Molecular design and synthesis of HIV protease inhibitors with structurally constrained active conformation

基于HIV蛋白酶抑制剂的底物-过渡态概念，我们成功地设计、研究了酶相互作用的最小位点，并合成了一种高选择性和有效的HIV蛋白酶抑制剂（kni272）。这些成功的结果归功于专注于酶反应机制的酶-底物（或抑制剂）复合物的分析，以及酶活性位点构象约束的底物过渡态模拟的分子设计。我们进一步发展了这些方法来设计和合成具有结构约束活性构象的抑制剂。突变型HIV蛋白酶的合成。已有报道称，在突变的HIV蛋白酶中，84-Ile被与Pl和Pl'亚位相互作用的Val所取代。为此，我们合成了84-Val的HIV蛋白酶衍生物，并对其酶活性进行了检测。HIV蛋白酶活性位点衍生物的合成。具有25-Asp等同氨基酸的HIV蛋白酶衍生物，参与与底物的相互作用。由于HIV蛋白酶作为二聚体起作用，我们研究了两种类型的组合：天然/等容和等容/等容。HIV蛋白酶抑制剂的合成。根据底物过渡态的概念，将裂解位点的酰胺键、ph - pro和Tyr-Pro （Pl-Pl’）特征转化为逆转录酶底物的等异位还原型、羟乙基型、二羟乙基型等。合成了具有刚性结构的二肽衍生物。利用核磁共振和分子模型对酶-底物配合物进行构象分析。酶抑制剂复合物的形成和分析。酶-抑制剂复合物的x射线晶体学和核磁共振分析表明，水分子在酶与抑制剂的相互作用中起重要作用，高效抑制剂具有高度约束的构象。

Based on the substrate-transition state concept of HIV protease inhibitors, we have succeeded to design, investigate the minimum site for enzyme-interaction and synthesize a highly selective and potent HIV protease inhibitor (KNI-272). These successful results are attributable to the analysis of enzyme-substrate (or-inhibitor) complexes focusing on enzyme reaction mechanism, and the molecular design of conformationally constrained mimics of substrate transition state in the enzyme active site. We further developed these methods to molecular design and synthesis of inhibitors with structurally constrained active conformation.1. Synthesis of mutant HIV protease.It has been already reported that in mutated HIV proteases 84-Ile is replaced with Val which interacts with Pl and Pl' subsites. Therefore we synthesized 84-Val HIV protease derivatives and examined the enzyme activity.2. Synthesis of active site derivatives of HIV proteases.HIV protease derivatives with isosteric amino acids at 25-Asp which participates in the interaction with the substrates. Since the HIV protease works as a dimer, we examined two types of combinations : native/isosteric and isosteric/isosteric.3. Synthesis of HIV protease inhibitors.Based on the substrate transition state concept, the amide bond of the cleavage sites, Phe-Pro and Tyr-Pro (Pl-Pl') characteristic to retroviral enzyme substrate was converted to the isosteric reduced-type, hydroxyethylene-type, dihydroxyethylene-type and others. Dipeptide derivatives with rigid structure were also synthesized.4. Conformational analysis of the enzyme-substrate complex was carried out by using NMR and molecular modeling.5. Fomation and analysis of enzyme-inhibitor complexes.X-ray crystallography and NMR analysis of the enzyme-inhibitor complex revealed that water molecules play an important role in the interaction of enzyme and inhibitors and that the highly potent inhibitor has highly constrained conformation.