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1.
Bioorg Med Chem Lett ; 18(11): 3251-5, 2008 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-18462940

RESUMO

A computer-aided drug design strategy leads to the identification of a new class of p38 inhibitors based on the 2-tolyl-(1,2,3-triazol-1-yl-4-carboxamide) scaffold. The tolyl triazole amides provided a potent platform amenable to optimization. Further exploration leads to compounds with greater than 100-fold improvement in binding affinity to p38. Derivatives prepared to alter the physicochemical properties produced inhibitors with IC(50)'s in human whole blood as low as 83 nM.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Triazóis/síntese química , Triazóis/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Sítios de Ligação , Desenho Assistido por Computador , Inibidores Enzimáticos/sangue , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Relação Estrutura-Atividade , Triazóis/sangue , Triazóis/química
3.
Protein Sci ; 6(1): 242-5, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9007996

RESUMO

The recombinant human p38 MAP kinase has been expressed and purified from both Escherichia coli and SF9 cells, and has been crystallized in two forms by the hanging drop vapor diffusion method using PEG as precipitant. Both crystal forms belong to space group P2(1)2(1)2(1). The cell parameters for crystal form 1 are a = 65.2 A, b = 74.6 A and c = 78.1 A. Those for crystal form 2 are a = 58.3 A, b = 68.3 A and c = 87.9 A. Diffraction data to 2.0 A resolution have been collected on both forms.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Proteínas Quinases Ativadas por Mitógeno , Animais , Baculoviridae/genética , Linhagem Celular , Cristalografia por Raios X , Escherichia coli/genética , Humanos , Proteínas Recombinantes/química , Spodoptera , Proteínas Quinases p38 Ativadas por Mitógeno
4.
J Med Chem ; 38(10): 1751-61, 1995 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-7752198

RESUMO

The on and off rate constants (kon and koff) were determined for a series of peptidomimetic, competitive inhibitors of human renin using a novel binding assay. The method entails analyzing a pair of ligand exchange reactions in which a dansylated inhibitor serves as the fluorescent probe. The first in the pair of reactions involves preincubating renin with the probe and initiating the reaction by addition of a sample inhibitor; the second reaction involves preincubating renin with the sample inhibitor and initiating the reaction by addition of probe. Both reactions yield progress curves which contain complementary information concerning the kon and koff of each ligand. The two curves are fitted simultaneously using models derived from the differential rate equations describing the ligand exchange process. The kon and koff rate constants for the probe were 6.85 x 10(6) M-1 s-1 and 2.96 x 10(-4) s-1, respectively, giving a calculated Kd of 43.2 pM. The Kd values for the inhibitor series varied over 2 orders of magnitude (27-2320 pM), while the individual kon (10(6)-10(7) M-1 s-1) and koff (10(-4)-10(-3) s-1) constants varied only over 1 order of magnitude.


Assuntos
Renina/antagonistas & inibidores , Ligação Competitiva , Humanos , Cinética , Ligantes , Proteínas Recombinantes/antagonistas & inibidores , Espectrometria de Fluorescência , Fatores de Tempo
5.
Biochem Pharmacol ; 52(11): 1757-65, 1996 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-8986139

RESUMO

CD26 and ecto-adenosine deaminase (ADA) are found associated on the plasma membrane of T lymphocytes and each possess distinct catalytic activities. CD26 has a proteolytic activity identical to dipeptidylpeptidase IV (DPPIV; E.C. 3.4.14.5), and ecto-ADA (E.C. 3.5.4.4) degrades extracellular adenosine. The cell surface expression of CD26 and ecto-adenosine deaminase (ecto-ADA) is regulated on stimulated T lymphocytes, and ADA binding to CD26 produces a synergistic costimulatory response with T cell receptor activation. This study addresses the potential regulation by allosteric interactions of the catalytic activities of CD26 associated with ecto-ADA, which could define the mechanism of the synergism observed in T cell signaling. Cell lines genetically deficient in ADA, ligands for ADA such as adenosine, and a specific inhibitor of ADA, deoxycoformycin, were used to define the effect of ADA activity on CD26 DPPIV activity and affinity for dipeptide substrate. Conversely, a recombinant Chinese hamster ovary cell line expressing human CD26 with or without a mutation in the DPPIV catalytic domain, and the boronic acid inhibitor Val-boroPro, were used to determine the effect of DPPIV activity on ecto-ADA activity and association with CD26. These studies found no significant allosteric interaction between the catalytic activities of CD26 and ecto-ADA when associated. Therefore, signaling events in T cells involving costimulation with CD26 and ecto-ADA and the synergism observed upon ADA binding to CD26 occur independently of the catalytic activities of these cell surface molecules.


