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1.
Bioorg Med Chem Lett ; 22(23): 7019-23, 2012 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-23099094

RESUMO

A previously described aryl sulfonamide series, originally found through HTS, targets GlmU, a bifunctional essential enzyme involved in bacterial cell wall synthesis. Using structure-guided design, the potency of enzyme inhibition was increased in multiple isozymes from different bacterial species. Unsuitable physical properties (low LogD and high molecular weight) of those compounds prevented them from entering the cytoplasm of bacteria and inhibiting cell growth. Further modifications described herein led to compounds that possessed antibacterial activity, which was shown to occur through inhibition of GlmU. The left-hand side amide and the right-hand side sulfonamides were modified such that enzyme inhibitory activity was maintained (IC(50) <0.1 µM against GlmU isozymes from Gram-negative organisms), and the lipophilicity was increased giving compounds with LogD -1 to 3. Antibacterial activity in an efflux-pump deficient mutant of Haemophilus influenzae resulted for compounds such as 13.


Assuntos
Acetiltransferases/antagonistas & inibidores , Antibacterianos/química , Inibidores Enzimáticos/química , Nucleotidiltransferases/antagonistas & inibidores , Oxazinas/química , Sulfonamidas/química , Acetiltransferases/metabolismo , Amidas/química , Antibacterianos/síntese química , Antibacterianos/farmacologia , Sítios de Ligação , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/enzimologia , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Nucleotidiltransferases/metabolismo , Oxazinas/síntese química , Oxazinas/farmacologia , Estrutura Terciária de Proteína , Sulfonamidas/síntese química , Sulfonamidas/farmacologia
2.
Bioorg Med Chem Lett ; 22(4): 1510-9, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22297115

RESUMO

A novel arylsulfonamide-containing series of compounds represented by 1, discovered by highthroughput screening, inhibit the acetyltransferase domain of N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). X-ray structure determination confirmed that inhibitor binds at the site occupied by acetyl-CoA, indicating that series is competitive with this substrate. This letter documents our early hit-to-lead evaluation of the chemical series and some of the findings that led to improvement in in-vitro potency against Gram-negative and Gram-positive bacterial isozymes, exemplified by compound 40.


Assuntos
Domínio Catalítico/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Nucleotidiltransferases/antagonistas & inibidores , Sulfonamidas/farmacologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/farmacologia , Sequência de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/genética , Ligação Competitiva , Cristalografia por Raios X , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Concentração Inibidora 50 , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Nucleotidiltransferases/química , Alinhamento de Sequência , Sulfonamidas/química
3.
J Biol Chem ; 286(47): 40734-42, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-21984832

RESUMO

GlmU is a bifunctional enzyme that is essential for bacterial growth, converting D-glucosamine 1-phosphate into UDP-GlcNAc via acetylation and subsequent uridyl transfer. A biochemical screen of AstraZeneca's compound library using GlmU of Escherichia coli identified novel sulfonamide inhibitors of the acetyltransferase reaction. Steady-state kinetics, ligand-observe NMR, isothermal titration calorimetry, and x-ray crystallography showed that the inhibitors were competitive with acetyl-CoA substrate. Iterative chemistry efforts improved biochemical potency against gram-negative isozymes 300-fold and afforded antimicrobial activity against a strain of Haemophilus influenzae lacking its major efflux pump. Inhibition of precursor incorporation into bacterial macromolecules was consistent with the antimicrobial activity being caused by disruption of peptidoglycan and fatty acid biosyntheses. Isolation and characterization of two different resistant mutant strains identified the GlmU acetyltransferase domain as the molecular target. These data, along with x-ray co-crystal structures, confirmed the binding mode of the inhibitors and explained their relative lack of potency against gram-positive GlmU isozymes. This is the first example of antimicrobial compounds mediating their growth inhibitory effects specifically via GlmU.


