Covalent drugs have emerged as a powerful class of therapeutics in targeted therapy. Their advantages include high selectivity through precise binding to specific amino acid residues, prolonged activity through irreversible binding, and the potential to retain efficacy against certain genetic mutations.
A representative application of mass spectrometry in covalent-binding analysis is the identification of KRAS-G12C and AMG-510 conjugates. KRAS-G12C is a cancer-relevant mutant form of KRAS, while AMG-510 (sotorasib) is a covalent inhibitor designed to bind specifically and irreversibly to the cysteine residue of KRAS-G12C.
Mass spectrometry detects the resulting covalent conjugate through specific mass shifts that indicate bond formation between AMG-510 and KRAS-G12C.
Covalent binding analysis of acetylated KRAS-G12C. The mass spectrometry profiles show acetylated KRAS-G12C before and after covalent modification. The comparison indicates the presence of acetylated KRAS-G12C with AMG-510 covalently attached.
ICE Bioscience also provides covalent-binding sample data using expressed EGFR protein with Afatinib, WRN with reference compounds, and P53 or P53 Y220C with KG13. Contact us for detailed data and further information.
Our Kinact/Ki Assay Services combine mass spectrometry data analysis and model fitting to determine key kinetic parameters for covalent inhibitors. These parameters help characterize inhibitor reactivity, potency, and binding kinetics.
The assay measures the inactivation rate constant (Kinact) and inhibition constant (Ki) for covalent inhibitors. In a representative study, the covalent interaction between KRAS-G12C and AMG-510 (sotorasib) was analyzed through the following workflow.
Incubate the target protein with the covalent inhibitor and collect samples at defined time points.
Analyze intact protein mass and covalent-conjugate formation using mass spectrometry.
Fit the time-dependent data to determine Kinact and Ki.
Our services identify the specific amino acid residues involved in covalent drug binding, supporting mechanistic understanding, lead optimization, and development of effective covalent therapeutics.
In an analysis of KRAS-G12C with AMG-510, the exact binding site of AMG-510 on the KRAS-G12C protein was identified through protein digestion, peptide separation, peptide detection, and data interpretation.
Digest the protein sample to generate peptides for site-level analysis.
Separate peptides before high-resolution mass analysis.
Detect modified peptides and fragment ions associated with covalent binding.
Interpret peptide coverage and fragment-ion data to assign the binding residue.
The peptide coverage map illustrates AMG-510 binding to the KRAS-G12C peptide sequence LVVVGACGVGK. The cysteine residue (Cys7) forms a covalent bond with AMG-510. Fragment ions are color-coded according to ion intensity, providing structural resolution at the residue level.
Access a comprehensive protein library for rapid, customized assay development tailored to specific protein targets.
Experienced scientists optimize assays for sensitivity and accuracy in detecting covalent-binding interactions.
Process approximately 200 samples daily to support fast and reliable research timelines.
Use instrumentation including the Vanquish Flex LC system and QE Plus HRMS for precise covalent-binding analysis.
The GSH binding assay evaluates the formation of drug–GSH adducts and supports characterization of the covalent-binding properties of potential therapeutic agents. Using high-resolution mass spectrometry, the assay provides information on covalent-interaction formation and stability to guide lead-compound optimization. Afatinib is used as an example of a covalent inhibitor targeting EGFR.
Reaction samples were analyzed at different time intervals to monitor the formation of Afatinib–GSH adducts. This experiment helps evaluate the rate of covalent-bond formation and provides information about compound reactivity and stability.
High-resolution mass spectrometry was used to analyze fragmentation-ion spectra of the Afatinib–GSH adduct. This analysis supports identification of binding sites and characterization of the covalent bond. The QE Plus HRMS provides molecular information for evaluating the binding mechanism.
Afatinib levels were measured at different time points after reaction with GSH to evaluate compound stability and persistence. The assay tracks compound degradation or modification over time and supports characterization in a biological context.
We value your inquiries and are here to provide you with tailored solutions for your drug discovery and development needs. Whether you have questions, require more information, or are interested in discussing potential collaborations, our team of experts is just a message away.
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