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

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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.