High Resolution Crystal Structures of HIV-1 Protease with a Potent Non-peptide Inhibitor (UIC-94017) Active Against Multi-drug-resistant Clinical Strains

doi:10.1016/j.jmb.2004.02.052

Journal of Molecular Biology

Volume 338, Issue 2, 23 April 2004, Pages 341-352

https://doi.org/10.1016/j.jmb.2004.02.052 Get rights and content

Abstract

The compound UIC-94017 (TMC-114) is a second-generation HIV protease inhibitor with improved pharmacokinetics that is chemically related to the clinical inhibitor amprenavir. UIC-94017 is a broad-spectrum potent inhibitor active against HIV-1 clinical isolates with minimal cytotoxicity. We have determined the high-resolution crystal structures of UIC-94017 in complexes with wild-type HIV-1 protease (PR) and mutant proteases PR_V82A and PR_I84V that are common in drug-resistant HIV. The structures were refined at resolutions of 1.10–1.53 Å. The crystal structures of PR and PR_I84V with UIC-94017 ternary complexes show that the inhibitor binds to the protease in two overlapping positions, while the PR_V82A complex had one ordered inhibitor. In all three structures, UIC-94017 forms hydrogen bonds with the conserved main-chain atoms of Asp29 and Asp30 of the protease. These interactions are proposed to be critical for the potency of this compound against HIV isolates that are resistant to multiple protease inhibitors. Other small differences were observed in the interactions of the mutants with UIC-94017 as compared to PR. PR_V82A showed differences in the position of the main-chain atoms of residue 82 compared to PR structure that better accommodated the inhibitor. Finally, the 1.10 Å resolution structure of PR_V82A with UIC-94017 showed an unusual distribution of electron density for the catalytic aspartate residues, which is discussed in relation to the reaction mechanism.

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, PR_V82A and PR_I84V, 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.

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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

Abstract

Introduction

Section snippets

Chemical synthesis of UIC-94017

Discussion

Chemical synthesis of UIC-94017

Acknowledgements

Bioorg. Med. Chem. Letters

Tetrahedron Letters

Advan. Pharmacol.

Methods Enzymol.

Methods Enzymol.

Prevalence of drug resistant mutants and virological response to combination therapy in patients with primary HIV-1 infection

J. Med. Virol.

Multiple concurrent reverse transcriptase and protease mutations and multidrug resistance of HIV-1 isolates from heavily treated patients

Ann. Intern. Med.

Phenotypic and genotypic analysis of clinical HIV-1 isolates reveals extensive protease inhibitor cross-resistance: a survey of over 6000 samples

AIDS

Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments

J. Virol.

Drug-resistant HIV-1 proteases identify enzyme residues important for substrate selection and catalytic rate

Biochemistry

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease

Eur. J. Biochem.

Combining mutations in HIV-1 protease to understand mechanisms of resistance

Proteins: Struct. Funct. Genet.

Altered substrate specificity of drug-resistant human immunodeficiency virus type 1 protease

J. Virol.

Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH

Protein Sci.

Crystal structure of an in vivo HIV-1 protease mutant in complex with saquinavir: insights into the mechanisms of drug resistance

Protein Sci.

Structural implications of drug resistant mutants of HIV-1 protease: high resolution crystal structures of the mutant protease/substrate analog complexes

Proteins: Struct. Funct. Genet.