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1.
ChemMedChem ; 7(2): 248-61, 2012 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-22213702

RESUMO

Small highly soluble probe molecules such as aniline, urea, N-methylurea, 2-bromoacetate, 1,2-propanediol, nitrous oxide, benzamidine, and phenol were soaked into crystals of various proteins to map their binding pockets and to detect hot spots of binding with respect to hydrophobic and hydrophilic properties. The selected probe molecules were first tested at the zinc protease thermolysin. They were then applied to a wider range of proteins such as protein kinase A, D-xylose isomerase, 4-diphosphocytidyl-2C-methyl-D-erythritol synthase, endothiapepsin, and secreted aspartic protease 2. The crystal structures obtained clearly show that the probe molecules populate the protein binding pockets in an ordered fashion. The thus characterized, experimentally observed hot spots of binding were subjected to computational active site mapping using HotspotsX. This approach uses knowledge-based pair potentials to detect favorable binding positions for various atom types. Good agreement between the in silico hot spot predictions and the experimentally observed positions of the polar hydrogen bond forming functional groups and hydrophobic portions was obtained. Finally, we compared the observed poses of the small-molecule probes with those of much larger structurally related ligands. They coincide remarkably well with the larger ligands, considering their spatial orientation and the experienced interaction patterns. This observation confirms the fundamental hypothesis of fragment-based lead discovery: that binding poses, even of very small molecular probes, do not significantly deviate or move once a ligand is grown further into the binding site. This underscores the fact that these probes populate given hot spots and can be regarded as relevant seeds for further design.


Assuntos
Proteínas/química , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Desenho de Fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Ligação Proteica , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/química , Software , Termolisina/química , Termolisina/metabolismo
2.
J Med Chem ; 54(22): 7784-96, 2011 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21972967

RESUMO

Druglike molecules are defined by Lipinski's rule of 5, to characterize fragment thresholds, they have been reduced from 5 to 3 (Astex's rule of 3). They are applied to assemble fragment libraries, and providers use them to select fragments for commercial offer. We question whether these rules are too stringent to compose fragment libraries with candidates exhibiting sufficient room for chemical subsequent growing and merging modifications as appropriate functional groups for chemical transformations are required. Usually these groups exhibit properties as hydrogen bond donors/acceptors and provide entry points for optimization chemistry. We therefore designed a fragment library (364 entries) without strictly applying the rule of 3. For initial screening for endothiapepsin binding, we performed a biochemical cleavage assay of a fluorogenic substrate at 1 mM. "Hits" were defined to inhibit the enzyme by at least 40%. Fifty-five hits were suggested and subsequently soaked into endothiapepsin crystals. Eleven crystal structures could be determined covering fragments with diverse binding modes: (i) direct binding to the catalytic dyad aspartates, (ii) water-mediated binding to the aspartates, (iii) no direct interaction with the dyad. They occupy different specificity pockets. Only 4 of the 11 fragments are consistent with the rule of 3. Restriction to this rule would have limited the fragment hits to a strongly reduced variety of chemotypes.


Assuntos
Ácido Aspártico Endopeptidases/química , Desenho de Fármacos , Modelos Moleculares , Relação Quantitativa Estrutura-Atividade , Bibliotecas de Moléculas Pequenas , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Fluorescência , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Estrutura Molecular , Ligação Proteica , Solubilidade , Estereoisomerismo
3.
PLoS One ; 5(5): e10647, 2010 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-20498719

RESUMO

As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD) is a member of the non-heme iron(II)-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe(3+) at a resolution of 1.85 A. Like other non-heme iron(II) and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded beta-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family.


Assuntos
Diamino Aminoácidos/síntese química , Diamino Aminoácidos/metabolismo , Bacillus/enzimologia , Dioxigenases/química , Heme/metabolismo , Ferro/metabolismo , Ácidos Cetoglutáricos/metabolismo , Motivos de Aminoácidos , Aminoácidos/metabolismo , Diamino Aminoácidos/química , Bacillus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Dioxigenases/metabolismo , Genoma Bacteriano/genética , Hidroxilação , Ligantes , Modelos Moleculares , Maleabilidade , Estrutura Secundária de Proteína , Eletricidade Estática
4.
EMBO J ; 29(13): 2101-13, 2010 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-20461057

RESUMO

The time course of inactivation of voltage-activated potassium (Kv) channels is an important determinant of the firing rate of neurons. In many Kv channels highly unsaturated lipids as arachidonic acid, docosahexaenoic acid and anandamide can induce fast inactivation. We found that these lipids interact with hydrophobic residues lining the inner cavity of the pore. We analysed the effects of these lipids on Kv1.1 current kinetics and their competition with intracellular tetraethylammonium and Kvbeta subunits. Our data suggest that inactivation most likely represents occlusion of the permeation pathway, similar to drugs that produce 'open-channel block'. Open-channel block by drugs and lipids was strongly reduced in Kv1.1 channels whose amino acid sequence was altered by RNA editing in the pore cavity, and in Kv1.x heteromeric channels containing edited Kv1.1 subunits. We show that differential editing of Kv1.1 channels in different regions of the brain can profoundly alter the pharmacology of Kv1.x channels. Our findings provide a mechanistic understanding of lipid-induced inactivation and establish RNA editing as a mechanism to induce drug and lipid resistance in Kv channels.


Assuntos
Ácidos Graxos Insaturados/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Edição de RNA , Tetraetilamônio/farmacologia , Animais , Ácido Araquidônico/metabolismo , Sítios de Ligação , Humanos , Modelos Moleculares , Mutação , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Ligação Proteica , Ratos , Xenopus laevis
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