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)

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.
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PMID:Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease. 210 21

We evaluated saquinavir, an orally active, selective inhibitor of HIV proteinase, in a randomised, double-blind, dose-ranging study in 49 zidovudine-naive HIV-positive patients with few or no symptoms and CD4 cell counts of 500 or less. The study was designed to assess the antiviral activity and tolerability of saquinavir. Patients were randomised to receive 25, 75, 200, or 600 mg of saquinavir three times daily for 16 weeks. No serious adverse events occurred. CD4 cell counts showed a trend indicative of a dose response in favour of the 600 mg dosage, the maximum increase being seen around week 4. In none of the 8 patients with positive plasma viraemia at baseline did cultures become negative after treatment; peripheral blood mononuclear cell and plasma-viral load by culture and DNA and RNA PCR all showed a trend towards reduction at higher doses of saquinovir. Saquinavir was well tolerated in this group of previously untreated patients with few or no symptoms; this study shows that an HIV-proteinase inhibitor is active in HIV-infected patients.
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PMID:Safety and activity of saquinavir in HIV infection. 771 88

HIV PROTEINASE INHIBITORS: The HIV proteinase enzyme has been identified as a potential target for antiretroviral therapy, as inhibition of this enzyme leads to the generation of immature, non-infectious virions. There are several proteinase inhibitors in development; the first to enter clinical trials was saquinavir. DEVELOPMENT OF SAQUINAVIR: Saquinavir, a transition-stage analogue of an HIV proteinase cleavage site, was developed using computer-led rational design techniques. It is a highly specific inhibitor of HIV-1 and -2 proteinases, with antiviral activity at concentrations 1000-fold less than those causing cytotoxicity. EUROPEAN CLINICAL EXPERIENCE WITH SAQUINAVIR: Three European clinical studies involving 202 patients have been conducted with saquinavir at doses of 25, 75, 200 and 600 mg three times a day. Two studies were dose-ranging monotherapy trials, one in asymptomatic or mildly symptomatic patients not previously treated with zidovudine, the other in patients with advanced HIV infection who had been treated with zidovudine. The third study was a combination therapy trial with zidovudine in previously untreated patients with advanced infection. Saquinavir was well tolerated either alone or in combination with zidovudine. In the monotherapy studies, CD4 cell counts and estimates of viral load showed the best results with the 600-mg dose. The combination of saquinavir and zidovudine resulted in higher and more sustained increases in CD4 cell counts than with either drug alone. The CD4 cell counts favoured saquinavir at 200 and 600 mg in combination with zidovudine, although plasma viraemia and the RNA polymerase chain reaction indicated that the 600-mg dose (in combination) produced better responses.
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PMID:HIV therapy advances. Update on a proteinase inhibitor. 784 Sep 13

An in-vitro assay was developed to screen for HIV-1 protease inhibitors using a non-infectious proviral clone (X19) with a deletion in the envelope gene (Ratner et al., 1991). The proviral clone, X19, was expressed transiently in the COS 7 cell line. The virus was able to replicate as assessed by the presence of p24 in the supernatants, yet the virions produced did not infect CD4 positive cells. To determine the effect of a known protease inhibitor on p24 antigen production, PD 148310 (a Ro 31-8959 analog) was added immediately after transfection of the COS 7 cells. Virus particles were produced maximally after 24 h and cell supernatants were assayed for p24 antigen production using a p24 ELISA assay. PD 148310 inhibited p24 release in a dose-dependent manner with an IC50 of 23.6 nM. Western blot analysis of the supernatants using a mouse monoclonal antibody against p24 confirmed the presence of a well-defined p24 band in the control lane. At 1000 nM of PD 148310 the p24 band was not detectable, leaving only the unprocessed p55 Gag precursor. This technique is a useful tool to screen for potential HIV-1 protease inhibitors.
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PMID:An HIV-1 protease screening assay using a non-infectious proviral clone. 786 43

A novel series of omega-aminoalkanoic acid derivatives of betulinic acid were synthesized and evaluated for their activity against human immunodeficiency virus (HIV). The anti-HIV-1 activity of several members of this new series was found to be in the nanomolar range in CEM 4 and MT-4 cell cultures. The optimization of the omega-aminoalkanoic acid side chain is described. The presence of an amide function within the side chain was found important for optimal activity. RPR 103611 (14g), a statine derivative, was found to be inactive against HIV-1 protease, reverse transcriptase, and integrase as well as on gp120/CD4 binding. "Time of addition" experiments suggested interaction with an early step of HIV-1 replication. As syncytium formation, but not virus-cell binding, seems to be affected, betulinic acid derivatives are assumed to interact with the postbinding virus-cell fusion process.
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PMID:Betulinic acid derivatives: a new class of specific inhibitors of human immunodeficiency virus type 1 entry. 867 42

