ICE Bioscience has pioneered studies utilizing mass spectrometry (MS) for covalent screening through techniques such as intact protein MS or peptide mapping. These cutting-edge methods enable comprehensive analysis of protein interactions and modifications, crucial for drug discovery and development. Through intact protein MS, we accurately identify and characterize covalent binding events between small molecules and target proteins. Additionally, peptide mapping allows for detailed analysis of protein structure and post-translational modifications, providing valuable insights into drug-protein interactions. With our expertise in MS-based covalent screening and the Q Exactive Plus system, ICE Bioscience offers tailored solutions to accelerate drug discovery efforts and enhance understanding of molecular mechanisms underlying therapeutic interventions.

KRAS-G12C and AMG-510 Conjugates Identification

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.

Covalent inhibitors are recognized as an important component in drug discovery and therapeutics. Since the first appearance of covalent inhibitors in the late 18th century, the field has advanced significantly and currently about 30% of the marketed drugs are covalent inhibitors. The numerous advantages of covalent inhibitors are counteracting the initial concerns regarding potential off-target toxicity. Thus, continuous research, especially for cancer targets is reported[1].

Covalent Inhibitors are usually small molecules that bind to enzymes and inactivate them temporarily or permanently. In general, covalent inhibition is a two-step process[2].First, an inhibitor reversibly associates with the target enzyme, by virtue of  which the chemical warhead of the inhibitor comes within a close  proximity of a targeted reactive amino acid residue of the enzyme. In the second step, reaction occurs between the two reactive entities in the inhibitor and the enzyme respectively to form a covalent bond. Reversible inhibitors differ from covalent inhibitors in that they do not involve the second step[3].

The use of small molecules as covalent inhibitors to target functionally critical enzymes in cells has found its implementation since late 19th century when Bayer started manufacturing aspirin as a painkiller and an anti-inflammatory drug.

1.Irreversible Covalent Inhibitors of Epidermal growth factor receptor (EGFR) 

Epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase family. It is involved in embry ogenesis and stem cell division, and is implicated in cell proliferation, mitosis, and cancer development. Over expression or increased activity of wild-type EGFR protein can lead to cell proliferation, migration, survival, and anti-apoptosis through signaling cascades, which are strongly associated with the occurrence and development of many cancers, such as NSCLC, breast cancer, glioma, head and neck cancer, cervical cancer, and bladder cancer.Therefore, EGFR has become a promising target for the design and development of anticancer drugs.

 Afatinib is the first covalent EGFR inhibitor approved by the FDA for lung cancer, and is a second-generation EGFR TKI, which was marketed in July 2013. Similar to the first-generation EGFR TKI, afatinib forms hydrogen bonds to the main chain of Met793 in the hinge region and interacts with hydrophobic regions.The furanyl group is exposed to the solvent, and the 3-chloro-4-fluorophenyl group is located near the “gatekeeper” residue.

2.Covalent Inhibitors for KRAS mutants

Mutations of the two KRAS isoforms occur in 60% of pancreatic, 34% of colorectal, and 16% of lung cancers Being one of the most common KRAS mutations leading to cancer, the G12C mutation is particularly interesting in the sense that it has an active non-native cysteine residue that can be easily targeted for covalent inhibition without affecting wild type KRAS. 

Sotorasib (AMG-510). In May 2021, the U.S. Food and Drug Administration (FDA) granted accelerated approval to Lumakras (sotorasib, AMG-510), a targeted anticancer drug, for the treatment of NSCLC patients with a KRASG12C mutation. It also became the first targeted drug for the treatment of KRAS gene mutation in the world, breaking the “undruggable” dilemma and marking a milestone in medical history. 

3.Covalent p53 modulators

P53 is a crucial protein that regulates the cell cycle and acts as a tumor suppressor.205 Studies have shown that approximately half of all human cancers, including serous ovarian cancer, lung squamous cell cancer, lung small cell cancer, triple-negative breast cancer, and squamous esophageal cancer, have alterations in the p53 gene, resulting in a loss of p53 function or decreased p53.

Expression P53 can be categorized as mutant type or wild type, with mutant p53 promoting tumorigenesis and wild-type p53 having broadspectrum tumor inhibition.TP53 mutations typically reduce  the expression of p53 protein or produce inactive variants, thus  compromising its cancer-inhibiting properties. In 2022, Kevan M.Shokat’s team continued their research and development work on the KRASG12S mutant and developed a small molecule covalent inhibitor of p53-Y220C mutant, known as KG13 . This inhibitor is specifically designed to bind to the p53 Y220C mutant, which restores the thermal stability of p53 protein to the level of wild-type p53 protein and activates the expression of downstream genes[4].

The irreversibility of covalent conjugation makes MS a suitable tool for covalent screening  characterization. MS allows label-free, direct detection of the native protein and the covalent adduct in the sample, providing relative quantification of target engagement. 

Several studies using MS to covalent screening by intact protein MS or peptide mapping have developed by ICE Bioscience.

[1]Covalent inhibitors: a rational approach to drug discovery

[2] T. A. Baillie, Angew Chem Int Ed Engl 2016, 55(43), 13408-13421.

[3]Boike, L. et al. Advances in covalent drug discovery. Nat. Rev. Drug Discov. 21, 881–898 (2022).

[4]Xie, X., Yu, T., Li, X., Zhang, N., Foster, L. J., Peng, C., Huang, W., & He, G. (2023). Recent advances in targeting the "undruggable" proteins: from drug discovery to clinical trials. Signal transduction and targeted therapy, 8(1), 335. https://doi.org/10.1038/s41392-023-01589-z