Screening/Small-Molecule–Protein Interactions

The screening of compound libraries by mass spectrometry is particularly attractive because of its direct readout and the fact that it is truly label-free. In the past few years, covalent enzyme inhibitors have seen a resurgence in drug discovery. Our state-of the-art covalent warhead platform is focused on the discovery and characterization of novel covalent inhibitors.

  • Mass spectrometry as the readout for biochemical assays monitors the conversion of substrates with native sequences to products without the requirement for substrate modifications or indirect detection methods, just by measuring their unique masses. The high-throughput capabilities of MALDI MS allow for screening of larger library sizes (100,000 to 1 million), whereas LC–MS (RapidFire™) is ideally used as an orthogonal screening method or for smaller compound libraries.
  • Comparison of screening results on dose-response (DR) level of MALDI MS and HTRF assay

  • Our covalent warhead platform covers the complete screening process:
    We use MALDI MS and short model peptides containing the reactive amino acid of interest (e.g., cysteine) to test members of a new or unknown covalent warhead library for reactivity. The actual screening process is based on intact mass measurement of the target protein after the incubation of covalent warhead library members at fixed incubation times and protein-to-compound ratio. A robust electrospray time-of-flight instrument is used for this purpose. We further validate hits in a time course fashion and use—if available—the protein of interest, with the targeted amino acid mutated as a negative control. Alternatively, we perform peptide mapping experiments to pinpoint the modified amino acids on the target protein. In order to prioritize hits for the lead-optimization process, the determination of the kinact/Ki value acts as a critical parameter, which is basically a rate constant describing the efficiency of covalent bond formation.

  • Native mass spectrometry is a powerful method of analyzing non-covalent-binding events. While non-covalent interactions are lost in conventional mass spectrometry, they are preserved in native mass spectrometry, allowing for the measurement of protein–ligand and protein–protein interactions. Native MS allows us to analyze binding stoichiometry to determine competitive versus cooperative binding modes and also to study protein complexes.

Using native mass spectrometry to characterize non-covalent protein–small molecule interactions.

References

Eaton, JK., et al., Selective covalent targeting of GPX4 using masked nitrile–oxide electrophiles. Nat Chem Biol (2020)

Mortier, J., et al., Computationally Empowered Workflow Identifies Novel Covalent Allosteric Binders for KRASG12C. ChemMedChem (2020)

Lemos, C., et al., Identification of Small Molecules that Modulate Mutant p53 Condensation. iScience (2020)

Orsi, DL., et al., Discovery and Structure-Based Design of Potent Covalent PPARγ Inverse-Agonists BAY-4931 and BAY-0069. J Med Chem (2022)

Orsi, DL., et al., Discovery and characterization of orally bioavailable 4-chloro-6-fluoroisophthalamides as covalent PPARG inverse-agonists. Bioorg Med Chem (2023)