Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effects of point mutations of the conserved Asp443, Glu478, Asn494, and Asp498 residues in the RNase H domain of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) have been analyzed. The mutants fell into two classes: (i) functional RT, but not detectable ribonuclease H activity, and (ii) uncharacterizable phenotype due to protein instability in the context of the RT/protease Escherichia coli co-expression system (Mizrahi, V., Lazarus, G. M., Miles, L. M., Meyers, C. A., and Debouck, C. (1989) Arch. Biochem. Biophys. 273, 347-358). The only mutation in the former class was D443A, whereas those in the latter included D443E, E478D, E478Q, D498E, D443A/D498N, D443E/D498N, D443Q/D498N, N494A, N494D, and N494Q. The results were interpreted in terms of the x-ray crystal structure of the HIV-1 RNase H domain (Davies, J. F., II, Hostomaska, Z., Hostomsky, Z., Jordan, S. R., and Matthews, D. A. (1991) Science 252, 88-95) and a general acid-general base hydrolysis mechanism (Katayanagi, K., Okumura, M., and Morikawa, K. (1993) Proteins Struct. Funct. Genet. 17, 337-346). The data suggested that structural perturbations within the RNase H domain interfered with maturation of the pol precursor by HIV-1 protease. Analysis of selected D443/D498 double mutants suggested that the destabilization caused by the D498N mutation could be suppressed by the formation of a new hydrogen bond between Asn498 and Asn443.
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PMID:Mutagenesis of the conserved aspartic acid 443, glutamic acid 478, asparagine 494, and aspartic acid 498 residues in the ribonuclease H domain of p66/p51 human immunodeficiency virus type I reverse transcriptase. Expression and biochemical analysis. 751 54

Over 25 selected naphthalenesulfonic acid derivatives were evaluated for their inhibitory effect on two different functional domains of the HIV-1 reverse transcriptase (RT), namely the ribonuclease H and DNA polymerase activities. Most of the analogues were found to be either specific toward the DNA polymerase activity or showed nonselective inhibition of both catalytic functions. The most active compounds are either symmetrical derivatives or nonsymmetrical derivatives containing a lipophilic appendage consisting of a palmitoyl or cholesteryl moiety. The six most active compounds in the preliminary screen, derivatives 6, 16, 17, 23, 26, and 27, were subjected to experiments to determine their 50% inhibitory concentration (IC50) values in the assays that measure RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H (RNase H) functions of HIV-1 RT. The most potent derivative was a nonsymmetric cholesterol-linked 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid analogue, compound 23, which demonstrated an IC50 value of 0.06 microM for inhibiting RDDP activity. Inhibition of DDDP and RNase H activity for this compound was demonstrated at concentrations that were over 100-fold of that for inhibiting RDDP activity. However, the potency of this active compound does not correlate in the whole virus assay, probably due to a lack of cellular entry. The cholesterol derivative, 23, also possesses HIV-1 protease inhibitory activity and belongs to a unique class of multifunctional HIV-1 inhibitors.
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PMID:Synthesis of naphthalenesulfonic acid small molecules as selective inhibitors of the DNA polymerase and ribonuclease H activities of HIV-1 reverse transcriptase. 752 80

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.
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PMID:Analysis of HIV type 1 reverse transcriptase expression in a human cell line. 753 25

Human plasma impairs the activity of the human immunodeficiency virus (HIV-1) proteinase to cleave the HIV-1 gag-polyprotein precursor. The inhibition is due to the entrapment of the proteinase by plasma alpha 2-macroglobulin (alpha 2M). In methylamine-treated plasma, where alpha 2M is inactivated, HIV proteinase is not blocked. The interaction of alpha 2M and HIV-1 proteinase resulting in covalent complexes of proteinase and alpha 2M was demonstrated by immunoblotting with antiserum either to alpha 2M or to the HIV proteinase. We suggest if HIV-1 proteinase would be released in vivo from infected patients' cells, alpha 2M entrapment may prevent or minimize a conceivable cleavage of extracellular matrix or plasma proteins by the HIV-1 enzyme.
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PMID:Human immunodeficiency virus type 1 proteinase is rapidly and efficiently inactivated in human plasma by alpha 2-macroglobulin. 753 87

