The study provides compelling evidence that BRB forms covalent adducts with CYP2D6 cysteine residues, resulting in irreversible enzyme inactivation. Specific cysteine residues, such as Cys443 and Cys296, are proposed as potential adduction sites based on prior literature. Could the authors clarify whether these specific sites were directly identified in this study through experimental validation, such as site-directed mutagenesis or advanced proteomics? If not, are there plans to employ techniques like tandem mass spectrometry or partial proteolysis coupled with high-resolution LC-MS/MS to pinpoint the exact modification sites in future investigations? Furthermore, while the study rules out ROS-mediated pathways using catalase and superoxide dismutase, could the authors elaborate on whether other ROS-related mechanisms or alternative adduction sites were systematically excluded to confirm the proposed metabolic pathway?
In Figure 2, you present two complementary experiments to identify the P450s involved in converting berberine to BRB:
A: Uses individual human recombinant P450 enzymes (CYP1A2, 2A6, etc.).
B: Uses rat liver microsomes (RLM) with chemical inhibitors selective for human P450s.
My concern is the cross-species comparison between these two panels. The conclusion that “CYP2D6 played a major role” (Section 3.1) is drawn from integrating results from a human recombinant system (Panel A) and a rat liver system (Panel B). However, the activity, abundance, and inhibitor sensitivity of P450 isoforms can differ significantly between human and rat systems.
Could you please help me understand the reasoning behind this experimental design?
Was there a specific rationale for using RLM in the inhibitor study instead of human liver microsomes or the same set of recombinant human enzymes?
Are the chemical inhibitors (e.g., quinidine for CYP2D6) known to have identical potency and selectivity in rat microsomes as they do in human systems? If not, could this affect the interpretation of the 80% inhibition seen in Figure 2B?
Without confirming that the rat ortholog of CYP2D6 is the primary metabolizer in the RLM system, is it possible that the strong inhibition by quinidine in rats reflects a different isoform involvement than in humans?
If the inhibitor data from the rat system does not directly translate to the human recombinant enzyme system, it might weaken the combined evidence that human CYP2D6 is the predominant enzyme for this biotransformation. This, in turn, could impact the foundational premise of the entire study, which focuses on the interaction between BRB and human CYP2D6.