Met degradation upon treatment with HGF, PROTAC 7 or DMSO control in the presence of cycloheximide (CHX). B – Table of calculated half-lives. C – Representative CHX time course of WT c-Met degradation and signalling in MDA-MB-231 cells treated with HGF. D – Representative CHX time course of exon 14 deleted c-Met degradation and signalling in Hs746T cells treated with HGF. E - Representative CHX time course of exon 14-deleted c-Met degradation in Hs746T cells treated with PROTAC 7. F and G – Both MDA-MB-231 and Hs746T cells were treated with either DMSO or PROTAC for 18 hours before the addition of HGF and lysis at the indicated time points following stimulation. H – Immunoprecipitation of c-Met from PROTAC 7 (1μM) or DMSO treated Hs746T cells followed by immunoblotting for Ubiquitin. I – Tandem Ubiquitin Binding Entity 1 (TUBE1) pulldown from PROTAC 7 (1μM) or DMSO treated Hs746T cells followed by immunoblotting for c-Met See also Figure S6.