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1.
ACS Med Chem Lett ; 10(3): 318-323, 2019 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-30891133

RESUMO

The atypical protein kinase C-iota (PKC-ι) enzyme is implicated in various cancers and has been put forward as an attractive target for developing anticancer therapy. A high concentration biochemical screen identified pyridine fragment weakly inhibiting PKC-ι with IC50 = 424 µM. Driven by structure-activity relationships and guided by docking hypothesis, the weakly bound fragment was eventually optimized into a potent inhibitor of PKC-ι (IC50= 270 nM). Through the course of the optimization, an intermediate compound was crystallized with the protein, and careful analysis of the X-ray crystal structure revealed a unique binding mode involving the post-kinase domain (C-terminal tail) of PKC-ι.

2.
Eur J Med Chem ; 157: 610-621, 2018 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-30125722

RESUMO

Even though many GyrB and ParE inhibitors have been reported in the literature, few possess activity against Gram-negative bacteria. This is primarily due to limited permeability across Gram-negative bacterial membrane as well as bacterial efflux mechanisms. Permeability of compounds across Gram-negative bacterial membranes depends on many factors including physicochemical properties of the inhibitors. Herein, we show the optimization of pyridylureas leading to compounds with potent activity against Gram-negative bacterial species such as P.aeruginosa, E.coli and A.baumannii.


Assuntos
Antibacterianos/farmacologia , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , Descoberta de Drogas , Escherichia coli/efeitos dos fármacos , Inibidores da Topoisomerase/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , DNA Topoisomerase IV/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pneumoniae/enzimologia , Relação Estrutura-Atividade , Inibidores da Topoisomerase/síntese química , Inibidores da Topoisomerase/química
3.
J Med Chem ; 61(10): 4386-4396, 2018 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-29688013

RESUMO

Protein kinase C iota (PKC-ι) is an atypical kinase implicated in the promotion of different cancer types. A biochemical screen of a fragment library has identified several hits from which an azaindole-based scaffold was chosen for optimization. Driven by a structure-activity relationship and supported by molecular modeling, a weakly bound fragment was systematically grown into a potent and selective inhibitor against PKC-ι.


Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Isoenzimas/antagonistas & inibidores , Neoplasias Hepáticas/tratamento farmacológico , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Carcinoma Hepatocelular/patologia , Humanos , Neoplasias Hepáticas/patologia , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Células Tumorais Cultivadas
4.
ACS Omega ; 2(11): 7881-7891, 2017 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-30023565

RESUMO

Overexpression of the eukaryotic initiation factor 4E (eIF4E) is linked to a variety of cancers. Both mitogen-activated protein kinases-interacting kinases 1 and 2 (Mnk1/2) activate the oncogene eIF4E through posttranslational modification (phosphorylating it at the conserved Ser209). Inhibition of Mnk prevents eIF4E phosphorylation, making the Mnk-eIF4E axis a potential therapeutic target for oncology. Recently, the design and synthesis of a series of novel potent compounds inhibiting the Mnk1/2 kinases were carried out in-house. Here, we describe computational models of the interactions between Mnk1/2 kinases and these inhibitors. Molecular modeling combined with free energy calculations show that these compounds bind to the inactive forms of the kinases. All compounds adopt similar conformations in the catalytic sites of both kinases, stabilized by hydrogen bonds with the hinge regions and with the catalytic Lys78 (Mnk1) and Lys113 (Mnk2). These hydrogen bond interactions clearly play a critical role in determining the conformational stability and potency of the compounds. We also find that van der Waals interactions with an allosteric pocket are key to their binding and potency. Two distinct hydration sites that appear to further stabilize the ligand binding/interactions were observed. Critically, the inclusion of explicit water molecules in the calculations results in improving the agreement between calculated and experimental binding free energies.

