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1.
J Am Chem Soc ; 140(21): 6596-6603, 2018 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-29668265

RESUMO

CRISPR-Cas RNA-guided endonucleases hold great promise for disrupting or correcting genomic sequences through site-specific DNA cleavage and repair. However, the lack of methods for cell- and tissue-selective delivery currently limits both research and clinical uses of these enzymes. We report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9-ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Uptake of such fluorescently labeled proteins in liver-derived cell lines HEPG2 (ASGPr+) and SKHEP (control; diminished ASGPr) was studied by live cell imaging and demonstrates increased accumulation of Cas9-ASGPrL RNP in HEPG2 cells as a result of effective ASGPr-mediated endocytosis. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor-facilitated and cell-type specific gene editing that did not rely on electroporation or the use of transfection reagents. Overall, these in vitro results validate the receptor-mediated delivery of genome-editing enzymes as an approach for cell-selective gene editing and provide a framework for future potential applications to hepatoselective gene editing in vivo.


Assuntos
Sistemas CRISPR-Cas , Endonucleases/metabolismo , Edição de Genes , Linhagem Celular Tumoral , Endonucleases/genética , Células Hep G2 , Humanos , Estrutura Molecular , Engenharia de Proteínas
2.
J Am Chem Soc ; 139(9): 3528-3536, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28230359

RESUMO

A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.


Assuntos
Receptor de Asialoglicoproteína/metabolismo , Compostos Bicíclicos com Pontes/química , Hepatócitos/metabolismo , Cetonas/química , Fígado/metabolismo , Polímeros/química , Compostos Bicíclicos com Pontes/metabolismo , Cristalografia por Raios X , Portadores de Fármacos/química , Humanos , Cetonas/metabolismo , Fígado/citologia , Modelos Moleculares , Estrutura Molecular , Polímeros/metabolismo
3.
J Am Chem Soc ; 134(4): 1978-81, 2012 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-22280495

RESUMO

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.


Assuntos
Acetilgalactosamina/química , Receptor de Asialoglicoproteína/química , Acetilgalactosamina/análogos & derivados , Humanos , Ligantes , Estrutura Molecular , Estereoisomerismo
4.
Nat Struct Mol Biol ; 14(2): 106-13, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17237796

RESUMO

Cholesteryl ester transfer protein (CETP) shuttles various lipids between lipoproteins, resulting in the net transfer of cholesteryl esters from atheroprotective, high-density lipoproteins (HDL) to atherogenic, lower-density species. Inhibition of CETP raises HDL cholesterol and may potentially be used to treat cardiovascular disease. Here we describe the structure of CETP at 2.2-A resolution, revealing a 60-A-long tunnel filled with two hydrophobic cholesteryl esters and plugged by an amphiphilic phosphatidylcholine at each end. The two tunnel openings are large enough to allow lipid access, which is aided by a flexible helix and possibly also by a mobile flap. The curvature of the concave surface of CETP matches the radius of curvature of HDL particles, and potential conformational changes may occur to accommodate larger lipoprotein particles. Point mutations blocking the middle of the tunnel abolish lipid-transfer activities, suggesting that neutral lipids pass through this continuous tunnel.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/química , Ésteres do Colesterol/química , Modelos Moleculares , Fosfatidilcolinas/química , Triglicerídeos/química , Animais , Sítios de Ligação , Células CHO , Proteínas de Transferência de Ésteres de Colesterol/genética , Cricetinae , Cricetulus , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Mutação Puntual , Ligação Proteica , Conformação Proteica
5.
J Lipid Res ; 51(5): 967-74, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-19965592