Active, recombinant p68 reverse transcriptase (RT) from human immunodeficiency virus type 2 (HIV-2), with an NH2-terminal extension containing a hexahistidine sequence was isolated from extracts of Escherichia coli by immobilized metal affinity chromatography. Treatment of the purified p68/p68 homodimer of HIV-2 RT with recombinant HIV-2 protease generates stable, active heterodimer (p68/p58) that is resistant to further hydrolysis. Analysis of this p68/p58 HIV-2 RT heterodimer revealed that while one subunit is intact p68, the p58 subunit is COOH-terminally truncated by cleavage, not at Phe440 as is seen in processing of the p66/p66 HIV-1 RT homodimer by HIV-1 protease, but at Met484. The expected COOH-terminal p10 fragment resulting from hydrolysis of p68 at Met484 is not released intact, but undergoes further cleavage at Asn494, Met503, and Tyr532. Processing of p68/p68 HIV-2 RT with the HIV-1 protease led to cleavage of the Phe440-Tyr441 bond, exactly as is seen with p66/p66 HIV-1 RT, to give the analogous p53 subunit. Studies of a peptide substrate modeled after residues 437-444 in HIV-2 RT showed that while the HIV-1 protease was able to cleave the Phe440 bond, this bond was resistant to cleavage by the HIV-2 enzyme. Our findings provide a rationale for the previous observation that the RT heterodimer isolated from HIV-2 lysates is larger than that from HIV-1. We conclude that the p68/p58 HIV-2 RT heterodimer, containing the Met484 truncated p58 subunit, is a biologically relevant form of the enzyme in vivo.
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PMID:The differential processing of homodimers of reverse transcriptases from human immunodeficiency viruses type 1 and 2 is a consequence of the distinct specificities of the viral proteases. 753 31

Viral resistance limits the value of drugs that act through competitive inhibition of HIV reverse transcriptase (RT). Thus, the emphasis of current research is on agents with other mechanisms. The possibilities include noncompetitive RT inhibitors, drugs against an entirely different enzyme--HIV proteinase, and measures to enhance immunity, either globally or against HIV specifically.
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PMID:An approach to antiretroviral treatment of HIV disease. New approaches. 754 85

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.
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PMID:Inhibition of the human immunodeficiency virus-1 protease and human immunodeficiency virus-1 replication by bathocuproine disulfonic acid Cu1+. 757 66

Comparative Molecular Field Analysis (CoMFA), a three-dimensional quantitative structure-activity relationship (3D-QSAR) paradigm (Cramer, R.D.; et al. (1988), J. Am. Chem. Soc., 110, 5959-5967), correlates variations in the (experimental) biological activity with 3D variance in the steric and electrostatic field of modeled compounds. Of general interest to the drug design area is the interpretation of CoMFA results, in order to gain maximum benefit from an established 3D-QSAR model. CoMFA studies report results using the standard deviation (stdev) times(*) coefficient (beta) field and its contributions to make SAR statements. This field is the scalar product of the absolute stdev of the CoMFA field at a lattice point and the QSAR equation coefficient (beta) at the same point. Negative beta values yield detrimental contributions, while positive beta values are considered beneficial. The QSAR equation is based on actual field values, therefore both positive and negative field values can have beneficial effect to the target property (Y), depending on the sign of beta. The results of a CoMFA model on 59 HIV-1 protease (HIV-PR) inhibitors (Waller, C.L.; et al. (1993), J. Med. Chem., 36, 4152-4160) were compared with the HIV-PR crystal structure to analyze the correspondence between CoMFA fields and ligand binding regions in the enzyme. Local steric and electrostatic interactions were analyzed in terms of various field values and beta coefficients. While redundant for some regions, other field contours besides stdev* beta bring additional information. Using this method, we observed a unique region with negative beta values for the electrostatic field (based on a -1 charged probe) located opposite of the scissile bond, between P1 and P1', where steric stdev* beta values are positive. Four hydrophobic residues in the HIV-PR crystal delimit the region, which is suggested as a new potential hydrophobic binding site for the inhibitors. The same region was confirmed using the stdev* beta contours of a HINT (Kellogg, G.; et al. (1991), J. Comput.-Aided Mol. Design, 5, 545-552) calculation on the same model. The steric, electrostatic and lipophilic fields of the CoMFA and HINT models are presented in various forms, and the information extracted is detailed.
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PMID:3D-QSAR of human immunodeficiency virus (I) protease inhibitors. III. Interpretation of CoMFA results. 757 6

Eleven different recombinant, drug-resistant HIV-1 protease (HIV PR) mutants--R8Q, V32I, M46I, V82A, V82F, V82I, I84V, V32I/I84V, M46I/V82F, M46I/I84V, and V32I/K45I/F53L/A71V/I84V/L89M--were generated on the basis of results of in vitro selection experiments using the inhibitors A-77003, A-84538, and KNI-272. Kinetic parameters of mutant and wild-type (WT) enzymes were measured along with inhibition constants (Ki) toward the inhibitors A-77003, A-84538, KNI-272, L-735,524, and Ro31-8959. The catalytic efficiency, kcat/Km, for the mutants decreased relative to WT by a factor of 1.2-14.8 and was mainly due to the elevation of Km. The effects of specific mutations on Ki values were unique with respect to both inhibitor and mutant enzyme. A new property, termed vitality, defined as the ratio (Kikcat/Km)mutant/(Kikcat/Km)WT was introduced to compare the selective advantage of different mutants in the presence of a given inhibitor. High vitality values were generally observed with mutations that emerged during in vitro selection studies. The kinetic model along with the panel of mutants described here should be useful for evaluating and predicting patterns of resistance for HIV PR inhibitors and may aid in the selection of inhibitor combinations to combat drug resistance.
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PMID:Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. 762 98

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


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