Postis, V. | Search bioRxiv

bioRxiv2025

Spatially Resolved Determination of Small Molecule Protein Affinities by Hydrogen Deuterium Exchange Mass Spectrometry

Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) is an established tool in drug discovery, used to characterize target engagement and conformational dynamics, frequently used in both biopharmaceutical and small molecule development. Conventional HDX-MS experiments are performed at saturating ligand concentrations to generate a binding "footprint", where decreased solvent exchange reflects a local structural stabilization and/or reduced solvent accessibility upon binding. Here, we present an extended HDX-MS and HDX-MS/MS titration workflow with electron capture dissociation (ECD) fragmentation capable of estimating apparent dissociation constants (KDapp) at global, peptide and single amino acid resolution by fitting uptake-concentration relationships under EX2 exchange and Langmuir binding assumptions. The ability to determine affinity constants in a spatially resolved manner combined with the automation available in HDX-MS sample handling and data analysis enables quantitative mapping of ligand-protein interactions and provides a scalable approach for structure-activity relationship studies in drug discovery.

biophysics↗

bioRxiv2024

Characterisation of RNA guanine-7 methyltransferase (RNMT) using a small molecule approach

The maturation of the RNA cap involving guanosine N-7 methylation, catalyzed by the HsRNMT (RNA guanine-7 methyltransferase)-RAM (RNA guanine-N7 methyltransferase activating subunit) complex, is currently under investigation as a novel strategy to combat PIK3CA mutant breast cancer. However, the development of effective drugs is hindered by a limited understanding of the enzymes mechanism and a lack of small molecule inhibitors. Following the elucidation of the HsRNMT-RAM molecular mechanism, we report the biophysical characterization of two small molecule hits. Biophysics, biochemistry and structural biology confirm that both compounds bind competitively with cap and bind effectively to HsRNMT-RAM in the presence of the co-product SAM, with a binding affinity (KD) of approximately 1 M. This stabilisation of the enzyme-product complex results in uncompetitive inhibition. Finally, we describe the properties of the cap pocket and provided suggestions for further development of the tool compounds.

biochemistry↗