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1.
PLoS One ; 10(6): e0128381, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26030924

RESUMO

The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/química , Epitopos/imunologia , Receptores CCR5/química , Receptores CCR5/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Células CHO , Quimiotaxia , Cricetinae , Cricetulus , Cristalografia por Raios X , Feminino , Humanos , Imunização , Ligantes , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Especificidade da Espécie
2.
Br J Pharmacol ; 171(14): 3364-75, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24628038

RESUMO

BACKGROUND AND PURPOSE: The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance. EXPERIMENTAL APPROACH: Using [(3) H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc. KEY RESULTS: Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor-ligand complex (RA), which transitioned to a more stable complex, R'A, with at least a 13-fold increase in affinity. The dissociation rate from R'A, k-2 , was 1.2 × 10(-3) min(-1) . The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5. CONCLUSIONS AND IMPLICATIONS: The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc-receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Antagonistas dos Receptores CCR5/farmacologia , Cicloexanos/farmacologia , Receptores CCR5/metabolismo , Triazóis/farmacologia , Sítios de Ligação/efeitos dos fármacos , Antagonistas dos Receptores CCR5/química , Cicloexanos/química , Relação Dose-Resposta a Droga , Humanos , Cinética , Ligantes , Maraviroc , Relação Estrutura-Atividade , Fatores de Tempo , Triazóis/química
3.
Hepatology ; 56(6): 2027-38, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22684948

RESUMO

UNLABELLED: During antiviral therapy, specific delivery of interferon-α (IFNα) to infected cells may increase its antiviral efficacy, trigger a localized immune reaction, and reduce the side effects caused by systemic administration. Two T-cell receptor-like antibodies (TCR-L) able to selectively bind hepatitis B virus (HBV)-infected hepatocytes of chronic hepatitis B patients and recognize core (HBc18-27) and surface (HBs183-91) HBV epitopes associated with different human leukocyte antigen (HLA)-A*02 alleles (A*02:01, A*02:02, A*02:07, A*02:11) were generated. Each antibody was genetically linked to two IFNα molecules to produce TCR-L/IFNα fusion proteins. We demonstrate that the fusion proteins triggered an IFNα response preferentially on the hepatocytes presenting the correct HBV-peptide HLA-complex and that the mechanism of the targeted IFNα response was dependent on the specific binding of the fusion proteins to the HLA/HBV peptide complexes through the TCR-like variable regions of the antibodies. CONCLUSION: TCR-L antibodies can be used to target cytokines to HBV-infected hepatocytes in vitro. Fusion of IFNα to TCR-L decreased the intrinsic biological activity of IFNα but preserved the overall specificity of the protein for the cognate HBV peptide/HLA complexes. This induction of an effective IFNα response selectively in HBV-infected cells might have a therapeutic advantage in comparison to the currently used native or pegylated IFNα.


Assuntos
Anticorpos Antivirais/farmacologia , Antivirais/farmacologia , Antígenos HLA-A/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Interferon-alfa/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Antivirais/imunologia , Fusão Gênica Artificial , Linfócitos T CD8-Positivos/efeitos dos fármacos , Quimiocinas/metabolismo , Portadores de Fármacos/farmacologia , Células Hep G2 , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Vírus da Hepatite B/genética , Humanos , Ativação Linfocitária/efeitos dos fármacos , Camundongos
6.
Antimicrob Agents Chemother ; 54(2): 734-41, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19995923

RESUMO

In passaging experiments, we isolated HIV strains resistant to MAb3952, a chemokine (C-C motif) receptor 5 (CCR5) monoclonal antibody (MAb) that binds to the second extracellular domain (extracellular loop 2 [ECL-2]) of CCR5. MAb3952-resistant viruses remain CCR5-tropic and are cross-resistant to a second ECL-2-specific antibody. Surprisingly, MAb3952-resistant viruses were more susceptible to RoAb13, a CCR5 antibody binding to the N terminus of CCR5. Using CCR5 receptor mutants, we show that MAb3952-resistant virus strains preferentially use the N terminus of CCR5, while the wild-type viruses preferentially use ECL-2. We propose this switch in the CCR5 binding site as a novel mechanism of HIV resistance.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Epitopos/imunologia , Infecções por HIV/tratamento farmacológico , HIV/efeitos dos fármacos , Receptores CCR5/imunologia , Fármacos Anti-HIV/farmacologia , Sítios de Ligação/genética , Linhagem Celular Tumoral , Células Cultivadas , Farmacorresistência Viral/genética , Epitopos/química , Epitopos/genética , HIV/genética , Infecções por HIV/virologia , Humanos , Receptores CCR5/química , Receptores CCR5/genética
7.
Bioorg Med Chem Lett ; 19(18): 5401-6, 2009 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-19674898

RESUMO

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.


