Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.16 (HIV-1 protease)
2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Eleven different recombinant, drug-resistant HIV-1 protease (HIV PR) mutants--R8Q, V32I, M46I, V82A, V82F, V82I, I84V, V32I/I84V, M46I/V82F, M46I/I84V, and V32I/K45I/F53L/A71V/I84V/L89M--were generated on the basis of results of in vitro selection experiments using the inhibitors A-77003, A-84538, and KNI-272. Kinetic parameters of mutant and wild-type (WT) enzymes were measured along with inhibition constants (Ki) toward the inhibitors A-77003, A-84538, KNI-272, L-735,524, and Ro31-8959. The catalytic efficiency, kcat/Km, for the mutants decreased relative to WT by a factor of 1.2-14.8 and was mainly due to the elevation of Km. The effects of specific mutations on Ki values were unique with respect to both inhibitor and mutant enzyme. A new property, termed vitality, defined as the ratio (Kikcat/Km)mutant/(Kikcat/Km)WT was introduced to compare the selective advantage of different mutants in the presence of a given inhibitor. High vitality values were generally observed with mutations that emerged during in vitro selection studies. The kinetic model along with the panel of mutants described here should be useful for evaluating and predicting patterns of resistance for HIV PR inhibitors and may aid in the selection of inhibitor combinations to combat drug resistance.
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PMID:Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. 762 98

The pharmacokinetic (PK) characteristics of KNI-272, a potent and selective HIV-1 protease inhibitor, were evaluated in rats after intravenous (IV) administration. The effect of dose on KNI-272 plasma kinetics, and the urinary and biliary elimination kinetics of KNI-272, were examined. After IV administration of 10.0 mg kg-1 KNI-272, the mean terminal elimination half-life, t1/2 lambda zeta, was 3.49 +/- 0.19 (SE) h, the total plasma clearance, CLtot, was 15.1 +/- 1.2 mL min-1 and the distribution volume at steady state, Vd,ss, was 3790 +/- 280 mL kg-1. On the other hand, after 1.0 mg kg-1 IV administration, t1/2 lambda zeta was 3.04 +/- 0.11 h, CLtot was 15.9 +/- 0.2 mL min-1, and Vd,ss was 6950 +/- 600 mL kg-1. The PK parameters of KNI-272 after IV administration showed that the disposition of KNI-272 in the rat plasma is linear within the dose range from 1.0 to 10.0 mg kg-1. Using an equilibrium dialysis method, the plasma binding of KNI-272 was measured in vitro. The free fractions were 17.7 +/- 0.6%, 12.1 +/- 1.5%, and 13.8 +/- 1.4% at the total concentration ranges of 9.898 +/- 0.097 microgram mL-1, 0.888 +/- 0.008 microgram mL-1, and 0.470 +/- 0.55 microgram mL-1, respectively. The percentages of the dose excreted into the urine and bile as the unchanged form were 1.20 +/- 1.06% and 1.61 +/- 0.32% at 1.0 mg kg-1 dose, and 0.164 +/- 0.083% and 1.42 +/- 0.26% at 10.0 mg kg-1 dose, respectively. The renal clearance (CLR) and the biliary clearance (CLB) were calculated to be 0.191 and 0.256 mL min-1 for 1.0 mg kg-1, and 0.0248 and 0.215 mL min-1 for 10.0 mg kg-1, respectively. When comparing these values with the CLtot values, the urinary and biliary excretion of KNI-272 are minor disposition routes.
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PMID:Plasma pharmacokinetics and urinary and biliary excretion of a new potent tripeptide HIV-1 protease inhibitor, KNI-272, in rats after intravenous administration. 784 37

Recently, a series of KNI compounds such as KNI-227 and KNI-272 has been synthesized and shows potent and selective HIV-1 protease inhibitory activity in vitro. In this study, we developed an HPLC assay system for KNI-227 and KNI-272 in rat plasma and examined the pharmacokinetic characteristics in rats after both intravenous (i.v.) and intraduodenal (i.d.) administrations to obtain the disposition characteristics and bioavailabilities of these new anti-AIDS drugs. After i.v. administration of KNI-227, 10.0 mg kg-1, the mean terminal elimination half-life, t1/2 lambda zeta, was 0.808 +/- 0.161(SE) h, the total body clearance, CLtot, was 11.7 +/- 3.3 ml min-1 and the distribution volume at steady state (Vd,ss) was 1410 +/- 460 ml kg-1. On the other hand, after i.v. administration of KNI-272, 10.0 mg kg-1, t1/2 lambda zeta was 2.86 +/- 0.78 h, CLtot was 15.3 +/- 1.4 ml min-1 and Vd,ss was 3440 +/- 670 ml kg-1. In the case of the i.d. administration of drugs, the mean peak plasma concentrations, Cmax, of KNI-227 and KNI-272 were 0.374 +/- 0.110 microgram ml-1 and 0.900 +/- 0.093 micrograms ml-1, respectively. The bioavailabilities (BA) of KNI-227 and KNI-272 to infinity, BA(0-infinity), were 5.90% and 42.3%, respectively. As compared with the lead compound, KNI-174, the BA of KNI-272 was improved about 10 times. Although the anti-AIDS virus activity of these two drugs has not been investigated in vivo, KNI-272 is expected to be a better candidate for oral anti-AIDS therapies.
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PMID:Comparison of a new orally potent tripeptide HIV-1 protease inhibitor (anti-AIDS drug) based on pharmacokinetic characteristics in rats after intravenous and intraduodenal administrations. 830 29

