RESUMO
The mTOR kinase regulates a variety of critical cellular processes and has become a target for the treatment of various cancers. Using a combination of property-based drug design and Free-Wilson analysis, we further optimized a series of selective mTOR inhibitors based on the (S)-6a-methyl-6a,7,9,10-tetrahydro[1,4]oxazino[3,4-h]pteridin-6(5H)-one scaffold. Our efforts resulted in 14c, which showed similar in vivo efficacy compared to previous lead 1 at 1/15 the dose, a result of its improved drug-like properties.
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Target-engagement pharmacodynamic (PD) biomarkers are valuable tools in the prioritization of drug candidates, especially for novel, first-in-class mechanisms whose robustness to alter disease outcome is unknown. Methionine aminopeptidase 2 (MetAP2) is a cytosolic metalloenzyme that cleaves the N-terminal methionine from nascent proteins. Inhibition of MetAP2 leads to weight loss in obese rodents, dogs and humans. However, there is a need to develop efficacious compounds that specifically inhibit MetAP2 with an improved safety profile. The objective of this study was to identify a PD biomarker for selecting potent, efficacious compounds and for predicting clinical efficacy that would result from inhibition of MetAP2. Here we report the use of NMet14-3-3γ for this purpose. Treatment of primary human cells with MetAP2 inhibitors resulted in an approx. 10-fold increase in NMet14-3-3γ levels. Furthermore, treatment of diet-induced obese mice with these compounds reduced body weight (approx. 20%) and increased NMet14-3-3γ (approx. 15-fold) in adipose tissues. The effects on target engagement and body weight increased over time and were dependent on dose and administration frequency of compound. The relationship between compound concentration in plasma, NMet14-3-3γ in tissue, and reduction of body weight in obese mice was used to generate a pharmacokinetic-pharmacodynamic-efficacy model for predicting efficacy of MetAP2 inhibitors in mice. We also developed a model for predicting weight loss in humans using a target engagement PD assay that measures inhibitor-bound MetAP2 in blood. In summary, MetAP2 target engagement biomarkers can be used to select efficacious compounds and predict weight loss in humans. SIGNIFICANCE STATEMENT: The application of target engagement pharmacodynamic biomarkers during drug development provides a means to determine the dose required to fully engage the intended target and an approach to connect the drug target to physiological effects. This work exemplifies the process of using target engagement biomarkers during preclinical research to select new drug candidates and predict clinical efficacy. We determine concentration of MetAP2 antiobesity compounds needed to produce pharmacological activity in primary human cells and in target tissues from an appropriate animal model and establish key relationships between pharmacokinetics, pharmacodynamics, and efficacy, including the duration of effects after drug administration. The biomarkers described here can aid decision-making in early clinical trials of MetAP2 inhibitors for the treatment of obesity.
Assuntos
Clorobenzenos/farmacologia , Cinamatos/farmacologia , Cicloexanos/farmacologia , Compostos de Epóxi/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Metionil Aminopeptidases/antagonistas & inibidores , Metionil Aminopeptidases/metabolismo , Sesquiterpenos/farmacologia , Inibidores da Angiogênese/química , Inibidores da Angiogênese/farmacologia , Animais , Biomarcadores/metabolismo , Clorobenzenos/química , Cinamatos/química , Cicloexanos/química , Relação Dose-Resposta a Droga , Compostos de Epóxi/química , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Valor Preditivo dos Testes , Sesquiterpenos/química , Resultado do TratamentoRESUMO
[This corrects the article DOI: 10.1021/acsmedchemlett.6b00481.].
RESUMO
Guided by co-crystal structural information obtained from a different series we were exploring, a scaffold morphing and SBDD approach led to the discovery of the 1,4-disubstituted indazole series as a novel class of GKAs that potently activate GK in enzyme and cell assays. anti-diabetic OGTT efficacy was demonstrated with 29 in a rodent models of type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Descoberta de Drogas , Ativadores de Enzimas/farmacologia , Glucoquinase/metabolismo , Indazóis/farmacologia , Administração Oral , Regulação Alostérica/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Ativadores de Enzimas/administração & dosagem , Ativadores de Enzimas/química , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Canais de Potássio Éter-A-Go-Go/metabolismo , Teste de Tolerância a Glucose , Humanos , Indazóis/administração & dosagem , Indazóis/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Modelos Moleculares , Estrutura Molecular , Bloqueadores dos Canais de Potássio/farmacologia , Relação Estrutura-AtividadeRESUMO
Apoptosis signal-regulating kinase 1 (ASK1/MAP3K) is a mitogen-activated protein kinase family member shown to contribute to acute ischemia/reperfusion injury. Using structure-based drug design, deconstruction, and reoptimization of a known ASK1 inhibitor, a lead compound was identified. This compound displayed robust MAP3K pathway inhibition and reduction of infarct size in an isolated perfused heart model of cardiac injury.
