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1.
J Med Chem ; 56(22): 9275-95, 2013 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-24164581

RESUMO

Preclinical antithrombotic efficacy and bleeding models have demonstrated that P2Y1 antagonists are efficacious as antiplatelet agents and may offer a safety advantage over P2Y12 antagonists in terms of reduced bleeding liabilities. In this article, we describe the structural modification of the tert-butyl phenoxy portion of lead compound 1 and the subsequent discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular, spiropiperidine indoline-substituted diaryl ureas are described as potent, orally bioavailable small-molecule P2Y1 antagonists with improved activity in functional assays and improved oral bioavailability in rats. Homology modeling and rat PK/PD studies on benchmark compound 3l will also be presented. Compound 3l was our first P2Y1 antagonist to demonstrate a robust oral antithrombotic effect with mild bleeding liability in the rat thrombosis and hemostasis models.


Assuntos
Desenho de Fármacos , Conformação Molecular , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/farmacocinética , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacocinética , Receptores Purinérgicos P2Y1/metabolismo , Compostos de Espiro/farmacologia , Compostos de Espiro/farmacocinética , Ureia/farmacologia , Ureia/farmacocinética , Animais , Disponibilidade Biológica , Humanos , Indóis/química , Modelos Moleculares , Compostos de Fenilureia/química , Compostos de Fenilureia/metabolismo , Antagonistas do Receptor Purinérgico P2Y/química , Antagonistas do Receptor Purinérgico P2Y/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2Y1/química , Homologia de Sequência de Aminoácidos , Compostos de Espiro/química , Compostos de Espiro/metabolismo , Ureia/química , Ureia/metabolismo
2.
Bioorg Med Chem Lett ; 23(14): 4107-11, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23747226

RESUMO

The design, synthesis and characterization of a phosphonate inhibitor of N-acetylneuraminate-9-phosphate phosphatase (HDHD4) is described. Compound 3, where the substrate C-9 oxygen was replaced with a nonlabile CH2 group, inhibits HDHD4 with a binding affinity (IC50 11µM) in the range of the native substrate Neu5Ac-9-P (compound 1, Km 47µM). Combined SAR, modeling and NMR studies are consistent with the phosphonate group in inhibitor 3 forming a stable complex with native Mg(2+). In addition to this key interaction, the C-1 carboxylate of the sugar interacts with a cluster of basic residues, K141, R104 and R72. Comparative NMR studies of compounds 3 and 1 with Ca(2+) and Mg(2+) are indicative of a highly dynamic process in the active site for the HDHD4/Mg(2+)/3 complex. Possible explanations for this observation are discussed.


Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Ácidos Siálicos/síntese química , Fosfatos Açúcares/síntese química , Animais , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Estrutura Terciária de Proteína , Ratos , Ácidos Siálicos/química , Ácidos Siálicos/metabolismo , Fosfatos Açúcares/química , Fosfatos Açúcares/metabolismo
3.
J Med Chem ; 54(19): 6548-62, 2011 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-21882820

RESUMO

Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of tyrosine residues, a process that involves a conserved tryptophan-proline-aspartate (WPD) loop in catalysis. In previously determined structures of PTPs, the WPD-loop has been observed in either an "open" conformation or a "closed" conformation. In the current work, X-ray structures of the catalytic domain of receptor-like protein tyrosine phosphatase γ (RPTPγ) revealed a ligand-induced "superopen" conformation not previously reported for PTPs. In the superopen conformation, the ligand acts as an apparent competitive inhibitor and binds in a small hydrophobic pocket adjacent to, but distinct from, the active site. In the open and closed WPD-loop conformations of RPTPγ, the side chain of Trp1026 partially occupies this pocket. In the superopen conformation, Trp1026 is displaced allowing a 3,4-dichlorobenzyl substituent to occupy this site. The bound ligand prevents closure of the WPD-loop over the active site and disrupts the catalytic cycle of the enzyme.


Assuntos
Modelos Moleculares , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/antagonistas & inibidores , Tiofenos/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Proteínas Tirosina Fosfatases Classe 5 Semelhantes a Receptores/química , Relação Estrutura-Atividade , Tiofenos/síntese química
4.
Proc Natl Acad Sci U S A ; 108(37): 15366-71, 2011 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-21896751

RESUMO

Influenza nucleoprotein (NP) plays multiple roles in the virus life cycle, including an essential function in viral replication as an integral component of the ribonucleoprotein complex, associating with viral RNA and polymerase within the viral core. The multifunctional nature of NP makes it an attractive target for antiviral intervention, and inhibitors targeting this protein have recently been reported. In a parallel effort, we discovered a structurally similar series of influenza replication inhibitors and show that they interfere with NP-dependent processes via formation of higher-order NP oligomers. Support for this unique mechanism is provided by site-directed mutagenesis studies, biophysical characterization of the oligomeric ligand:NP complex, and an X-ray cocrystal structure of an NP dimer of trimers (or hexamer) comprising three NP_A:NP_B dimeric subunits. Each NP_A:NP_B dimeric subunit contains two ligands that bridge two composite, protein-spanning binding sites in an antiparallel orientation to form a stable quaternary complex. Optimization of the initial screening hit produced an analog that protects mice from influenza-induced weight loss and mortality by reducing viral titers to undetectable levels throughout the course of treatment.