Assuntos
Antagonistas dos Receptores CCR5 , Piperidinas/química , Piperidinas/farmacologia , Receptores CCR5/metabolismo , Animais , Células CACO-2 , Cães , Haplorrinos , Humanos , Piperidinas/farmacocinética , Ratos , Compostos de Espiro/química , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade
8.
Antiviral Res ; 83(3): 257-66, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19559732

RESUMO

Resistance to antiretroviral drugs is a common problem in the treatment of HIV-1-infected patients. To overcome resistance, we generated a novel, bifunctional HIV-1 entry inhibitor by combining the anti-CD4 monoclonal antibody (mAb) 6314 with a fusion inhibitor similar to T-651 (anti-CD4 mAb based BiFunctional Fusion Inhibitor, CD4-BFFI). CD4-BFFI has potent antiviral activity against a multitude of HIV-1 isolates independent of their co-receptor usage and genetic background. It has higher antiviral potency compared to the fusion inhibitor T-651 or the anti-CD4 mAb 6314 used independently. More importantly, every HIV-1 strain tested was fully inhibited by CD4-BFFI while many strains were only partially inhibited by 6314. CD4-BFFI also retained antiviral potency against virus strains resistant to two fusion inhibitors, a CCR5 antagonist and an anti-CCR5 mAb. Pre-incubation of cells with a saturating concentration of anti-CD4 mAbs reduced the antiviral potency of CD4-BFFI, suggesting that binding of CD4-BFFI to the cell surface via its CD4 mAb portion is required for the antiviral potency of its fusion inhibitor moiety. Collectively, we present a novel HIV-1 inhibitor with a dual mode of action and excellent antiviral potency against wildtype and entry-inhibitor resistant virus strains suggesting that CD4-BFFI may have a high barrier to resistance.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD4/imunologia , Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Linhagem Celular , Humanos , Proteínas Recombinantes de Fusão/farmacologia
9.
J Biol Chem ; 284(8): 5175-85, 2009 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-19097993

RESUMO

In this study, we describe a novel CD4-targeting bifunctional human immunodeficiency virus (HIV-1) fusion inhibitor (CD4-BFFI) that blocks HIV-1 entry by inhibiting both HIV-1 attachment and fusion and is highly potent against both R5 and X4 HIV-1 viruses in various antiviral assays, including peripheral blood mononuclear cell (PBMC) infection assays. Previously, we have reported a CCR5 antibody-based bifunctional HIV-1 fusion inhibitor (BFFI) that was highly active in blocking R5 HIV-1 infection but was ineffective against X4 viruses infecting human PBMCs (Kopetzki, E., Jekle, A., Ji, C., Rao, E., Zhang, J., Fischer, S., Cammack, N., Sankuratri, S., and Heilek, G. (2008) Virology J. 5, 56-65). CD4-BFFI, which consists of two HIV-1 fusion inhibitor (FI) T-651 variant peptides recombinantly fused to the Fc end of a humanized anti-CD4 monoclonal antibody, has demonstrated more than 100-fold greater antiviral activity than T-651 variant or the parental CD4 monoclonal antibody. Mechanistic studies revealed that CD4-BFFI primarily blocks the HIV-1-cell fusion step through its FI peptide moieties. The enhanced antiviral activity of CD4-BFFI is most likely due to avid binding of the bivalent FI peptides as well as the increased local concentration of CD4-BFFI via attachment to the target cell surface receptor CD4. In vivo pharmacokinetic studies demonstrated that CD4-BFFI was stable in monkey blood, and a dose of 10 mg/kg maintained serum concentrations greater than 2,000-fold over the IC(90) value for 7 days postdosing. This novel bifunctional inhibitor with improved potency and favorable pharmacokinetic properties may offer a novel approach for HIV-1 therapy.


Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD4 , Inibidores da Fusão de HIV/farmacologia , HIV-1/imunologia , Fragmentos Fc das Imunoglobulinas/farmacologia , Leucócitos Mononucleares/metabolismo , Peptídeos/farmacologia , Internalização do Vírus/efeitos dos fármacos , Animais , Anticorpos Monoclonais/farmacocinética , Antagonistas dos Receptores CCR5 , Células CHO , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Inibidores da Fusão de HIV/farmacocinética , Humanos , Leucócitos Mononucleares/virologia , Macaca fascicularis , Peptídeos/farmacocinética , Receptores CCR5/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética , Proteínas Recombinantes de Fusão/farmacologia
10.
Bioorg Med Chem Lett ; 19(1): 209-13, 2009 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19014885

RESUMO

Replacement of the cyclic carbamate in our previously disclosed 1-oxa-3,9-diazaspiro[5.5]undecan-2-one template led to the discovery of two novel series of 3,9-diazaspiro[5.5]undecane and undeca-2-one CCR5 antagonists. The synthesis, SAR, and antiviral activities of these two series are described. One compound (32) was found to have attractive combination of antiviral potency, selectivity, and pharmacokinetic profile. The asymmetric synthesis of 32 was also accomplished and both enantiomers were equally potent.


Assuntos
Alcanos/síntese química , Antivirais/síntese química , Antagonistas dos Receptores CCR5 , Compostos de Espiro/síntese química , Administração Oral , Alcanos/farmacologia , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Disponibilidade Biológica , Descoberta de Drogas , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade
11.
Virol J ; 5: 56, 2008 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-18452606

RESUMO

We describe a novel strategy in which two inhibitors of HIV viral entry were incorporated into a single molecule. This bifunctional fusion inhibitor consists of an antibody blocking the binding of HIV to its co-receptor CCR5, and a covalently linked peptide which blocks envelope mediated virus-cell fusion. This novel bifunctional molecule is highly active on CCR5- and X4-tropic viruses in a single cycle assay and a reporter cell line with IC50 values of 0.03-0.05 nM. We demonstrated that both inhibitors contribute to the antiviral activity. In the natural host peripheral blood mononuclear cells (PBMC) the inhibition of CXCR4-tropic viruses is dependant on the co-expression of CCR5 and CXCR4 receptors. This bifunctional inhibitor may offer potential for improved pharmacokinetic parameters for a fusion inhibitor in humans and the combination of two active antiviral agents in one molecule may provide better durability in controlling the emergence of resistant viruses.


Assuntos
Inibidores da Fusão de HIV/farmacologia , HIV-1/efeitos dos fármacos , Receptores CCR5/metabolismo , Ligação Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Antagonistas dos Receptores CCR5 , Linhagem Celular , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Concentração Inibidora 50 , Fusão de Membrana/efeitos dos fármacos , Camundongos , Receptores CCR5/imunologia , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Proteínas Recombinantes de Fusão/farmacologia
12.
Mol Pharmacol ; 73(3): 789-800, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18096812

RESUMO

In addition to being an important receptor in leukocyte activation and mobilization, CCR5 is the essential coreceptor for human immunodeficiency virus (HIV). A large number of small-molecule CCR5 antagonists have been reported that show potent activities in blocking chemokine function and HIV entry. To facilitate the design and development of next generation CCR5 antagonists, docking models for major classes of CCR5 antagonists were created by using site-directed mutagenesis and CCR5 homology modeling. Five clinical candidates: maraviroc, vicriviroc, aplaviroc, TAK-779, and TAK-220 were used to establish the nature of the binding pocket in CCR5. Although the five antagonists are very different in structure, shape, and electrostatic potential, they were able to fit in the same binding pocket formed by the transmembrane (TM) domains of CCR5. It is noteworthy that each antagonist displayed a unique interaction profile with amino acids lining the pocket. Except for TAK-779, all antagonists showed strong interaction with Glu283 in TM 7 via their central basic nitrogen. The fully mapped binding pocket of CCR5 is being used for structure-based design and lead optimization of novel anti-HIV CCR5 inhibitors with improved potency and better resistance profile.


