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 high-molecular-weight dendritic cytoskeletal protein known as microtubule-associated protein (MAP)-2 displays the capacity to stimulate tubulin polymerization and to associate with microtubules. Serine proteases cleave MAP-2 into a C-terminal M(r) 28,000-35,000 microtubule-binding fragment and a larger N-terminal M(r) 240,000 projection-arm region. We now show that human immunodeficiency virus (HIV) proteinase also progressively degrades purified MAP-2 in vitro. This proteolysis reaction is characterized by transient accumulation of at least six intermediates, and most abundant of these is an M(r) 72,000 species that retains the ability to associate with taxol-stabilized microtubules. Treatment of this M(r) 72,000 species with thrombin releases the same M(r) 28,000 component as that derived from thrombin action on intact high-molecular-weight MAP-2, indicating that the viral aspartoproteinase action preferentially occurs further toward the N-terminus. The association of the M(r) 72,000 component with microtubules can be disrupted by the presence of a 21-amino acid peptide analogue of the second repeated sequence in the MAP-2 microtubule-binding region. We also studied HIV proteinase action on MAP-2 in the presence of tubulin and other MAPs that recycle with tubulin, and contrary to other published studies we found no effect of such treatment on microtubule self-assembly behavior. Cleavage of isolated MAP-2 by the HIV enzyme at high salt concentrations, followed by desalting and addition of tubulin, also resulted in microtubule assembly, albeit with slightly reduced efficiency.
J Neurochem 1992 Sep
PMID:Cleavage of bovine brain microtubule-associated protein-2 by human immunodeficiency virus proteinase. 149 13

Two octapeptides derived from the sequence of the N- and C-termini of HIV-1 protease were tested for their ability to inhibit HIV-1 reproduction. Weak inhibitory activity was found with each of the two peptides. It is assumed that HIV-1 protease is the target of the inhibitory action. In spite of the moderate inhibitory activity the results are encouraging since they may be improved by various means.
Biochem Biophys Res Commun 1991 Sep 16
PMID:HIV-1 reproduction is inhibited by peptides derived frm the N- and C-termini of HIV-1 protease. 168 Mar 23

The reverse transcriptase (RT) of human immunodeficiency virus type-1 (HIV-1) is comprised of two subunits of approximately 66kD and 51kD. We have defined the carboxyl terminus of the 51kD molecule using the 66kD RT and HIV-1 protease (PR) expressed in yeast. Precise constructs encoding the 66kD and 51kD molecules were expressed individually, in yeast, at high levels. The purified recombinant subunits were shown to associate into heterodimers that retained both RT and RNase H activities. Only the 66kD molecule could associate into homodimers. Such homodimers retained approximately 80% of the RT activity of the heterodimers. Our data demonstrates that the 51/66kD heterodimer, analogous to that found in vivo, can be reconstituted in vitro and is more efficient in both RT and RNase H activity than the homodimer.
Biochem Biophys Res Commun 1990 Sep 14
PMID:Characterization of the human immunodeficiency virus type-1 reverse transcriptase enzyme produced in yeast. 169 61

A novel class of HIV-1 protease inhibitors containing a hydroxymethylcarbonyl (HMC) isostere were designed from the substrate transition state and synthesized. Phenylnorstatine [Pns; (2R,3S)-3-amino-2-hydroxy-4-phenylbutyric acid] and the 2S diastereomer, (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid, named allophenylnorstatine (Apns) were effective transition-state mimics, and incorporation of Pns-Pro or Apns-Pro at the P1-P1' site gave potent and specific HIV-1 protease inhibitors. In the inhibitory assays, the chemically synthesized [Ala67,95] HIV-1 protease was used.
Chem Pharm Bull (Tokyo) 1991 Sep
PMID:Rational design and synthesis of a novel class of active site-targeted HIV protease inhibitors containing a hydroxymethylcarbonyl isostere. Use of phenylnorstatine or allophenylnorstatine as a transition-state mimic. 180 62

By using a structure-based computer-assisted search, we have found a butyrophenone derivative that is a selective inhibitor of the human immunodeficiency virus 1 (HIV-1) protease. The computer program creates a negative image of the active site cavity using the crystal structure of the HIV-1 protease. This image was compared for steric complementarity with 10,000 molecules of the Cambridge Crystallographic Database. One of the most interesting candidates identified was bromperidol. Haloperidol, a closely related compound and known antipsychotic agent, was chosen for testing. Haloperidol inhibits the HIV-1 and HIV-2 proteases in a concentration-dependent fashion with a Ki of approximately 100 microM. It is highly selective, having little inhibitory effect on pepsin activity and no effect on renin at concentrations as high as 5 mM. The hydroxy derivative of haloperidol has a similar effect on HIV-1 protease but a lower potency against the HIV-2 enzyme. Both haloperidol and its hydroxy derivative showed activity against maturation of viral polypeptides in a cell assay system. Although this discovery holds promise for the generation of nonpeptide protease inhibitors, we caution that the serum concentrations of haloperidol in normal use as an antipsychotic agent are less than 10 ng/ml (0.03 microM). Thus, concentrations required to inhibit the HIV-1 protease are greater than 1000 times higher than the concentrations normally used. Haloperidol is highly toxic at elevated doses and can be life-threatening. Haloperidol is not useful as a treatment for AIDS but may be a useful lead compound for the development of an antiviral pharmaceutical.
Proc Natl Acad Sci U S A 1990 Sep
PMID:Structure-based design of nonpeptide inhibitors specific for the human immunodeficiency virus 1 protease. 220 60

Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein in Escherichia coli using a double-plasmid expression system in which an RT precursor protein was expressed and processed in vivo by HIV-1 protease produced in trans. The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to greater than 90% homogeneity and were found to share a common NH2 terminus as judged by sequence analysis of the polypeptide mixture. The observed sequence confirmed correct in vivo cleavage by protease at the protease-RT polyprotein junction to yield an NH2 terminus identical to that of genuine viral RT (M. M. Lightfoote et al. (1986) J. Virol. 60, 771-775; F. diMarzo Veronese et al. (1986) Science 231, 1289-1291). The bacterially expressed RT had a specific activity similar to that of viral RT and inhibition studies with phosphonoformate confirmed that it was indistinguishable from the viral enzyme with respect to sensitivity to this inhibitor. Polymerase activated gel analysis of the mixture indicated that p66 was associated with a higher level of RT activity than p51. RNase H activated gel analysis suggested that the purified preparation of recombinant RT was free of endogenous E. coli RNase H, and that the RNase H activity of RT was exclusively associated with the p66 polypeptide, supporting the hypothesis that the RNase H domain is located in the COOH-terminal region of the molecule.
Arch Biochem Biophys 1989 Sep
PMID:Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities. 247 69

The functional analysis of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) subunits on transient and constitutive expression, in the absence or presence of the HIV-1 protease (PR) expression, in a human cell line is described. HIV-1 RT is a heterodimer composed of a 51-kDa subunit (p51) and a 66-kDa subunit (p66). Cloning and expression of the RT region of the HIV-1 pol gene in the HT-1080 human fibrosarcoma cell line yielded p66 without any detectable p51 and a low level of RT activity could be measured. Transient expression of PR and RT in cis generated p51 and p66, but when RT and PR were expressed in trans only p66 was produced. Attempts to establish a stable cell line expressing the PR-RT region of the pol gene were hampered by an apparent intolerance of HT-1080 cells to the HIV-1 PR expression. Therefore, to generate p51 independent of PR expression, the 51-kDa subunit was cloned separately. p51 lacked detectable RT activity. Coexpression of p51 and p66 resulted in a dramatic increase in RT activity. Stable HT-1080 cells producing both p51 and p66 exhibited on average a 15-fold increase in RT activity compared to the parental cell line. Immunofluorescence revealed a diffuse cytoplasmic localization of p51 and p66. To date, this is the first example of a human cell line that is constitutively expressing HIV-1 RT in the absence of HIV-1 infection.
AIDS Res Hum Retroviruses 1994 Sep
PMID:Analysis of HIV type 1 reverse transcriptase expression in a human cell line. 753 25

We have used the vaccinia virus-T7 RNA polymerase-based expression system for studies on the activity of proteases from various retroviruses on homologous and heterologous Gag polyproteins in eukaryotic cells. Proteases from human immunodeficiency virus (HIV) types 1 and 2, equine infectious anaemia virus, human T cell leukaemia virus type 1 and human spumavirus were produced and were shown to cleave their cognate Gag substrates produced in trans. Analysis of cross reactivity revealed that lentivirus proteases cleaved only lentivirus Gag proteins and oncovirus proteases acted primarily on oncovirus Gag proteins. The HIV-2 protease cleaved the HIV-1 Gag precursor almost as efficiently as HIV-1 protease. Expression of the 5' end of the human spumavirus pol gene revealed that it encodes a functional protease that acts specifically on the human spumavirus Gag polyprotein. This assay will allow further investigation on the activity and specificity of retrovirus proteases in eukaryotic cells.
J Gen Virol 1995 Sep
PMID:Analysis of cross reactivity of retrovirus proteases using a vaccinia virus-T7 RNA polymerase-based expression system. 756 54

The protease encoded by the human immunodeficiency virus-1 (HIV-1) is essential for processing viral polyproteins which contain the enzymes and structural proteins required for the infectious virus. It was previously found that cupric chloride, in the presence of dithiothreitol or ascorbic acid, could inhibit the HIV-1 protease. It was suggested that a Cu1+ chelate was the moiety responsible for inhibition of the protease. This hypothesis has now been investigated directly by utilizing the stable Cu1+ chelate, bathocuproine disulfonic acid Cu1+ (BCDS-Cu1+). BCDS-Cu1+ inhibited the HIV-1 wild type protease as well as a mutant HIV-1 protease lacking cysteines. BCDS-Cu1+ was a competitive inhibitor of the mutant HIV-1 protease with an apparent Ki of 1 microM. Replication of HIV-1 in human lymphocytes and the cytotoxic effect of HIV-1 in CEM cells was inhibited by micromolar BCDS-Cu1+. Inhibition of the protease and of HIV replication by BCDS-Cu1+ was dependent on the presence of Cu1+ as BCDS alone was ineffective. EDTA blocked the inhibition of the protease by Cu1+ but was unable to block inhibition of the protease by BCDS-Cu1+, indicating that the Cu1+ complex was the inhibitory agent. The apparent IC50 for BCDS-Cu1+ on the inhibition of replication by primary isolates of HIV-1 was 5 microM. However, BCDS-Cu1+ did not affect polyprotein processing in an H9 cell line chronically infected with HIV-1, indicating that BCDS-Cu1+ acts by yet another mechanism to block HIV infection. Other possible targets for BCDS-Cu1+ include inhibition of viral adsorption and/or inhibition of the HIV-1 integrase.
Arch Biochem Biophys 1995 Sep 10
PMID:Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+. 757 66

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.
J Virol 1995 Sep
PMID:Analysis of resistance to human immunodeficiency virus type 1 protease inhibitors by using matched bacterial expression and proviral infection vectors. 763 88


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