Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California.
Amgen Research, Pharmacokinetics and Drug Metabolism, Amgen Inc., South San Francisco, California
Drug Metab Dispos. 2020 Jun;48(6):508-514. doi: 10.1124/dmd.119.089813. Epub 2020 Mar 19.
Experiments designed to identify the mechanism of cytochrome P450 inactivation are critical to drug discovery. Small molecules irreversibly inhibit P450 enzymatic activity via two primary mechanisms: apoprotein adduct formation or heme modification. Understanding the interplay between chemical structures of reactive electrophiles and the impact on CYP3A4 structure and function can ultimately provide insights into drug design to minimize P450 inactivation. In a previous study, raloxifene and N-(1-pyrene) iodoacetamide (PIA) alkylated CYP3A4 in vitro; however, only raloxifene influenced enzyme activity. Here, two alkylating agents with cysteine selectivity, PIA and pyrene maleimide (PM), were used to investigate this apparent compound-dependent disconnect between CYP3A4 protein alkylation and activity loss. The compound's effect on 1) enzymatic activity, 2) carbon monoxide (CO) binding capacity, 3) intact heme content, and 4) protein conformation were measured. Results showed that PM had a large time-dependent loss of enzyme activity, whereas PIA did not. The differential effect on enzymatic activity between PM and PIA was mirrored in the CO binding data. Despite disruption of CO binding, neither compound affected the heme concentrations, inferring there was no destruction or alkylation of the heme. Lastly, differential scanning fluorescence showed PM-treated CYP3A4 caused a shift in the onset temperature required to induce protein aggregation, which was not observed for CYP3A4 treated with PIA. In conclusion, alkylation of CYP3A4 apoprotein can have a variable impact on catalytic activity, CO binding, and protein conformation that may be compound-dependent. These results highlight the need for careful interpretation of experimental results aimed at characterizing the nature of P450 enzyme inactivation. SIGNIFICANCE STATEMENT: Understanding the mechanism of CYP3A4 time-dependent inhibition is critical to drug discovery. In this study, we use two cysteine-targeting electrophiles to probe how subtle variation in inhibitor structure may impact the mechanism of CYP3A4 time-dependent inhibition and confound interpretation of traditional diagnostic experiments. Ultimately, this simplified system was used to reveal insights into CYP3A4 biochemical behavior. The insights may have implications that aid in understanding the susceptibility of CYP enzymes to the effects of electrophilic intermediates generated via bioactivation.
设计用于鉴定细胞色素 P450 失活机制的实验对于药物发现至关重要。小分子通过两种主要机制不可逆地抑制 P450 酶活性：脱辅基蛋白加合物的形成或血红素修饰。了解反应性亲电试剂的化学结构之间的相互作用以及对 CYP3A4 结构和功能的影响，最终可以为最小化 P450 失活的药物设计提供深入了解。在之前的研究中，雷洛昔芬和 N-(1-蒽基)碘乙酰胺（PIA）在体外使 CYP3A4 烷基化；然而，只有雷洛昔芬影响酶活性。在这里，使用两种具有半胱氨酸选择性的烷化剂，PIA 和蒽马来酰亚胺（PM），来研究 CYP3A4 蛋白烷基化和活性丧失之间这种明显的化合物相关的不相关。测量了化合物对 1）酶活性，2）一氧化碳（CO）结合能力，3）完整血红素含量和 4）蛋白质构象的影响。结果表明，PM 具有很大的时间依赖性酶活性丧失，而 PIA 则没有。PM 和 PIA 之间对酶活性的不同影响反映在 CO 结合数据中。尽管 CO 结合受到破坏，但两种化合物均不影响血红素浓度，推断血红素没有被破坏或烷基化。最后，差示扫描荧光显示 PM 处理的 CYP3A4 导致诱导蛋白质聚集所需的起始温度发生变化，而用 PIA 处理的 CYP3A4 则没有观察到这种变化。总之，CYP3A4 脱辅基蛋白的烷基化可能对催化活性、CO 结合和蛋白质构象产生不同的影响，这可能与化合物有关。这些结果强调了在旨在表征 P450 酶失活性质的实验结果的解释时需要谨慎。意义陈述：了解 CYP3A4 时间依赖性抑制的机制对于药物发现至关重要。在这项研究中，我们使用两种半胱氨酸靶向亲电试剂来探究抑制剂结构的细微变化如何可能影响 CYP3A4 时间依赖性抑制的机制，并混淆对传统诊断实验的解释。最终，这个简化的系统被用来揭示对 CYP3A4 生化行为的深入了解。这些见解可能有助于理解 CYP 酶对通过生物活化产生的亲电中间体的影响的敏感性。