Assuntos
Fármacos Anti-HIV/classificação , Fármacos Anti-HIV/metabolismo , Antagonistas dos Receptores CCR5 , HIV-1/efeitos dos fármacos , Receptores CCR5/química , Amidas/química , Amidas/metabolismo , Amidas/farmacologia , Sequência de Aminoácidos , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Benzoatos/química , Benzoatos/metabolismo , Benzoatos/farmacologia , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Cicloexanos/química , Cicloexanos/metabolismo , Cicloexanos/farmacologia , Dicetopiperazinas , Inibidores da Fusão de HIV/farmacologia , Humanos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Maraviroc , Fusão de Membrana/efeitos dos fármacos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacologia , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pirimidinas/química , Pirimidinas/metabolismo , Pirimidinas/farmacologia , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , Compostos de Amônio Quaternário/farmacologia , Ensaio Radioligante , Receptores CCR5/genética , Receptores CCR5/metabolismo , Homologia de Sequência de Aminoácidos , Compostos de Espiro/química , Compostos de Espiro/metabolismo , Compostos de Espiro/farmacologia , Eletricidade Estática
13.
Mol Pharmacol ; 72(1): 18-28, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17392523

RESUMO

A panel of four CCR5 monoclonal antibodies (mAbs) recognizing different epitopes on CCR5 was examined in CCR5-mediated cell-cell fusion assay, alone or in combination with a variety of small molecule CCR5 antagonists. Although no antagonism was observed between any of the CCR5 inhibitors, surprisingly potent synergy was observed between CCR5 mAbs and antagonists, and the synergistic activity was confirmed in other antiviral assays. Strong synergy was also observed between CCR5 inhibitors and the human immunodeficiency virus (HIV) fusion inhibitor enfuvirtide. There was no synergy observed between small molecule CCR5 inhibitors; however, potent synergy was observed between mAbs recognizing different parts of CCR5. In all synergistic combinations, greater synergy was achieved at higher percent inhibition levels. A negative correlation was found between the degree of synergy between the two classes of CCR5 inhibitors and the ability to compete each other for binding to the receptor. For example, the greatest synergy, observed between the mAb ROAb13 and the small molecule inhibitor maraviroc, did not interfere with binding to CCR5 for either inhibitor, whereas no synergy was found between mAb 45523 and maraviroc, which do compete for binding to CCR5. In addition, in contrast to a recent report, the CCR5 inhibitors tested here were found to inhibit the same stage of HIV entry. Based on the data presented here, we hypothesize that CCR5 inhibitors exert synergistic antiviral actions through a cobinding mechanism.


Assuntos
Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais/farmacologia , Antagonistas dos Receptores CCR5 , Animais , Fármacos Anti-HIV/metabolismo , Anticorpos Monoclonais/metabolismo , Células CHO , Cricetinae , Cricetulus , Sinergismo Farmacológico , Enfuvirtida , Proteína gp41 do Envelope de HIV/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores CCR5/metabolismo
14.
Antimicrob Agents Chemother ; 51(4): 1386-97, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17242138

RESUMO

Six mouse anti-human CCR5 monoclonal antibodies (mAbs) that showed potent antiviral activities were identified from over 26,000 mouse hybridomas. The epitopes for these mAbs were determined by using various CCR5 mutants, including CCR5/CCR2B chimeras. One mAb, ROAb13, was found to bind to a linear epitope in the N terminus of CCR5. Strikingly, the other five mAbs bind to epitopes derived from extracellular loop 2 (ECL2). The three most potent mAbs, ROAb12, ROAb14, and ROAb18, require residues from both the N-terminal (Lys171 and Glu172) and C-terminal (Trp190) halves of ECL2 for binding; two other mAbs, ROAb10 and ROAb51, which also showed potent antiviral activities, require Lys171 and Glu172 but not Trp190 for binding. Binding of the control mAb 2D7 completely relies on Lys171 and Glu172. Unlike 2D7, the novel mAbs ROAb12, ROAb14, and ROAb18 do not bind to the linear peptide 2D7-2SK. In addition, all three mAbs bind to monkey CCR5 (with Arg at position 171 instead of Lys); however, 2D7 does not. Since five of the six most potent CCR5 mAbs derived from the same pool of immunized mice require ECL2 as epitopes, we hypothesize that CCR5 ECL2 contains the dominant epitopes for mAbs with potent antiviral activities. These dominant epitopes were found in CCR5 from multiple species and were detected in large proportions of the total cell surface CCR5. mAbs recognizing these epitopes also showed high binding affinity. A homology model of CCR5 was generated to aid in the interpretation of these dominant epitopes in ECL2.