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Since the 1990s, Tanzania has been implementing health sector reforms including decentralization of primary healthcare services to districts and users. The impact of the reforms on the access, quality and appropriateness of primary healthcare services from the viewpoint of users is, however, not clearly documented. This article draws on a gendered users' perspective to address the question of whether the delivery of gender-sensitive primary health services has improved after the reforms. The article is based on empirical data collected through a household survey, interviews, focus group discussions, case studies and analysis of secondary data in two rural districts in Tanzania. The analysis shows that the reforms have generated mixed effects: they have contributed to improving the availability of health facilities in some villages but have also reinforced inter-village inequalities. Men and women hold similar views on the perceived changes and appropriateness to women on a number of services. Gender inequalities are, however, reflected in the significantly low membership of female-headed households in the community health fund and their inability to pay the user fees and in the fact that women's reproductive and maternal health needs are as yet insufficiently addressed. Although over half of users are satisfied with the services, more women than men are dissatisfied. The reforms appear to have put much emphasis on building health infrastructure and less on quality issues as perceived by users.
Assuntos
Política , Atenção Primária à Saúde/organização & administração , Serviços de Saúde Rural/organização & administração , Atitude Frente a Saúde , Custo Compartilhado de Seguro , Honorários Médicos , Feminino , Reforma dos Serviços de Saúde/organização & administração , Acessibilidade aos Serviços de Saúde/organização & administração , Humanos , Entrevistas como Assunto , Masculino , Satisfação do Paciente , Atenção Primária à Saúde/economia , Atenção Primária à Saúde/normas , Qualidade da Assistência à Saúde/organização & administração , Serviços de Saúde Rural/economia , Serviços de Saúde Rural/normas , Fatores Sexuais , Inquéritos e Questionários , TanzâniaRESUMO
Guided by co-crystal structures of compounds 15, 22 and 30, an SBDD approach led to the discovery of the 6-methyl pyridone series as a novel class of GKAs that potently activate GK in enzyme and cell assays. Anti-diabetic OGTT efficacy was demonstrated with 54 in a mouse model of type 2 diabetes.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Desenho de Fármacos , Ativadores de Enzimas/farmacologia , Glucoquinase/metabolismo , Hipoglicemiantes/farmacologia , Piridonas/farmacologia , Animais , Cristalografia por Raios X , Diabetes Mellitus Experimental/tratamento farmacológico , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Ativadores de Enzimas/administração & dosagem , Ativadores de Enzimas/química , Teste de Tolerância a Glucose , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Modelos Moleculares , Estrutura Molecular , Piridonas/administração & dosagem , Piridonas/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND AND AIM: Acute hepatitis B course may be significantly modified by underlying chronic hepatitis C. The aim of this study was to compare clinical and virological characteristics of acute hepatitis B in patients with or without chronic hepatitis C virus (HCV) infection. MATERIALS AND METHODS: Twenty-seven patients with symptomatic acute hepatitis B were enrolled: 14 with underlying chronic HCV (Group A) and 13, matched by age and gender, with single hepatitis B (Group B). All patients were followed-up until HBsAg negativization. RESULTS: Group A patients were HCV-RNA-negative on hospital admission and all but one remained negative during follow-up. HBeAg tested positive in 92.9% and 84.6% of Groups A and B patients, respectively. ALT, bilirubin, prothrombin time values and HBsAg titer were similar in both groups. Nevertheless, lower mean HBV-DNA levels (p=0.03), a shorter duration of HBsAg positivity (p<0.01) and of symptoms before ALT peak (p=0.014), and significantly lower peak ALT values (p=0.03) were observed in Group A compared to Group B patients. CONCLUSIONS: Acute HBV infection suppressed HCV replication. Conversely, the underlying HCV infection exerted a modulatory effect on HBV replication which influenced the course, though not the outcome, of the acute disease. Although acute hepatitis B showed a mild clinical course in both groups of patients, HBV vaccination should be suggested to risk subjects.
