Journal
ACS CHEMICAL BIOLOGY
Volume 14, Issue 5, Pages 882-892Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.8b01016
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Funding
- Medical Research Council [MC_UU_12016/2]
- European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007-2013) [ERC-2012-StG-311460 DrugE3CRLs]
- GlaxoSmithKline
- Merck KGaA
- AstraZeneca BBSRC Studentship
- Boehringer Ingelheim
- BBSRC [1644353] Funding Source: UKRI
- MRC [MC_U127015387, MC_UU_12016/2, MC_UU_00018/1] Funding Source: UKRI
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Inducing post-translational protein knockdown is an important approach to probe biology and validate drug targets. An efficient strategy to achieve this involves expression of a protein of interest fused to an exogenous tag, allowing tag directed chemical degraders to mediate protein ubiquitylation and proteasomal degradation. Here, we combine improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing technology to trigger rapid degradation of endogenous target proteins. Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity VHL binder VH298, induced reversible degradation of two endosomally localized proteins, SGK3 and VPS34, with a DC50 of 3-10 nM. HaloPROTAC-E induced rapid (similar to 50% degradation after 30 min) and complete (D-max of similar to 95% at 48 h) depletion of Halo tagged SGK3, blocking downstream phosphorylation of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater steady state degradation of Halo tagged endogenous VPS34 than the previously reported HaloPROTAC3 compound. Quantitative global proteomics revealed that HaloPROTAC-E is remarkably selective inducing only degradation of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1, and ATG14) and no other proteins were significantly degraded. This study exemplifies the combination of HaloPROTACs with CRISPR/Cas9 endogenous protein tagging as a useful method to induce rapid and reversible degradation of endogenous proteins to interrogate their function.
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