Covalent drugs have emerged as a powerful class of therapeutics in targeted therapy, offering several significant advantages, including high selectivity due to their precise binding to specific amino acid residues, prolonged duration of action through irreversible binding, and reduced likelihood of drug resistance by maintaining efficacy despite genetic mutations.
Extensive Protein Library: We possess a comprehensive library of target proteins, enabling us to establish and customize assays for new and unique protein targets quickly and efficiently.
Assay Development and Optimization: Our expert team can develop and optimize assay conditions to suit specific research needs, ensuring high sensitivity and accuracy in detecting covalent binding interactions.
High Throughput Capacity: With the capability to process approximately 200 samples per day, we provide rapid and reliable analysis to support your research timelines and goals.
Advanced Instrumentation: Utilizing state-of-the-art equipment, including the Vanquish Flex LC system and QE Plus high-resolution mass spectrometer (HRMS), we deliver detailed and high-quality data on covalent binding interactions.
The precise identification and quantification of covalent interactions between small molecules and target proteins are crucial for understanding drug efficacy and safety. A key aspect of this approach involves assessing the reactivity of candidate compounds with cellular nucleophiles. Among these, glutathione (GSH) is a critical tripeptide that plays a central role in cellular detoxification and drug metabolism.
The GSH binding assay allows researchers to evaluate the formation of drug-GSH adducts and to better understand the covalent binding properties of potential therapeutic agents. By leveraging advanced mass spectrometry techniques, this assay provides detailed insights into the covalent interactions and stability of drug candidates, guiding the optimization of lead compounds for enhanced therapeutic efficacy. Afatinib, a covalent inhibitor targeting EGFR.
To determine the kinetics of the interaction, we monitored the reaction time between GSH and Afatinib. Samples were analyzed at various time intervals to observe the formation of Afatinib-GSH adducts. This assay helped in understanding the rate at which Afatinib forms covalent bonds with GSH, providing insights into its reactivity and stability.
We utilized HRMS to analyze the fragmentation ion spectra of the Afatinib-GSH adduct. This detailed spectral analysis allowed us to identify and confirm the specific binding sites and the nature of the covalent bond formed. The high-resolution capabilities of the QE Plus HRMS provided precise molecular details, essential for understanding the binding mechanism at the atomic level.
To assess the stability and persistence of Afatinib in the presence of GSH, we measured the levels of Afatinib at different time points post-reaction. This assay enabled us to track the degradation or modification of Afatinib over time, ensuring a comprehensive understanding of its pharmacokinetic properties in a biological context.
A prime example of the power of mass spectrometry in covalent binding analysis is the identification of KRAS-G12C and AMG-510 conjugates. KRAS-G12C, a mutant form of the KRAS protein, is a key target in cancer therapy due to its role in driving tumor growth in various cancers. AMG-510 (sotorasib) is a pioneering covalent inhibitor designed to specifically bind to the KRAS-G12C mutation, irreversibly inhibiting its activity. Using mass spectrometry, we are able to accurately identify the covalent conjugates formed between AMG-510 and KRAS-G12C. This identification process involved detecting the specific mass shifts indicative of covalent bond formation between the inhibitor and the target cysteine residue on KRAS-G12C.
Covalent Binding Analysis of Acetylated KRAS-G12C. The mass spectrometry profiles reveal acetylated KRAS-G12C both before and after covalent modification with a compound. The graph shows the mass of the acetylated KRAS-G12C with and without the compound, indicating the acetylated KRAS-G12C with the AMG510 compound covalently attached.
We also provide covalent binding sample data using our expressed EGFR protein with Afatinib, WRN with reference compounds, and P53 and P53 Y220C with KG13. Contact us for detailed data and further information.
Our Kinact/Ki Assay Services provide comprehensive evaluation of covalent inhibitors, combining advanced mass spectrometry data analysis and model fitting to determine key kinetic parameters. This assay is crucial for understanding the reactivity and efficiency of covalent drugs.
The Kinact/Ki assay measures the inactivation rate constant (Kinact) and the inhibition constant (Ki) for covalent inhibitors. These parameters are essential for characterizing the potency and binding kinetics of a drug, providing insights into its potential efficacy and selectivity.
In a demonstration of our Kinact/Ki assay capabilities, we analyzed the covalent interaction between the KRAS-G12C mutant protein and the covalent inhibitor AMG510 (sotorasib). The assay involved the following steps:
Incubation and Sampling: KRAS-G12C was incubated with AMG510 at various time points (0s to 100s), allowing us to monitor the binding over time.
Mass Spectrometry Analysis: Samples were analyzed using HRMS, with data showing the formation of KRASG12C-AMG510 adducts at different incubation times.
Data Analysis and Model Fitting: The binding data were fitted to a kinetic model to determine the Kinact and Ki values. The final results indicated a Kinact/Ki value of 12961.04 M⁻¹s⁻¹, highlighting the high reactivity and efficiency of AMG510 in binding to KRAS-G12C.
Our services provide detailed analysis of specific amino acid residues involved in covalent drug binding. This service is essential for understanding the precise interactions between covalent inhibitors and their target proteins, aiding in the optimization and development of effective therapeutics.
In a recent analysis of the KRASG12C mutant protein with the covalent inhibitor AMG510, our services identified the exact binding site of AMG510 on the KRASG12C protein. The process involved:
Protein Digestion: KRASG12C protein was enzymatically digested into peptides.
Peptide Separation: High-resolution separation techniques isolated the relevant peptides.
Peptide Detection: Mass spectrometry detected the peptides, highlighting those modified by AMG510.
Data Interpretation: Analysis confirmed the covalent binding site on the cysteine residue (Cys12) of KRASG12C, crucial for the inhibitor's efficacy.
The accompanying Peptide Coverage Map illustrates the binding site of AMG510 on the KRASG12C peptide. This map shows the peptide sequence LVVVGACGVGK, where the cysteine residue (Cys7) forms a covalent bond with AMG510. The fragment ions are color-coded based on their ion intensity, providing a detailed structural resolution of 1.0 residues.
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