Kessl Jacques J, Eidahl Jocelyn O, Shkriabai Nikolozi, Zhao Zhuojun, McKee Christopher J, Hess Sonja, Burke Terrence R, Kvaratskhelia Mamuka
Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, the Ohio State University, Columbus, Ohio 43210, USA.
Mol Pharmacol. 2009 Oct;76(4):824-32. doi: 10.1124/mol.109.058883. Epub 2009 Jul 28.
HIV-1 integrase (IN) is a validated target for developing antiretroviral inhibitors. Using affinity acetylation and mass spectrometric (MS) analysis, we previously identified a tetra-acetylated inhibitor (2E)-3-[3,4-bis(acetoxy)phenyl]-2-propenoate-N-[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propenyl]-L-serine methyl ester; compound 1] that selectively modified Lys173 at the IN dimer interface. Here we extend our efforts to dissect the mechanism of inhibition and structural features that are important for the selective binding of compound 1. Using a subunit exchange assay, we found that the inhibitor strongly modulates dynamic interactions between IN subunits. Restricting such interactions does not directly interfere with IN binding to DNA substrates or cellular cofactor lens epithelium-derived growth factor, but it compromises the formation of the fully functional nucleoprotein complex. Studies comparing compound 1 with a structurally related IN inhibitor, the tetra-acetylated-chicoric acid derivative (2R,3R)-2,3-bis[[(2E)-3-[3,4-bis(acetyloxy)phenyl]-1-oxo-2-propen-1-yl]oxy]-butanedioic acid (compound 2), indicated striking mechanistic differences between these agents. The structures of the two inhibitors differ only in their central linker regions, with compounds 1 and 2 containing a single methyl ester group and two carboxylic acids, respectively. MS experiments highlighted the importance of these structural differences for selective binding of compound 1 to the IN dimer interface. Moreover, molecular modeling of compound 1 complexed to IN identified a potential inhibitor binding cavity and provided structural clues regarding a possible role of the central methyl ester group in establishing an extensive hydrogen bonding network with both interacting subunits. The proposed mechanism of action and binding site for the small-molecule inhibitor identified in the present study provide an attractive venue for developing allosteric inhibitors of HIV-1 IN.
HIV-1整合酶（IN）是开发抗逆转录病毒抑制剂的一个经过验证的靶点。利用亲和乙酰化和质谱（MS）分析，我们之前鉴定出一种四乙酰化抑制剂（2E）-3-[3,4-双（乙酰氧基）苯基]-2-丙烯酸酯-N-[(2E)-3-[3,4-双（乙酰氧基）苯基]-1-氧代-2-丙烯基]-L-丝氨酸甲酯；化合物1]，它能选择性地修饰IN二聚体界面处的赖氨酸173。在此，我们进一步努力剖析抑制机制以及对化合物1选择性结合至关重要的结构特征。通过亚基交换试验，我们发现该抑制剂强烈调节IN亚基之间的动态相互作用。限制这种相互作用不会直接干扰IN与DNA底物或细胞辅因子晶状体上皮衍生生长因子的结合，但会损害完全功能性核蛋白复合物的形成。将化合物1与结构相关的IN抑制剂四乙酰化咖啡酸衍生物（2R,3R）-2,3-双[[(2E)-3-[3,4-双（乙酰氧基）苯基]-1-氧代-2-丙烯-1-基]氧基]-丁二酸（化合物2）进行比较的研究表明，这些药物之间存在显著的机制差异。这两种抑制剂的结构仅在其中心连接区域有所不同，化合物1和2分别含有一个甲酯基团和两个羧酸基团。MS实验突出了这些结构差异对化合物1与IN二聚体界面选择性结合的重要性。此外，与IN复合的化合物1的分子模拟确定了一个潜在的抑制剂结合腔，并提供了有关中心甲酯基团在与两个相互作用亚基建立广泛氢键网络中可能作用的结构线索。本研究中鉴定的小分子抑制剂的作用机制和结合位点为开发HIV-1 IN的变构抑制剂提供了一个有吸引力的途径。