Assuntos
Adenosina Desaminase/metabolismo , Ácidos Borônicos/farmacologia , Dipeptidil Peptidase 4/metabolismo , Inibidores Enzimáticos/farmacologia , Pentostatina/farmacologia , Inibidores de Adenosina Desaminase , Regulação Alostérica , Animais , Células CHO , Linhagem Celular , Cricetinae , Humanos , Linfócitos/enzimologia
6.
Arch Biochem Biophys ; 254(2): 421-9, 1987 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-3579312

RESUMO

Pentalenene synthase, an enzyme which catalyzes the cyclization of farnesyl pyrophosphate to the sesquiterpene hydrocarbon pentalenene, has been partially purified from the supernatant fraction of Streptomyces UC5319 by a combination of anion-exchange, hydroxylapatite, and gel-filtration chromatography. The molecular weight of the partially purified synthase was estimated by gel filtration chromatography to be 57,000 and the cyclase activity was shown to be associated with a major protein band among eight visible by nondenaturing polyacrylamide disc gel electrophoresis. The Km for farnesyl pyrophosphate was 0.77 +/- 0.21 microM and the Vmax for the partially purified synthase was 287 +/- 21 nmol of pentalenene/mg protein per hour. Cyclase activity required the presence of a divalent metal cation. Although either Mg2+ or Mn2+ could be used, Mn2+ was inhibitory at concentrations above 2.5 mM. No other cofactors were required. Whereas neither product, pentalenene nor inorganic pyrophosphate, showed significant inhibition of cyclase activity at concentrations of ca 10 microM, the combination of the two resulted in an approximate sevenfold increase in the apparent Km for farnesyl pyrophosphate, suggesting that both products can bind cooperatively at the active site to inhibit pentalenene synthase competitively.


Assuntos
Liases Intramoleculares , Isomerases/metabolismo , Streptomyces/enzimologia , Cátions Bivalentes , Cromatografia , Ciclopentanos/farmacologia , Estabilidade de Medicamentos , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Isomerases/antagonistas & inibidores , Isomerases/isolamento & purificação , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Fosfatos de Poli-Isoprenil/farmacologia , Sesquiterpenos
7.
J Biol Chem ; 271(47): 29599-604, 1996 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-8939889

RESUMO

Integrase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and a type I topoisomerase. Upon modification with N-ethylmaleimide (NEM), a sulfhydryl-directed reagent, Int loses its capacity to bind "arm-type" DNA sequences and, consequently, to carry out recombination; however, its ability to bind "core-type" sequences and its topoisomerase activity are unaffected. In this report, the NEM-sensitive site was identified by modifying Int with [14C]NEM. Following cleavage by formic acid, which cleaves Asp-Pro bonds, and fractionation on a Fractogel HW-50 (F) sizing column, the fragment containing the primary site of [14C]NEM incorporation was subjected to amino acid sequencing. The results indicate that the primary site of [14C]NEM incorporation is in the peptide-spanning amino acid residues 1-28, which contains a cysteine at position 25. To confirm that Cys-25 is the target of NEM reactivity, site-directed mutagenesis was used to change this cysteine to alanine or serine. The mutant protein is not chemically modified by NEM and shows no loss of activity after NEM treatment. The fact that C25A and C25S both retain full recombination activity indicates that the SH group of Cys-25 does not provide any critical contacts, either with arm-type DNA or with other parts of the Int protein to form the arm-type recognition pocket. The loss of arm-type DNA binding and the concomitant loss of recombination function as a result of NEM modification must be due to the presence of the maleimide moiety and not due to loss of a critical cysteine contact.