5.
J Biol Chem ; 291(34): 17743-53, 2016 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-27365392

RESUMO

Bacterial topoisomerases are attractive antibacterial drug targets because of their importance in bacterial growth and low homology with other human topoisomerases. Structure-based drug design has been a proven approach of efficiently developing new antibiotics against these targets. Past studies have focused on developing lead compounds against the ATP binding pockets of both DNA gyrase and topoisomerase IV. A detailed understanding of the interactions between ligand and target in a solution state will provide valuable information for further developing drugs against topoisomerase IV targets. Here we describe a detailed characterization of a known potent inhibitor containing a 9H-pyrimido[4,5-b]indole scaffold against the N-terminal domain of the topoisomerase IV E subunit from Escherichia coli (eParE). Using a series of biophysical and biochemical experiments, it has been demonstrated that this inhibitor forms a tight complex with eParE. NMR studies revealed the exact protein residues responsible for inhibitor binding. Through comparative studies of two inhibitors of markedly varied potencies, it is hypothesized that gaining molecular interactions with residues in the α4 and residues close to the loop of ß1-α2 and residues in the loop of ß3-ß4 might improve the inhibitor potency.


Assuntos
DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/química , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Inibidores da Topoisomerase/química , Humanos , Indóis/química , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Estrutura Secundária de Proteína
6.
J Med Chem ; 59(7): 3063-78, 2016 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-27011159

RESUMO

Clinically used BCR-ABL1 inhibitors for the treatment of chronic myeloid leukemia do not eliminate leukemic stem cells (LSC). It has been shown that MNK1 and 2 inhibitors prevent phosphorylation of eIF4E and eliminate the self-renewal capacity of LSCs. Herein, we describe the identification of novel dual MNK1 and 2 and BCR-ABL1 inhibitors, starting from the known kinase inhibitor 2. Initial structure-activity relationship studies resulted in compound 27 with loss of BCR-ABL1 inhibition. Further modification led to orally bioavailable dual MNK1 and 2 and BCR-ABL1 inhibitors 53 and 54, which are efficacious in a mouse xenograft model and also reduce the level of phosphorylated eukaryotic translation initiation factor 4E in the tumor tissues. Kinase selectivity of these compounds is also presented.


Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Técnicas de Química Sintética , Relação Dose-Resposta a Droga , Fator de Iniciação 4E em Eucariotos/metabolismo , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Camundongos SCID , Terapia de Alvo Molecular/métodos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
7.
Biophys J ; 109(9): 1969-77, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26536273

RESUMO

Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G(+) strain (Streptococcus pneumoniae (sParE)) and a G(-) strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between ß2 and α3, and the ß2 and ß6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher KD than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.


Assuntos
DNA Topoisomerase IV/química , Sequência de Aminoácidos , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/genética , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Estrutura Secundária de Proteína , Pseudomonas aeruginosa , Soluções , Streptococcus pneumoniae , Ressonância de Plasmônio de Superfície , Temperatura
8.
Biochem Biophys Res Commun ; 467(4): 961-6, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26471301

RESUMO

Bacterial topoisomerase IV (ParE) is essential for DNA replication and serves as an attractive target for antibacterial drug development. The X-ray structure of the N-terminal 24 kDa ParE, responsible for ATP binding has been solved. Due to the accessibility of structural information of ParE, many potent ParE inhibitors have been discovered. In this study, a pyridylurea lead molecule against ParE of Escherichia coli (eParE) was characterized with a series of biochemical and biophysical techniques. More importantly, solution NMR analysis of compound binding to eParE provides better understanding of the molecular interactions between the inhibitor and eParE.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Topoisomerase IV/metabolismo , DNA Topoisomerase IV/farmacologia , Escherichia coli/enzimologia , Trifosfato de Adenosina/antagonistas & inibidores , Sequência de Aminoácidos , Antibacterianos/farmacologia , Ligação Competitiva , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/química , Desenho de Fármacos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular
9.
FEBS Lett ; 589(19 Pt B): 2683-9, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26272827

RESUMO

The N-terminal ATP binding domain of the DNA gyrase B subunit is a validated drug target for antibacterial drug discovery. Structural information for this domain (pGyrB) from Pseudomonas aeruginosa is still missing. In this study, the interaction between pGyrB and a bis-pyridylurea inhibitor was characterized using several biophysical methods. We further carried out structural analysis of pGyrB using NMR spectroscopy. The secondary structures of free and inhibitor bound pGyrB were obtained based on backbone chemical shift assignment. Chemical shift perturbation and NOE experiments demonstrated that the inhibitor binds to the ATP binding pocket. The results of this study will be helpful for drug development targeting P. aeruginosa.


