Slow-Onset Inhibition of Mycobacterium tuberculosis InhA: Revealing Molecular Determinants of Residence Time by MD Simulations

An important kinetic parameter for drug efficacy is the residence time of a compound at a drug target, which is related to the dissociation rate constant koff. For the essential antimycobacterial target InhA, this parameter is most likely governed by the ordering of the flexible substrate binding loop (SBL). Whereas the diphenyl ether inhibitors 6PP and triclosan (TCL) do not show loop ordering and thus, no slow-binding inhibition and high koff values, the slightly modified PT70 leads to an ordered loop and a residence time of 24 minutes. To assess the structural differences of the complexes from a dynamic point of view, molecular dynamics (MD) simulations with a total sampling time of 3.0 µs were performed for three ligand-bound and two ligand-free (perturbed) InhA systems. The individual simulations show comparable conformational features with respect to both the binding pocket and the SBL, allowing to define five recurring conformational families. Based on their different occurrence frequencies in the simulated systems, the conformational preferences could be linked to structural differences of the respective ligands to reveal important determinants of residence time. The most abundant conformation besides the stable EI* state is characterized by a shift of Ile202 and Val203 toward the hydrophobic pocket of InhA. The analyses revealed potential directions for avoiding this conformational change and, thus, hindering rapid dissociation: (1) an anchor group in 2'-position of the B-ring for scaffold stabilization, (2) proper occupation of the hydrophobic pocket, and (3) the introduction of a barricade substituent in 5'-position of the diphenyl ether B-ring.

药物效力的一项关键动力学参数为化合物在药物靶点上的滞留时间，该参数与解离速率常数（dissociation rate constant, koff）直接相关。对于抗结核分枝杆菌的关键靶点InhA而言，该参数大概率由柔性底物结合环（substrate binding loop, SBL）的有序化程度所调控。二苯醚类抑制剂6PP与三氯生（triclosan, TCL）不会引发环结构有序化，因此不表现出慢结合抑制现象，且解离速率常数koff值较高；而经过小幅修饰的PT70则可诱导环结构有序化，使化合物的滞留时间达到24分钟。为从动态视角评估各复合物的结构差异，本研究针对3个配体结合体系与2个无配体（扰动型）InhA体系开展了分子动力学（molecular dynamics, MD）模拟，总采样时长达3.0微秒。各独立模拟在结合口袋与SBL的构象特征上均表现出相似性，据此可定义5类重复出现的构象家族。基于各构象家族在模拟体系中的出现频率差异，可将构象偏好性与对应配体的结构差异相关联，从而揭示滞留时间的关键决定因素。除稳定的EI*状态外，丰度最高的构象表现为异亮氨酸202（Ile202）与缬氨酸203（Val203）向InhA的疏水口袋发生位移。本研究的分析结果为规避此类构象变化、进而阻碍快速解离指明了潜在方向：（1）在二苯醚B环的2'-位引入锚定基团以稳定配体骨架；（2）充分占据疏水口袋；（3）在二苯醚B环的5'-位引入位阻取代基。