Assuntos
Acetiltransferases/antagonistas & inibidores , Acetiltransferases/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/metabolismo , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/enzimologia , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Sulfonamidas/farmacologia , Acetilcoenzima A/metabolismo , Acetiltransferases/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Ligação Competitiva , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Concentração Inibidora 50 , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Multimerização Proteica , Estrutura Quaternária de Proteína , Reprodutibilidade dos Testes
4.
Bioorg Med Chem Lett ; 17(2): 394-9, 2007 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-17095214

RESUMO

A series of substituted 3,4-dihydro-2-quinolone glycogen phosphorylase inhibitors, which have potential as antidiabetic agents, is described. Initial members of the series showed good enzyme inhibitory potency but poor physical properties. Optimisation of the 1-substituent led to 2,3-dihydroxypropyl compounds which showed good in vitro potency and improved physical properties, together with good DMPK profiles and acute in vivo efficacy in a rat model. X-ray crystallographic data are presented, showing an unexpected variety of binding orientations at the dimer interface site.


Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Glicogênio Fosforilase/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Quinolinas/síntese química , Quinolinas/farmacologia , Animais , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Células CACO-2 , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Indicadores e Reagentes , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Músculo Esquelético/enzimologia , Coelhos , Ratos
5.
J Med Chem ; 49(22): 6465-88, 2006 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-17064066

RESUMO

Src family kinases (SFKs) are nonreceptor tyrosine kinases that are reported to be critical for cancer progression. We report here a novel subseries of C-5-substituted anilinoquinazolines that display high affinity and specificity for the tyrosine kinase domain of the c-Src and Abl enzymes. These compounds exhibit high selectivity for SFKs over a panel of recombinant protein kinases, excellent pharmacokinetics, and in vivo activity following oral dosing. N-(5-Chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530) inhibits c-Src and Abl enzymes at low nanomolar concentrations and is highly selective over a range of kinases. AZD0530 displays excellent pharmacokinetic parameters in animal preclinically and in man (t(1/2) = 40 h). AZD0530 is a potent inhibitor of tumor growth in a c-Src-transfected 3T3-fibroblast xenograft model in vivo and led to a significant increase in survival in a highly aggressive, orthotopic model of human pancreatic cancer when dosed orally once daily. AZD0530 is currently undergoing clinical evaluation in man.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Benzodioxóis/síntese química , Benzodioxóis/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Quinazolinas/síntese química , Quinazolinas/farmacologia , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/química , Células 3T3 , Animais , Antineoplásicos/farmacocinética , Benzodioxóis/farmacocinética , Proliferação de Células/efeitos dos fármacos , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Cães , Inibidores Enzimáticos/farmacocinética , Feminino , Humanos , Indicadores e Reagentes , Masculino , Camundongos , Camundongos Nus , Modelos Moleculares , Invasividade Neoplásica/prevenção & controle , Quinazolinas/farmacocinética , Ratos , Solubilidade , Relação Estrutura-Atividade , Termodinâmica , Transplante Heterólogo , Quinases da Família src/biossíntese
6.
Bioorg Med Chem Lett ; 16(21): 5567-71, 2006 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16945526

RESUMO

Two series of novel thienopyrrole inhibitors of recombinant human liver glycogen phosphorylase a (GPa) which are effective in reducing glucose output from rat hepatocytes are described. Representative compounds have been shown to bind at the dimer interface site of the rabbit muscle enzyme by X-ray crystallography.


Assuntos
Glicogênio Fosforilase/antagonistas & inibidores , Pirróis/farmacologia , Animais , Cristalografia por Raios X , Humanos , Pirróis/síntese química , Pirróis/química , Coelhos , Ratos , Relação Estrutura-Atividade
7.
FEMS Yeast Res ; 5(6-7): 635-46, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15780663

RESUMO

Pycnoporus cinnabarinus lac1 gene was expressed in Yarrowia lipolytica. Different secretion signals and culture media were tested. Production was correlated to both culture growth rate and cell morphology (highest at low growth rate, without mycelium). Recombinant laccase was characterized (immunodetection, N-terminal sequencing) and purified. Production was estimated to 20 mgl(-1) in a bioreactor. Thus, complex metalloenzymes can be produced in Yarrowia, assuming some control of host physiology. Lac1p production was compared in Yarrowia, Pichia and Aspergillus: recombinant proteins were active, but host systems differed in transformation efficiency, production, and glycosylation. If not the best producer, Yarrowia offers very high transformation efficiencies, allowing the genetic engineering of laccases for industrial applications.


