The host lab has developed a bump-and-hole system for the BET BD, pairing a leucine/alanine mutation with an ethyl-derived analogue of an established benzodiazepine scaffold based on I-BET762 [1]. Building on top of this, the system is optimized with the introduction of a more conservative and less disruptive leucine/valine (L/V) mutation. The L/V BD mutant retains good binding to chromatin peptide comparable to the WT protein. An extensive SAR study of diverse benzodiazepine analogues is performed, which identifies potent, mutant-selective inhibitors with desirable physiochemical properties. Along with the SAR study, eleven crystal structures are solved for the L/V mutant alone and in complex with the representative benzodiazepine analogues providing precious information for structure-guided analogue design (figure 1B). The best compound shows about 200 nM potency, with >100-fold selectivity for the L/V mutant over WT. Through a variety of in vitro and cellular assays the capabilities of the optimized system is validated. Work for the bump-and-hole until this point is written up in an article which is submitted to a peer-reviewed journal and is currently under revision. Generation of cell line incorporating these mutation by CRISPR is on the way which will provide a setting closer to physiological condition.
In the PROTAC approach project, BET proteins are targeted by linking BET-inhibitor, JQ1, to E3 ligase VHL (Von Hippel–Lindau) ligand [2]. The researcher characterized and profiled the activity of these PROTAC molecules in cell and in vitro. The first PROTAC molecule reported by the host lab, MZ1, although based on a pan-selective BET-inhibitor scaffold, demonstrates a selective substrate degradation preference against BRD4 over BRD2 and BRD3. In a collaborative work within the Ciulli lab, first crystal structure of a PROTAC induced complex of E3-ligase with its target protein is reported [3]. A model explaining the observed selectivity is proposed and leads to the development structure-designed PROTAC, e.g. AT1, which shows exquisite selectivity for BRD4.
The researcher also leads a SAR study of another series of BET-targeting PROTAC molecules [4]. The researcher showed that by substituting the targeting recruitment warhead would result in a completely different selectivity profile against BET proteins. Extensive work on in vitro E3-PROTAC-BD complex formation was performed and the results were backed up by cellular protein degradation study.
Overview of the results
During the project period, an optimized bump-and-hole system on the BET BD has been established. It provides selective chemical tools for future cellular and in vivo target validation studies. The PROTAC approach demonstrates a strategy to achieve selective and efficient protein knock-down by small molecule. Structural basis for efficient PROTAC activity is established, supported by extensive SAR study both in vitro and in cell. This provides valuable knowledge for PROTAC design in the developing field.
The researcher’s work contributes to two peer-reviewed research papers on PROTAC [3,4] and one paper under revision on bump-and-hole . His contribution is also recognized by the field as he is invited as speaker at the 12th Annual Drug Discovery Chemistry Conference (2017) held in San Diego on Targeted Protein Degradation by Small Molecules. Knowledge gains in the project also enables him to co-author a review paper on the topic of chemical genetics in epigenetics [5].
1. Baud M.G.J. et al., Science 2014, 346(6209), 638-641.
2. Zengerle M. et al., ACS Chemical Biology 2015, 10(8):1770-7
3. Gadd M.S. et al. Nature Chemical Biology 2016, 13, 514–521.
4. Chan K.-H. et al. Journal of Medicinal Chemistry 2017, DOI: 10.1021/acs.jmedchem.6b01912
5. Runcie A. C. et al. Current Opinion in Chemical Biology 2016, 33, 186–194.