In order to improve the design of HIV-1 protease inhibitors, it is essential to understand how they interact with active site residues, particularly the catalytic Asp25 and Asp125 residues. KNI-272 is a promising, potent HIV-1 protease inhibitor (K(i) approximately 5 pM), currently undergoing phase 1 clinical trials. Because KNI-272 is asymmetric, the complex it forms with the homodimeric HIV-1 protease also lacks symmetry, and the two protease monomers can have distinct NMR spectra. Monomer specific signal assignments were obtained for amino acid residues in the drug binding site as well as for six of the eight Asp residues in the protease/KNI-272 complex. Using these assignments, the ionization states of the Asp carboxyl groups were determined from measurements of (a) the pD dependence of the chemical shifts of the Asp carboxyl carbons and (b) the H/D isotope effect upon the Asp carboxyl carbon chemical shifts. The results of these measurements indicate that the carboxyl of Asp25 is protonated while that of Asp125 is not protonated. These findings provide not only the first experimental evidence regarding the distinct protonation states of Asp25/125 in HIV-1 protease/drug complexes, but also shed light on interactions responsible for inhibitor binding that should form the basis for improved drug designs.
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PMID:Solution NMR evidence that the HIV-1 protease catalytic aspartyl groups have different ionization states in the complex formed with the asymmetric drug KNI-272. 875 55

The human immunodeficiency (HIV) codes for an aspartic protease known to be essential for retroviral maturation and replication. The HIV protease can recognize Phe-Pro and Tyr-Pro sequences as the virus-specific cleavage site. These features provided a basis for the rational design of selective HIV protease-targeted drugs for the treatment of acquired immunodeficiency syndrome (AIDS). HIV protease is formed from two identical 99 amino acid peptides. We replaced the two Cys residues by L-Ala to synthesize [Ala67,95]-HIV-1 protease by the solid phase method and then prepared [Tyr6,42, Nle36,46, (NHCH2COSCH2CO)51-52, Ala67,95] HIV-1 protease (NY-5 isolate) using the thioester chemical ligation method. Based on the substrate transition state, we designed and synthesized a novel class of HIV protease inhibitors containing an unnatural amino acid, (2S, 3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) with a hydroxymethylcarbonyl (HMC) isostere. Among them, the conformationally constrained tripeptide kynostatin (KNI)-272 (iQoa-Mta-Apns-Thz-NHBut) was a highly selective and superpotent HIV protease inhibitor (Ki = 0.0055 nM). KNI-272 exhibited potent antiviral activities against both AZT-sensitive and -insensitive clinical HIV-1 isolates as well as HIV-2 with low cytotoxicity. After i.d. administration, bioavailability of KNI-272 was 42.3% in rats. Also, KNI-272 exhibited in vivo anti-HIV activities in human PBMC-SCID mice. The x-ray crystallography and molecular modeling studies showed that the HMC group in KNI-272 interacted excellently with the aspartic acid carboxyl groups of HIV protease active site in the essentially same hydrogen-bonding mode as the transition state. This result implies that the HMC isostere is an ideal transition-state mimic and contributes to the high activity of KNI-272.
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PMID:Design and synthesis of substrate-based peptidomimetic human immunodeficiency virus protease inhibitors containing the hydroxymethylcarbonyl isostere. 878 65

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.
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PMID:Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology. 914 62