Assuntos
Antivirais/farmacologia , Nucleoproteínas/química , Nucleoproteínas/metabolismo , Orthomyxoviridae/fisiologia , Bibliotecas de Moléculas Pequenas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Antivirais/uso terapêutico , Cristalografia por Raios X , Modelos Animais de Doenças , Ensaios de Triagem em Larga Escala , Hidrodinâmica , Camundongos , Modelos Moleculares , Nucleoproteínas/ultraestrutura , Orthomyxoviridae/efeitos dos fármacos , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Bibliotecas de Moléculas Pequenas/uso terapêutico , Soluções
5.
Artigo em Inglês | MEDLINE | ID: mdl-21795790

RESUMO

Protein tyrosine phosphatase γ is a membrane-bound receptor and is designated RPTPγ. RPTPγ and two mutants, RPTPγ(V948I, S970T) and RPTPγ(C858S, S970T), were recombinantly expressed and purified for X-ray crystallographic studies. The purified enzymes were crystallized using the hanging-drop vapor-diffusion method. Crystallographic data were obtained from several different crystal forms in the absence and the presence of inhibitor. In this paper, a description is given of how three different crystal forms were obtained that were used with various ligands. An orthorhombic crystal form and a trigonal crystal form were obtained both with and without ligand, and a monoclinic crystal form was only obtained in the presence of a particularly elaborated inhibitor.


Assuntos
Domínio Catalítico , Proteínas Tirosina Fosfatases Semelhantes a Receptores/química , Sequência de Aminoácidos , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Proteínas Tirosina Fosfatases Semelhantes a Receptores/genética , Proteínas Tirosina Fosfatases Semelhantes a Receptores/isolamento & purificação
6.
Anal Biochem ; 392(1): 59-69, 2009 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-19497292

RESUMO

Eg5 is a kinesin whose inhibition leads to cycle arrest during mitosis, making it a potential therapeutic target in cancers. Circular dichroism and isothermal titration calorimetry of our pyrrolotriazine-4-one series of inhibitors with Eg5 motor domain revealed enhanced binding in the presence of adenosine 5'-diphosphate (ADP). Using this information, we studied the interaction of this series with ADP-Eg5 complexes using a thermal shift assay. We measured up to a 7 degrees C increase in the thermal melting (T(m)) of Eg5 for an inhibitor that produced IC(50) values of 60 and 130 nM in microtubule-dependent adenosine triphosphatase (ATPase) and cell-based cytotoxicity assays, respectively. In general, the inhibitor potency of the pyrrolotriazine-4-one series in in vitro biological assays correlated with the magnitude of the thermal stability enhancement of ADP-Eg5. The thermal shift assay also confirmed direct binding of Eg5 inhibitors identified in a high-throughput screen and demonstrated that the thermal shift assay is applicable to a range of chemotypes and can be useful in evaluating both potent (nM) and relatively weakly binding (microM) leads. Overall, the thermal shift assay was found to be an excellent biophysical method for evaluating direct binding of a large number of compounds to Eg5, and it complemented the catalytic assay screens by providing an alternative determination of inhibitor potency.


Assuntos
Bioquímica/métodos , Cinesina/química , Pirróis/análise , Pirróis/química , Triazinas/análise , Triazinas/química , Difosfato de Adenosina/metabolismo , Fenômenos Biofísicos , Calorimetria , Linhagem Celular Tumoral , Dicroísmo Circular , Humanos , Cinesina/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Ligação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Temperatura
7.
J Med Chem ; 51(19): 6225-9, 2008 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-18771253

RESUMO

Fragment-like inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK2) include 5-hydroxyisoquinoline (IC50 approximately 85 microM). Modeling studies identified four possible binding modes for this compound. Two-dimensional (1)H-(1)H NOESY data obtained with selectively protonated samples of MK2 in complex with 5-hydroxyisoquinoline demonstrated that two of the four predicted binding modes are well populated. A second small isoquinoline was subsequently shown to bind in a single mode. NMR and modeling studies using this general approach are expected to facilitate "scaffold hopping" and structure-guided elaborations of fragment-like kinase inhibitor cores.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Isoquinolinas/farmacologia , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sítios de Ligação/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Isoquinolinas/química , Espectroscopia de Ressonância Magnética/normas , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Prótons , Padrões de Referência , Relação Estrutura-Atividade
8.
J Med Chem ; 49(16): 5013-7, 2006 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-16884313

