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1.
Biochemistry ; 46(10): 2899-908, 2007 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-17298035

RESUMO

LEDGF/p75 is known to enhance the integrase strand transfer activity in vitro, but the underlying mechanism is unclear. Using an integrase assay with a chemiluminescent readout adapted to a 96-well plate format, the effect of LEDGF/p75 on both the 3'-processing and strand transfer steps was analyzed. Integrase inhibitors of the strand transfer reaction remained active in the presence of LEDGF/p75, but displayed 3- to 7-fold higher IC50 values. Our analyses indicate that, in the presence of 150 nM LEDGF/p75, active integrase/donor DNA complexes were increased by 5.3-fold during the 3'-processing step. In addition, these integrase/donor DNA complexes showed a 4.5-fold greater affinity for the target DNA during the subsequent strand transfer step. We also observed a 3.7-fold increase in the rate constant of catalysis of the strand transfer step when 150 nM LEDGF/p75 was present during the 3'-processing step. In contrast, when LEDGF/p75 was added at the beginning of the strand transfer step, no increase in either the concentration of active integrase/donor DNA complex or its rate constant of strand transfer catalysis was observed. This observation suggested that the integrase/donor DNA formed in the absence of LEDGF/p75 became refractory to the stimulatory effect of LEDGF/p75. Instead, this LEDGF/p75 added at the start of the strand transfer step was able to promote the formation of a new cohort of active integrase/donor DNA complexes which became functional with a delay of 45 min after LEDGF/p75 addition. We propose a model whereby LEDGF/p75 can only bind integrase before the latter binds donor DNA whereas donor DNA can engage either free or LEDGF/p75-bound integrase.


Assuntos
DNA/metabolismo , Integrase de HIV/metabolismo , HIV-1/enzimologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Substâncias Macromoleculares/metabolismo
2.
J Mol Biol ; 363(3): 635-47, 2006 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-16979654

RESUMO

The introduction of human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) markedly improved the clinical outcome and control of HIV-1 infection. However, cross-resistance among PIs due to a wide spectrum of mutations in viral protease is a major factor limiting their broader clinical use. Here we report on the suppression of PI resistance using a covalent attachment of a phosphonic acid motif to a peptidomimetic inhibitor scaffold. The resulting phosphonate analogs maintain high binding affinity to HIV-1 protease, potent antiretroviral activity, and unlike the parent molecules, display no loss of potency against a panel of clinically important PI-resistant HIV-1 strains. As shown by crystallographic analysis, the phosphonate moiety is highly exposed to solvent with no discernable interactions with any of the enzyme active site or surface residues. We term this effect "solvent anchoring" and demonstrate that it is driven by a favorable change in the inhibitor binding entropy upon the interaction with mutant enzymes. This type of thermodynamic behavior, which was not found with the parent scaffold fully buried in the enzyme active site, is a result of the increased degeneracy of inhibitor binding states, allowing effective molecular adaptation to the expanded cavity volume of mutant proteases. This strategy, which is applicable to various PI scaffolds, should facilitate the design of novel PIs and potentially other antiviral therapeutics.


Assuntos
Desenho de Fármacos , Farmacorresistência Viral Múltipla , Inibidores da Protease de HIV/química , Protease de HIV/química , Organofosfonatos/química , Solventes , Sulfato de Atazanavir , Sítios de Ligação , Infecções por HIV/tratamento farmacológico , Protease de HIV/metabolismo , Inibidores da Protease de HIV/metabolismo , Inibidores da Protease de HIV/uso terapêutico , Humanos , Modelos Moleculares , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Piridinas/química , Piridinas/metabolismo , Termodinâmica
3.
Antivir Ther ; 11(2): 155-63, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16640096

RESUMO

The HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs) tenofovir (TFV), abacavir, didanosine and stavudine can select for K65R, whereas zidovudine (AZT) and stavudine can select for thymidine analogue mutations (TAMs) in HIV-1 reverse transcriptase (RT). HIV-1 with TAMs shows reduced susceptibility to all NRTIs, most notably AZT, whereas HIV-1 with K65R shows reduced susceptibility to all NRTIs except AZT. K65R and TAMs rarely occur together in patients. However, when present together, K65R can restore susceptibility to AZT. This study characterizes the underlying mechanisms of resistance of these RT mutants to TFV and AZT. K65R mediated decreased binding/incorporation of TFV and AZT (increased Ki/Km of 7.1- and 4.3-fold, respectively), but also decreased excision of TFV and AZT (0.7- and 0.3-fold, respectively) when compared with wild-type RT. By contrast, TAMs mediated increased TFV and AZT excision (11- and 5.4-fold, respectively), and showed no changes in binding/incorporation. When these mutations were combined, K65R reversed TAM-mediated AZT resistance by strongly reducing AZT excision. Molecular modelling studies suggest that K65R creates additional hydrogen bonds that reduce the conformational mobility of RT, resulting in reduced polymerization and excision. Thus, consistent with clinical HIV-1 genotyping data, there appears to be no net NRTI resistance benefit for TAMs and K65R to develop together in patients taking AZT and TFV disoproxil fumarate, where the TAM pathway alone provides the greatest resistance for both drugs.