RESUMO

The CETP inhibitor, torcetrapib, was prematurely terminated from phase 3 clinical trials due to an increase in cardiovascular and noncardiovascular mortality. Because nearly half of the latter deaths involved patients with infection, we have tested torcetrapib and other CETPIs to see if they interfere with lipopolysaccharide binding protein (LBP) or bactericidal/permeability increasing protein (BPI). No effect of these potent CETPIs on LPS binding to either protein was detected. Purified CETP itself bound weakly to LPS with a Kd >or= 25 microM compared with 0.8 and 0.5 nM for LBP and BPI, respectively, and this binding was not blocked by torcetrapib. In whole blood, LPS induced tumor necrosis factor-alpha normally in the presence of torcetrapib. Furthermore, LPS had no effect on CETP activity. We conclude that the sepsis-related mortality of the ILLUMINATE trial was unlikely due to a direct effect of torcetrapib on LBP or BPI function, nor to inhibition of an interaction of CETP with LPS. Instead, we speculate that the negative outcome seen for patients with infections might be related to the changes in plasma lipoprotein composition and metabolism, or alternatively to the known off-target effects of torcetrapib, such as aldosterone elevation, which may have aggravated the effects of sepsis.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Infecções/imunologia , Quinolinas/farmacologia , Proteínas de Fase Aguda/imunologia , Proteínas de Fase Aguda/metabolismo , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/metabolismo , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Humanos , Lipopolissacarídeos/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Ligação Proteica/efeitos dos fármacos , Ressonância de Plasmônio de Superfície
6.
Bioconjug Chem ; 19(8): 1604-13, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18646836

RESUMO

Cholesteryl ester transfer protein (CETP) transfers neutral lipids between different types of plasma lipoprotein. Inhibitors of CETP elevate the fraction of plasma cholesterol associated with high-density lipoproteins and are being developed as new agents for the prevention and treatment of cardiovascular disease. The molecular basis of their function is not yet fully understood. To aid in the study of inhibitor interactions with CETP, a torcetrapib-related compound was coupled to different biotin-terminated spacer groups, and the binding of CETP to the streptavidin-bound conjugates was monitored on agarose beads and in a surface plasmon resonance biosensor. CETP binding was poor with a 2.0 nm spacer arm, but efficient with polyethyleneglycol spacers of 3.5 or 4.6 nm. The conjugate based on a 4.6 nm spacer was used for further biosensor experiments. Soluble inhibitor blocked the binding of CETP to the immobilized drug, as did preincubation with a disulfide-containing covalent inhibitor. To provide a first estimate of the binding site for torcetrapib-like inhibitors, CETP was modified with a disulfide-containing agent that modifies Cys-13 of CETP. Mass spectrometry of the modified protein indicated that a single half-molecule of the disulfide was covalently bound to CETP, and peptide mapping after digestion with pepsin confirmed previous reports based on mutagenesis that Cys-13 was the site of modification. Modified CETP was unable to bind to the biosensor-mounted torcetrapib analog, indicating that the binding site on CETP for torcetrapib is in the lipid-binding pocket near the N-terminus of the protein. The crystal structure of CETP shows that the sulfhydryl group of Cys-13 resides at the bottom of this pocket.


Assuntos
Proteínas de Transferência de Ésteres de Colesterol/antagonistas & inibidores , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Marcadores de Afinidade/química , Marcadores de Afinidade/metabolismo , Sítios de Ligação , Ligação Competitiva , Biotina/metabolismo , Proteínas de Transferência de Ésteres de Colesterol/química , Proteínas de Transferência de Ésteres de Colesterol/genética , Ligantes , Mutagênese , Ligação Proteica , Quinolinas/química , Quinolinas/metabolismo
7.
Nat Commun ; 8(1): 1908, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29199275

RESUMO

Lysosomal integral membrane protein-2 (LIMP-2/SCARB2) contributes to endosomal and lysosomal function. LIMP-2 deficiency is associated with neurological abnormalities and kidney failure and, as an acid glucocerebrosidase receptor, impacts Gaucher and Parkinson's diseases. Here we report a crystal structure of a LIMP-2 luminal domain dimer with bound cholesterol and phosphatidylcholine. Binding of these lipids alters LIMP-2 from functioning as a glucocerebrosidase-binding monomer toward a dimeric state that preferentially binds anionic phosphatidylserine over neutral phosphatidylcholine. In cellular uptake experiments, LIMP-2 facilitates transport of phospholipids into murine fibroblasts, with a strong substrate preference for phosphatidylserine. Taken together, these biophysical and cellular studies define the structural basis and functional importance of a form of LIMP-2 for lipid trafficking. We propose a model whereby switching between monomeric and dimeric forms allows LIMP-2 to engage distinct binding partners, a mechanism that may be shared by SR-BI and CD36, scavenger receptor proteins highly homologous to LIMP-2.


