DrugBank:EXPT00572 - FACTA Search

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Query: DrugBank:EXPT00572 (Asn)
11,732 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3' region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF/VVNGLVK-NH(2) (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2' position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1', FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2' subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1' subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF/VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVF Psi(CH(2)NH)VVNGL-NH(2.) This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors.
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PMID:Molecular basis for the relative substrate specificity of human immunodeficiency virus type 1 and feline immunodeficiency virus proteases. 1153 8

In the present work we have constructed a series of backbone cyclic peptides, which differed in the amino acid residues located at the C-terminal position of the previously described BCvir peptide (A. Friedler, N. Zakai, O. Karni, Y.C. Broder, L. Baraz, M. Kotler, A. Loyter, C. Gilon, Biochemistry 37 (1998)). BCvir is a cyclic peptide, derived from the nuclear localization signal (NLS) of the human immunodeficiency virus type 1 matrix protein. The majority of the cyclic peptides described here inhibited nuclear import in vitro. The most potent inhibitors were those bearing bulky hydrophobic amino acids such as Leu, Phe or Nal (naphthyl Ala) at the C-terminus. On the other hand, peptides bearing polar amino acid residues such as Asn, Cys or a reduced amide bond were not inhibitory. The present studies demonstrate the importance of a bulky hydrophobic C-terminal side chain and an exocyclic amide bond preceding it, to the inhibitory activity of the NLS-derived BC peptides. Being only inhibitory, these BC peptides resemble classic receptor antagonists.
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PMID:Inhibition of nuclear import by backbone cyclic peptidomimetics derived from the HIV-1 MA NLS sequence. 1190 19

CXCR4 is a G protein-coupled receptor for stromal-derived factor 1 (SDF-1) that plays a critical role in leukocyte trafficking, metastasis of mammary carcinoma, and human immunodeficiency virus type-1 infection. To elucidate the mechanism for CXCR4 activation, a constitutively active mutant (CAM) was derived by coupling the receptor to the pheromone response pathway in yeast. Conversion of Asn-119 to Ser or Ala, but not Asp or Lys, conferred autonomous CXCR4 signaling in yeast and mammalian cells. SDF-1 induced signaling in variants with substitution of Asn-119 to Ser, Ala, or Asp, but not Lys. These variants had similar cell surface expression and binding affinity for SDF-1. CXCR4-CAMs were constitutively phosphorylated and present in cytosolic inclusions. Analysis of antagonists revealed that exposure to AMD3100 or ALX40-4C induced G protein activation by CXCR4 wild type, which was greater in the CAM, whereas T140 decreased autonomous signaling. The affinity of AMD3100 and ALX40-4C binding to CAMs was less than to wild type, providing evidence of a conformational shift. These results illustrate the importance of transmembrane helix 3 in CXCR4 signaling. Insight into the mechanism for CXCR4 antagonists will allow for the development of a new generation of agents that lack partial agonist activity that may induce toxicities, as observed for AMD3100.
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PMID:A point mutation that confers constitutive activity to CXCR4 reveals that T140 is an inverse agonist and that AMD3100 and ALX40-4C are weak partial agonists. 1192 1

It has previously been reported that mutations in the Gln(151) residue of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) greatly enhance RT fidelity. In this study, we employed pre-steady state kinetic assays to elucidate the mechanistic role of residue Gln(151) in highly error prone DNA synthesis by HIV-1 RT. Using our Q151N high fidelity mutant, which is structurally altered in its ability to interact with the 3'-OH on the sugar moiety of the incoming deoxynucleotide triphosphate (dNTP), we examined how this change in RT-dNTP interaction affects HIV-1 RT fidelity. First, we found the binding affinity (K(D)) of wild type and Q151N RT proteins to different template/primers to be similar. These results indicate that the Gln(151) residue is not involved in the formation of the binary complex (RT.template/primer) during DNA polymerization. We also found that by changing residue 151 from a Gln-->Asn, the maximum rate of dNTP incorporation (k(pol)) for both correct and incorrect dNTPs was not affected. In contrast, the ability of the Q151N mutant to bind both correct and incorrect dNTPs (K(d)) was diminished. The Q151N mutant was 120-fold less efficient at binding correct dNTP than wild type RT, and its decrease in binding was such that we were unable to measure the actual binding affinity of Q151N for incorrect dNTPs. Presumably, the fidelity increase observed during the steady state is explained by this defect in Q151N binding to incorrect dNTP. In wild type RT, residue Gln(151) is important for tight binding of incorrect dNTPs and may contribute to the low fidelity nature of HIV-1 RT. Since the Q151N mutation also alters RT binding to correct dNTPs, the wild type Gln(151) residue may play an important role in efficient binding of RT to correct dNTPs. Our findings suggest that residue Gln(151) is an important element for the execution of both highly error prone and efficient DNA synthesis by HIV-1 RT.
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PMID:Mechanistic role of residue Gln151 in error prone DNA synthesis by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Pre-steady state kinetic study of the Q151N HIV-1 RT mutant with increased fidelity. 1192 82

