Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Cell Chem Biol ; 26(5): 662-673.e7, 2019 05 16.
Article in English | MEDLINE | ID: mdl-30827936

ABSTRACT

The G protein-coupled receptor (GPCR) CXCR4 is a co-receptor for HIV and is involved in cancers and autoimmune diseases. We characterized five purine or quinazoline core polyamine pharmacophores used for targeting CXCR4 dysregulation in diseases. All were neutral antagonists for wild-type CXCR4 and two were biased antagonists with effects on ß-arrestin-2 only at high concentrations. These compounds displayed various activities for a constitutively active mutant (CAM). We use the IT1t-CXCR4 crystal structure and molecular dynamics (MD) simulations to develop two hypotheses for the activation of the N1193.35A CAM. The N1193.35A mutation facilitates increased coupling of TM helices III and VI. IT1t deactivates the CAM by disrupting the coupling between TM helices III and VI, mediated primarily by residue F872.53. Mutants of F872.53 in N1193.35A CXCR4 precluded constitutive signaling and prevented inverse agonism. This work characterizes CXCR4 ligands and provides a mechanism for N1193.35A constitutive activation.


Subject(s)
Molecular Dynamics Simulation , Receptors, CXCR4/antagonists & inhibitors , Small Molecule Libraries/metabolism , Benzylamines , Chemokine CXCL12/pharmacology , Cyclams , HEK293 Cells , HIV Infections/metabolism , HIV Infections/pathology , HIV Infections/virology , HIV-1/drug effects , Heterocyclic Compounds/pharmacology , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Mutagenesis, Site-Directed , Protein Conformation, alpha-Helical , Protein Structure, Tertiary , Receptors, CXCR4/genetics , Receptors, CXCR4/metabolism , Signal Transduction/drug effects , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , beta-Arrestin 2/metabolism
2.
J Med Chem ; 58(3): 1452-65, 2015 Feb 12.
Article in English | MEDLINE | ID: mdl-25584630

ABSTRACT

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC50 of 0.5 nM against HIV-1 entry into host cells and an IC50 of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4(+)/CD34(+) stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.


Subject(s)
CCR5 Receptor Antagonists/pharmacology , HIV Fusion Inhibitors/pharmacology , HIV Infections/drug therapy , HIV-1/drug effects , Receptors, CXCR4/antagonists & inhibitors , Virus Internalization/drug effects , Animals , CCR5 Receptor Antagonists/chemical synthesis , CCR5 Receptor Antagonists/chemistry , Cell Line , Dose-Response Relationship, Drug , HIV Fusion Inhibitors/chemical synthesis , HIV Fusion Inhibitors/chemistry , HIV Infections/virology , HIV-1/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Microbial Sensitivity Tests , Molecular Dynamics Simulation , Molecular Structure , Receptors, CXCR4/metabolism , Structure-Activity Relationship
3.
Anal Chem ; 83(2): 463-6, 2011 Jan 15.
Article in English | MEDLINE | ID: mdl-21114271

ABSTRACT

A selective, oligonucleotide-based, label-free, turn-on fluorescence detection method for 3' → 5' exonuclease activity has been developed using crystal violet as a G-quadruplex-binding probe. The assay is highly simple and rapid, does not require the use of gel-based equipment or radioisotopic labeling, and is amenable to high-throughput and real-time detection. A proof-of-concept of this assay has been demonstrated for prokaryotic Exonuclease III (ExoIII) and human TREX1.


Subject(s)
Exonucleases/metabolism , Fluorescent Dyes/chemistry , G-Quadruplexes , Gentian Violet/chemistry , Spectrometry, Fluorescence/methods , Circular Dichroism , Exodeoxyribonucleases/metabolism , Humans , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Phosphoproteins/metabolism
4.
Antimicrob Agents Chemother ; 55(2): 895-903, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21078952

ABSTRACT

Individual variation in response to antiretroviral therapy is well-known, but it is not clear if demographic characteristics such as gender, age, and ethnicity are responsible for the variation. To optimize anti-HIV therapy and guide antiretroviral drug discovery, determinants that cause variable responses to therapy need to be evaluated. We investigated the determinants of intracellular concentrations of nucleoside analogs using peripheral blood mononuclear cells from 40 healthy donors. We observed individual differences in the concentrations of the intracellular nucleoside analogs; the mean concentrations of the triphosphate metabolite of ethynylstavudine (4'-Ed4T), zidovudine (AZT), and lamivudine (3TC) were 0.71 pmol/10(6) cells (minimum and maximum, 0.10 and 3.00 pmol/10(6) cells, respectively), 0.88 pmol/10(6) cells (minimum and maximum, 0.10 and 15.18 pmol/10(6) cells, respectively), and 1.70 pmol/10(6) cells (minimum and maximum, 0.20 and 7.73 pmol/10(6) cells, respectively). Gender and ethnicity had no effect on the concentration of 4'-Ed4T and 3TC metabolites. There was a trend for moderation of the concentrations of AZT metabolites by gender (P = 0.17 for gender·metabolite concentration). We observed variability in the activity and expression of cellular kinases. There was no statistically significant correlation between thymidine kinase 1 (TK-1) activity or expression and thymidine analog metabolite concentrations. The correlation between the activity of deoxycytidine kinase (dCK) and the 3TC monophosphate metabolite concentration showed a trend toward significance (P = 0.1). We observed an inverse correlation between the multidrug-resistant protein 2 (MRP2) expression index and the concentrations of AZT monophosphate, AZT triphosphate, and total AZT metabolites. Our findings suggest that the observed variation in clinical response to nucleoside analogs may be due partly to the individual differences in the intracellular concentrations, which in turn may be affected by the cellular kinases involved in the phosphorylation pathway and ATP-binding cassette (ABC) transport proteins.