Assuntos
Anticorpos Anti-Hepatite B/imunologia , Hepatite B/imunologia , Anticorpos Anti-Hepatite C/imunologia , Hepatite C Crônica/imunologia , RNA Viral/análise , Superinfecção/virologia , Replicação Viral , Doença Aguda , DNA Viral/imunologia , Feminino , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite B/complicações , Hepatite B/epidemiologia , Antígenos de Superfície da Hepatite B/sangue , Anticorpos Anti-Hepatite C/sangue , Hepatite C Crônica/complicações , Hepatite C Crônica/epidemiologia , Humanos , Masculino , RNA Viral/imunologia , Estudos Soroepidemiológicos , Superinfecção/epidemiologiaRESUMO
UNLABELLED: Hepatitis C virus (HCV) infection is associated with a significant reduction of health related quality of life (QOL), the causes and mechanisms of which are still unknown. To explore whether treatment history could affect QOL, we examined patients with detectable HCV viraemia who had a different therapeutic background. Two hundred sixty-four consecutive subjects with chronic HCV infection and detectable viraemia were enrolled. Of these, 163 were untreated patients, 43 were relapsers, 58 were nonresponders (NR) to nonpegylated interferon (IFN) therapy. To assess QOL, three self-report instruments were employed: the Short Form-36 (SF-36), the Chronic Liver Disease Questionnaire (CLDQ-I) and the World Health Organization Quality of Life assessment (WHOQOL-BREF). Clinical and demographic data were collected, and the QOL scores of HCV-positive patients were compared with those of an Italian normative sample and healthy controls. Further antiviral treatment was offered to untreated and relapsed patients but not to NR. All patient groups displayed lower QOL scores compared with the normative sample and controls. NR displayed lower QOL scores in several areas compared with untreated patients and relapsers. In multivariate regression analyses, being NR and having a physical comorbidity were significantly associated with poorer QOL. CONCLUSIONS: Treatment history and expectations and physical comorbidity may affect QOL in HCV-positive patients. Untreated and relapsed patients have comparable levels of QOL and higher scores than NR.
Assuntos
Hepatite C Crônica/tratamento farmacológico , Interferons/uso terapêutico , Qualidade de Vida , Adulto , Idoso , Comorbidade , Feminino , Nível de Saúde , Hepatite C Crônica/virologia , Humanos , Modelos Lineares , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
Diethyldithiodicarbonate (DDD), a cheap and easily prepared compound, is found to be a rapid and efficient sulfurizing reagent in solid phase synthesis of phosphorothioate oligodeoxyribonucleotides via the phosphoramidite approach. Product yield and quality based on IP-LC-MS compares well with high quality oligonucleotides synthesized using phenylacetyl disulfide (PADS) which is being used for manufacture of our antisense drugs.
Assuntos
Carbonatos/química , Oligonucleotídeos/síntese química , Sulfetos/química , Enxofre/química , Tionucleotídeos/síntese química , Indicadores e Reagentes , Oligonucleotídeos/química , Tionucleotídeos/químicaRESUMO
A new reagent immobilized on solid support allowing for solid-phase synthesis of oligonucleotides with a 3'-terminal phosphorothioate monoester is described. The support is compatible with phosphoramidite chemistry for automated oligonucleotide synthesis. Final deprotection with ammonia under standard conditions leads to oligonucleotide 3'-terminal phosphorothioate.
Assuntos
Oligonucleotídeos/síntese química , Compostos Organotiofosforados/síntese química , Amônia/química , Eletroforese Capilar , Indicadores e Reagentes , Espectroscopia de Ressonância MagnéticaRESUMO
Rat and porcine galanin and their fragments inhibited cholecystokinin-8 (CCK-8)-stimulated amylase secretion with the following activities: rat galanin-(1-29) = porcine galanin-(1-29) = galanin-(1-15) = rat galanin-(3-29) > rat galanin-(2-29) = porcine galanin-(2-29) > galanin-(1-10). Fragments of rat galanin-(9-29) and N alpha-acetyl-galanin-(9-29) were able to inhibit CCK-8-stimulated pancreatic amylase secretion but only at higher dose levels. Porcine galanin-(15-29) and rat galanin-(21-29) were unable to produce significant inhibition. Rat and porcine galanin-(1-29), galanin-(1-15) and rat N alpha-acetyl-galanin-(9-29) also inhibited basal pancreatic amylase secretion. In the rat jejunal strip contraction model, rat galanin-(1-29) and porcine galanin-(1-29) have similar potencies. Galanin-(1-15) and galanin-(1-10) stimulate rat jejunal strip contraction with decreasing potencies. Elimination of Gly1 from the N-terminus of both rat and porcine galanin had no significant effect either on pancreatic amylase secretion or on jejunal strip contraction. The rat galanin-(3-29) and (9-29) are not active in the stimulation of rat jejunal strip contraction. Acetylation of porcine galanin-(9-29) created a peptide that was a powerful stimulator of rat jejunal strip contraction. The present data indicate that N-terminal rat galanin amino acid residues are crucial for rat jejunal strip contraction but are not required for inhibition of pancreatic amylase. These results suggest that the galanin amino acid sequence contains several specific domains, which can be recognized by specific galanin receptor subsets.
Assuntos
Amilases/metabolismo , Músculo Liso/efeitos dos fármacos , Neuropeptídeos/farmacologia , Pâncreas/enzimologia , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Galanina , