In vitro metabolism of targeted covalent inhibitors and their thiol conjugates by gut microbiome from rats, mice, and humans.

Abstract

Targeted covalent inhibitor (TCI) represents a noncanonical class of small molecules that function via "inactivating" the target protein through the formation of drug-protein adducts. The electrophilic groups (warheads) embedded in the TCIs are essential for their activity while also being recognized as sites susceptible to metabolism by various enzymes and endogenous nucleophiles. Given the growing knowledge of gut microbiome-mediated drug metabolism and its impact on drug absorption, distribution, metabolism, and excretion, the fate of the reactive warhead-containing TCIs in the gut warrants further understanding. In this study, we selected unsubstituted terminal acrylamides (ibrutinib, sotorasib, and divarasib), β-substituted acrylamides (afatinib, neratinib, and dacomitinib), an α-substituted acrylamide (adagrasib), an alkynamide (acalabrutinib), and a salicylaldehyde (voxelotor) to investigate. An anaerobic in vitro approach was utilized using both fecal slurry and feces-outgrown bacteria from rats, mice, and humans. The results showed that double bond reduction was the major metabolism captured for terminal acrylamides, but the activity decreases significantly when α or β substitutions are present; acalabrutinib was stable; and voxelotor was efficiently reduced to a benzyl alcohol metabolite. Synthesized TCI-GSH adducts can be efficiently hydrolyzed sequentially to cysteine adducts, which are rather stable from further microbiome modifications. There were no apparent species differences between rats, mice, and humans qualitatively, while the reductase activity observed was generally higher in the human gut microbiome. This study provides insights into both enzymatic and nonenzymatic reactions of TCIs and their thiol conjugates in the gut environment, which can be translated to the understanding of their absorption, distribution, metabolism, and excretion behavior during drug development. SIGNIFICANCE STATEMENT: Understanding the gut microbiome metabolism of targeted covalent inhibitors and their thiol conjugates will help interpret absorption, distribution, metabolism, and excretion studies for new targeted covalent inhibitors in delineating that from human metabolism, predicting clearance pathways, and assessing the impact on absorption/reabsorption. The species difference information can inform proper preclinical species for better human translation in overall drug behavior. The experimental conditions developed from this work can also be adapted to study gut microbiome metabolism in general across different species.