Covalent Drugs DMPK Services

Intact protein technology uses high-resolution mass spectroscopy to determine the precise molecular weight of protein. When the target protein covalently binds to small molecular compounds, the molecular weight of the protein increases, which can directly characterize the binding situation. The method has the advantages of simple development, high throughput and good reproducibility. WuXi AppTec DMPK can provide screening services for covalent drugs based on HRMS, including screening related compounds such as covalent molecular glue and covalent PROTAC* (Proteolysis-targeting chimera), and identifying binding sites. 

Because of the possible reactivity between the chemical warhead and plasma protein, the conventional test methods may lead to low reliability of f_u value, especially those covalent drugs with serious nonspecific binding problems. By optimizing methods, such as low temperature, diluting plasma, ultracentrifugation or flux dialysis, we can determine the plasma protein binding rate of covalent drugs with low stability in plasma, and get a more reliable f_u value.

According to the characteristics of covalent drugs, other experiments were established to evaluate the distribution and metabolism of covalent drugs in vitro, such as HSA binding experiment, HSA stability experiment, GSH binding experiment and GSH stability experiment catalyzed by GST enzyme.

WuXi AppTec DMPK can provide comprehensive services of metabolite profiling and structure identification, analyze, separate and identify metabolites in biological matrix, and deeply understand the possible metabolic and clearance pathways of covalent drugs. Combined with the characteristics of covalent drugs, we mainly use GSH-containing liver microsomal incubation system to detect the metabolites of covalent drugs under the action of CYP enzyme, electrophilic warheads and/or products of reactive metabolites combined with GSH. Liver S9/cytosol and blood incubation system were used to investigate the electrophilic reaction products mediated by glutathione transferase. Plasma or albumin incubation system was used to evaluate the degree of covalent binding between covalent drugs and albumin.

WuXi AppTec DMPK can accurately determine the binding rate of covalent drugs and their metabolites or reactive metabolites intermediates with proteins by using radioactive labeling technology. Combined with HRMS and enzymatic hydrolysis technology, the possible forms of binding moiety can be analyzed, such as the binding of original drugs or metabolites, the types of proteins bound, and the binding sites.

Moreover, the ¹⁴C-labeled drugs can be directly synthesized in WuXi AppTec, and a reasonable labeling site can be designed according to the chemical structure, synthesis process and early unlabeled metabolite identification results.