title: Hydroxyethylene isostere inhibitors of human immunodeficiency virus-1 protease: structure-activity analysis using enzyme kinetics, X-ray crystallography, and infected T-cell assays.
abstract: Analogues of peptides ranging in size from three to six amino acids and containing the hydroxyethylene dipeptide isosteres Phe psi Gly, Phe psi Ala, Phe psi NorVal, Phe psi Leu, and Phe psi Phe, where psi denotes replacement of CONH by (S)-CH(OH)CH2, were synthesized and studied as HIV-1 protease inhibitors. Inhibition constants (Ki) with purified HIV-1 protease depend strongly on the isostere in the order Phe psi Gly greater than Phe psi Ala greater than Phe psi NorVal greater than Phe psi Leu greater than Phe psi Phe and decrease with increasing length of the peptide analogue, converging to a value of 0.4 nM. Ki values are progressively less dependent on inhibitor length as the size of the P1' side chain within the isostere increases. The structures of HIV-1 protease complexed with the inhibitors Ala-Ala-X-Val-Val-OMe, where X is Phe psi Gly, Phe psi Ala, Phe psi NorVal, and Phe psi Phe, have been determined by X-ray crystallography (resolution 2.3-3.2 A). The crystals exhibit symmetry consistent with space group P6(1) with strong noncrystallographic 2-fold symmetry, and the inhibitors all exhibit 2-fold disorder. The inhibitors bind in similar conformations, forming conserved hydrogen bonds with the enzyme. The Phe psi Gly inhibitor adopts an altered conformation that places its P3' valine side chain partially in the hydrophobic S1' pocket, thus suggesting an explanation for the greater dependence of the Ki value on inhibitor length in the Phe psi Gly series. From the kinetic and crystallographic data, a minimal inhibitor model for tight-binding inhibition is derived in which the enzyme subsites S2-S2' are optimally occupied. The Ki values for several compounds are compared with their potencies as inhibitors of proteolytic processing in T-cell cultures chronically infected with HIV-1 (MIC values) and as inhibitors of acute infectivity (IC50 values). There is a rank-order correspondence, but a 20-1000-fold difference, between the values of Ki and those of MIC or IC50. IC50 values can approach those of Ki but are highly dependent on the conditions of the acute infectivity assay and are influenced by physiochemical properties of the inhibitors such as solubility.
authors.affiliation: Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
authors.forename: G B
authors.initials: GB
authors.lastname: Dreyer
authors.forename: D M
authors.initials: DM
authors.lastname: Lambert
authors.forename: T D
authors.initials: TD
authors.lastname: Meek
authors.forename: T J
authors.initials: TJ
authors.lastname: Carr
authors.forename: T A
authors.initials: TA
authors.lastname: Tomaszek
authors.forename: A V
authors.initials: AV
authors.lastname: Fernandez
authors.forename: H
authors.initials: H
authors.lastname: Bartus
authors.forename: E
authors.initials: E
authors.lastname: Cacciavillani
authors.forename: A M
authors.initials: AM
authors.lastname: Hassell
authors.forename: M
authors.initials: M
authors.lastname: Minnich
mesh_terms: D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000138:chemical synthesis*; D015153:Blotting, Western; D002460:Cell Line; D005030:Ethylenes / Q000737:chemistry / Q000494:pharmacology; D016333:HIV Protease / Q000737:chemistry / Q000302:isolation & purification; D017320:HIV Protease Inhibitors; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000138:chemical synthesis*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013601:T-Lymphocytes; D014779:Virus Replication / Q000187:drug effects*; D014961:X-Ray Diffraction
chemical_list: D000998:Antiviral Agents; D005030:Ethylenes; D017320:HIV Protease Inhibitors; D007202:Indicators and Reagents; D009842:Oligopeptides; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D016333:HIV Protease