Journal of Molecular Biology
High Resolution Crystal Structures of HIV-1 Protease with a Potent Non-peptide Inhibitor (UIC-94017) Active Against Multi-drug-resistant Clinical Strains
Introduction
Inhibitors of the HIV-1 protease (PR) are effective antiviral drugs and have dramatically improved the survival of patients infected with HIV-1. However, the long-term therapeutic efficacy is compromised by the rapid selection of drug-resistant variants of the PR.1 Cross-resistance and multi-drug resistance have been reported in AIDS patients on combination therapy.2., 3. The clinical inhibitors select for different mutations, and complex patterns of mutations have been observed.4 HIV PR is catalytically active as a dimer, and the catalytic Asp25 residues from both subunits interact closely at the subunit interface. The substrate-binding site consists of subsites S3 to S4′ for peptide substrates extending from residues P3 to P4′. The clinical inhibitors are observed to bind in PR subsites S2–S2′. Mutations that alter the inhibitor-binding site are common, including mutations of residues D30, M46, I50, V82 and I84. Other mutations alter residues located far from the inhibitor-binding site, such as L90M. Mutants with either increased or decreased catalytic activity, inhibition or stability relative to the wild-type enzyme have been observed.5., 6., 7., 8., 9. Crystal structures of resistant mutants have provided the molecular basis for the altered enzymatic properties and the resistant phenotype.7., 10., 11., 12. The result of detailed structural and kinetic studies is that individual mutations, and the tested pairs of mutations, show a range of effects that depend on the specific combination of mutant with substrate or inhibitor. Therefore, it is important to obtain high-resolution crystal structures in order to observe the small structural changes associated with the mutations.
Novel inhibitors that target critical conserved regions of the PR different from those of the current drugs are expected to be more active against resistant variants of HIV-1. A novel non-peptide inhibitor UIC-94017 (TMC114) has been developed that is extremely potent in vivo against a wide spectrum of HIV strains including multi-drug-resistant clinical strains.13 This promising non-peptidic inhibitor is in Phase IIB clinical trials. This compound is chemically related to the clinical inhibitor amprenavir, and was designed with a novel bis-tetrahydrofuranylurethane (bis-THF) group to incorporate additional polar interactions with main-chain atoms of the PR dimer. Crystal structures of complexes of the UIC-94017 with PR and two of the most common drug-resistant mutants, PRV82A and PRI84V, have been determined at high resolution to understand the molecular basis for the potency of this compound.
Section snippets
Chemical synthesis of UIC-94017
The synthesis of UIC-94017 (1) is outlined in Figure 1. Commercially available optically active epoxide 314 was reacted with isobutylamine in 2-propanol at 84 °C for six hours. The resulting amino alcohol was reacted with 4-nitrobenzenesulfonyl chloride to provide the sulfonamide derivative 3.15 Reduction of nitro group of 4 by catalytic hydrogenation over 10% Pd–C in ethyl acetate provided the corresponding aromatic amine. Trifluoroacetic acid promoted removal of BOC-group followed by reaction
Discussion
The UIC-94017 inhibitor is in clinical trials for further development as a new antiviral agent for treatment of primary and multi-PR inhibitor-resistant HIV infections due to its favorable pharmacokinetics.13 The hydrogen bond interactions of UIC-94017 with HIV PR can be compared to those of substrate analogs. PR recognizes peptide substrates and peptidic inhibitors by means of a series of hydrogen bond interactions with the main-chain atoms of the substrate, as observed for crystal structures
Chemical synthesis of UIC-94017
The synthesis is shown in Figure 1. UIC-94017 (compound 1) was formed by the reaction of the intermediate compounds (1S)-benzyl-3-[isobutyl-(4-nitrobenzenesulfonyl)amino]-(2R)-hydroxy-propyl]-carbamic acid tert-butylester (compound 4) and bis-tetrahydrofuranylsuccinimidyl carbonate (compound 5). Synthesis of the intermediate compounds is described.
Compound 4 was prepared by heating a stirred solution of tert-butyl[S-(R,R)]-(−)-(1-oxiranyl-2-phenylethyl)carbamate 3 (100 mg, 0.38 mmol) and iso
Acknowledgements
This research was supported in part by the National Institutes of Health grants GM62920 (I.T.W. and R.W.H.) and GM53386 (A.G.), Hungarian OTKA F35191, the Georgia Cancer Coalition, and the Georgia Research Alliance. We thank the staff at the SER-CAT beamline at the Advanced Photon Source, Argonne National Laboratory, for assistance during X-ray data collection. Use of the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under
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Y.T. and P.I.B. contributed equally to this work.