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)

Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.
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PMID:Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease. 137 37

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.
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PMID:Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease. 188 29

Rationally designed synthetic inhibitors of retroviral proteases inhibit the processing of viral polypeptides in cultures of human T lymphocytes infected with human immunodeficiency virus type 1 (HIV-1) and therefore suppress the infectivity of HIV-1 in vitro. We have previously reported the antiviral activity in vitro of HIV-1 protease inhibitors against the C-type retrovirus Rauscher murine leukemia virus (RMuLV) and the lentivirus simian immunodeficiency virus (SIV). The same compounds which blocked the infectivity of HIV-1 also inhibited the infectivity of RMuLV and SIV in vitro. This report extends these findings by testing the antiviral activity of HIV-1 protease inhibitors in vivo in the RMuLV model. RMuLV-infected mice were treated twice a day (bid) with either an active (SKF 108922) or inactive (SKF 109273) compound for fourteen days by the intraperitoneal (i.p.) route. Compared with excipient control, SKF 108922, formulated with hydroxypropyl-beta-cyclodextrin (HPB), reduced virus-induced splenomegaly, viremia, and serum reverse transcriptase (RT) levels, while SKF 109273 was inactive. The HPB vehicle by itself enhanced replication of RMuLV. The effects of changing the formulation and the route of administration were examined. SKF 108922, formulated in HPB, had similar antiviral activity when administered by the i.p. or subcutaneous (SC) routes. However, SKF 108922 administered as a colloidal suspension in cholesterol sulfate (CS) had no detectable antiviral effect. Measurements of the circulating levels of the protease inhibitor in plasma explained this result. Plasma concentrations of SKF 108922 exceeded 1000 nM within 10 min after SC administration of the compound solubilized in HPB, but SKF 108922 was not detected in plasma after SC administration of the same dose formulated with CS. Information on optimal conditions for administering these agents should prove useful in guiding their clinical application Therefore, RMuLV should provide a good model for the preclinical evaluation and development of this class of agents for the treatment of HIV.
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PMID:Effects of SKF 108922, an HIV-1 protease inhibitor, on retrovirus replication in mice. 873 97

Until recently, treatment for human immunodeficiency virus type 1 (HIV-1) infection was limited to the use of nucleoside inhibitors of the viral enzyme reverse transcriptase. While these agents initially offered promise, they have only modest antiviral activity and the benefits of treatment are limited by the emergence of drug resistance and dose-limiting toxic effects. Development of more potent drugs that target different stages of the virus life cycle has thus been aggressively pursued. Efforts to develop inhibitors of HIV-1 protease have yielded a potent new class of compounds that suppress HIV-1 replication to an extent far greater than was previously attainable. Four protease inhibitors, saquinavir mesylate, ritonavir, nelfinavir, and indinavir sulfate, have been approved by the Food and Drug Administration. Other agents are undergoing active investigation. The purpose of this article is to review the currently available data on those agents that have been approved for clinical use.
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PMID:Human immunodeficiency virus type 1 protease inhibitors. 914 Feb 65

Drug-resistant mutants of HIV-1 protease limit the long-term effectiveness of current anti-viral therapy. In order to study drug resistance, the wild-type HIV-1 protease and the mutants R8Q, V32I, M46I, V82A, V82I, V82F, I84V, V32I/I84V and M46I/I84V were modeled with the inhibitors saquinavir and indinavir using the program AMMP. A new screen term was introduced to reproduce more correctly the electron distribution of atoms. The atomic partial charge was represented as a delocalized charge distribution instead of a point charge. The calculated protease-saquinavir interaction energies showed the highly significant correlation of 0.79 with free energy differences derived from the measured inhibition constants for all 10 models. Three different protonation states of indinavir were evaluated. The best indinavir model included a sulfate and gave a correlation coefficient of 0.68 between the calculated interaction energies and free energies from inhibition constants for nine models. The exception was R8Q with indinavir, probably due to differences in the solvation energy. No significant correlation was found using the standard molecular mechanics terms. The incorporation of the new screen correction resulted in better prediction of the effects of inhibitors on resistant protease variants and has potential for selecting more effective inhibitors for resistant virus.
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PMID:Molecular mechanics analysis of drug-resistant mutants of HIV protease. 1038 43

Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.
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PMID:Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion. 1112 Jun 45

A protease designated pleureryn, with an N-terminal sequence dissimilar from previously reported mushroom metalloendopeptidases and showing only limited resemblance to aspartic proteinases, albeit considerable homology to DNA replication licensing factor, was isolated from fresh fruiting bodies of the edible mushroom Pleurotus eryngii. The purification protocol entailed ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel Blue gel, ion exchange chromatography on CM-Sepharose, and FPLC-gel filtration on Superdex 75. The protease was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel Blue gel and CM-Sepharose. It demonstrated a single band with a molecular weight of 11.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Pleureryn demonstrated a protease activity of 9364 U/mg toward casein. It exhibited a pH optimum of 5.0 and a temperature optimum of 45 degrees C, with substantial activity remaining at high temperatures and pH 4 and 12. The activity of the protease was adversely affected by pepstatin A, indicating that it is an aspartic protease. PMSF, trypsin inhibitor, and EDTA exerted no striking effect, suggesting that it is neither a serine protease nor a metalloprotease. It inhibited translation in a rabbit reticulocyte lysate system with an IC(50) of 20 nM. Pleureryn also exhibited some inhibitory activity against HIV-1 reverse transcriptase, reminiscent of a suppressive action of HIV-1 protease on its homologous reverse transcriptase but was devoid of ribonuclease, deoxyribonuclease, and antifungal activities.
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PMID:Pleureryn, a novel protease from fresh fruiting bodies of the edible mushroom Pleurotus eryngii. 1172 12