Assuntos
Antígenos CD36/metabolismo , Colesterol/metabolismo , Proteínas de Membrana Lisossomal/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidilserinas/metabolismo , Receptores Depuradores/metabolismo , Animais , Cristalografia por Raios X , Fibroblastos/metabolismo , Células HEK293 , Humanos , Camundongos , Fosfolipídeos/metabolismo
8.
J Med Chem ; 60(18): 7835-7849, 2017 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-28853885

RESUMO

Increased fructose consumption and its subsequent metabolism have been implicated in hepatic steatosis, dyslipidemia, obesity, and insulin resistance in humans. Since ketohexokinase (KHK) is the principal enzyme responsible for fructose metabolism, identification of a selective KHK inhibitor may help to further elucidate the effect of KHK inhibition on these metabolic disorders. Until now, studies on KHK inhibition with small molecules have been limited due to the lack of viable in vivo pharmacological tools. Herein we report the discovery of 12, a selective KHK inhibitor with potency and properties suitable for evaluating KHK inhibition in rat models. Key structural features interacting with KHK were discovered through fragment-based screening and subsequent optimization using structure-based drug design, and parallel medicinal chemistry led to the identification of pyridine 12.


Assuntos
Desenho de Fármacos , Frutoquinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Cristalografia por Raios X , Frutoquinases/química , Frutoquinases/metabolismo , Humanos , Masculino , Simulação de Acoplamento Molecular , Piridinas/química , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley
9.
Sci Rep ; 6: 30859, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27527709

RESUMO

Interleukin-17A (IL-17A) is a principal driver of multiple inflammatory and immune disorders. Antibodies that neutralize IL-17A or its receptor (IL-17RA) deliver efficacy in autoimmune diseases, but no small-molecule IL-17A antagonists have yet progressed into clinical trials. Investigation of a series of linear peptide ligands to IL-17A and characterization of their binding site has enabled the design of novel macrocyclic ligands that are themselves potent IL-17A antagonists.


Assuntos
Interleucina-17/antagonistas & inibidores , Interleucina-17/química , Peptídeos Cíclicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Algoritmos , Sítios de Ligação , Células Cultivadas , Desenho de Fármacos , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Compostos Macrocíclicos/química , Compostos Macrocíclicos/farmacologia , Simulação de Dinâmica Molecular , Peptídeos Cíclicos/química , Ligação Proteica , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
10.
Sci Rep ; 6: 26071, 2016 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-27184415

RESUMO

IL-17A is a pro-inflammatory cytokine that has been implicated in autoimmune and inflammatory diseases. Monoclonal antibodies inhibiting IL-17A signaling have demonstrated remarkable efficacy, but an oral therapy is still lacking. A high affinity IL-17A peptide antagonist (HAP) of 15 residues was identified through phage-display screening followed by saturation mutagenesis optimization and amino acid substitutions. HAP binds specifically to IL-17A and inhibits the interaction of the cytokine with its receptor, IL-17RA. Tested in primary human cells, HAP blocked the production of multiple inflammatory cytokines. Crystal structure studies revealed that two HAP molecules bind to one IL-17A dimer symmetrically. The N-terminal portions of HAP form a ß-strand that inserts between two IL-17A monomers while the C-terminal section forms an α helix that directly blocks IL-17RA from binding to the same region of IL-17A. This mode of inhibition suggests opportunities for developing peptide antagonists against this challenging target.


