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)

Inhibitors of HIV-1 protease represent a new class of antiretroviral compounds. Here, we report the design and synthesis of two novel C2 symmetry-based inhibitors, MP-134 and MP-167, specifically targeted against HIV-1 variants with reduced sensitivity to another related protease inhibitor, A-77003. In addition, we describe the in vitro selection of viral variants with reduced sensitivity of these two protease inhibitors. An isoleucine-to-valine substitution at residue 84 (I84V) of the HIV-1 protease confers resistance to MP-134, whereas a glycine-to-valine substitution at residue 48 (G48V) confers resistance to MP-167. Testing other protease inhibitors against these variants has revealed specific overlapping patterns of resistance among these agents. These findings have important implications in the design of combination regimens using multiple protease inhibitors and underscore the need to develop non-cross-resistant compounds to be used toward this goal.
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PMID:Design, synthesis, and resistance patterns of MP-134 and MP-167, two novel inhibitors of HIV type 1 protease. 882 19

Patient human immunodeficiency virus type 1 (HIV-1) isolates that are resistant to protease inhibitors may contain amino acid substitutions L10I/V, M46L/I, G-48V, L63P, V82A/F/T, I84V, and L90M in the protease gene. Substitutions at positions 82 and/or 90 occur in variants that display high levels of resistance to certain protease inhibitors. Nucleotide substitutions at these two sites also lead to the loss of two HindII restriction enzyme digestion sites, and these changes make possible a rapid procedure for the detection of drug-resistant variants in patients on protease inhibitor therapy. This procedure was used to detect the emergence of mutated viruses at various times after the initiation of therapy with the HIV-1 protease inhibitor indinavir. The method includes viral RNA isolation from plasma and reverse transcription PCR amplification of the protease gene with fluorescence-tagged primers. The PCR product is digested with HindII, the cleavage products are separated on a urea-acrylamide gel in a DNA sequencer, and the extent of cleavage is automatically analyzed with commercially available software. In viruses from 34 blood samples from four patients, mutations leading to an amino acid change at residue 82 appeared as early as 6 weeks after the start of therapy and persisted throughout the course of the study period (48 weeks). Mutations leading to double substitutions at residues 82 and 90 were seen at a lower frequency and appeared later than the change at position 82. The changes detected by restriction enzyme cleavage were confirmed by DNA sequencing of the cloned protease genes by reverse transcription PCR amplification of viral RNA from isolates in plasma. In addition to the changes at positions 82 and 90, we have identified M46L/I, G48V, and I54V substitutions in isolates derived from indinavir-treated patients. HindII analysis of uncloned, PCR-amplified DNA offers a rapid screening procedure for the detection of virus isolates containing mutations at amino acid residues 82 and 90 in the HIV-1 protease gene. By using other restriction enzymes, the same method can be used to detect additional protease drug-resistant variants and is generally applicable for the detection of mutations.
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PMID:Emergence of protease inhibitor resistance mutations in human immunodeficiency virus type 1 isolates from patients and rapid screening procedure for their detection. 891 59

The hydroxyethylurea human immunodeficiency virus type 1 (HIV-1) protease inhibitors SC-55389A and SC-52151 were used to select drug-resistant variants in vitro. One clinical HIV-1 strain (89-959) and one laboratory HIV-1 strain (LAI) were passaged in peripheral blood mononuclear cells or CEMT4 cells in the presence of SC-55389A. Resistant isolates from both strains consistently had a mutation to serine for asparagine at amino acid 88 (N88S) in the protease gene either alone or in combination with a change to phenylalanine at position 10. The N88S mutation, recreated by oligonucleotide-mediated site-directed mutagenesis in HXB2, was sufficient to confer resistance to SC-55389A. In contrast, SC-52151-resistant variants selected from the monocytotropic strain SF162 had multiple substitutions in the protease gene (I11V, M461, F53L, A71V, and N88D), and the N88D mutation, re-created by oligonucleotide-mediated site-directed mutagenesis in HXB2, did not confer resistance to SC-52151. The potencies of L735,524 and Ro31-8959 were not reduced when these compounds were assayed against variants with either the N88S or N88D substitution. Position 88 is in a helix that lies behind the substrate binding pocket and may indirectly influence inhibitor binding through interactions with the amino acid at position 31. The selected mutations were persistent in the viral populations after more than 20 passages in the absence of drugs. Passaging of virus first in SC-55389A alone and then in combination with SC-52151 resulted in the accumulation of more mutations in the protease gene (L10F, D35E, D37M, I47V, 154L, A71V, V82I, and S88D) and in the selection of a variant that was cross-resistant to multiple protease inhibitors. These results indicate that a mutation in the HIV-1 protease at a position that is located outside of the substrate binding pocket confers resistance to a protease inhibitor and that mutations in the protease gene accumulate with increasing selection pressure and can persist in the absence of selection pressure.
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PMID:A mutation in human immunodeficiency virus type 1 protease at position 88, located outside the active site, confers resistance to the hydroxyethylurea inhibitor SC-55389A. 905 85

