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)

In human skin fibroblasts microinjected with purified human immunodeficiency virus type 1 protease (HIV-1 PR), stress fibers were lost and alterations in nuclear morphology and condensation of nuclear chromatin were observed. Thereafter, the vimentin intermediate filament (IF) network collapsed. No effect was seen on the microtubules. While complicated by loss of affected cells from the substratum, a minimum estimate of the proportion of cells demonstrating these effects is 50%. Observation of single cells demonstrated that these effects were largely irreversible and were steps leading to the death of the HIV-1 PR-injected cells. After microinjection of various dilutions of the HIV-1 PR, it was observed that the changes in nuclear morphology and chromatin condensation were detectable under conditions where little or no effect was observed on both stress fibers and the IF network. Proteins of cells labelled with [35S]methionine and microinjected with either HIV-1 PR or BSA were subjected to two-dimensional gel electrophoresis. The major differences in the gel patterns were a diminution in the amount of vimentin and the appearance of novel products comigrating with cleavage products obtained after treatment of vimentin with HIV-1 PR in vitro. Thus, the HIV-1 PR is capable not only of cleaving IF subunit proteins in vivo, but also can catalyze alterations in other cellular structures.
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PMID:Human immunodeficiency virus type 1 protease microinjected into cultured human skin fibroblasts cleaves vimentin and affects cytoskeletal and nuclear architecture. 172 1

There are already reports, from clinical trials with human immunodeficiency virus type 1 protease inhibitors, of the emergence of drug-resistant mutants which have one or more point mutations in their protease genes. To examine roles of individual and multiple amino acid substitutions in terms of altered enzyme and virus drug sensitivities, we have produced matched vectors for bacterial expression and virus production. Both vectors accept the same restriction enzyme fragment, produced by PCR or PCR-mutagenesis of the protease gene, allowing parallel expression of mutant enzymes in Escherichia coli and in recombinant viruses. The utility of this vector system was demonstrated by using protease variants glycine to valine at amino acid 48 (G48V) and leucine to methionine at amino acid 90 (L90M) identified after passage of HIV-1 in the Roche phase II clinical trial protease inhibitor Ro 31-8959 (H. Jacobsen, K. Yasargil, D. L. Winslow, J. C. Craig, A. Krohn, I. B. Duncan, and J. Mous, Virology 206:527, 1995). G48V, L90M, and G48V/L90M exhibited successively less processing in vitro than the wild-type enzyme, and the purified enzymes were 220-, 20-, and 720-fold, respectively, less sensitive to Ro 31-8959. The reduced enzyme sensitivity correlated directly with the sensitivities of the matched recombinant viruses, in that individual mutations L90M and G48V conferred 2-fold and 4- to 6-fold increases in 50% inhibitory concentration, respectively, whereas G48V/L90M was 8 to 10 times less sensitive to Ro 31-8959. A proviral vector with the entire protease gene deleted was constructed for use as an in vivo recombination target for an overlapping protease PCR fragment, generating wild-type infectious virus. Finally, direct ligation of restriction fragments, generated from random PCR mutagenesis, into the proviral vector should provide a library of protease mutations that allow extremely rapid selection of highly resistant viral variants.
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PMID:Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors. 763 88

We have used an 'activated' molecular dynamics approach to simulate flap opening in HIV-1 protease. An initial impulse for flap opening was provided by applying harmonic constraints to non-flap residues. After an initial 'melting' phase, the two beta-hairpin structures that constitute the flaps opened to a 25 A gap within 200 ps of simulation. Analysis of backbone torsion angles suggests that flap opening is related to conformational changes at Lys 45, Met 46, Gly 52 and Phe 53. In contrast, similar molecular dynamics simulations on the M46I mutant, which is associated with drug resistance, indicates that this mutation stabilizes the flaps in a closed conformation.
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PMID:Flap opening in HIV-1 protease simulated by 'activated' molecular dynamics. 779 68

The irreversible inhibition of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteases by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and eight haloperidol derivatives has been studied. EPNP specifically inhibits HIV-1 and HIV-2 proteases with a stoichiometry of one EPNP molecule/dimeric enzyme. The site of modification of HIV-2 protease by EPNP has been unambiguously identified as Asp-25 using high performance tandem mass spectrometry. The haloperidol derivatives assayed consist of epoxides, ynones, and alpha,beta-unsaturated ketones. The Kinact values for these haloperidol derivatives range from 10.7 to 521 microM for HIV-1 protease and from 8.6 to 283 microM for the HIV-2 enzyme, being in some cases approximately 1000-fold more potent irreverisble inhibitors of HIV proteases than EPNP. This potency results from the haloperidol character of the compounds and the chemical reactivity of the groups capable of forming a covalent bond with the enzyme. Covalent modification of HIV-2 protease by a radiolabeled epoxide derivative of haloperidol, UCSF 84, is prevented by EPNP and the peptidomimetic transition state analog U-85548. In similar experiments, incorporation of UCSF 84 into HIV-1 protease is partially prevented by these active-site inhibitors. In contrast, a mutant HIV-1 protease, HIV-1 PR C95M, in which Cys-95 has been replaced by Met, is labeled 50% less than HIV-1 protease and is fully protected by EPNP and U-85548. These results indicate the presence of 2 reactive residues in HIV-1 protease: Cys-95 and another located in the active site of the enzyme. The alpha,beta-unsaturated ketone derivative of haloperidol, UCSF 191, which is stable over a broad pH range, was used to study the pH profile of inactivation of HIV-1 and HIV-2 proteases. Comparison of the profiles of inactivation of wild-type HIV-1 protease, HIV-1 PR C95M, and HIV-1 PR C67L as well as HIV-2 protease (which has no cysteine residues) reveals the contribution of Cys-95 to the reactivity of these irreversible inhibitors. The inhibitors UCSF 70, UCSF 84, UCSF 115, UCSF 142, and UCSF 191 reduce p55gag polyprotein processing when assayed in a mammalian cell line that produces HIV-1 viral particles lacking the envelope.
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PMID:In vitro characterization of nonpeptide irreversible inhibitors of HIV proteases. 814 59

