Lead Generation Strategies

Quantifying ternary complex formation, essential for targeted protein degradation by PROTACs and molecular glues, remains challenging. We here present a proximity binding assay that simultaneously measures binary and ternary interaction kinetics using a biosensor. A Y-shaped DNA scaffold brings target proteins and E3 ligase receptors into proximity, enabling detection of ternary complexes by FRET and binary binding by fluorescence quenching, supporting real-time analysis with minimal sample consumption.

Despite the importance of the POI-PROTAC-E3 ternary complex and higher-order interactions (e.g., the POI-PROTAC-E3-E2-Ub multiprotein complex) in PROTAC discovery and development, methods to capture these species at scale are lacking. We recently leveraged the benefits of native mass spectrometry to establish a ‘show all’ approach to address this gap. This presentation will describe the approach and the potential complementarity with medicinal chemistry and other structural biology and biochemical methods.

PARP-mediated poly(ADP-ribose) formation recruits and organises DNA repair proteins, while PARG-mediated PAR degradation terminates repair and preserves genomic integrity. Guided by x-ray structures of PDD00017272 bound to PARG, we pursued covalent inhibition of nearby Cys872 to address the challenging catalytic pocket and long PARG half-life. Structure-guided hydrazide acrylamide inhibitors showed biochemical activity but limited cellular potency and plasma stability. Subsequent scaffold hopping, new exit-vector design, and incorporation of a bicyclo[1.1.0]butane carboxamide warhead yielded FORX-425, a potent covalent PARG inhibitor with strong cellular activity, high plasma stability, and promising mouse pharmacokinetics, highlighting BCB warheads for challenging pockets.

The kinase MPS1 is a pivotal component in the cell division process and reversible inhibitors have been evaluated in clinical trials for the treatment of aggressive types of solid tumors. This talk will cover the development and comprehensive structural, biochemical, and cellular characterization of the first MPS1 covalent inhibitor. This proof of covalent ligandability alongside promising biological results will guide future drug discovery efforts towards MPS1-targeted cancer therapies.

The basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor MITF is a melanoma oncogene, but considered undruggable as direct targeting is unprecedented. We have identified fragments binding to the DNA binding domain of MITF and optimised them to sub-micromolar ligands by fragment merging. Detailed structural and biophysical validation will be presented. NMR studies and MD simulations indicate that the interconversion of kinked and straight helices is slowed down by compound binding.