The synthetic peptide DP178, derived from the carboxyl-terminal heptad repeat region of human immunodeficiency virus type 1 GP41 protein is a potent inhibitor of viral-mediated fusion and contains the sequence ELDKWA, which constitutes the recognition epitope for the broadly neutralizing human monoclonal antibody 2F5. Efforts at eliciting a 2F5-like immune response by immunization with peptides or fusion proteins containing this sequence have not met with success, possibly because of incorrect structural presentation of the epitope. Although the structure of the carboxyl-terminal heptad repeat on the virion is not known, several recent reports have suggested a propensity for alpha-helical conformation. We have examined DP178 in the context of a model for optimized alpha-helices and show that the native sequence conforms poorly to the model. Solution conformation of DP178 was studied by circular dichroism and NMR spectroscopy and found to be predominantly random, consistent with previous reports. NMR mapping was used to show that the low percentage of alpha-helix present was localized to residues Glu(662) through Asn(671), a region encompassing the 2F5 epitope. Using NH(2)-terminal extensions derived from either GP41 or the yeast GCN4 leucine zipper dimerization domain, we designed peptide analogs in which the average helicity is significantly increased compared with DP178 and show that these peptides exhibit both a modest increase in affinity for 2F5 using a novel competitive solution-based binding assay and an increased ability to inhibit viral entry in a single-cycle infectivity model. Selected peptides were conjugated to carrier protein and used for guinea pig immunizations. High peptide-specific titers were achieved using these immunogens, but the resulting sera were incapable of viral neutralization. We discuss these findings in terms of structural and immunological considerations as to the utility of a 2F5-like response.
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PMID:Enhancement of alpha -helicity in the HIV-1 inhibitory peptide DP178 leads to an increased affinity for human monoclonal antibody 2F5 but does not elicit neutralizing responses in vitro. Implications for vaccine design. 1223 96

Cyanovirin-N (CV-N) is under development as a topical (vaginal or rectal) microbicide to prevent sexual transmission of human immunodeficiency virus (HIV), and an economically feasible means for very large-scale production of the protein is an urgent priority. We observed that N-glycosylation of CV-N in yeast eliminated the anti-HIV activity, and that dimeric forms and aggregates of CV-N occurred under certain conditions, potentially complicating the efficient, large-scale manufacture of pure monomeric CV-N. We therefore expressed and tested CV-N homologs in which the glycosylation-susceptible Asn residue at position 30 was replaced with Ala, Gln, or Val, and/or the Pro at position 51 was replaced by Gly to eliminate potential conformational heterogeneity. All homologs exhibited anti-HIV activity comparable to wild-type CV-N, and the Pro51Gly homologs were significantly more stable proteins. These glycosylation-resistant, functional cyanovirins should be amenable to large-scale production either in bacteria or in eukaryotic hosts.
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PMID:Functional homologs of cyanovirin-N amenable to mass production in prokaryotic and eukaryotic hosts. 1235 69

The human immunodeficiency virus type I (HIV-1) transmembrane glycoprotein gp41 mediates viral entry through fusion of the target cellular and viral membranes. A segment of gp41 containing the sequence Glu-Leu-Asp-Lys-Trp-Ala has previously been identified as the epitope of the HIV-1 neutralizing human monoclonal antibody 2F5 (MAb 2F5). The 2F5 epitope is highly conserved among HIV-1 envelope glycoproteins. Antibodies directed at the 2F5 epitope have neutralizing effects on a broad range of laboratory-adapted HIV-1 variants and primary isolates. Recently, a crystal structure of the epitope bound to the Fab fragment of MAb 2F5 has shown that the 2F5 peptide adopts a beta-turn conformation [Pai, E. F., Klein, M. H., Chong, P., and Pedyczak, A. (2000) World Intellectual Property Organization Patent WO-00/61618]. We have designed cyclic peptides to adopt beta-turn conformations by the incorporation of a side-chain to side-chain lactam bridge between the i and i + 4 residues containing the Asp-Lys-Trp segment. Synthesis of extended, nonconstrained peptides encompassing the 2F5 epitope revealed that the 13 amino acid sequence, Glu-Leu-Leu-Glu-Leu-Asp-Lys-Trp-Ala-Ser-Leu-Trp-Asn, maximized MAb 2F5 binding. Constrained analogues of this sequence were explored to optimize 2F5 binding affinity. The solution conformations of the constrained peptides have been characterized by NMR spectroscopy and molecular modeling techniques. The results presented here demonstrate that both inclusion of the lactam constraint and extension of the 2F5 segment are necessary to elicit optimal antibody binding activity. The ability of these peptide immunogens to stimulate a high titer, peptide-specific immune response incapable of viral neutralization is discussed in regard to developing an HIV-1 vaccine designed to elicit a 2F5-like immune response.
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PMID:HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb. 1264 52

