Pharmacokinetics, Dynamics and Metabolism and ‡Worldwide Medicinal Chemistry, Pfizer Inc. , Groton, Connecticut 06340, United States.
Chem Res Toxicol. 2013 Nov 18;26(11):1739-45. doi: 10.1021/tx400301q. Epub 2013 Oct 28.
Despite several advantages of covalent inhibitors (such as increased biochemical efficiency, longer duration of action on the target, and lower efficacious doses) over their reversible binding counterparts, there is a reluctance to use covalent inhibitors as a drug design strategy in pharmaceutical research. This reluctance is due to their anticipated reactions with nontargeted macromolecules. We hypothesized that there may be a threshold limit for nonspecific covalent binding, below which a covalent binding drug may be less likely to cause toxicity due to irreversible binding to off-target macromolecules. Estimation of in vivo covalent binding burden from in vitro data has previously been used as an approach to distinguish those agents more likely to cause toxicity (e.g., hepatotoxicity) via metabolic activation to reactive metabolites. We have extended this approach to nine covalent binding drugs to determine in vitro covalent binding burden. In vitro covalent binding burden was determined by incubating radiolabeled drugs with pooled human hepatocytes. These data were scaled to an estimate of in vivo covalent binding burden by combining the in vitro data with daily dose. Scaled in vivo daily covalent binding burden of marketed covalent drugs was found to be under 10 mg/day, which is in agreement with previously reported threshold value for metabolically activated reversible drugs. Covalent binding was also compared to the intrinsic reactivities of the covalent inhibitors assessed using nucleophiles glutathione and N-α-acetyl lysine. The intrinsic reactivity did not correlate with observed in vitro covalent binding, which demonstrated that the intrinsic reactivity of the electrophilic groups of covalent drugs does not exclusively account for the extent of covalent binding. The ramifications of these findings for consideration of using a covalent strategy in drug design are discussed.
尽管共价抑制剂（如增加生化效率、对靶标的作用持续时间更长、有效剂量更低）相对于它们的可逆结合对应物具有几个优势，但制药研究中仍不愿将共价抑制剂用作药物设计策略。这种不情愿是由于它们预期会与非靶向大分子发生反应。我们假设可能存在非特异性共价结合的阈值限制，低于该限制，由于与非靶标大分子的不可逆结合，共价结合药物不太可能引起毒性。先前已经使用从体外数据估计体内共价结合负担作为区分那些更有可能通过代谢活化产生反应性代谢物而导致毒性（例如肝毒性）的药物的方法。我们已经将这种方法扩展到九种共价结合药物，以确定体外共价结合负担。通过将放射性标记的药物与混合人肝细胞孵育来确定体外共价结合负担。通过将体外数据与每日剂量相结合，将这些数据扩展到体内共价结合负担的估计值。已发现上市共价药物的体内每日共价结合负担低于 10mg/天，这与先前报道的代谢激活可逆药物的阈值值一致。还比较了共价抑制剂的固有反应性与使用亲核试剂谷胱甘肽和 N-α-乙酰赖氨酸评估的共价抑制剂的固有反应性。固有反应性与观察到的体外共价结合没有相关性，这表明共价药物的亲电基团的固有反应性并不能完全说明共价结合的程度。讨论了这些发现对在药物设计中考虑使用共价策略的影响。