Assuntos
Domínio Catalítico , DNA Girase/química , DNA Girase/metabolismo , Pseudomonas aeruginosa/enzimologia , Inibidores da Topoisomerase II/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Ureia/química , Ureia/metabolismo , Ureia/farmacologia
10.
PLoS One ; 9(8): e105222, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25141257

RESUMO

PA-824 is a bicyclic 4-nitroimidazole, currently in phase II clinical trials for the treatment of tuberculosis. Dose fractionation pharmacokinetic-pharmacodynamic studies in mice indicated that the driver of PA-824 in vivo efficacy is the time during which the free drug concentrations in plasma are above the MIC (fT>MIC). In this study, a panel of closely related potent bicyclic 4-nitroimidazoles was profiled in both in vivo PK and efficacy studies. In an established murine TB model, the efficacy of diverse nitroimidazole analogs ranged between 0.5 and 2.3 log CFU reduction compared to untreated controls. Further, a retrospective analysis was performed for a set of seven nitroimidazole analogs to identify the PK parameters that correlate with in vivo efficacy. Our findings show that the in vivo efficacy of bicyclic 4-nitroimidazoles correlated better with lung PK than with plasma PK. Further, nitroimidazole analogs with moderate-to-high volume of distribution and Lung to plasma ratios of >2 showed good efficacy. Among all the PK-PD indices, total lung T>MIC correlated the best with in vivo efficacy (rs = 0.88) followed by lung Cmax/MIC and AUC/MIC. Thus, lung drug distribution studies could potentially be exploited to guide the selection of compounds for efficacy studies, thereby accelerating the drug discovery efforts in finding new nitroimidazole analogs.


Assuntos
Nitroimidazóis/farmacologia , Nitroimidazóis/farmacocinética , Tuberculose/tratamento farmacológico , Animais , Células CACO-2 , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/efeitos dos fármacos , Estudos Retrospectivos
12.
ACS Chem Biol ; 7(7): 1190-7, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22500615

RESUMO

Growing evidence suggests that the presence of a subpopulation of hypoxic non-replicating, phenotypically drug-tolerant mycobacteria is responsible for the prolonged duration of tuberculosis treatment. The discovery of new antitubercular agents active against this subpopulation may help in developing new strategies to shorten the time of tuberculosis therapy. Recently, the maintenance of a low level of bacterial respiration was shown to be a point of metabolic vulnerability in Mycobacterium tuberculosis. Here, we describe the development of a hypoxic model to identify compounds targeting mycobacterial respiratory functions and ATP homeostasis in whole mycobacteria. The model was adapted to 1,536-well plate format and successfully used to screen over 600,000 compounds. Approximately 800 compounds were confirmed to reduce intracellular ATP levels in a dose-dependent manner in Mycobacterium bovis BCG. One hundred and forty non-cytotoxic compounds with activity against hypoxic non-replicating M. tuberculosis were further validated. The resulting collection of compounds that disrupt ATP homeostasis in M. tuberculosis represents a valuable resource to decipher the biology of persistent mycobacteria.


Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Antituberculosos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Homeostase/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Trifosfato de Adenosina/fisiologia , Animais , Antituberculosos/química , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Cricetinae , Cricetulus , Células HeLa , Homeostase/fisiologia , Humanos , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/crescimento & desenvolvimento
13.
Structure ; 20(1): 101-12, 2012 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-22244759

RESUMO

Tuberculosis continues to be a global health threat, making bicyclic nitroimidazoles an important new class of therapeutics. A deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis catalyzes the reduction of nitroimidazoles such as PA-824, resulting in intracellular release of lethal reactive nitrogen species. The N-terminal 30 residues of Ddn are functionally important but are flexible or access multiple conformations, preventing structural characterization of the full-length, enzymatically active enzyme. Several structures were determined of a truncated, inactive Ddn protein core with and without bound F(420) deazaflavin coenzyme as well as of a catalytically competent homolog from Nocardia farcinica. Mutagenesis studies based on these structures identified residues important for binding of F(420) and PA-824. The proposed orientation of the tail of PA-824 toward the N terminus of Ddn is consistent with current structure-activity relationship data.


Assuntos
Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Nitrorredutases/química , Nitrorredutases/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Flavinas/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese , Nitroimidazóis/metabolismo , Nitrorredutases/genética , Ligação Proteica , Espécies Reativas de Nitrogênio/metabolismo
14.
FEBS J ; 279(1): 113-25, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22023140

RESUMO

The bicyclic 4-nitroimidazoles PA-824 and OPC-67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro-drugs that are reductively activated by a deazaflavin (F(420)) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA-824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo-oxazoles bearing only short alkyl substituents at the C-7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA-824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA-824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover-independent assessment of affinity. Our results indicate that (R)-PA-824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C-3 of the imidazole ring orienting in a non-productive position with respect to the incoming hydride from F(420). The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.


Assuntos
Antituberculosos/farmacologia , Flavinas/metabolismo , Mycobacterium tuberculosis/enzimologia , Nitroimidazóis/farmacologia , Nitrorredutases/metabolismo , Oxazóis/farmacologia , Clonagem Molecular , Cinética , Óxido Nítrico/metabolismo , Nitrorredutases/genética , Nitrorredutases/isolamento & purificação , Conformação Proteica , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por Substrato
15.
J Med Chem ; 54(16): 5639-59, 2011 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-21755942

RESUMO

The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 µM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.


Assuntos
Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Nitroimidazóis/farmacologia , Nitrorredutases/metabolismo , Pró-Fármacos/farmacologia , Animais , Antituberculosos/química , Antituberculosos/farmacocinética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Cinética , Taxa de Depuração Metabólica , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Nitroimidazóis/química , Nitroimidazóis/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Tuberculose/tratamento farmacológico , Tuberculose/microbiologia
16.
Cell Host Microbe ; 3(3): 137-45, 2008 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-18329613

RESUMO

Antibiotics are typically more effective against replicating rather than nonreplicating bacteria. However, a major need in global health is to eradicate persistent or nonreplicating subpopulations of bacteria such as Mycobacterium tuberculosis (Mtb). Hence, identifying chemical inhibitors that selectively kill bacteria that are not replicating is of practical importance. To address this, we screened for inhibitors of dihydrolipoamide acyltransferase (DlaT), an enzyme required by Mtb to cause tuberculosis in guinea pigs and used by the bacterium to resist nitric oxide-derived reactive nitrogen intermediates, a stress encountered in the host. Chemical screening for inhibitors of Mtb DlaT identified select rhodanines as compounds that almost exclusively kill nonreplicating mycobacteria in synergy with products of host immunity, such as nitric oxide and hypoxia, and are effective on bacteria within macrophages, a cellular reservoir for latent Mtb. Compounds that kill nonreplicating pathogens in cooperation with host immunity could complement the conventional chemotherapy of infectious disease.