Assuntos
Bacteriófago lambda/enzimologia , Etilmaleimida/farmacologia , Inibidores de Integrase/farmacologia , Integrases/metabolismo , Sequência de Aminoácidos , Cisteína/metabolismo , Integrases/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida
8.
J Enzyme Inhib ; 11(3): 151-69, 1997 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-9204385

RESUMO

Peptide boronic acid dipeptide compounds were analyzed for their ability to inhibit recombinant human dipeptidylpeptidase IV (CD26, DPPIV). Rate constants for the peptide boronates are difficult to obtain because the active boronic acid dipeptide exists in equilibrium with a cyclic inactive species in aqueous solution. Rate constants were determined for the inhibition of DPPIV using several peptide boronates at different pH values. Val-boroPro forms the most tightly bound complex with DPPIV; the first order half life for dissociation of the inactive enzyme-inhibitor complex at 23 degrees C is approximately 27 days.


Assuntos
Ácidos Borônicos/farmacologia , Dipeptídeos/farmacologia , Dipeptidil Peptidase 4/efeitos dos fármacos , Inibidores de Serina Proteinase/farmacologia , Ligação Competitiva , Dipeptidil Peptidase 4/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Fluorescência
9.
J Biol Chem ; 263(16): 7678-85, 1988 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-2836392

RESUMO

High levels of covalent integrase-DNA complexes accumulate when suicide substrates containing a medial nick within the overlap region are nicked by lambda integrase protein. The tyrosine residue at position 342 is shown to form a covalent bond with DNA at the sites of strand exchange. A mutant integrase in which this tyrosine is changed to phenylalanine is devoid of both topoisomerase and recombinase activity but still binds to both core- and arm-type DNA binding sites with an affinity comparable to wild-type integrase. Tyrosine-342 is located within a 40-amino acid region that is conserved among 15 known recombinases comprising the "integrase family." The present results show that this small region of homology participates in catalysis of strand transfer.


Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , DNA Topoisomerases Tipo I/metabolismo , Integrases , Dados de Sequência Molecular
10.
Proteins ; 20(1): 98-102, 1994 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-7824526

RESUMO

For therapeutically relevant targets, the evaluation of enzymes in complex with their inhibitors by cocrystallization and high resolution structural analysis has become a vital component of structure-driven drug design and development. Two approaches, hanging drop vapor diffusion and a novel microtube batch method, were utilized in parallel to grow crystals of recombinant HIV-2 protease and recombinant human renin in complex with inhibitors. In the case of HIV-2 protease in complex with a reduced amide inhibitor, crystallization was achieved only by the microbatch method. In the case of human renin, the addition of precipitant was required for crystal growth. The microbatch method described here is a useful supplementary or alternative approach for screening parameters and generating crystals suitable for high resolution structural analysis.


Assuntos
Ácido Aspártico Endopeptidases/química , Cristalização , HIV-2/enzimologia , Renina/química , Cristalografia por Raios X , Protease de HIV , Humanos
11.
Proc Natl Acad Sci U S A ; 90(18): 8387-91, 1993 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-8378311

RESUMO

The crystal structure of HIV-2 protease in complex with a reduced amide inhibitor [BI-LA-398; Phe-Val-Phe-psi (CH2NH)-Leu-Glu-Ile-amide] has been determined at 2.2-A resolution and refined to a crystallographic R factor of 17.6%. The rms deviation from ideality in bond lengths is 0.018 A and in bond angles is 2.8 degrees. The largest structural differences between HIV-1 and HIV-2 proteases are located at residues 15-20, 34-40, and 65-73, away from the flap region and the substrate binding sites. The rms distance between equivalent C alpha atoms of HIV-1 and HIV-2 protease structures excluding these residues is 0.5 A. The shapes of the S1 and S2 pockets in the presence of this inhibitor are essentially unperturbed by the amino acid differences between HIV-1 and HIV-2 proteases. The interaction of the inhibitor with HIV-2 protease is similar to that observed in HIV-1 protease structures. The unprotected N terminus of the inhibitor interacts with the side chains of Asp-29 and Asp-30. The glutamate side chain of the inhibitor forms hydrogen bonds with the main-chain amido groups of residues 129 and 130.


Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Protease de HIV/química , Protease de HIV/metabolismo , Oligopeptídeos/metabolismo , Inibidores de Proteases/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , HIV-1/enzimologia , HIV-2/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/química , Inibidores de Proteases/química , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Difração de Raios X/métodos
12.
Arch Biochem Biophys ; 328(2): 317-23, 1996 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-8645010

RESUMO

A numerical method was applied to a system of differential rate equations describing the monomer-dimer-inhibitor (M-D-I) interaction involving human immunodeficiency virus type 1 protease and a peptidomimetic, competitive inhibitor. Two pairs of progress curves were obtained, one involving the M-D interaction and the other the M-D-I interaction. Each pair of reactions was designed to begin with extreme conditions and end at the identical equilibrium position. The results were compared with analytical (exact mathematical) methods reported previously. Good agreement between the two methods was observed at high- and low-salt conditions for the rates of monomer association and dimer dissociation. Not surprisingly, however, the major difference was observed in the analyses involving the M-D-I interaction, since analytical methods cannot account for dimer dissociation in the presence of inhibitor. While the estimates for the inhibitor off rate were comparable for high-salt conditions (where dimer dissociation is minimized), the analytical method underestimated this parameter for low-salt conditions by an order of magnitude, the consequence of mistaking inactive M for inactive DI.


Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/química , Protease de HIV/metabolismo , HIV-1/enzimologia , Matemática , Modelos Teóricos , Ligação Competitiva , Escherichia coli , Humanos , Cinética , Substâncias Macromoleculares , Proteínas Recombinantes/metabolismo
13.
Cell ; 54(7): 923-9, 1988 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-2843292

RESUMO

The 40 kd lambda Integrase protein is shown to contain two autonomous DNA binding domains with different sequence specificities. Competition experiments in which the binding activity of Int is assayed through nuclease protection demonstrate the functional independence of the two DNA recognition specificities. Proteolytic cleavage of Int and footprinting analysis of the resulting two major peptides allow the physical separation and identification of two DNA binding domains: an amino-terminal peptide that interacts with "arm-type" sites and a carboxy-terminal peptide that binds to "core-type" sequences. In addition, the data suggest that the two domains can bind DNA simultaneously, consistent with a model in which Integrase would link two disparate DNA sequences.


Assuntos
Bacteriófago lambda/enzimologia , DNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Quimotripsina , Enzimas de Restrição do DNA , DNA Recombinante/metabolismo , Desoxirribonucleases , Integrases , Modelos Genéticos , Plasmídeos
14.
Antimicrob Agents Chemother ; 41(5): 965-71, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9145853

RESUMO

Palinavir is a potent inhibitor of the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteases. Replication of laboratory strains (HIV-1, HIV-2, and simian immunodeficiency virus) and HIV-1 clinical isolates is inhibited by palinavir with 50% effective concentrations ranging from 0.5 to 30 nM. The average cytotoxic concentration of palinavir (35 microM) in the various target cells indicates a favorable therapeutic index. Potent antiviral activity is retained with increased doses of virus and with clinical isolates resistant to zidovudine (AZT), didanosine (ddI), or nevirapine. Combinations of palinavir with either AZT, ddI, or nevirapine demonstrate synergy or additivity in the inhibition of HIV-1 replication. Palinavir retains anti-HIV-1 activity when administered postinfection until times subsequent to the reverse transcription step. In chronically infected CR-10 cells, palinavir blocks Gag precursor polyprotein processing completely, reducing greater than 99% of infectious particle production. The results indicate that the antiviral activity of palinavir is specific to inhibition of the viral protease and occurs at a late stage in the replicative cycle of HIV-1. On the basis of the potent in vitro activity, low-level cytotoxicity, and other data, palinavir was selected for in-depth preclinical evaluation.


