(M1030-01-03) Integrated High-Throughput Platform for Covalent Inhibitor Screening and Characterization

Purpose: Covalent inhibitors contain specific functional groups that can form covalent bonds with the corresponding sites of key mediating proteins in the formation of diseases^[1], causing their conformational changes, inhibiting their activity, and losing their pathological functions. The covalent bond with the target protein is more persistent and can achieve complete target occupancy at relatively low concentration. Therefore, covalent inhibitors have the advantages of prolonged efficacy, reduced dosing frequency, and higher selectivity. Covalently bound drugs are a rapidly developing discipline in the field of drug discovery. We have established a high-throughput screening and characterization platform for covalent binding drugs to provide technical support for drug researchers.

Methods: We take the KARS^G12C protein and its inhibitor sotorasib as an example. First, the differential scanning fluorescence (DSF) method was established for high-throughput screening of covalent inhibitors. By using programmed temperature increase, the protein unfolds at high temperature, and the fluorescent dye will bind with the exposed hydrophobic part. The data will be fitted with the Boltzmann equation to calculate the melting temperature (Tm) of the protein. Second, after the target protein and candidate drug bind, the shift of molecular weight can be used to determine whether a covalent bond has been formed by HRMS. Finally, the peptide mapping method using high-resolution mass spectrometry was established to identify which amino acid was binding to the candidate drug.

Results: KRAS^G12C and sotorasib results showed that ΔTm was observed from the melt curve plot. When the covalent drug binds with the target protein, the stability of the target protein is enhanced, and the melting temperature increases. The difference in melting temperatures of the two is expressed as ΔTm. The principle graph of DSF is shown in Figure 1. The DSF method has the advantages of low protein sample requirements, high throughput, and short analysis time. It is suitable for high-throughput candidate drug screening and target discovery. The MW of KRAS^G12C is 23034 Da. A mass shift of 560 Da was observed by using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) after incubating with sotorasib (as shown in Figure 2). Under this incubation condition, it was found that 100% of the protein was modified by sortorasib. The intact protein method is reproducible. LC-HRMS can provide whether there is binding, binding ratio, and other detailed information. The KRAS^G12C protein and sotorasib were incubated at 37°C. After digestion with trypsin, 100% sequence coverage can be achieved using LC-HRMS. The result is shown in Figure 3. The result indicates that sotorasib covalently binds to the cysteine of peptide LVVVGACGVGK. The retention time of this peptide is 58.37 min, and the confidence score is 100 points.

Conclusion: In this work, we have established a high-throughput screening and characterization platform based on DSF, intact protein and peptide mapping analysis, which applies to covalent inhibitors. The platforms show high-throughput, accuracy, stability, and rapidity.

References: [1] Lonsdale R, Ward R A. Structure-based design of targeted covalent inhibitors[J]. Chemical Society Reviews, 2018:10.1039.

Figure 1. Covalent binding of drug induces protein melting temperature (Tm) migration.

Figure 2. Intact protein result using LC-HRMS.

Figure 3. Peptide mapping of KRASG12C after covalent binding with sotorasib.