Subject(s)
Anti-HIV Agents/metabolism , Lamivudine/metabolism , Leukocytes, Mononuclear/metabolism , Nucleosides/metabolism , Stavudine/analogs & derivatives , Zidovudine/metabolism , Anti-HIV Agents/chemistry , CD4-Positive T-Lymphocytes/enzymology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cross-Sectional Studies , Deoxycytidine Kinase/metabolism , Female , HIV Seronegativity , Humans , Lamivudine/analogs & derivatives , Leukocytes, Mononuclear/enzymology , Leukocytes, Mononuclear/immunology , Lymphocyte Activation , Male , Nucleosides/chemistry , Polyphosphates/metabolism , Sex Factors , Stavudine/metabolism , Thymidine Kinase/metabolism , Treatment Outcome , Zidovudine/analogs & derivatives
5.
Antimicrob Agents Chemother ; 53(11): 4640-6, 2009 Nov.
Article in English | MEDLINE | ID: mdl-19704131

ABSTRACT

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4'-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4'-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4'-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4'-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4'-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4'-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4'-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4'-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4'-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4'-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/genetics , HIV-1/drug effects , Mutation , Stavudine/analogs & derivatives , Computer Simulation , Drug Resistance, Viral , Humans , Microbial Sensitivity Tests , Reverse Transcriptase Inhibitors/pharmacology , Stavudine/pharmacology
6.
DNA Repair (Amst) ; 8(10): 1179-89, 2009 Oct 02.
Article in English | MEDLINE | ID: mdl-19617005

ABSTRACT

The major mammalian exonuclease TREX1 has been proposed to play a role in DNA repair and drug resistance. However, no cellular evidence substantiates this claim. Recent reports indicate TREX1's involvement in autoimmunity. To further understand its role, we studied TREX1 expression and functionality in anticancer drug-treated tumor cells. We report that the expression and localization of TREX1 are cell-type dependent. Camptothecin and other DNA damaging agents induced both TREX1 protein and its mRNA in a dose- and time-dependent manner. Using a TREX1-inducible cell line, we performed clonogenic assays and found no change in sensitivity of the cells to the agents upon TREX1 induction, suggesting that TREX1 may not play a role in DNA repair or drug sensitivity. Nevertheless, TREX1 serves as a key enzyme in the degradation of DNA from dying cells leading to less cellular DNA. Ubiquitously expressed in normal tissues, TREX1 may act in degrading DNA in all cell types undergoing a dying process before phagocytosis occurs.


Subject(s)
Antineoplastic Agents/pharmacology , DNA Damage , Exodeoxyribonucleases/metabolism , Neoplasms/drug therapy , Neoplasms/pathology , Phosphoproteins/metabolism , Apoptosis/drug effects , Camptothecin/pharmacology , Cell Line, Tumor , DNA/genetics , DNA/metabolism , DNA Repair/drug effects , Exodeoxyribonucleases/genetics , Gene Expression Regulation, Neoplastic/drug effects , Humans , Neoplasms/genetics , Phosphoproteins/genetics , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism
7.
Antiviral Res ; 73(3): 185-91, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17109975

ABSTRACT

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), is a recently discovered nucleoside reverse transcriptase inhibitor (NRTI) showing a 5- to 10-fold greater anti-human immunodeficiency virus type 1 (HIV-1) activity and less cellular and mitochondrial toxicity than its parental compound, stavudine (D4T). It is also active against a variety of NRTI-resistant HIV-1 mutants under non-cytotoxic concentrations. In this study, the effects of 4'-Ed4TTP, which is the triphosphate metabolite of 4'-Ed4T, on HIV-1 reverse transcriptase (RT) activity were investigated. We found that 4'-Ed4TTP was a substrate of HIV-1 RT serving as a DNA chain terminator, and it inhibited the DNA polymerase activity of RT more efficiently than D4TTP. The value of Ki(4'-Ed4TTP)/Km(dTTP) is 0.15 for DNA/RNA primer/template duplex (P/T), but 0.7 for DNA/DNA P/T, suggesting 4'-Ed4TTP inhibits RT more efficiently during RNA-dependent DNA synthesis than DNA-dependent DNA synthesis. 4'-Ed4TTP was also found to inhibit the 3TC (Lamivudine)-resistant RT mutant, M184V, with 3-fold less efficiency than the wild type (wt) RT. 4'-Ed4TTP showed much less inhibitory effects toward major host DNA polymerases. Overall, our results suggest that 4'-Ed4TTP is the active form for anti-HIV-1 activity via its inhibitory effect against RT.


Subject(s)
Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/enzymology , Nucleic Acid Synthesis Inhibitors/pharmacology , Polyphosphates/pharmacology , Reverse Transcriptase Inhibitors/pharmacology , Stavudine/analogs & derivatives , Animals , Cattle , DNA/biosynthesis , DNA/genetics , DNA/metabolism , DNA-Directed DNA Polymerase/metabolism , HIV Reverse Transcriptase/metabolism , HeLa Cells , Humans , RNA/genetics , RNA/metabolism , Reverse Transcriptase Inhibitors/chemistry , Ribonuclease H/metabolism , Stavudine/chemistry , Stavudine/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...