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.
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PMID:Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein. 867 50

Antiretroviral chemotherapeutic agents exhibit complex immunoregulatory changes in HIV-1 infected individuals that reflect the summation of the direct effects of the agents on the immune response and indirect effects through the amelioration of viral replication. Immunological parameters are useful in the context of clinical investigation and in the management of HIV-1 infected individuals in that they serve as prognostic indicators of disease, markers of biologic activity of antiretroviral drugs, and, to a lesser extent, predictors of clinical outcome. Many questions remain in the evaluation of these parameters in the context of clinical trials and in the management of individual patients. Although it is frequently tempting to measure all available parameters in any setting, it is important to be cognizant both of cost and of the fact that many of these parameters are codependent variables (29). Most evaluations that have been undertaken to date involve nucleoside analogs, either as single agents or in combination. It is clear that CD4 cells rise in association with the initiation of non-nucleoside reverse transcriptase inhibitors and HIV-1 protease inhibitors (30-32). It is not yet clear whether the CD4 cell changes observed with these classes of drugs exhibit the same relationships with other immunologic markers or with virologic or clinical parameters. Evidence has begun to emerge that CD4 cell changes in association with HIV-1 protease inhibitor therapy may be more durable than changes in viral load as assessed by plasma HIV-1 RNA. Given the more robust antiviral potency of HIV-1 protease inhibitors alone or in combination with nucleoside analogs, it is quite likely that many of the ambiguities with respect to prior studies of immunologic markers will be resolved in that the changes observed will likely be of a much greater magnitude and duration than those seen in association with nucleoside analog monotherapy. Finally, with the advent of more potent antiviral regimens, and with the more reproducible and sensitive techniques for measuring viral replication in vivo, the cogent investigation of immunologic parameters will provide important insights into the pathogenesis of HIV-1 infection (33, 34).
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PMID:Immunomodulatory effects of antiretroviral chemotherapy. 878 19

L-735,524 is a potent inhibitor of the HIV-1 protease. In cell culture, the compound interferes with virus replication by causing production of noninfectious immature viral particles containing an unprocessed gag-pol polyprotein. Initial human clinical studies demonstrated that treatment with the inhibitor caused circulating viral levels to decline and this decline was associated with increases in the CD4 count of varying magnitude. However, in most patients, antiviral activity is lost as viral variants with reduced susceptibility to the inhibitor are selected. The resistant phenotype appears to require an amino acid substitution at protease codon 82. However, this amino acid alteration alone is insufficient for expression of the resistance phenotype. Co-expression of various additional alterations seems to be required, but the nature of these additional substitutions differs among resistant isolates. HIV-1 variants, cross-resistant to a panel of structurally diverse protease inhibitors, were isolated from patients following prolonged L-735,524 therapy.
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PMID:In vivo selection of HIV-1 variants with reduced susceptibility to the protease inhibitor L-735,524 and related compounds. 881 97

Prostratin, a non-tumor-promoting phorbol ester, inhibited human immunodeficiency virus (HIV)-induced cell killing and viral replication in a variety of acutely-infected cell systems. The potency and degree of cytoprotection was dependent on both viral strain and host cell type. Prostratin activated viral expression in two latently-infected cell lines, but had little or no effect on chronically-infected cell lines. Prostratin caused a dose-dependent, but reversible, decrease in CD4 expression in the CEM-SS and MT-2 cell lines. This down-regulation of CD4 was inhibited in a dose-dependent manner by the protein kinase C (PKC) antagonist, staurosporine. In addition, the cytoprotective and cytostatic effects of prostratin in CEM-SS cells acutely infected with HIV-1RF were reversed by bryostatin-1, a PKC agonist. Prostratin had no effect on reverse transcriptase or HIV-1 protease, nor did it inhibit the binding of gp120 to CD4. We conclude that prostratin inhibits HIV cytopathicity and replication through mechanism(s) involving PKC enzyme(s).
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PMID:Antireplicative and anticytopathic activities of prostratin, a non-tumor-promoting phorbol ester, against human immunodeficiency virus (HIV). 902 Oct 50

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


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