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

Two different responses to the therapy were observed in a group of patients receiving the protease inhibitor indinavir. In one, suppression of virus replication occurred and has persisted for 90 weeks (bDNA, < 500 human immunodeficiency virus type 1 [HIV-1] RNA copies/ml). In the second group, a rebound in virus levels in plasma followed the initial sharp decline observed at the start of therapy. This was associated with the emergence of drug-resistant variants. Sequence analysis of the protease gene during the course of therapy revealed that in this second group there was a sequential acquisition of protease mutations at amino acids 46, 82, 54, 71, 89, and 90. In the six patients in this group, there was also an identical mutation in the gag p7/p1 gag protease cleavage site. In three of the patients, this change was seen as early as 6 to 10 weeks after the start of therapy. In one patient, a second mutation occurred at the gag p1/p6 cleavage site, but it appeared 18 weeks after the time of appearance of the p7/p1 mutation. Recombinant HIV-1 variants containing two or three mutations in the protease gene were constructed either with mutations at the p7/p1 cleavage site or with wild-type (WT) gag sequences. When recombinant HIV-1-containing protease mutations at 46 and 82 was grown in MT2 cells, there was a 68% reduction in its rate of replication compared to the WT virus. Introduction of an additional mutation at the gag p7/p1 protease cleavage site compensated for the partially defective protease gene. Similarly, rates of replication of viruses with mutations M46L/I, I54V, and V82A in protease were enhanced both in the presence and in the absence of Indinavir when combined with mutations in the gag p7/p1 and the gag p1/p6 cleavage sites. Optimal rates of virus replication require protease cleavage of precursor polyproteins. A mutation in the cleavage site that enhanced the availability of a protein that was rate limiting for virus maturation would confer on that virus a significant growth advantage and may explain the uniform emergence of viruses with alterations at the p7/p1 cleavage site. This is the first report of the emergence of mutations in the gag p7/p1 protease cleavage sites in patients receiving protease therapy and identifies this change as an important determinant of HIV-1 resistance to protease inhibitors in patient populations.
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PMID:Drug resistance during indinavir therapy is caused by mutations in the protease gene and in its Gag substrate cleavage sites. 926 88

The effect of antiretroviral therapy on both T-cell numbers and T-cell function in peripheral blood was studied. CD4+ and CD8+ T-cell numbers, T-cell reactivity to CD3 monoclonal antibodies (mAb), and viral RNA load date were obtained from patients treated for at least 28 weeks with either the HIV-1 protease inhibitor ritonavir, the nonnucleoside HIV-1 reverse transcriptase (RT) inhibitor nevirapine, or the nucleoside-analogue RT inhibitor zidovudine. Compared with both RT inhibitors, treatment with the protease inhibitor ritonavir resulted in the most significant and persistent elevation of CD4+ and CD8+ T-cell counts. However, in vitro T-cell functional improvement was of limited duration in the ritonavir-treated group and was inversely correlated with viral RNA load changes during the entire follow-up period. Thus, despite what can be assumed of responses during RT inhibitor therapy, quantitative responses on therapy did not necessarily correlate with qualitative immunologic responses, as can be seen during treatment with ritonavir. For optimal immune reconstitution, both numeric and functional immunologic improvements are essential. During antiretroviral therapy, measurement of in vitro improvement in immune function will be useful as a correlate for transient drug-induced alteration of immunodeficiency.
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PMID:Patterns of T-cell repopulation, virus load reduction, and restoration of T-cell function in HIV-infected persons during therapy with different antiretroviral agents. 942 Mar 8