DC-SIGN is a C-type lectin that binds to endogenous adhesion molecules ICAM-2 and ICAM-3 as well as the viral envelope glycoprotein human immunodeficiency virus, type 1, glycoprotein (gp) 120. We wished to determine whether DC-SIGN binds differently to its endogenous ligands ICAM-2 and ICAM-3 versus HIV-1 gp120. We found that recombinant soluble DC-SIGN bound to gp120-Fc more than 100- and 50-fold better than ICAM-2-Fc and ICAM-3-Fc, respectively. This relative difference was maintained using DC-SIGN expressed on three different CD4-negative cell lines. Although the cell surface affinity for gp120 varied by up to 4-fold on the cell lines examined, the affinity for gp120 was not a correlate of the ability of the cell line to transfer virus. Monosaccharides with equatorial 4-OH groups competed as well as D-mannose for gp120 binding to DC-SIGN, regardless of how the other hydroxyl groups were positioned. Disaccharide competitors and glycan chip analysis showed that DC-SIGN has a preference for oligosaccharides linked in an alpha-anomeric configuration. Alanine-scanning mutagenesis of DC-SIGN revealed that highly conserved residues that coordinate calcium (Asp-366) and/or are involved in both calcium and specific carbohydrate interactions (Glu-347, Asn-349, Glu-354, and Asp-355) significantly compromised binding to all three ligands. Mutating non-conserved residues (Asn-311, Arg-345, Val-351, Gly-352, Glu-353, Ser-360, Gly-361, and Asn-362) minimally affected binding except for the Asp-367 mutant, which enhanced gp120 binding but diminished ICAM-2 and ICAM-3 binding. Conversely, mutating the moderately conserved residue (Gly-346) abrogated gp120 binding but enhanced ICAM-2 and ICAM-3 binding. Thus, DC-SIGN appears to bind in a distinct but overlapping manner to gp120 when compared with ICAM-2 and ICAM-3.
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PMID:DC-SIGN binds to HIV-1 glycoprotein 120 in a distinct but overlapping fashion compared with ICAM-2 and ICAM-3. 1497 Feb 26

We previously described the adaptation of the neutralization-sensitive human immunodeficiency virus type 1 (HIV-1) strain IIIB to a neutralization-resistant phenotype in an accidentally infected laboratory worker. During long-term propagation of this resistant isolate, designated FF3346, on primary peripheral blood leukocytes in vitro, an HIV-1 variant appeared that had regained sensitivity to neutralization by soluble CD4 (sCD4) and the broadly neutralizing monoclonal antibody b12. When an early passage of FF3346 was subjected to limiting-dilution culture in peripheral blood mononuclear cells, eight virus variants with various degrees of neutralization resistance were isolated. Two of them, the sCD4 neutralization-resistant variant LW_H8(res) and the sCD4 neutralization-sensitive variant LW_G9(sens), were selected for further study. Interestingly, these two viruses were equally resistant to neutralization by agents that recognize domains other than the CD4 binding site. Site-directed mutagenesis revealed that the increased neutralization sensitivity of variant LW_G9(sens) resulted from only two changes, an Asn-to-Ser substitution at position 164 in the V2 loop and an Ala-to-Glu substitution at position 370 in the C3 domain of gp120. In agreement with this notion, the affinity of b12 for monomeric gp120 containing the N164S and A370E substitutions in the background of the molecular clone LW_H8(res) was higher than its affinity for the parental gp120. Surprisingly, no correlation was observed between CD4 binding affinity for monomeric gp120 and the level of neutralization resistance, suggesting that differences in sCD4 neutralization sensitivity between these viruses are only manifested in the context of the tertiary or quaternary structure of gp120 on the viral surface. The results obtained here indicate that the neutralization-sensitive strain IIIB can become neutralization resistant in vivo under selective pressure by neutralizing antibodies but that this resistance may be easily reversed in the absence of immunological pressure.
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PMID:Increased sensitivity to CD4 binding site-directed neutralization following in vitro propagation on primary lymphocytes of a neutralization-resistant human immunodeficiency virus IIIB strain isolated from an accidentally infected laboratory worker. 1514 Sep 62

A two-serine insertion at position 69 (i69SS) of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) appears to be critical to enhance multi-nucleoside RT inhibitor resistance (MNR) in the sequence context of multiple zidovudine (AZT) resistance mutations (i.e., M41L, L210W, T215Y). In this study, we measured the replication capacity relative to the wild-type (WT) HIV-1 of a series of recombinant viruses carrying the i69SS in the background of a clinical isolate with MNR in which we introduced mutations D67N, Y215T, Y215S, or Y215N. In vitro measurements included replication kinetics and growth competition assays at different multiplicities of infection (MOI). While the addition of D67N had a minor effect on replication capacity, the reversion of Tyr-215 to Thr, Ser, or Asn was sufficient to increase the virus ability to replicate in a drug-free environment. The same genotypic changes at position 215 rendered the MNR virus susceptible to AZT and stavudine. Interestingly, the presence of the insertion together with mutation T215Y in an otherwise WT sequence background was not sufficient to confer high-level resistance to AZT, although its replication capacity was clearly impaired. Therefore, the RT residue 215 plays a critical role in both replication capacity and drug resistance of multidrug-resistant viruses containing the i69SS.
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PMID:Relative replication fitness of multi-nucleoside analogue-resistant HIV-1 strains bearing a dipeptide insertion in the fingers subdomain of the reverse transcriptase and mutations at codons 67 and 215. 1526 99

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