Assuntos
Inibidores Enzimáticos/metabolismo , Interleucina-17/antagonistas & inibidores , Peptídeos/metabolismo , Receptores de Interleucina-17/metabolismo , Substituição de Aminoácidos , Células Cultivadas , Cristalografia por Raios X , Inibidores Enzimáticos/isolamento & purificação , Humanos , Interleucina-17/química , Programas de Rastreamento , Modelos Moleculares , Mutagênese , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/isolamento & purificação , Ligação Proteica , Conformação Proteica
11.
Protein Sci ; 24(1): 20-6, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25287857

RESUMO

Undecaprenyl pyrophosphate synthase (UPPs) is an essential enzyme in a key bacterial cell wall synthesis pathway. It catalyzes the consecutive condensations of isopentenyl pyrophosphate (IPP) groups on to a trans-farnesyl pyrophosphate (FPP) to produce a C55 isoprenoid, undecaprenyl pyrophosphate (UPP). Here we report the discovery and co-crystal structures of a drug-like UPPs inhibitor in complex with Streptococcus pneumoniae UPPs, with and without substrate FPP, at resolutions of 2.2 and 2.1 Å, respectively. The UPPs inhibitor has a low molecular weight (355 Da), but displays potent inhibition of UPP synthesis in vitro (IC50 50 nM) that translates into excellent whole cell antimicrobial activity against pathogenic strains of Streptococcal species (MIC90 0.4 µg mL(-1) ). Interestingly, the inhibitor does not compete with the substrates but rather binds at a site adjacent to the FPP binding site and interacts with the tail of the substrate. Based on the structures, an allosteric inhibition mechanism of UPPs is proposed for this inhibitor. This inhibition mechanism is supported by biochemical and biophysical experiments, and provides a basis for the development of novel antibiotics targeting Streptococcus pneumoniae.


Assuntos
Alquil e Aril Transferases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Transferases/antagonistas & inibidores , Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Antibacterianos/química , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Infecções Pneumocócicas/tratamento farmacológico , Infecções Pneumocócicas/microbiologia , Fosfatos de Poli-Isoprenil/metabolismo , Sesquiterpenos/metabolismo , Streptococcus pneumoniae/química , Streptococcus pneumoniae/metabolismo , Transferases/química , Transferases/metabolismo
12.
Nat Commun ; 4: 1888, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23695682

RESUMO

The constituent polypeptides of the interleukin-17 family form six different homodimeric cytokines (IL-17A-F) and the heterodimeric IL-17A/F. Their interactions with IL-17 receptors A-E (IL-17RA-E) mediate host defenses while also contributing to inflammatory and autoimmune responses. IL-17A and IL-17F both preferentially engage a receptor complex containing one molecule of IL-17RA and one molecule of IL-17RC. More generally, IL-17RA appears to be a shared receptor that pairs with other members of its family to allow signaling of different IL-17 cytokines. Here we report crystal structures of homodimeric IL-17A and its complex with IL-17RA. Binding to IL-17RA at one side of the IL-17A molecule induces a conformational change in the second, symmetry-related receptor site of IL-17A. This change favors, and is sufficient to account for, the selection of a different receptor polypeptide to complete the cytokine-receptor complex. The structural results are supported by biophysical studies with IL-17A variants produced by site-directed mutagenesis.


Assuntos
Interleucina-17/química , Receptores de Interleucina-17/química , Regulação Alostérica , Sequência de Aminoácidos , Sequência Conservada , Cristalização , Cristalografia por Raios X , Células HEK293 , Humanos , Interleucina-17/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores de Interleucina-17/metabolismo , Ressonância de Plasmônio de Superfície
13.
J Biol Chem ; 284(19): 13193-201, 2009 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-19244237

RESUMO

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Quinase 2 de Adesão Focal/química , Naftalenos/farmacologia , Conformação Proteica , Pirazóis/farmacologia , Sequência de Aminoácidos , Calcificação Fisiológica , Clonagem Molecular , Cristalografia por Raios X , Quinase 2 de Adesão Focal/antagonistas & inibidores , Quinase 2 de Adesão Focal/metabolismo , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Ligação Proteica , Homologia de Sequência de Aminoácidos
14.
J Med Chem ; 52(11): 3576-85, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19438227

