VH032

PROTACs represent a promising modality that has gained significant attention for the treatment of cancer, Alzheimer's disease, and so forth. Due to limited structural information of the POI-PROTAC-E3 ligase ternary complex, the discovery of active PROTACs relies on the screening of diversity-oriented PROTAC libraries. VH032 amine is a key building block for the synthesis of VHL E3 ligase-based PROTACs. To construct VHL PROTAC libraries rapidly, the availability of VH032 amine is crucial. In this paper, we report a column chromatography-free process which enables the production of 42.5 g of VH032 amine hydrochloride in 65% overall yield with 97% purity in a week.

The von Hippel-Lindau (VHL) tumor suppressor associates with transcription factors elongin-C and elongin-B to form the VHL-elongin-C-elongin-B protein complex and carry out its functions, such as degradation of hypoxia-inducible factors. VHL ligands are used not only to modulate hypoxia-signaling pathways and potentially treat chronic anemia or ischemia but also to form bivalent ligands as proteolysis-targeting chimeras to degrade proteins for potential therapeutic applications. Sensitive and selective VHL-based binding assays are critical for identifying and characterizing VHL ligands with high-throughput screening approaches. VHL ligand-binding assays, such as isothermal titration calorimetry, surface plasmon resonance, and fluorescence polarization assays, are reported but with limitations. Isothermal titration calorimetry requires higher protein concentrations with a lower throughput than fluorescence-based assays do. Surface plasmon resonance requires protein immobilization, which introduces variation and is not suitable for testing a large number of ligands. Fluorescence polarization can be sensitive with high-throughput capability but is susceptible to assay interference, and small-molecule-based fluorescent probes are not available. We developed the first small-molecule-based high-affinity VHL fluorescent probe BODIPY FL VH032 (5), with a K d of 3.01 nM, for a time-resolved fluorescence resonance energy-transfer assay. This new assay is sensitive, selective, resistant to assay interference, and capable of characterizing VHL ligands with a wide range of affinities. It is also suitable for VHL ligand identification and characterization with high-throughput screening.

Proteolysis targeting chimeras (PROTACs) are catalytic heterobifunctional molecules that can selectively degrade a protein of interest by recruiting a ubiquitin E3 ligase to the target, leading to its ubiquitylation and degradation by the proteasome. Most degraders lie outside the chemical space associated with most membrane-permeable drugs. Although many PROTACs have been described with potent activity in cells, our understanding of the relationship between structure and permeability in these compounds remains limited. Here, we describe a label-free method for assessing the permeability of several VH032-based PROTACs and their components by combining a parallel artificial membrane permeability assay (PAMPA) and a lipophilic permeability efficiency (LPE) metric. Our results show that the combination of these two cell-free membrane permeability assays provides new insight into PROTAC structure-permeability relationships and offers a conceptual framework for predicting the physicochemical properties of PROTACs in order to better inform the design of more permeable and more effective degraders.