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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 proteolysis-targeting chimeras (PROTAC). Homo-PROTACs are bivalent small molecules and are intended to dimerize an E3 ligase and then induce its self-degradation. Creative Biolabs has constructed a professional Homo-PROTAC platform to help our clients design and synthesize highly customized PROTAC for drug development.
The human genome comprises > 600 predicted E3 ligases that play important roles in normal cellular physiology and disease states, making them attractive targets for inhibitor discovery. Moreover, E3 ligases are multi-domain and multi-subunit enzymes, and targeting an individual binding site leaves other scaffolding regions untouched and other interactions functional. As a result, E3 ligase inhibition may be ineffective or fail to recapitulate genetic knockout or knockdown. The blockade of E3 ligase activity requires targeting of protein-protein interactions (PPIs). Homo-PROTACs were described as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells.
Fig.1 Schematic diagram of Homo-PROTACs.
Homo-PROTACs were first reported for the VHL, based on the structures of two potent VHL ligands (VH032 and VH298). The linker was attached to different positions and the length of the polyethylene glycol chains varied from 3 to 5 ethylene glycol units. The biological evaluation of the homo-PROTACs revealed that the most active compounds (CM11) were selectively degrading the long isoform of VHL. The position of the linker and the stereochemistry were crucial for degradation. The CM11 dimerizes VHL with high avidity in vitro and induces proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion. 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.
Homobifunctional PROTACs that utilize CRBN as the hijacked degrader and at the same time, as the protein targeted for degradation, were synthesized, aiming at the chemical-induced CRBN degradation. Two pomalidomide moieties were conjugated via linear linkers with varying lengths, varying hydrophobicity, and different attachment positions. The compound with a linker length of eight atoms (CC15a) was identified as the most potent CRBN degrader. The most active PROTAC degraded specifically CRBN and showed only weak effects on the neo-substrates, with no effect on other members of the CRL3 ligase family.
The E3 ligases could be hijacked against each other using a heterodimerizing PROTAC, leading either to degradation of both E3 ligases or preferential degradation of one E3 ligase. A library of CRBN-VHL PROTACs was designed and synthesized, using pomalidomide as CRBN handle and as for the VHL handle, structural modifications were designed on two known VHL ligands, including different attachment points of the linker. A few compounds led to significant degradation of CRBN, whereas none of the compounds showed significant degradation of VHL. In this ‘double-hijacking’ approach, the VHL-CRBN PROTACs resulted in preferential degradation of CRBN over VHL.
Fig.2 Chemical structures of different ligands used in Homo-PROTACs.
Homo-PROTACs is a powerful new strategy for drugging E3 ligases. With years of research and development, Creative Biolabs is experienced in PROTAC techniques. By using different design strategies, we can generate both homo-bivalent PROTAC and heterodimeric PROTAC for customers’ specific demands.
Based on the Homo-PROTAC platform, Creative Biolabs systematically deploys a series of services to fully explore the potential of PROTAC as a new generation of small molecule drugs. If you are interested in our services, please directly contact us and consult our technical supports for more details.