Protein–Small Molecule Interactions in Native Mass Spectrometry

Abstract

Small molecule drug discovery has been propelled by the continual development of novel scientific methodologies to occasion therapeutic advances. Although established biophysical methods can be used to obtain information regarding the molecular mechanisms underlying drug action, these approaches are often inefficient, low throughput, and ineffective in the analysis of heterogeneous systems including dynamic oligomeric assemblies and proteins that have undergone extensive post-translational modification. Native mass spectrometry can be used to probe protein–small molecule interactions with unprecedented speed and sensitivity, providing unique insights into polydisperse biomolecular systems that are commonly encountered during the drug discovery process. In this review, we describe potential and proven applications of native MS in the study of interactions between small, drug-like molecules and proteins, including large multiprotein complexes and membrane proteins. Approaches to quantify the thermodynamic and kinetic properties of ligand binding are discussed, alongside a summary of gas-phase ion activation techniques that have been used to interrogate the structure of protein–small molecule complexes. We additionally highlight some of the key areas in modern drug design for which native mass spectrometry has elicited significant advances. Future developments and applications of native mass spectrometry in drug discovery workflows are identified, including potential pathways toward studying protein–small molecule interactions on a whole-proteome scale.

Multiplexed Screening of Thousands of Natural Products for Protein–Ligand Binding in Native Mass Spectrometry

“…post-translational modifications, salt/ligand binding) on macromolecular structure. 30 It is expected that this method can be readily applied within target-based NP drug discovery workflows to confidently identify small molecule binders without the need for the fractionation of extracts prior to (or following) screening 4 . This should facilitate highly efficient hit dereplication and potentially enable the rapid and rational prioritisation of specific NPs for further investigation based on their structural features.…”

Multiplexed screening of thousands of natural products for protein-ligand binding in native mass spectrometry

“…30 It is expected that this method can be readily applied within target-based NP drug discovery workflows to confidently identify small molecule binders without the need for the fractionation of extracts prior to (or following) screening. 4 This should facilitate highly efficient hit dereplication and potentially enable the rapid and rational prioritisation of specific NPs for further investigation based on their structural features. Using this approach, it is anticipated that large groups of NP extracts that collectively contain more than 100,000 compounds could be screened per day, accelerating target-based drug discovery campaigns without the resource intensive collection, curation, and storage of large libraries of pure natural products or fractionated NP extracts.…”

“…Native, non-denaturing mass spectrometry (MS) has served as a valuable approach for the rapid, direct, and sensitive measurement of protein-small molecule complexes, revealing valuable information regarding the stoichiometry, thermodynamics and kinetics of ligand binding. [4][5][6][7] Due to the ability to directly detect proteinligand complexes, native MS measurements are highly resistant to common assay interferences encountered during pharmaceutical screening such as cytotoxicity, compound aggregation, and redox cycling. 8 Consequently, native MS experiments are typically employed for hit validation.…”

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

“…Native mass spectrometry (MS) has the potential to distinguish allosteric mechanisms by more directly probing bound-state distributions. 6,[16][17][18] Unlike aggregate measurements of average fractional saturation, native MS can directly probe the populations of states with different numbers of bound ligands. 18 The mass (and mass-to-charge ratios, m/z) of a macromolecular complex is exactly determined by the molecular weight of its constituents.…”