Introducing BromoTAG and AGB1: our new inducible degron system for potent, rapid, selective protein degradation

  • ToC graphics
  • Figure 1. (A) Pan-selective BET degraders, MZ1 and ARV-771. (B) Pan-selective BET inhibitors, (+)-JQ1 and I-BET762 (top). Allele-specific bumped BET inhibitors, ME, ET, 9-ME-1, and 9-ET-1 (bottom). (C) Tailoring the “bump-and-hole” approach to BET bromodomains to produce a high-affinity selective pairing that can be utilized as a degron system. (D) Conceptualization of the BromoTag degron approach.
  • Figure 2. Design and development of a heterozygous knock-in BromoTag-Brd2 HEK293 cell line.
  • Figure 3. (A) Ternary complex between Brd4BD2 (green, cartoon/surface representation), MZ1 (1, stick, gray carbons), and VCB (VHL: blue; elongin C: pink; elongin B—pale orange; and cartoon/surface representations). Leu387 (stick, green) is indicated by an arrow (PDB code: 5T35).
  • Scheme 1. Synthesis of I-BET762-Based B&H–PROTACs and Non-bumped Control Compound
  • Scheme 2. Synthesis of Racemic Bumped JQ1 Ligands
  • Scheme 3. Conjugation of Linkers to VHL Ligands
  • Scheme 4. Synthesis of JQ1-Based B&H–PROTACs as Mixtures of Two Diastereomers
  • Figure 5. Biological evaluation of second-generation B&H–PROTACs in BromoTag-Brd2 HEK293 cells.
  • Scheme 5. Synthesis of Enantiomerically Pure AGB1, AGB2, and AGB3
  • Figure 6. Biological evaluation of AGB1 (46), AGB2 (47), and AGB3 (48) in BromoTag-Brd2 HEK293 cells.
  • Figure 7. FP of B&H–PROTAC binary and ternary complex binding.
  • Figure 8. Cellular mechanistic characterization of AGB1 (46) degradation activity.
  • Scheme 6. Synthesis of Negative Control cis-AGB1 (52)
  • Figure 9. Proteomics of AGB1 (46) and cis-AGB1 (52) treated heterozygous BromoTag-Brd2 HEK293 cells.
  • Figure 10. Plasma stability and in vivo PK studies of AGB1 (46) in mice.

Great work by Adam and Conner, who led chemistry and biology on this study, and in collaboration with Dario Alessi's group! 

Read the Open Access full article here.

Authors: Adam G. Bond, Conner Craigon, Kwok-Ho Chan, Andrea Testa, Athanasios Karapetsas, Rotimi Fasimoye, Thomas Macartney, J. Julian Blow, Dario R. Alessi, and Alessio Ciulli*

Title: Development of BromoTag: A “Bump-and-Hole”–PROTAC System to Induce Potent, Rapid, and Selective Degradation of Tagged Target Proteins

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.

 

Our PROTAC degrader AGB1 and BromoTAG-specific antibody are available with no strings-attached upon request. Feel free to contact Alessio if you'd like to try the BromoTag out with your favourite protein!