Assuntos
Basidiomycota/enzimologia , Lacase/metabolismo , Proteínas Recombinantes/metabolismo , Yarrowia/enzimologia , Yarrowia/genética , Basidiomycota/genética , Meios de Cultura , Microbiologia Industrial , Lacase/genética , Lacase/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
8.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 10 Pt 2): 1882-5, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12351846

RESUMO

Mlc1p is a calmodulin-like protein from the budding yeast Saccharomyces cerevisiae, where it has been identified as a subunit of a class V myosin, Myo2p, and a binding partner of an IQGAP-like protein, Iqg1p. Through its interactions with these two proteins, Mlc1p plays a role in polarized growth and cytokinesis. Mlc1p has been crystallized in complexes with four different IQ target motifs from the neck region of Myo2p: IQ2, IQ3, IQ4 and IQ2-IQ3 (referred to as IQ2,3). Electron-density maps for two of the complexes (Mlc1p-IQ4 and Mlc1p-IQ2,3) were obtained from multiple anomalous dispersion (MAD) experiments based on selenomethionine derivatives. The other two structures (Mlc1p-IQ2 and Mlc1p-IQ3) were determined by molecular replacement using the partially refined structure of Mlc1p-IQ2,3 as a search model.


Assuntos
Cadeias Leves de Miosina/química , Miosina Tipo V/química , Sequência de Aminoácidos , Sítios de Ligação , Divisão Celular , Clonagem Molecular , Escherichia coli , Modelos Moleculares , Dados de Sequência Molecular , Cadeias Leves de Miosina/isolamento & purificação , Cadeias Leves de Miosina/metabolismo , Miosina Tipo V/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Difração de Raios X/métodos
9.
Proc Natl Acad Sci U S A ; 99(12): 8003-8, 2002 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-12048248

RESUMO

Actin is the most abundant protein in eukaryotic cells, but its release from cells into blood vessels can be lethal, being associated with clinical situations including hepatic necrosis and septic shock. A homeostatic mechanism, termed the actin-scavenger system, is responsible for the depolymerization and removal of actin from the circulation. During the first phase of this mechanism, gelsolin severs the actin filaments. In the second phase, the vitamin D-binding protein (DBP) traps the actin monomers, which accelerates their clearance. We have determined the crystal structures of DBP by itself and complexed with actin to 2.1 A resolution. Similar to its homologue serum albumin, DBP consists of three related domains. Yet, in DBP a strikingly different organization of the domains gives rise to a large actin-binding cavity. After complex formation the three domains of DBP move slightly to "clamp" onto actin subdomain 3 and to a lesser extent subdomain 1. Contacts between actin and DBP throughout their extensive 3,454-A(2) intermolecular interface involve a mixture of hydrophobic, electrostatic, and solvent-mediated interactions. The area of actin covered by DBP within the complex approximately equals the sum of those covered by gelsolin and profilin. Moreover, certain interactions of DBP with actin mirror those observed in the actin-gelsolin complex, which may explain how DBP can compete effectively with gelsolin for actin binding. Formation of the strong actin-DBP complex proceeds with limited conformational changes to both proteins, demonstrating how DBP has evolved to become an effective actin-scavenger protein.


Assuntos
Actinas/química , Actinas/metabolismo , Proteína de Ligação a Vitamina D/química , Proteína de Ligação a Vitamina D/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Gelsolina/química , Gelsolina/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Coelhos
10.
Structure ; 10(4): 557-67, 2002 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11937060

RESUMO

S100A6 is a member of the S100 family of Ca(2+) binding proteins, which have come to play an important role in the diagnosis of cancer due to their overexpression in various tumor cells. We have determined the crystal structures of human S100A6 in the Ca(2+)-free and Ca(2+)-bound states to resolutions of 1.15 A and 1.44 A, respectively. Ca(2+) binding is responsible for a dramatic change in the global shape and charge distribution of the S100A6 dimer, leading to the exposure of two symmetrically positioned target binding sites. The results are consistent with S100A6, and most likely other S100 proteins, functioning as Ca(2+) sensors in a way analogous to the prototypical sensors calmodulin and troponin C. The structures have important implications for our understanding of target binding and cooperativity of Ca(2+) binding in the S100 family.


Assuntos
Cálcio/metabolismo , Proteínas de Ciclo Celular , Estrutura Terciária de Proteína , Proteínas S100/química , Proteínas S100/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/genética
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