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) approximately 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.
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PMID:Large scale purification and refolding of HIV-1 protease from Escherichia coli inclusion bodies. 839 90

Human immunodeficiency virus type 1 (HIV-1) expresses its structural and functional proteins within Gag-Pol precursor polyproteins. Specific proteolytic processing of the precursors by the viral protease is critical for the maturation and infectivity of viral particles. To observe the influence of autoprocessing on the activation of recombinant HIV-1 protease, we constructed different HIV-1 protease forms, with or without the Phe-Pro bond directly upstream of the protease domain, and expressed them in Escherichia coli systems. We found that the presence of a short upstream sequence of the protease domain, which could generate the original N-terminus of the protease by autoproteolysis of the Phe-Pro bond, resulted in processing of active protease, whereas for a wild-type protease extended only with the initiator methionine, the proteolytic activity was not recovered. Our results suggested that autoprocessing of the direct upstream sequence of the protease domain is an essential step for the activation of recombinant HIV-1 protease in the E. coli expression system. Expression of HIV-1 protease as fusion proteins revealed that the existence of a fusion portion increased the accumulation of expressed protease by affecting its homotypic dimer formation.
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PMID:Autoprocessing: an essential step for the activation of HIV-1 protease. 868 2

AG1343 ([3S-(3R*,4aR*,8aR*,2'S*,3'S*)]-2-[2' hydroxy-3'-phenylthiomethyl-4'-aza-5'-oxo-5'-(2''-methyl-3''-hydro xy-phenyl) pentyl]-decahydroiso-quinoline-3-N-t-butylcarboxamide methanesulfonic acid) is a selective, nonpeptidic inhibitor of human immunodeficiency virus (HIV) protease (Ki = 2 nM) that was discovered by protein structure-based drug design methodologies. AG1343 was effective against the replication of several laboratory and clinical HIV type 1 (HIV-1) or HIV-2 isolates including pyridinone- and zidovudine-resistant strains, with 50% effective concentrations ranging from 9 to 60 nM. In reversibility studies, inhibition of gag (p55) proteolytic processing in HIV-1 particles from cells treated with AG1343 was maintained for up to 36 h after drug removal. The ability of virus to develop resistance to AG1343 was studied by serial passage of HIV-1 NL4.3 in the presence of increasing concentrations of drug. After 28 passages, a variant with a 30-fold reduction in susceptibility to AG1343 was isolated. Molecular analysis of the protease from this variant indicated a double change from a Met to Ile at residue 46 and an Ile to Val or Ala at residue 84 (M46I+I84V, A). Consistent with these findings, reductions in susceptibility were observed for recombinant viruses constructed to contain the single I84V change or the double M46I+I84V substitutions. Resistance, however, was not detected for recombinant viruses containing other key mutations in HIV-1 protease, including a Val to Ile change at residue 32 or a Val to Ala or Phe at residue 82. The potent anti-HIV activity of AG1343 against several isolates suggests that AG1343 should perform well during ongoing human phase II clinical trials.
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PMID:Antiviral and resistance studies of AG1343, an orally bioavailable inhibitor of human immunodeficiency virus protease. 883 68

Molecular models of HIV-1 protease and 21 peptide substrates with single amino acid substitutions at positions from P4 to P3' were built and compared with kinetic measurements. The crystal structure of HIV-1 protease with a peptidic inhibitor was modified to model the peptide substrate Pro-Ala-Val-Ser-Leu-Ala-Met-Thr for the starting geometry. Models were built of two reaction intermediates, HIV protease with peptide substrate and with its tetrahedral intermediate. The energy minimization used a new algorithm that increased the speed and eliminated a cut-off for non-bonded interactions. After minimization the models for substrate and tetrahedral intermediate both had root mean square deviations of 0.48 A for all atoms of the HIV protease compared to the starting crystal structure. Differences in the model structures and interaction energies for HIV protease with different substrates were analyzed. The calculated interaction energies for the 21 HIV protease-tetrahedral intermediate models gave a correlation coefficient of 0.64 with the kinetic measurements. The eight substrates with changes in the P1 and P1' residues next to the scissile bond gave the highest correlation of 0.93, while the 14 substrates with changes in P2-P2' gave a correlation coefficient of 0.86. The catalytic mechanism and factors influencing the catalytic efficiency of the different substrates are discussed in relation to the models. The predictive ability of molecular mechanics calculations is discussed in the context of the statistical mechanics analysis of the differences in free energy.
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PMID:Molecular mechanics calculations on HIV-1 protease with peptide substrates correlate with experimental data. 887 45

Molecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3', where the scissile bond is between P1 and P1'. were built and compared with kinetic measurements. The unsubstituted peptide substrate. Pro-Ala-Val-Ser-Leu-Ala-Met-Thr, represents the NC-PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease-substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease-substrate models with changes in P1 to P1' next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in P1 and P1', and 0.56 for all substrates. These results are compared to those from similar calculations on HIV-1 protease and discussed in relation to the rate-limiting steps in the catalytic mechanism and the entropic contributions.
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PMID:Molecular mechanics calculations on Rous sarcoma virus protease with peptide substrates. 938 39

Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H(2)O(2) resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H(2)O(2) led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.
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PMID:HIV-2 protease is inactivated after oxidation at the dimer interface and activity can be partly restored with methionine sulphoxide reductase. 1067 47


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