We asked whether human immunodeficiency virus type 1 (HIV-1) protease plays a major role in the early stages of infection (i.e. from viral entry to reverse transcription) by using various protease inhibitors (saquinavir, ritonavir, and KNI-272). When assessed in the two-day multinuclear activation of a galactosidase indicator (MAGI) assay, involving a single cycle of HIV-1 replication, all protease inhibitors failed to block infection of HeLa-CD4-LTR-beta-gal cells by HIV-1, while reverse transcriptase (RT) inhibitors (AZT and ddI) completely blocked the infection. Moreover, when HIV-1 proviral DNA synthesis was examined by polymerase chain reaction in HeLa-CD4-LTR-beta-gal cells exposed to HIV-1 and cultured in the presence of protease inhibitors, a significant amount of proviral DNA was detected, while no proviral DNA synthesis was detected when the cells were cultured in the presence of RT inhibitors. Protease inhibitors also failed to block chloramphenicol acetyltransferase (CAT) expression in HLCD4-CAT cells exposed to HIV-1, while RT inhibitors completely suppressed CAT expression. These results strongly suggest, contrary to a previous report by Nagy et al. (1994), that HIV-1 protease does not play a major role in the early stages of infection.
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PMID:HIV-1 protease does not play a critical role in the early stages of HIV-1 infection. 944 67

We investigated the role of the two highly conserved cysteine residues, cysteines 67 and 95, of the human immunodeficiency virus type 1 (HIV-1) protease in regulating the activity of that protease during viral maturation. To this end, we generated four HIV-1 molecular clones: the wild type, containing both cysteine residues; a protease mutant in which the cysteine at position 67 was replaced by an alanine (C67A); a C95A protease mutant; and a double mutant (C67A C95A). When immature virions were produced in the presence of an HIV-1 protease inhibitor, KNI-272, and the inhibitor was later removed, limited polyprotein processing was observed for wild-type virion preparations over a 20-h period. Treatment of immature wild-type virions with the reducing agent dithiothreitol considerably improved the rate and extent of Gag processing, suggesting that the protease is, in part, reversibly inactivated by oxidation of the cysteine residues. In support of this, C67A C95A virions processed Gag up to fivefold faster than wild-type virions in the absence of a reducing agent. Furthermore, oxidizing agents, such as H2O2 and diamide, inhibited Gag processing of wild-type virions, and this effect was dependent on the presence of cysteine 95. Electron microscopy revealed that a greater percentage of double-mutant virions than wild-type virions developed a mature-like morphology on removal of the inhibitor. These studies provide evidence that under normal culture conditions the cysteines of the HIV-1 protease are susceptible to oxidation during viral maturation, thus preventing immature virions from undergoing complete processing following their release. This is consistent with the cysteines being involved in the regulation of viral maturation in cells under oxidative stress.
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PMID:Conserved cysteines of the human immunodeficiency virus type 1 protease are involved in regulation of polyprotein processing and viral maturation of immature virions. 988 17

The changes in the inhibitor binding constants due to the mutation of isoleucine to valine at position 84 of HIV-1 protease are calculated using molecular dynamics simulations. The calculations are done for three potent inhibitors--KNI-272, L-735,524 (indinavir or MK-639), and Ro 31-8959 (saquinavir). The calculations agree with the experimental data both in terms of an overall trend and in the magnitude of the resulting free energy change. HIV-1 protease is a homodimer, so each mutation causes two changes in the enzyme. The decrease in the binding free energy from each mutated side chain differs among the three inhibitors and correlates well with the size of the cavities induced in the protein interior near the mutated residue. The cavities are created as a result of a mutation to a smaller side chain, but the cavities are less than would be predicted from the wild-type structures, indicating that there is significant relaxation to partially fill the cavities.
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PMID:Molecular mechanisms of resistance: free energy calculations of mutation effects on inhibitor binding to HIV-1 protease. 1008 71

The aspartyl dyad of free HIV-1 protease has apparent pK(a)s of approximately 3 and approximately 6, but recent NMR studies indicate that the aspartyl dyad is fixed in the doubly protonated form over a wide pH range when cyclic urea inhibitors are bound, and in the monoprotonated form when the inhibitor KNI-272 is bound. We present computations and measurements related to these changes in protonation and to the thermodynamic linkage between protonation and inhibition. The Poisson-Boltzmann model of electrostatics is used to compute the apparent pK(a)s of the aspartyl dyad in the free enzyme and in complexes with four different inhibitors. The calculations are done with two parameter sets. One assigns epsilon = 4 to the solute interior and uses a detailed model of ionization; the other uses epsilon = 20 for the solute interior and a simplified representation of ionization. For the free enzyme, both parameter sets agree well with previously measured apparent pK(a)s of approximately 3 and approximately 6. However, the calculations with an internal dielectric constant of 4 reproduce the large pKa shifts upon binding of inhibitors, but the calculations with an internal dielectric constant of 20 do not. This observation has implications for the accurate calculation of pK(a)s in complex protein environments. Because binding of a cyclic urea inhibitor shifts the pK(a)s of the aspartyl dyad, changing the pH is expected to change its apparent binding affinity. However, we find experimentally that the affinity is independent of pH from 5.5 to 7.0. Possible explanations for this discrepancy are discussed.
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PMID:Thermodynamic linkage between the binding of protons and inhibitors to HIV-1 protease. 1021 Jan 96


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