Identification of Non-covalent Drug Pockets

Identification of Non-Covalent Drug Pockets

In the process of small molecule drug development, it is crucial to determine the binding mode between the drug and the target protein. On the one hand, it helps to understand the mechanism of the drug actions, On the other hand, it provides data support its subsequent structural optimization. Structural biology techniques, including X-ray, cryo-EM, NMR, etc., have been widely used in determining drug binding models, especially high-resolution drug-protein co-crystal structures, which can greatly help drug structure optimization. However, protein structure analysis has always been a challenging task in life science research, especially for membrane protein targets such as GPCRs and ion channels.

To obtain the structure of proteins, it requires a series of processes, including protein expression and purification, manual screening of drug-protein interactions, optimization of protein crystallization conditions, data collection from crystals, and structure resolution. These procedures demand significant time and financial investments.

At the cellular level, knowing the precise mechanism of action for drugs and target proteins is the aspiration of drug developers. This avoids the loss of protein structure during the purification process and the alse positives caused by artificially screened buffer systems and high concentration drug-protein conditions.

Technical Principles

The principle is to use bioactive photoaffinity chemical probes (similar to the activity of drug molecules) to incubate them with disease-related cells or tissues at the normal concentration of the drug. Then, the non-covalent interaction between the drug molecular probe and the protein target is converted into a covalent interaction through in-situ rapid photo-cross-linking. Finally, after target protein enrichment, enzymatic digestion to release unmodified peptides, and selective enrichment of modified peptides with drug molecular probes, peptide information can be quickly determined with the help of high-resolution biological macromolecule mass spectrometry.

Since chemical probes can only cross-link in situ with spatially adjacent peptides, that is, peptides near the binding pocket, not only can target information be determined through this peptide information, but drug binding pocket information can also be obtained, and then with the help of molecular docking, the binding model of the drug and the target protein can be quickly obtained for subsequent experimental verification and use.