Assuntos
Fármacos Anti-HIV/farmacologia , Inibidores da Protease de HIV/farmacologia , HIV-1/efeitos dos fármacos , Quinolinas/farmacologia , Valina/análogos & derivados , Combinação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , HIV-2/efeitos dos fármacos , Humanos , Nevirapina , Piridinas/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Vírus da Imunodeficiência Símia/efeitos dos fármacos , Valina/farmacologia , Replicação Viral/efeitos dos fármacos
15.
Nat Struct Biol ; 4(4): 311-6, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9095200

RESUMO

The crystal structure of human p38 mitogen-activated protein (MAP) kinase in complex with a potent and highly specific pyridinyl-imidazole inhibitor has been determined at 2.0 A resolution. The structure of the kinase, which is in its unphosphorylated state, is similar to that of the closely-related ERK2. The inhibitor molecule is bound in the ATP pocket. A hydrogen bond is made between the pyridyl nitrogen of the inhibitor and the main chain amido nitrogen of residue 109, analogous to the interaction from the N1 atom of ATP. The crystal structure provides possible explanations for the specificity of this class of inhibitors. Other protein kinase inhibitors may achieve their specificity through a similar mechanism. The structure also reveals a possible second binding site for this inhibitor, with currently unknown function.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Inibidores Enzimáticos/química , Imidazóis/química , Proteínas Quinases Ativadas por Mitógeno , Piridinas/química , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Simulação por Computador , Cristalografia por Raios X , Humanos , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Quinases p38 Ativadas por Mitógeno
16.
J Biol Chem ; 266(22): 14670-4, 1991 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-1713587

RESUMO

Nevirapine (BI-RG-587) is a potent and specific non-nucleoside inhibitor of human immunodeficiency virus type-1 reverse transcriptase. The compound is non-competitive with respect to template, primer, and nucleoside triphosphates indicating that BI-RG-587 does not act directly at the catalytic site. The binding site for this inhibitor was investigated by employing an azido photoaffinity analogue, BI-RJ-70, to covalently label the enzyme. The resulting photoadduct was subjected to enzymatic digestion by trypsin and endoproteinase lys-C and a single, highly labeled peptide was identified as residues 174-199. Sequencing of this peptide identified Tyr-181 and Tyr-188 as labeled residues.


Assuntos
Azepinas/metabolismo , HIV-1/enzimologia , Piridinas/metabolismo , Inibidores da Transcriptase Reversa , Marcadores de Afinidade , Sequência de Aminoácidos , Sítios de Ligação , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Nevirapina , Mapeamento de Peptídeos , Tripsina
17.
Biochemistry ; 33(41): 12527-34, 1994 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-7918476

RESUMO

Association and dissociation rate constants for a competitive inhibitor of HIV-1 protease were determined by a novel method employing a pair of integrated rate equations. This method, termed the paired progress curve method, is both rapid and reproducible. Progress curves, taken at a single concentration of inhibitor, are analyzed simultaneously to determine association and dissociation rate constants, the concentration of active sites, and the catalytic rate constant. The method is applied to BILA 398, a compound for which the cocrystal structure with HIV-2 protease has been reported recently [Tong, L., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8387-8391]. This compound exhibited an association constant of 1.6 x 10(7) M-1 s-1 and a dissociation constant of 1.0 x 10(-4) s-1 corresponding to a binding affinity constant of 6.4 x 10(-12) M. During the course of the analysis, nonlinearity was observed in control reactions containing enzyme and substrate only. This was subsequently shown to be due to a reversible inactivation process resulting from enzyme dilution. Integrated rate equations were developed on the basis of the dissociation of active dimeric enzyme during dilution and a reassociation of dilute monomers following the addition of substrate. The equations were modeled to the data, yielding a dissociation constant of 1.9 x 10(-3) s-1 and an association constant of 9.2 x 10(5) M-1 s-1 for the monomer-dimer interconversion process. This corresponds to an equilibrium constant of 4 x 10(-9) M for the dimerization of HIV-1 protease.


Assuntos
Inibidores da Protease de HIV/metabolismo , Protease de HIV/metabolismo , HIV-1/enzimologia , Ligação Competitiva , Protease de HIV/química , Cinética , Substâncias Macromoleculares , Análise de Regressão
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