The tryptophan time-resolved fluorescence intensity and anisotropy of the HIV-1 protease dimer is shown to be a quick and efficient method for the conformational characterization of protease inhibitor complexes. Four fluorescence lifetimes were needed to adequately describe the fluorescence decay of the two tryptophan residues, W6 and W42, per protease monomer. As a result of the wavelength dependence of the respective amplitudes, the 2.06 ns and the 4.46 ns decay constants were suggested to be the intrinsic fluorescence lifetimes of the more solvent-exposed W6 and the less exposed W42 residues, respectively. Analysis of the fluorescence anisotropy decay yielded a short correlation time of 250 ps corresponding to local chromophore motions, and a long correlation time of 12.96 ns resulting from overall rotation of the protease enzyme. Fluorescence lifetimes and rotational correlation times changed when inhibitors of the HIV-1 protease were added. The effects of 11 different inhibitors including statine-derived, hydroxyethylamine-derived, and 2 symmetrical inhibitors on the protease fluorescence dynamics were investigated. Inhibitor binding is shown to induce an increase of the mean fluorescence lifetime taumean, an increase of the short rotational correlation time phi1, as well as a decrease of the long rotational correlation time phi2. The mean rotational correlation time phimean was identified as the global dynamic parameter for a given molecular complex, which correlates with the inhibitor dissociation constant Ki, and therefore with the activity of the inhibitor.
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PMID:Time-resolved fluorescence anisotropy of HIV-1 protease inhibitor complexes correlates with inhibitory activity. 948 28

An understanding of the mechanisms of virologic cross-resistance between human immunodeficiency virus type 1 protease inhibitors is important for the establishment of effective treatment strategies for patients who no longer respond to their initial protease inhibitor. Protease gene sequencing results from patients treated with saquinavir showed significant increases in the frequency of the G48V protease mutation in patients receiving higher doses of the drug. In addition, all six patients who developed the G48V mutation during saquinavir therapy developed the V82A mutation either on continued saquinavir or after a switch to nelfinavir or indinavir. In vitro susceptibility assays showed that all 13 isolates with reduced susceptibilities to two or more protease inhibitors had either the G48V or L90M mutation, along with an average of six other protease mutations. Reduced susceptibility to nelfinavir was found in 14 isolates, but only 1 possessed the D30N mutation. These results suggest that mutations selected in vivo by initial saquinavir therapy may provide more cross-resistance to the other protease inhibitors than has been previously reported.
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PMID:Human immunodeficiency virus type 1 protease genotypes and in vitro protease inhibitor susceptibilities of isolates from individuals who were switched to other protease inhibitors after long-term saquinavir treatment. 957 9

Resistance of HIV-1 to protease inhibitors has been associated with changes at residues Val82 and Ile84 of HIV-1 protease (HIV PR). Using both an enzyme assay with a peptide substrate and a cell-based infectivity assay, we examined the correlation between the inhibition constants for enzyme activity (Ki values) and viral replication (IC90 values) for 5 active site mutants and 19 protease inhibitors. Four of the five mutations studied (V82F, V82A, I84V, and V82F/I84V) had been identified as conferring resistance during in vitro selection using a protease inhibitor. The mutant protease genes were expressed in Escherichia coli for preparation of enzyme, and inserted into the HXB2 strain of HIV for test of antiviral activity. The inhibitors included saquinavir, indinavir, nelfinavir, 141W94, ritonavir (all in clinical use), and 14 cyclic ureas with a constant core structure and varying P2, P2' and P3, P3' groups. The single mutations V82F and I84V caused changes with various inhibitors ranging from 0.3- to 86-fold in Ki and from 0.1- to 11-fold in IC90. Much larger changes compared to wild type were observed for the double mutation V82F/I84V both for Ki (10-2000-fold) and for IC90 (0.7-377-fold). However, there were low correlations (r2 = 0.017-0.53) between the mutant/wild-type ratio of Ki values (enzyme resistance) and the mutant/wild-type ratio of viral IC90 values (antiviral resistance) for each of the HIV proteases and the viruses containing the identical enzyme. Assessing enzyme resistance by "vitality values", which adjust the Ki values with the catalytic efficiencies (kcat/Km), caused no significant improvement in the correlation with antiviral resistance. Therefore, our data suggest that measurements of enzyme inhibition with mutant proteases may be poorly predictive of the antiviral effect in resistant viruses even when mutations are restricted to the protease gene.
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PMID:Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency. 962 35

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21-65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif21-65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif41-65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif21-65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif21-65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31-8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.
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PMID:Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif. 981 92


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