Structure-Based Design of a Macrocyclic PROTAC.

Andrea Testa,Scott J Hughes,Alessio Ciulli,Xavier Lucas,Jane E Wright

doi:10.1002/anie.201914396

Andrea Testa, Scott J Hughes + Show 3 more

Open Access

https://doi.org/10.1002/anie.201914396

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Abstract

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co‐crystal structure of macroPROTAC‐1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12‐fold loss of binary binding affinity for Brd4, macroPROTAC‐1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.

Highlights

Targeted protein degradation is a powerful new modality of chemical biology and drug discovery.[1,2] proteolysis-targeting chimeras (PROTACs) are bifunctional molecules composed of a ligand for a target protein of interest, and a ligand for an E3 ubiquitin ligase, joined by a flexible linker
Using isothermal titration calorimetry (ITC), fluorescence polarization (FP), and X-ray crystallography, we show that macroPROTAC-1 better discriminates between the recruitment of the second and the first bromodomain and extra-terminal motif (BET) bromodomains to VHL
Inspired and guided by the crystal structure of the ternary complex between our BET degrader MZ1, VHL, and Brd4BD2,[10] we designed a series of macrocyclic PROTACs with the aim to lock the PROTAC in the bound conformation (Figure 1 A)

Summary

Introduction

Knowledge of the PROTAC binding mode in a ternary complex, ideally from co-crystal structures, provides an opportunity to rationally design compounds to better fit and stabilize the newly created interface within the ternary complex, using structure-based drug design.[10,13,19,20] Our first crystal structure of the PROTAC MZ1 in complex with the E3 ligase VHL and its target, the second bromodomain (BD2) of the bromodomain and extra-terminal motif (BET) protein, Brd, revealed that the two ligand moieties of MZ1 (the VHL ligand, VH032, and the BET inhibitor, JQ1) lay in close spatial proximity within the ternary complexes.[10] We imagined that a macrocyclic PROTAC could be designed based on the crystal structure as a strategy to lock the PROTAC conformation in the bound state. Despite a greater than 10-fold loss in binary binding affinity for Brd, macroPROTAC-1 exhibits rapid and potent intracellular degradation of Brd, and cytotoxicity in BET-sensitive cancer cell lines that are comparable to MZ1

Results and Discussion

1: Brd4BD2

Conclusion

Conflict of interest