The Development and Application of Biophysical Assays for Evaluating Ternary Complex Formation Induced by Proteolysis Targeting Chimeras (PROTACS)

Summary

Here we describe protocols for the biophysical characterization of ternary complex formation induced by proteolysis targeting chimeras (PROTACS) that involve the ubiquitin ligases Von Hippel-Lindau E3 ligase (VHL) and Cereblon (CRBN). Biophysical methods illustrated herein include surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC).

Abstract

E3 ligases and proteins targeted for degradation can be induced to form complexes by heterobifunctional molecules in a multi-step process. The kinetics and thermodynamics of the interactions involved contribute to efficiency of ubiquitination and resulting degradation of the protein. Biophysical techniques such as surface plasmon resonance (SPR), biolayer interferometry (BLI), and isothermal titration calorimetry (ITC) provide valuable information that can be used in the optimization of those interactions. Using two model systems, a biophysical assay tool kit for understanding the cooperativity of ternary complex formation and the impact of the 'hook effect' on binding kinetics was established. In one case, a proteolysis targeting chimera (PROTAC) molecule that induced ternary complex formation between Brd4^BD2 and VHL was evaluated. The heterobifunctional molecule, MZ1, has nM affinities for both the Brd4^BD2 protein (SPR K_D = 1 nM, ITC K_D = 4 nM) and the VHL complex (SPR K_D = 29 nM, ITC K_D = 66 nM). For this system, robust SPR, BLI, and ITC assays were developed that reproduced published results demonstrating the cooperativity of ternary complex formation. In the other case, a molecule that induced ternary complexes between a 46.0 kDa protein, PPM1D, and cereblon [CRBN (319-442)] was studied. The heterobifunctional molecule, BRD-5110, has an SPR K_D = 1 nM for PPM1D but much weaker binding against the truncated CRBN (319-442) complex (SPR K_D= ~ 3 µM). In that case, the binding for CRBN in SPR was not saturable, resulting in a "hook-effect". Throughput and reagent requirements for SPR, BLI, and ITC were evaluated, and general recommendations for their application to PROTAC projects were provided.

Introduction

The polyubiquitination of proteins in the cell is a tightly regulated process that involves enzymes in the Ubiquitin Ligase family^1,2. The terminal enzymes in the pathway are the E3 ubiquitin ligases that covalently attach ubiquitin molecules to their protein-binding partners³. The polyubiquitination of those protein binding partners targets them for proteolytic degradation by the proteasome⁴. This system is part of the protein homeostasis process that has been therapeutically leveraged to induce the degradation of proteins involved in disease^5<....

Protocol

All the proteins were overexpressed in E.coli with good yield and purity (>80%) following the literature protocols¹⁸. Biotinylation was carried out using a BirA-catalyzed reaction¹⁸. All small molecules were prepared at 1 mM stock solutions in 100% DMSO. The procedures described herein do not require specialized laboratory safety equipment or precautions. Standard laboratory personal protective equipment (PPE) should be used (i.e., lab coat, safety goggles, and.......

Discussion

Biophysical characterization of the binary and ternary interactions between PROTAC molecules and their protein binding partners can provide unique and complementary insights relative to widely used cellular systems. Understanding the affinity between each warhead of a PROTAC molecule and its protein binding partners can help guide medicinal chemistry efforts toward the optimization of those interactions. Previously published crystal structures of ternary PROTAC complexes have revealed that atoms in the linker region can .......

Materials

Name	Company	Catalog Number	Comments
96-plate	Greiner	655076	flat-bottom, black plates used In BLI experiments
96-well plate	Nunc	73520-120	Plate use for ITC sample preparation
96-well plate	Greiner	650101	Plate used to prepare samples for SPR experiments
Auto iTC200 micro-calorimeter	Malvern Panalytical		Instrument used to perform ITC experiments. Product discontinued.
Biacore S200	Cytiva	29136649	Instrument used to perform SPR experiments
MZ1	ProbeChem	PC-60099	PROTAC that binds to VHL and Brd4BD2
NTA sensor chip	Cytiva	BR100532	SPR chip used to perform SPR experiments involving PPM1D
Octet Red-384	Sartorius		Instrument used to perform BLI experiments. Product discontinued.
Plate cover	Malvern	PQA0001	Cover for Nunc 96-well plate (73520-120)
Plate cover	Cytiva	28975816	Plate cover for Greiner plate (650101)
Series S SA sensor chip	Cytiva	BR100531	SPR chip used to perform SPR experiments involving MZ1:VHL:BRD4
Streptavidin (SA) Dip and Read Biosensors	Sartorius	18-509	Coated sensors used in BLI experiments