Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader

Abstract

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 (∼50% degradation after 30 min) and complete (Dmax of ∼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.

Systematic exploration of different E3 ubiquitin ligases: an approach towards potent and selective CDK6 degraders

“…In a second VHL-based series, a distinct functionalization site of the VHL ligand was chosen as an exit vector ( Table 2 and Scheme 2). 44,45 As reported, different points of attachment to the VHL ligand can result in two contrasting E3 ligase recruitment geometries and an isoform-selective degradation of two closely related proteins. 44 To unambiguously investigate the impact of the linker attachment point on the target degradation, the same linkers as before were realized.…”

“…The activity of 29, when compared with its close relative 30 was unexpected, also in the light of the same lipophilicity of both compounds (Table 2). In the subsequent optimization step, inspired by previous reports, 43,45,61,63 the valine-isoindolinone moiety of 29 was replaced by tert-leucine acylated with a cyanocyclopropanecarbonyl (33) or uorocyclopropanecarbonyl group (34). These two structural modications led to CDK4/6d with outstanding properties, combining strong degrading potency with remarkable CDK6 selectivity, as evidenced by their selectivity indices of 46 and 31, respectively.…”

An activity-guided map of electrophile-cysteine interactions in primary human immune cells

“…More generally, we should emphasize that some of the cysteine ligandability events mapped in this study, including those showing good tractability, may fail to produce direct functional effects on proteins. Such so-called "silent" compounds still have the potential to be converted into heterobifunctional small-molecule degraders of proteins, an approach that has been successfully used with other covalent ligands (Buckley et al, 2015;Burslem et al, 2018;Tovell et al, 2019).…”

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…The reports above demonstrated degradation of overexpressed HaloTag‐fused proteins, which, whilst conceptually useful, is limited in its application to study protein function. To address this issue, the Ciulli and Alessi laboratories recently reported that CRISPR/Cas9 genome editing can be used to insert a HaloTag at the N terminus of VPS34 and the C terminus of SGK3, whilst preserving endogenous expression levels and biological functions . These HaloTag fusion proteins were successfully degraded by a VHL‐based HaloPROTAC, with degradation potency in the n m range and high selectivity.…”