RESUMO

3-Hydroxyquinolin-2(1H)-one (2) was discovered by high throughput screening in a functional assay to be a potent inhibitor of human DAAO, and its binding affinity was confirmed in a Biacore assay. Cocrystallization of 2 with the human DAAO enzyme defined the binding site and guided the design of new analogues. The SAR, pharmacokinetics, brain exposure, and effects on cerebellum D-serine are described. Subsequent evaluation against the rat DAAO enzyme revealed a divergent SAR versus the human enzyme and may explain the high exposures of drug necessary to achieve significant changes in rat or mouse cerebellum D-serine.


Assuntos
D-Aminoácido Oxidase/antagonistas & inibidores , Hidroxiquinolinas/farmacologia , Hidroxiquinolinas/farmacocinética , Animais , Cerebelo/metabolismo , Cristalografia por Raios X , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidroxiquinolinas/síntese química , Masculino , Camundongos , Ratos , Ratos Sprague-Dawley , Serina/metabolismo , Relação Estrutura-Atividade
15.
J Biol Chem ; 278(2): 1067-74, 2003 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-12414808

RESUMO

The structural flexibility and thermostability of glutamate dehydrogenase (GDH) from Clostridium symbiosum were examined by limited proteolysis using three proteinases with different specificities, trypsin, chymotrypsin, and endoproteinase Glu-C. Clostridial GDH resisted proteolysis by any of these enzymes at 25 degrees C. Above 30 degrees C, however, GDH became cleavable by chymotrypsin, apparently at a single site. SDS-PAGE indicated the formation of one large fragment with a molecular mass of approximately 44 kDa and one small one of <10 kDa. Proteolysis was accompanied by the loss of enzyme activity, which outran peptide cleavage, suggesting a cooperative conformational change. Proteolysis was prevented by either of the substrates 2-oxoglutarate or l-glutamate but not by the coenzymes NAD(+) or NADH. Circular dichroism spectroscopy indicated that the protective effects of these ligands resulted from fixation of flexible regions of the native structure of the enzyme. Size-exclusion chromatography and SDS-PAGE studies of chymotrypsin-treated GDH showed that the enzyme retained its hexameric structure and all of its proteolytic fragments. However, circular dichroism spectroscopy and analytical ultracentrifugation showed global conformational changes affecting the overall compactness of the protein structure. Chymotrypsin-catalyzed cleavage also diminished the thermostability of GDH and the cooperativity of the transition between its native and denatured states. N-terminal amino acid sequencing and mass spectrometry showed that heat-induced sensitivity to chymotrypsin emerged in the loop formed by residues 390-393 that lies between helices alpha(15) and alpha(16) in the folded structure of the enzyme.


Assuntos
Proteínas de Bactérias/química , Clostridium/enzimologia , Glutamato Desidrogenase/química , Sequência de Aminoácidos , Quimotripsina/farmacologia , Dicroísmo Circular , Espectrometria de Massas , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Quaternária de Proteína
16.
Bioorg Med Chem Lett ; 13(3): 379-82, 2003 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-12565933

RESUMO

In this communication, we wish to describe the discovery of a novel series of 6-azauracil-based thyromimetics that possess up to 100-fold selectivities for binding and functional activation of the beta(1)-isoform of the thyroid receptor family. Structure-activity relationship studies on the 3,5- and 3'-positions provided compounds with enhanced TR beta affinity and selectivity. Key binding interactions between the 6-azauracil moiety and the receptor have been determined through of X-ray crystallographic analysis.


Assuntos
Receptores dos Hormônios Tireóideos/efeitos dos fármacos , Hormônios Tireóideos/farmacologia , Uracila/análogos & derivados , Uracila/química , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Indicadores e Reagentes , Ligantes , Modelos Moleculares , Mimetismo Molecular , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Uracila/farmacologia
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