Abstract
Small-molecule-induced protein depletion technologies, also called inducible degrons, allow degradation of genetically engineered target proteins within cells and animals. Here, we design and develop the BromoTag, a new inducible degron system comprising a Brd4 bromodomain L387A variant as a degron tag that allows direct recruitment by heterobifunctional bumped proteolysis targeting chimeras (PROTACs) to hijack the VHL E3 ligase. We describe extensive optimization and structure–activity relationships of our bump-and-hole–PROTACs using a CRISPR knock-in cell line expressing model target BromoTag-Brd2 at endogenous levels. Collectively, our cellular and mechanistic data qualifies bumped PROTAC AGB1 as a potent, fast, and selective degrader of BromoTagged proteins, with a favorable pharmacokinetic profile in mice. The BromoTag adds to the arsenal of chemical genetic degradation tools allowing us to manipulate protein levels to interrogate the biological function and therapeutic potential in cells and in vivo.
Highlights
Targeted protein degradation is rapidly established as a powerful modality of chemical biology and drug discovery
Examples of tag-based degron systems include the auxin-inducible degron (AID), whereby a target protein is fused with the AID/IAA17 degron sequence that is recognized by the plant cullin RING E3 ligase TIR1 in the presence of the molecular glue auxin6 or in recent developments of bumped analogues selectively targeting mutant TIR1;7,8 HaloPROTACs, bifunctional molecules that bear a chloroalkane warhead which forms a covalent bond with a HaloTag-fused target protein at one end and a von Hippel− Lindau (VHL) E3 ligase ligand at the other end;9,10 and dTAGs, bifunctional molecules which bind to a FKBP12F36V tag that is fused to the target protein at one end and either cereblon (CRBN) or VHL ligases at the other end
Through a careful structural guided design, we have developed AGB1 (46) and qualified it as a fast, highly selective, and potent B&H−Proteolysis targeting chimeras (PROTACs) degrader for our new inducible degron system, BromoTag
Summary
Targeted protein degradation is rapidly established as a powerful modality of chemical biology and drug discovery. Examples of tag-based degron systems include the auxin-inducible degron (AID), whereby a target protein is fused with the AID/IAA17 degron sequence that is recognized by the plant cullin RING E3 ligase TIR1 in the presence of the molecular glue auxin or in recent developments of bumped analogues selectively targeting mutant TIR1;7,8 HaloPROTACs, bifunctional molecules that bear a chloroalkane warhead which forms a covalent bond with a HaloTag-fused target protein at one end and a von Hippel− Lindau (VHL) E3 ligase ligand at the other end; and dTAGs, bifunctional molecules which bind to a FKBP12F36V tag that is fused to the target protein at one end and either cereblon (CRBN) or VHL ligases at the other end.11,12 These approaches have been used successfully to induce targeted protein degradation in cells and in vivo, but they all have disadvantages and limitations. AID methods can be leaky (background target degradation even prior to auxin dosage), require high concentrations of auxin to work, and require inconvenient additional engineering to allow for the expression of the non-native plant E3 ligase; all limitations lead
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