Assuntos
Anticorpos Monoclonais/imunologia , Antagonistas dos Receptores CCR5 , Epitopos/química , HIV-1/imunologia , Receptores CCR5/imunologia , Mapeamento de Epitopos , Epitopos/genética , Anticorpos Anti-HIV , HIV-1/patogenicidade , Humanos , Receptores CCR5/química , Receptores CCR5/genética , Relação Estrutura-Atividade
15.
Antiviral Res ; 74(2): 125-37, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17166600

RESUMO

To identify monoclonal antibodies (mAbs) with high potency and novel recognition sites, more than 25,000 of mouse hybridomas were screened and 4 novel anti-human CCR5 mAbs ROAb12, ROAb13, ROAb14, and ROAb18 showing potent activity in cell-cell fusion (CCF) assay were identified. These mAbs demonstrated potent antiviral activities in both single-cycle HIV infection (IC(50) range: 0.16-4.3 microg/ml) and PBMC viral replication (IC(50) range: 0.02-0.04 microg/ml) assays. These potent antiviral effects were donor-independent. All 4 mAbs were also highly potent in the PhenoSense assay against 29 HIV isolates covering clade A through G. In all antiviral assays, these mAbs showed potency superior to the previously reported mAb 2D7 in side-by-side comparison studies. All 4 mAbs were also fully active against viruses resistant to HIV fusion inhibitor enfuvirtide and CCR5 antagonist maraviroc. Although ROAb12, ROAb14, and ROAb18 inhibited RANTES, MIP1alpha and MIP1beta binding and cell activation, the other novel mAb ROAb13 was inactive in inhibiting cell activation by these three ligands. Furthermore, highly synergistic antiviral effects were found between mAb ROAb13 and 2D7 or ROAb12. In addition, none of these mAbs showed agonist activity or caused internalization of the CCR5 receptor.


Assuntos
Fármacos Anti-HIV/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , HIV/efeitos dos fármacos , HIV/fisiologia , Receptores CCR5/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Antagonistas dos Receptores CCR5 , Fusão Celular , Linhagem Celular , Quimiocina CCL5/metabolismo , Cricetinae , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Receptores CCR5/metabolismo
16.
J Biomol Screen ; 11(6): 652-63, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16844967

RESUMO

There has been increasing interest in the identification of novel HIV entry inhibitors. For the discovery of these entry inhibitors, robust surrogate anti-HIV assays are highly desired. The authors report a novel anti-HIV assay system using Moloney murine leukemia viruses (MMLVs) pseudotyped with cytoplasmic tail-truncated HIV envelope protein gp140. These pseudotyped MMLV-HIVgp140 viral particles carry luciferase transcripts; therefore, robust luciferase signal can be detected in cells infected by these pseudotypes. Polycationic agent polybrene and spinoculation markedly enhanced the infection efficiency of these pseudotypes. It was demonstrated that the tropism of these pseudotypes is dependent on the pseudotyped HIV envelope proteins. MMLV viruses pseudotyped with gp140 from an R5 HIV virus specifically infect CCR5-expressing cells, and viruses pseudotyped with gp140 from an X4 HIV virus specifically infect CXCR4-expressing cells. Furthermore, CCR5 antagonists inhibited only MMLV-gp140(R5) infections, and CXCR4 antagonists inhibited only MMLV-gp140(X4) infections. A variety of known HIV entry inhibitors were tested in both R5- and X4-dependent pseudotype antiviral assays, and the IC50 values generated were consistent with published results. The pseudotype antiviral assay was also used in the characterization of hundreds of novel CCR5 antagonists. The IC50 values determined in this assay were compared with those determined in HIV antiviral and cell-cell fusion (CCF) assays, and good correlation was found between pseudotype antiviral assay and HIV antiviral assay (R2 = 0.9) or CCF assay (R2 = 0.8).


Assuntos
Antivirais/análise , HIV-1/fisiologia , Vírus da Leucemia Murina de Moloney/química , Receptores de HIV/antagonistas & inibidores , Linhagem Celular , HIV-1/genética , Humanos , Vírus da Leucemia Murina de Moloney/genética
17.
J Biomol Screen ; 11(1): 65-74, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16314403

RESUMO

In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC50 data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSO on this assay system were assessed, and a 2- to 3-fold increase in luciferase activity was observed in the presence of 0.05% to 2% DMSO. Although cell-cell fusion efficiency was enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.


Assuntos
Bioensaio/métodos , Fusão Celular , Proteína gp160 do Envelope de HIV/genética , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Regulação para Cima , Animais , Antagonistas dos Receptores CCR5 , Linhagem Celular , Células Cultivadas , Técnicas de Cocultura , Dimetil Sulfóxido/farmacologia , Células Gigantes , Inibidores da Fusão de HIV , Humanos , Concentração Inibidora 50 , Relação Estrutura-Atividade , Fatores de Tempo
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