Inhibitors of proteolytic enzymes (proteases) are emerging as prospective treatments for diseases such as AIDS and viral infections, cancers, inflammatory disorders, and Alzheimer's disease. Generic approaches to the design of protease inhibitors are limited by the unpredictability of interactions between, and structural changes to, inhibitor and protease during binding. A computer analysis of superimposed crystal structures for 266 small molecule inhibitors bound to 48 proteases (16 aspartic, 17 serine, 8 cysteine, and 7 metallo) provides the first conclusive proof that inhibitors, including substrate analogues, commonly bind in an extended beta-strand conformation at the active sites of all these proteases. Representative superimposed structures are shown for (a) multiple inhibitors bound to a protease of each class, (b) single inhibitors each bound to multiple proteases, and (c) conformationally constrained inhibitors bound to proteases. Thus inhibitor/substrate conformation, rather than sequence/composition alone, influences protease recognition, and this has profound implications for inhibitor design. This conclusion is supported by NMR, CD, and binding studies for HIV-1 protease inhibitors/substrates which, when preorganized in an extended conformation, have significantly higher protease affinity. Recognition is dependent upon conformational equilibria since helical and turn peptide conformations are not processed by proteases. Conformational selection explains the resistance of folded/structured regions of proteins to proteolytic degradation, the susceptibility of denatured proteins to processing, and the higher affinity of conformationally constrained 'extended' inhibitors/substrates for proteases. Other approaches to extended inhibitor conformations should similarly lead to high-affinity binding to a protease.

High-resolution crystal structures are described for seven macrocycles complexed with HIV-1 protease (HIVPR). The macrocycles possess two amides and an aromatic group within 15-17 membered rings designed to replace N- or C-terminal tripeptides from peptidic inhibitors of HIVPR. Appended to each macrocycle is a transition state isostere and either an acyclic peptide, nonpeptide, or another macrocycle. These cyclic analogues are potent inhibitors of HIVPR, and the crystal structures show them to be structural mimics of acyclic peptides, binding in the active site of HIVPR via the same interactions. Each macrocycle is restrained to adopt a beta-strand conformation which is preorganized for protease binding. An unusual feature of the binding of C-terminal macrocyclic inhibitors is the interaction between a positively charged secondary amine and a catalytic aspartate of HIVPR. A bicyclic inhibitor binds similarly through its secondary amine that lies between its component N-terminal and C-terminal macrocycles. In contrast, the corresponding tertiary amine of the N-terminal macrocycles does not interact with the catalytic aspartates. The amine-aspartate interaction induces a 1.5 A N-terminal translation of the inhibitors in the active site and is accompanied by weakened interactions with a water molecule that bridges the ligand to the enzyme, as well as static disorder in enzyme flap residues. This flexibility may facilitate peptide cleavage and product dissociation during catalysis. Proteases [Aba67,95]HIVPR and [Lys7,Ile33,Aba67,95]HIVPR used in this work were shown to have very similar crystal structures.

Results are presented for inhibitors of HIV-1 protease that demonstrate a new strategy for developing peptidomimetics, involving the replacement of flexible segments of peptide substrates with conformationally constrained hydrolytically-stable macrocyclic structural mimics. A 15-membered macrocycle that imitates the tripeptide PheIle-Val was designed and incorporated into the C-terminus of Ac-Leu-Val-Phe-CHOHCH 2 -{Phe-Ile-Val}-NH 2 , an inhibitor of HIV-1 protease derived from a substrate sequence. Advantages of the macrocycle over the acyclic peptide include constraining its components into their bioactive conformation and protecting the amide bonds from enzymatic degradation, the cycle being stable to acid, gastric proteases, and plasma. Molecular modeling and X-ray structural studies reveal that the cyclic inhibitors have a unique enzyme-binding mode, the sterically unencumbered hydroxyethylamine isostere binds via both its hydroxyl and protonated nitrogen to the anionic Asp25 catalytic residues. The novel macrocycle superimposes well on the linear peptidic inhibitor for which it was designed as a structural mimic. Structural mimicry led to functional mimicry as shown by comparable inhibition of the protease by cyclic and acyclic molecules. Further modification of the acyclic N-terminus (Leu-Val-Phe) gave stable, water-soluble, potent inhibitors of HIV-1 protease. This approach may have general application to the development of mimetics of other bioactive peptides, including inhibitors of other enzymes.

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