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dTAG (degradation TAG) is an innovative approach to target validation, using heterobifunctional small molecule Degraders to harness a cell’s protein degradation system and eliminate a protein of interest. The dTAG system, developed by Dr Behnam Nabet and colleagues at the Dana Farber Cancer Center, is a generalizable approach and a form of Targeted Protein Degradation (TPD). Other TPD approaches, such as the PROTACs™ MZ 1 (Cat. No. 6154) and THAL SNS 032 (Cat. No. 6532), utilize an existing ligand for the protein of interest linked to an E3 ligase ligand in the development of the Degrader. dTAG technology removes the need for a known ligand, by combining Degrader technology with genome engineering.
Figure 1: dTAG mechanism of action
The target protein is expressed as a chimera with mutant FKBP12F36V, either by CRISPR/Cas9-mediated locus-specific knock-in or lentiviral transgene expression. The dTAG compound, e.g. dTAG-13 (Cat. No. 6605), is composed of an E3 ligase ligand linked to a highly selective FKBP12F36V ligand, which form a ternary complex between the fusion protein and E3 ligase causing polyubiquitination and degradation of the target protein.
In vitro, dTAG-13 has been shown to lead to rapid and potent degradation of BRD4-FKBP12F36V, with no effect on endogenous wild type FKBP12, BRD2 or BRD3 levels. To validate this method, the dTAG system has also been applied to protein chimeras of FKBP12F36V fused with EZH2, HDAC1, KRAS, MYC and PLK1. Efficacy of dTAG-13 has also been demonstrated in vivo, using a luciferase-FKBP12F36V chimera. The fusion protein was first expressed in a human leukemia cell line, then engrafted into bone marrow in mice. dTAG-13 treatment led to a rapid reduction in bioluminescence signal indicating the effective degradation of the luciferase-FKBP12F36V chimera.
Conventional strategies for target validation include disrupting gene expression, and therefore total cellular protein levels, with RNA-interference (RNAi) or CRISPR/Cas9, or inhibiting protein function with a small molecule antagonist. Pharmacological approaches like small molecules offer a number of advantages over purely genetic methods, including dose-dependent effects, as well as rapid and reversible action. In contrast, genetic approaches offer less dynamic control, don’t allow for titration of effect size and are most often completely irreversible. Using dTAG for target validation combines the key advantages of genetic and pharmacological strategies, providing rapid and dose-dependent effects on total cellular protein abundance, which are reversible on Degrader wash-out.
dTAG-13 has been used to identify and validate novel targets in cancer. The YEATS domain-containing protein ENL, is part of the super elongation complex (SEC) that orchestrates gene activity at the level of transcriptional elongation, by increasing the catalytic rate of RNA polymerase during transcription. This protein has been previously identified in acute myeloid leukemia (AML) as a key protein that supports pathogenesis via the maintenance of dysregulated gene expression, however there are no known ligands available for ENL.
To validate ENL as a target in AML, Erb et al. expressed ENL as a FKBP12F36V fusion protein in a human AML cell line and demonstrated selective degradation of ENL with nanomolar concentrations of dTAG-13. This resulted in genome-wide suppression of transcriptional initiation and elongation, and cell growth arrest. Further investigation using dTAG-13 to degrade ENL showed its role in recruitment of SEC and identified the YEATS domain as a chromatin-reader that is essential for ENL-dependent cell growth.
PROTAC is a registered trademark of Arvinas, Inc.
Erb et al. (2017) Transcription control by the ENL YEATS domain in acute leukemia. Nature. 543, 270. PMID: 28241139
Mayor-Ruiz & Winter (2019) Identification and characterization of cancer vulnerabilities via targeted protein degradation. Drug Discov Today Technol. 31, 81. PMID: 31200863
Nabet et al. (2018) The dTAG system for immediate and target-specific protein degradation. Nat Chem Biol. 14, 431. PMID: 29581585