Department of Molecular Medicine, Scripps Research, La Jolla, California, USA.
Department of Molecular Medicine, Scripps Research, La Jolla, California, USA.
J Biol Chem. 2022 May;298(5):101909. doi: 10.1016/j.jbc.2022.101909. Epub 2022 Apr 6.
Cytochrome P450 3A4 and 3A5 catalyze the metabolic clearance of a large portion of therapeutic drugs. Azamulin is used as a selective inhibitor for 3A4 and 3A5 to define their roles in metabolism of new chemical entities during drug development. In contrast to 3A4, 3A5 exhibits homotropic cooperativity for the sequential binding of two azamulin molecules at concentrations used for inhibition. To define the underlying sites and mechanisms for cooperativity, an X-ray crystal structure of 3A5 was determined with two azamulin molecules in the active site that are stacked in an antiparallel orientation. One azamulin resides proximal to the heme in a pose similar to the 3A4-azamulin complex. Comparison to the 3A5 apo structure indicates that the distal azamulin in 3A5 ternary complex causes a significant induced fit that excludes water from the hydrophobic surfaces of binding cavity and the distal azamulin, which is augmented by the stacking interaction with the proximal azamulin. Homotropic cooperativity was not observed for the binding of related pleuromutilin antibiotics, tiamulin, retapamulin, and lefamulin, to 3A5, which are larger and unlikely to bind in the distal site in a stacked orientation. Formation of the 3A5 complex with two azamulin molecules may prevent time-dependent inhibition that is seen for 3A4 by restricting alternate product formation and/or access of reactive intermediates to vulnerable protein sites. These results also contribute to a better understanding of sites for cooperative binding and the differential structural plasticity of 3A5 and 3A4 that contribute to differential substrate and inhibitor binding.
细胞色素 P450 3A4 和 3A5 催化了大部分治疗药物的代谢清除。阿扎美伦被用作 3A4 和 3A5 的选择性抑制剂，以在药物开发过程中定义它们在新化学实体代谢中的作用。与 3A4 不同，3A5 在用于抑制的浓度下表现出对两个阿扎美伦分子顺序结合的同型协同作用。为了定义协同作用的潜在位点和机制，确定了两个阿扎美伦分子在活性位点的 3A5 的 X 射线晶体结构，它们以反平行取向堆叠。一个阿扎美伦位于靠近血红素的位置，其构象类似于 3A4-阿扎美伦复合物。与 3A5 无配体结构的比较表明，3A5 三元复合物中的远端阿扎美伦导致显著的诱导契合，将水排除在疏水结合腔表面和远端阿扎美伦之外，这通过与近端阿扎美伦的堆叠相互作用得到增强。与相关的截短侧耳素抗生素、替米考星、瑞他帕林和莱福米特结合到 3A5 时，没有观察到同型协同作用，这些抗生素较大，不太可能以堆叠的方式结合到远端位点。形成两个阿扎美伦分子的 3A5 复合物可能会通过限制替代产物的形成和/或使易受攻击的蛋白质部位接触到反应性中间体，来防止 3A4 出现时间依赖性抑制。这些结果也有助于更好地理解协同结合的位点以及 3A5 和 3A4 的差异结构可塑性，这有助于不同的底物和抑制剂结合。
