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Trivalent PROTACs Enhance Protein Degradation Through Cooperativity and Avidity

preprint
submitted on 11.11.2020, 00:49 and posted on 12.11.2020, 13:04 by Satomi Imaide, Kristin M. Riching, Vesna Vetma, Claire Whitworth, Scott J. Hughes, Nicole Trainor, Sarah D. Mahan, Nancy Murphy, Kwok-Ho Chan, Andrea Testa, Chiara Maniaci, Marjeta Urh, Danette L. Daniels, Alessio Ciulli

Bivalent small-molecule degraders, or proteolysis targeting chimeras (PROTACs), work by simultaneously binding a target protein and E3 ubiquitin ligase to produce a ternary complex. To drive target ubiquitination and degradation at low catalytic concentrations, degraders must form appropriately positioned complexes of sufficient stability, aided by intra-complex interactions. We hypothesized these molecular recognition features could be enhanced by increasing binding valency. Here we present trivalent PROTACs as a strategy to boost protein degradation. Our design for a trivalent PROTAC consisted of two BET bromodomain inhibitors and an E3 ligase ligand, each separately tethered via a branched linker. In screening, we identified SIM1, a VHL-based PROTAC, as a highly potent BET degrader, capable of low picomolar degradation for all family members, with preference for BRD2. In functional comparison studies to bivalent PROTACs or inhibitors, SIM1 showed more sustained anti-cancer activity across numerous therapeutically relevant cell lines. Biophysical, biochemical, and cellular mechanistic studies showed SIM1 induces conformational changes upon binding to the BET protein to simultaneously engage with high avidity both its bromodomains in a cis intramolecular fashion. The resulting 1:1:1 complex showed positive cooperativity, high stability and prolonged cellular residence time. We provide proof-of-concept for augmenting the binding valency of proximity-induced modalities as a strategy to leverage both cooperativity and avidity within the ternary complex to advance functional outcomes.

Funding

European Research Council (ERC, Starting Grant ERC-2012-StG-311460 DrugE3CRLs to A.C.)

Ono Pharma (visiting scientist fund to S.I.)

Wellcome Trust strategic award to Dundee (100476/Z/12/Z)

Wellcome Trust strategic award to Dundee (094090/Z/10/Z)

History

Email Address of Submitting Author

a.ciulli@dundee.ac.uk

Institution

University of Dundee

Country

United Kingdom

ORCID For Submitting Author

0000-0002-8654-1670

Declaration of Conflict of Interest

The authors declare the following competing financial interest(s): The Ciulli laboratory receives or has received sponsored research support from Almirall, Amphista therapeutics, Boehringer Ingelheim, Eisai, Nurix therapeutics, and Ono Pharmaceuticals. A.C. is a scientific founder, shareholder, non-executive director and consultant of Amphista therapeutics, a company that is developing targeted protein degradation therapeutic platforms. K.M.R., S.D.M., N.M., M.U., and D.L.D. are employees of Promega Corporation. Promega Corporation is the commercial owner by assignment of patents of the HaloTag, NanoLuc, NanoBRET target engagement, and HiBiT technologies and their applications.

Version Notes

version no. 1

Licence

Exports