Homo-PROTACs: bivalent small-molecule dimerizers of the VHL E3 ubiquitin ligase to induce self-degradation

Abstract

E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for 'proteolysis-targeting chimeras'). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases.Targeting the ubiquitin proteasome system to modulate protein homeostasis using small molecules has promising therapeutic potential. Here the authors describe Homo-PROTACS: small molecules that can induce the homo-dimerization of E3 ubiquitin ligases and cause their proteasome-dependent degradation.

Fig. 1

Structure-guided design of Homo-PROTACs to induce VHL dimerization. a Crystal structures of VHL in complex with VH298 (PDB code 5LLI). VHL is shown as a pink surface and the bound ligand as sticks representation with purple carbons, nitrogen atoms in blue, oxygen in red and sulphur in dark yellow. b Chemical structure of VHL inhibitors VH032 and VH298. c–e General chemical structures and design of Homo-PROTACs compounds. Linkage sites at the acetyl and phenyl groups are indicated in red

Fig. 2

Synthesis of symmetric homo-PROTAC compounds derivatized from the terminal acetyl group. The shown route yielded symmetric trans-trans CM09, CM10 and CM11 and negative control symmetric cis-cis compound CMP98

Fig. 3

Synthesis of negative control homo-PROTAC compound derivatized from the terminal acetyl group. Compound CMP99 has cis-trans configuration

Fig. 4

Homo-PROTACs CM09, CM10 and CM11 induce selective knockdown of the long isoform of VHL. a HeLa cells were treated with 0.1% DMSO, VH032 (150 µM) and 1 µM of the indicated compounds for 10 h. Abundance of individual proteins was analyzed by western blotting using corresponding specific antibodies accordingly after SDS-PAGE. The data demonstrated is a representative example of experiments performed in biological duplicates. b Different cells lines were treated with siRNA targeting VHL proteins or negative control siRNA (for 48 h), as well as with CM11 (1 µM) or 0.1% v/v DMSO for 10 h. The bands observed to selectively disappear in the presence of CM09–11 are closely migrating isoforms of pVHL30 (see refs ^, ). The data demonstrated is a representative example of experiments performed as four biological replicates

Fig. 5

CM11 induces pVHL30 depletion in a concentration and time-dependent fashion. (left) Dose-response profile of HeLa cells treated with increasing concentration of CM11 for 4 or 24 h. (right) Time-course immunoblots of lysates from HeLa cells treated 1 µM of CM11 up to 48 h. Control treatments of 0.1% DMSO, CoCl₂ (100 µM), IOX2 (150 µM), and VH032 (250 µM or 1 µM) are included. All data demonstrated is a representative example of experiments performed in biological duplicates

Fig. 6

CM11 activity is CRL2^VHL and proteasome-dependent. HeLa cells treated with CM11 in the absence or presence of proteasome inhibitor MG132, neddylation inhibitor MLN4924, VHL inhibitor VH032 or PHD2 inhibitor IOX4 as negative control. The data demonstrated here is representative of one biological replicate

Fig. 7

Biophysical studies of Homo-PROTACs binding to VHL. a Superposition of the integrated ITC heat curves of CM11 (blue), CMP99 (black) or CMP98 (green) titrations against VCB. b SEC assay of complex formation after incubation of CM11 (red), CMP98 (pale green), CMP99 (purple), VH032 (dotted purple) or DMSO (black) with VCB. c AlphaLISA: intensity values titrating CM09, CM10, CM11 and CMP98 against VCB. Each point is mean ( ± SEM) intensity of four technical replicates

Fig. 8

Proposed model for the mechanism of action of Homo-PROTAC CM11