Assuntos
Aciltransferases/antagonistas & inibidores , Antituberculosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Rodanina/farmacologia , Aciltransferases/genética , Animais , Proteínas de Bactérias/genética , Células Cultivadas , Contagem de Colônia Microbiana , Inibidores Enzimáticos/farmacologia , Deleção de Genes , Teste de Complementação Genética , Cobaias , Hipóxia/imunologia , Pulmão/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Estrutura Molecular , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Óxido Nítrico/imunologia , Rodanina/química , Rodanina/toxicidade , Tuberculose/imunologia , Tuberculose/microbiologia , Virulência , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/genética
17.
Neurol India ; 55(2): 106-10, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17558112

RESUMO

OBJECTIVE: To derive population norms on the Malayalam adaptation of Addenbrooke's Cognitive Examination (M-ACE) and the inclusive Malayalam mini mental state examination (M-MMSE). MATERIALS AND METHODS: Education-stratified norms were obtained on randomly selected cognitively unimpaired community elders (n = 519). RESULTS: Valid data on norms was available on 488 subjects (age 68.5 +/- 7.1 and education 7.9 +/- 5.4). Education and age, but not gender had a significant effect on both M-ACE and M-MMSE. When compared to the effect of age, the effect of education was sevenfold more on the M-ACE and ninefold more on the M-MMSE. The mean composite score on the M-ACE (and the M-MMSE) was 42.8 +/- 9.8 (14.9 +/- 3.1) for those with 0 (n = 72), 55.9 +/- 12.5 (19.7 +/- 4.1) with 1-4 (n = 96), 62.6 +/- 11.4 (21.9 +/- 3.7) with 5-8 (n = 81), 77 +/- 10.2 (25.7 +/- 2.4) with 9-12 (n = 136) and 83.4 +/- 7.2 (26.7 +/- 1.6) with > 12 (n = 103) years of formal education. CONCLUSIONS: Education has the most potent effect on performance on both M-ACE and M-MMSE in the Indian cohort. Education-stratified scores on the M-ACE and the M-MMSE, will provide a more appropriate means of establishing the cognitive status of patients. It is also our feeling that these cut-off scores will be useful across India.


Assuntos
Cognição/fisiologia , Cultura , Educação , Testes Neuropsicológicos/normas , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Transtornos Cognitivos/diagnóstico , Transtornos Cognitivos/epidemiologia , Transtornos Cognitivos/psicologia , Feminino , Humanos , Índia/epidemiologia , Masculino , Pessoa de Meia-Idade , População , Padrões de Referência , Fatores Sexuais
18.
Nat Chem Biol ; 2(11): 618-26, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17028580

RESUMO

Cell-permeable small molecules that inhibit their targets on fast timescales are powerful probes of cell-division mechanisms. Such inhibitors have been identified using phenotype-based screens with chemical libraries. However, the characteristics of compound libraries needed to effectively span cell-division phenotype space, to find probes that target different mechanisms, are not known. Here we show that a small collection of 100 diaminopyrimidines (DAPs) yields a range of cell-division phenotypes, including changes in spindle geometry, chromosome positioning and mitotic index. Monopolar mitotic spindles are induced by four inhibitors, including one that targets Polo-like kinases (Plks), evolutionarily conserved serine/threonine kinases. Using chemical inhibitors and high-resolution live-cell microscopy, we found that Plk activity is needed for the assembly and maintenance of bipolar mitotic spindles. Plk inhibition destabilizes kinetochore microtubules while stabilizing other spindle microtubules, leading to monopolar spindles. Further testing of compounds based on 'privileged scaffolds', such as the DAP scaffold, could lead to new cell-division probes and antimitotic agents.


Assuntos
Benzotiazóis/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/fisiologia , Fenilenodiaminas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Pirimidinas/farmacologia , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/fisiologia , Animais , Benzotiazóis/química , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Haplorrinos , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Fenótipo , Fenilenodiaminas/química , Inibidores de Proteínas Quinases/química , Pirimidinas/química , Interferência de RNA , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Sensibilidade e Especificidade , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/genética , Fuso Acromático/metabolismo , Relação Estrutura-Atividade , Fatores de Tempo , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Xenopus laevis/metabolismo
19.
Mol Microbiol ; 60(5): 1109-22, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16689789

