Enhancing therapeutic potential with bivalent molecules

This article is based on a poster originally authored by Tayo Alleyne-Weir, James Craswell, William Richardson, Eleftheria Arvaniti, Phil Leonard, Phil Rawlins, Nicholas Bland and Trevor Askwith, which was presented at ELRIG Drug Discovery 2024 in affiliation with Domainex Ltd.

This poster is being hosted on this website in its raw form, without modifications. It has not undergone peer review but has been reviewed to meet AZoNetwork's editorial quality standards. The information contained is for informational purposes only and should not be considered validated by independent peer assessment.

Introduction

• Bivalent molecules are heterobifunctional molecules with three components: a protein-of-interest (POI) binding moiety, a linker, and an E3 ubiquitin ligase warhead.
• Bivalent molecules can hijack the ubiquitin-protease system (UPS) which results in the degradation of the POI.
• Mechanism of action:
  • Recruits POI and E3 ligase to form a ternary complex
  • Ternary complex formation facilitates the polyubiquitination of the POI through proximity to an E3 ligase
  • The ubiquitinated POI is then trafficked to the proteasome for degradation, recycling the bivalent molecule to target another copy of the POI.

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

• This case study uses MZ1 and dBET1; bivalent molecules that connect a ligand for BRD3-BD2 (or other BRD proteins) and either VHL or CRBN warheads
• BRD3 is a member of the bromodomain and extra-terminal motif (BET) protein family and a therapeutic target for various diseases, including cancer
• Cereblon (CRBN) and von Hippel-Lindau (VHL) commonly utilize E3 ligases in bivalent molecule development.
• BRD3-BD2, VHL, and CRBN proteins have all been prepared by Domainex for use in this work

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

Spectral shift

• Fluorescence-based biophysical technique used to determine ligand binding
• Requires modification of a target protein with NanoTemper’s 2^nd generation RED dye
• The dye’s chemical environment can be affected directly when the ligand binds in close proximity or through confirmational changes induced by ligand binding, which cause a shift in the emission wavelength
• K_D derived by plotting the ratiometric measurement (FI 670 nm / 650 nm) against ligand concentration
• Generally, a better S/N ratio than MST and TRIC with less sensitivity to aggregates

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

Grating-coupled interferometry (GCI)

• Label-free rapid kinetic assessment for binding characterization
• Creoptix WAVEdelta system uses (GCI) – analogous to Surface Plasmon Resonance (SPR)
  • Light travels over the entire length of the chip rather than a single point
  • More binding events contribute to the signal, making the system more sensitive
• Novel microfluidics that are part of the disposable biosensor cartridge
  • Enables measurements of very fast off-rates up to 10 s^-1
  • Amenable to crude samples, harsh chemicals, and particles up to 1000 nm
• Proprietary waveRAPID (Repeated Analyte Pulses of Increasing Duration) method

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

Proximity-based biochemical assays

• Homogenous, no-wash immunoassays with high sensitivity and wide dynamic ranges.
• Simple to adapt to high throughput and automated processes.
• Excitation of the donor bead at 680 nm generates singlet oxygen molecules.
• If the donor is near the acceptor, the generated singlet oxygen can cause a cascade of reactions leading to chemiluminescence at 615 nm.
• It can be used for sandwich or competition assays.

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

Image Credit: Image courtesy of Tayo Alleyne-Weir et al., in partnership with ELRIG (UK) Ltd.

Summary

• Targeted Protein Degradation (TPD) represents a transformative approach to drug discovery, offering an innovative alternative to traditional small-molecule inhibitors.
• Bivalent molecules in TPD have enhanced the efficiency and durability of this therapeutic method
• At Domainex, advanced biophysical and biochemical techniques have been successfully employed to characterize molecular interactions:
  • Spectral Shift
  • Grating-Coupled Interferometry (GCI)
  • Proximity-based FRET (Alpha/OMEGA)
• These techniques accurately quantify complex affinities, advancing understanding of TPD and its therapeutic potential.
• Domainex can use these techniques to support diverse drug discovery initiatives and therapeutic modalities.

About Domainex

Domainex is a fully integrated drug discovery CRO based near Cambridge, UK. It serves pharmaceutical, biotechnology, academic and patient foundations globally. Domainex’s drug discovery service business was established in 2001 and since that time has continued to expand to serve a wider range of international clients including UCB, FORMA Therapeutics, St George’s University, and The Institute of Cancer Research.

About ELRIG (UK) Ltd.

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