Assuntos
Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Mutação/genética , Zidovudina/farmacologia , Linhagem Celular , HIV-1/genética , Humanos , Modelos Moleculares , Fenótipo , Ligação Proteica , Inibidores da Transcriptase Reversa/farmacologia
4.
Mol Immunol ; 39(12): 719-27, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12531283

RESUMO

Our previous study showed that recombinant canine IL-13 (rcaIL-13) stimulated production of allergen-specific IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs. This has also been demonstrated using human IL-13 (huIL-13) and PBMC isolated from human allergy patients. The stimulatory activity of rcaIL-13 was specifically inhibited by a fusion protein of the extracellular domain of canine IL-13Ralpha2 and the Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). In this communication, we report the construction and expression of a non-fused recombinant extracellular domain of canine IL-13Ralpha2 (rcaIL-13Ralpha2) in an E. coli expression system. The E. coli expressed rcaIL-13Ralpha2 was isolated in inclusion bodies, then solubilized in buffer containing denaturants and reducing agents. After refolding and purification, the biological activity of rcaIL-13Ralpha2 was found in the monomer fraction resulting from gel filtration and ion exchange chromatographies. Biological activity of purified rcaIL-13Ralpha2 was demonstrated by the specific inhibition of rcaIL-13 activity in a TF-1 cell proliferation assay. Additionally, rcaIL-13Ralpha2 was found to be active in neutralizing rcaIL-13 induced upregulation of IgE mRNA levels in PBMCs of "high IgE" dogs, which have been bred to exhibit a predisposition for high IgE production and are used as a model for allergic asthma. The data confirm our previous report that the regulatory effects of IL-13 on IgE production in canine PBMCs are similar to those reported in humans. Thus, allergic dogs, such as the "high IgE" producing dogs, may be excellent models for research on IgE-mediated diseases in humans.


Assuntos
Receptores de Interleucina/genética , Alérgenos , Animais , Asma/genética , Asma/imunologia , Sequência de Bases , Linhagem Celular , DNA Complementar/genética , Cães , Escherichia coli/genética , Expressão Gênica , Humanos , Imunoglobulina E/biossíntese , Técnicas In Vitro , Interleucina-13/farmacologia , Subunidade alfa1 de Receptor de Interleucina-13 , Leucócitos Mononucleares/imunologia , Dobramento de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Interleucina/biossíntese , Receptores de Interleucina/química , Receptores de Interleucina-13 , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
5.
Vet Immunol Immunopathol ; 89(1-2): 13-27, 2002 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-12208047

RESUMO

Type I interferons (IFN) are important mediators of the host defense against viral infections in mammals. In humans multiple subtypes of IFN-alpha exist, most of which possess antiviral activity. Little is known about the type I IFN genes in cats and the role they may play in feline immunological responses to viruses. We have isolated cDNAs encoding five feline IFN-alpha (feIFN) subtypes that share from 95 to 99% amino acid sequence identity. FeIFN-alpha5 has five additional amino acids inserted at position 139, which are not present in the other four subtypes. Sequence identity of the feIFN proteins encoded by the five clones compared to human IFN-alpha2 is approximately 60%. Unlike most of the human subtypes, each of the five feline IFN sequences has an N-glycosylation recognition site. Expression of all five feIFN-alpha subtypes in Chinese hamster ovary (CHO) cells was confirmed by Western blot analysis, and all resulting proteins were glycosylated. The antiviral activity of each feIFN-alpha subtype produced in transiently transfected CHO cell cultures was tested in vitro. In addition, subtype feIFN-alpha6 was expressed in the yeast, Pichia pastoris. The resulting secreted mature recombinant protein was purified and demonstrated significant antiviral activity and induction of 2',5'-oligoadenylate synthetase activity in vitro.


Assuntos
Gatos/imunologia , Interferon Tipo I/genética , 2',5'-Oligoadenilato Sintetase/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting/veterinária , Células CHO , Gatos/genética , Clonagem Molecular , Cricetinae , Escherichia coli/genética , Escherichia coli/metabolismo , Interferon Tipo I/química , Interferon Tipo I/farmacologia , Dados de Sequência Molecular , Pichia/genética , Pichia/metabolismo , RNA/química , RNA/genética , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Vírus da Estomatite Vesicular Indiana/imunologia
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