RESUMO

Growth of bacteria and fungi on fatty acid substrates requires the catabolic beta-oxidation cycle and the anaplerotic glyoxylate cycle. Propionyl-CoA generated by beta-oxidation of odd-chain fatty acids is metabolized via the methylcitrate cycle. Mycobacterium tuberculosis possesses homologues of methylcitrate synthase (MCS) and methylcitrate dehydratase (MCD) but not 2-methylisocitrate lyase (MCL). Although MCLs share limited homology with isocitrate lyases (ICLs) of the glyoxylate cycle, these enzymes are thought to be functionally non-overlapping. Previously we reported that the M. tuberculosis ICL isoforms 1 and 2 are jointly required for growth on fatty acids, in macrophages, and in mice. ICL-deficient bacteria could not grow on propionate, suggesting that in M. tuberculosis ICL1 and ICL2 might function as ICLs in the glyoxylate cycle and as MCLs in the methylcitrate cycle. Here we provide biochemical and genetic evidence supporting this interpretation. The role of the methylcitrate cycle in M. tuberculosis metabolism was further evaluated by constructing a mutant strain in which prpC (encoding MCS) and prpD (encoding MCD) were deleted. The DeltaprpDC strain could not grow on propionate media in vitro or in murine bone marrow-derived macrophages infected ex vivo; growth under these conditions was restored by complementation with a plasmid containing prpDC. Paradoxically, bacterial growth and persistence, and tissue pathology, were indistinguishable in mice infected with wild-type or DeltaprpDC bacteria.


Assuntos
Carbono-Carbono Liases/metabolismo , Citrato (si)-Sintase/metabolismo , Ácido Cítrico , Isocitrato Liase/metabolismo , Isoenzimas/metabolismo , Mycobacterium tuberculosis/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Carbono-Carbono Liases/classificação , Carbono-Carbono Liases/genética , Células Cultivadas , Citrato (si)-Sintase/genética , Ácido Cítrico/química , Ácido Cítrico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Hidroliases/genética , Hidroliases/metabolismo , Isocitrato Liase/classificação , Isocitrato Liase/genética , Isoenzimas/classificação , Isoenzimas/genética , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Estrutura Molecular , Mycobacterium tuberculosis/genética , Filogenia , Propionatos/metabolismo
20.
J Clin Rheumatol ; 11(1): 56-8, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16357698

RESUMO

In patients at risk for pulmonary emboli, consideration is often given to placement of an inferior vena cava (IVC) filter to prevent propagation of a distal thrombus. However, long-term benefits remain controversial, and deep venous thrombosis and IVC thrombosis may result from the procedure itself. Whether a filter if beneficial or even detrimental in patients with the antiphospholipid syndrome (APS) is unclear. We reviewed clinical outcomes in 2 patients who had IVC filter placement years before the diagnosis of the APS and 1 who had a contraindication to anticoagulation. Recurrent pulmonary emboli were seen despite the presence of the filter. IVC pathology sometimes revealed thrombus both proximal and distal to the IVC filter. Pulmonary emboli in the APS may be secondary to deep venous thrombosis (DVT). They may also occur secondary to a cardiac source or in situ thrombosis in the pulmonary vessels. An IVC filter will not be of benefit if the heart or the lungs are the primary source for the emboli. It may also not protect against propagation of a more distal thrombus if collateral vessels develop around the filter or a thrombus is present on the proximal side of the filter. Recurrence of pulmonary emboli after a filter placement should alert the clinician to the possibility of a hypercoagulable state such as APS. Clinicians need to assess risks and benefits carefully before placing a permanent IVC filter in patients with APS. Whether a temporary or retrievable filter is safer in APS and more effective is unknown at the present time.


Assuntos
Síndrome Antifosfolipídica/complicações , Embolia Pulmonar/etiologia , Filtros de Veia Cava , Adulto , Anticoagulantes/uso terapêutico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Embolia Pulmonar/prevenção & controle , Recidiva , Trombose Venosa/diagnóstico , Trombose Venosa/etiologia , Trombose Venosa/prevenção & controle
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