RESUMO

The NMR structure is presented for compound 1 (BMS-480404) (Ki = 33 (+/-2) nM) bound to keratinocyte fatty acid-binding protein. This article describes interactions between a high affinity drug-like compound and a member of the fatty acid-binding protein family. A benzyl group ortho to the mandelic acid in 1 occupies an area of the protein that fatty acids do not normally contact. Similar to that in the kFABP-palmitic acid structure, the acid moiety in 1 is proximal to R129 and Y131. Computational modeling indicates that the acid moiety in 1 interacts indirectly via a modeled water molecule to R109.


Assuntos
Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Proteínas de Ligação a Ácido Graxo/química , Queratinócitos/metabolismo , Sítios de Ligação , Simulação por Computador , Proteínas de Ligação a Ácido Graxo/metabolismo , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular
9.
Bioorg Med Chem Lett ; 16(15): 3937-42, 2006 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-16730979

RESUMO

Synthesis and SAR of substituted pyrrolotriazine-4-one analogues as Eg5 inhibitors are described. Many of these analogues displayed potent inhibitory activities in the Eg5 ATPase and A2780 cell proliferation assays. In addition, pyrrolotriazine-4-one analogue 26 demonstrated in vivo efficacy in an iv P388 murine leukemia model. Both NMR and X-ray crystallographic studies revealed that these analogues bind to an allosteric site on the Eg5 protein.


Assuntos
Cinesina/antagonistas & inibidores , Pirróis/síntese química , Pirróis/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Pirróis/química , Relação Estrutura-Atividade
10.
J Mol Recognit ; 19(3): 227-33, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16583354

RESUMO

NF-kappaB activation is mediated by the IKK signalsome. Though this signalsome is comprised of IKK-1, IKK-2, and NEMO/IKKgamma, it is the interaction between IKK-2 and NEMO that is critical to formation of a functional signalsome. More specifically, previous reports have indicated that this interaction involves the C-terminal LDWSWL residues of IKK-2 (called the Nemo Binding Domain (NBD)) and the N-terminus of NEMO. In an effort to characterize the IKK-2:NEMO interaction, we have investigated several NBD-containing peptides for their ability to bind NEMO and inhibit the critical IKK-2:NEMO interaction. The six residue NBD peptide, LDWSWL, showed modest binding to NEMO and little inhibition of the IKK-2:NEMO interaction, whereas peptides containing the NBD plus additional flanking amino acids (NBD-containing peptides) more effectively bound NEMO and inhibited the interaction. These longer NBD-containing peptides may be required to give the NBD an appropriate conformation for recognition by NEMO and/or to provide for additional interactions with NEMO.


Assuntos
Aminoácidos/química , Quinase I-kappa B/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Western Blotting , Linhagem Celular , Vetores Genéticos/genética , Humanos , Quinase I-kappa B/efeitos dos fármacos , Quinase I-kappa B/genética , Espectroscopia de Ressonância Magnética , NF-kappa B/metabolismo , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/farmacologia , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Spodoptera
13.
Health Care Financ Rev ; 23(3): 131-59, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12500353

RESUMO

In this article, we estimate expenditures by businesses, households, and governments in providing financing for health care for 1987-2000 and track measures of burden that these costs impose. Although burden measures for businesses and the Federal Government have stabilized or improved since 1993, measures of burden for State and local governments are deteriorating slightly--a situation that is likely to worsen in the near future. As health care spending accelerates and an economy wide recession seems imminent, businesses, households, and governments that finance health care will face renewed health cost pressures on their revenue and income.


Assuntos
Efeitos Psicossociais da Doença , Custos de Cuidados de Saúde/estatística & dados numéricos , Gastos em Saúde/estatística & dados numéricos , Coleta de Dados , Custos de Saúde para o Empregador/estatística & dados numéricos , Custos de Saúde para o Empregador/tendências , Características da Família , Financiamento Governamental/classificação , Financiamento Governamental/estatística & dados numéricos , Financiamento Governamental/tendências , Custos de Cuidados de Saúde/tendências , Gastos em Saúde/classificação , Gastos em Saúde/tendências , Pesquisa sobre Serviços de Saúde , Humanos , Estados Unidos , Indenização aos Trabalhadores/estatística & dados numéricos
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