Molecular Recognition of Macrocyclic Peptidomimetic Inhibitors by HIV-1 Protease ²,‡ Jennifer L. Martin,* Jake Begun, § Aaron Schindeler, | Wasa A. Wickramasinghe, ⊥ Dianne Alewood, Paul F. Alewood, Douglas A. Bergman, Ross I. Brinkworth, Giovanni Abbenante, Darren R. March, @ Robert C. Reid, and David P. Fairlie Centre for Drug Design and DeVelopment, UniVersity of Queensland, Brisbane QLD 4072, Australia ReceiVed January 25, 1999; ReVised Manuscript ReceiVed April 8, 1999 ABSTRACT: 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 -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 Å 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 [Aba 67,95 ]HIVPR and [Lys 7 ,Ile 33 ,Aba 67,95 ]- HIVPR used in this work were shown to have very similar crystal structures. Human immunodeficiency virus type-1 (HIV-1) 1 is the causative agent of acquired immunodeficiency syndrome (AIDS). Since the aspartyl protease enzyme from HIV-1 (HIVPR) is essential for viral replication, it is a target for therapeutic intervention in the disease (1-3). The enzyme processes viral proteins from precursors encoded by the gag and gag-pol HIV genes, and its inhibition has been shown to prevent viral maturation and the production of infectious progeny virus (4-6). The crystal structure of the uncomplexed form of HIVPR was determined in 1989 (7, 8). The first cocrystal structure of an HIVPR-inhibitor complex was also reported in 1989 (9). Since then, the structures of many HIVPR-inhibitor complexes have been determined (for reviews, see refs 10- 12). Structures of HIVPR complexes have been deposited in the Protein Data Bank (PDB) (13), and more recently, the HIV protease database was established (14) (http://www- fbsc.ncifcrf.gov/HIVdb) as a repository for structures of HIVPR-inhibitor complexes. Computer modeling based on crystal structures of known HIVPR-peptide inhibitor complexes suggests that the enzyme can accommodate cyclized tripeptide analogues corresponding to the linear sequences of peptidomimetic inhibitors. Inhibitors designed in this way would have the advantage of being conformationally restrained and preor- ganized for protease binding, binding with reduced entropy. In addition, the cyclic inhibitors are more resistant to proteolytic degradation than linear peptidomimetic inhibitors (15). Furthermore, these constrained macrocycle templates can be used to independently optimize inhibitor components. Macrocycles corresponding to both the N- and C-termini of HIVPR inhibitors have been synthesized and are potent inhibitors of HIVPR (16, 17). Here we describe the crystal structures of complexes formed between HIVPR and seven macrocycles that each inhibit the enzyme at nanomolar concentrations (Chart 1). Four of these inhibitors contain macrocycles as mimics for ² This work was supported by an ARC Queen Elizabeth II Fellowship to J.L.M. and NHMRC Grant 33190 to D.P.F. ‡ Coordinates deposited under PDB file names 1b6j, 1b6k, 1b6l, 1b6m, 1b6n, 1b6o, and 1b6p. * To whom correspondence should be addressed. Telephone: +61 7 3365 4942. Fax: +61 7 3365 1990. E-mail: j.martin@ mailbox.uq.edu.au. § Present address: Health Science and Technology Program, Harvard Medical School, 270 Longwood Ave., Boston, MA 02115. | Present address: Victor Chang Cardiac Research Institute, 384 Victoria St., Darlinghurst NSW 2010, Australia. ⊥ Present address: National Research Centre for Environmental Toxicology, 39 Kessels Rd., Coopers Plains QLD 4108, Australia. @ Present address: Research School of Chemistry, Australian National University, Canberra ACT 2600 Australia. 1 Abbreviations: Aba, L-R-amino-n-butyric acid; DMSO, dimethyl sulfoxide; HIV, human immunodeficiency virus; HIVPR, chemically synthesized protease enzyme from HIV-1 (SF2 isolate) with Cys67 and Cys95 replaced with Aba ([Aba 67,95 ]HIVPR); HIVKI, as described for HIVPR but with two additional sequence changes (Gln7Lys and Leu33Ile) to limit autoproteolysis ([Lys 7 ,Ile 33 ,Aba 67,95 ]HIVPR); rmsd, root-mean-square deviation; RP-HPLC, reversed phase high-pressure liquid chromatography. 7978 Biochemistry 1999, 38, 7978-7988 10.1021/bi990174x CCC: $18.00 © 1999 American Chemical Society Published on Web 06/22/1999