Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules

Abstract

Chemical biology strategies for directly perturbing protein homeostasis including the degradation tag (dTAG) system provide temporal advantages over genetic approaches and improved selectivity over small molecule inhibitors. We describe dTAGV-1, an exclusively selective VHL-recruiting dTAG molecule, to rapidly degrade FKBP12F36V-tagged proteins. dTAGV-1 overcomes a limitation of previously reported CRBN-recruiting dTAG molecules to degrade recalcitrant oncogenes, supports combination degrader studies and facilitates investigations of protein function in cells and mice.

An in vivo CRISPR screening platform for prioritizing therapeutic targets in AML

“…By contrast, MARCH5 mutations (H43W and C68S) that disrupt its RING domain and thus ubiquitinase function ablated the rescuing ability (25,26), indicating the requirement for the catalytic function of MARCH5 in AML ( Figure 3B and Figure S6C ). Additionally, for MARCH5 validation we deployed a dTAG system, which uses a hetero-bifunctional small molecule that binds the FKBP12 F36V -fused target protein (i.e., MARCH5) and an E3 ligase complex (i.e., VHL), bringing the two in close proximity and leading to the ubiquitination and proteasome-mediated degradation of the target protein ( Figure S6D )(27). In this case, we deleted endogenous MARCH5 by CRISPR and expressed exogenous MARCH5 with a FKBP12 F36V -tag, showing that the defective growth can be recapitulated by MARCH5 degradation with the dTAG molecule dTAG V -1 ( Figure 3C-D ).…”

Charting a New Path Towards Degrading Every Protein

“…The system that my colleagues and I developed is called the degradation tag (dTAG) system, which uses highly selective FKBP12 F36V degraders (dTAG molecules) that coordinate an interaction between an E3 ubiquitin ligase and an FKBP12 F36V ‐tagged protein, triggering destruction of the tagged protein (Figure 1). [7–8] While I hoped the technology would be enabling for biological investigation, particularly for chemically controlling recalcitrant targets, the amount of positive global interest I received exceeded all of my expectations. Since the initial description of the technology, including my demonstration that the system works in vivo , [7] we and many others have applied dTAG to study divergent targets in development and disease [8–23] .…”

“… [7–8] While I hoped the technology would be enabling for biological investigation, particularly for chemically controlling recalcitrant targets, the amount of positive global interest I received exceeded all of my expectations. Since the initial description of the technology, including my demonstration that the system works in vivo , [7] we and many others have applied dTAG to study divergent targets in development and disease [8–23] . The speed of protein loss afforded by dTAG is a unique advantage over loss‐of‐function genetic approaches and is critical for evaluating cellular switches, transcriptional regulators, and essential proteins.…”

Major advances in targeted protein degradation: PROTACs, LYTACs, and MADTACs

“…In addition, PROTACs have been shown to successfully degrade major disease-causing proteins that have traditionally eluded the pharmaceutical industry such as tau ( 20 , 21 ) and KRAS ( 22 , 23 , 24 ). Furthermore, the establishment of Halo-PROTACs ( 25 ) and dTAG ( 26 , 27 ) systems, which allow targeted degradation of HaloTag7 and FKBP fusion proteins respectively, has allowed for degradation of proteins without established ligands. The breadth of proteins targeted by PROTACs has been extensively reviewed elsewhere and thus will not be the focus of this article ( 28 , 29 , 30 ).…”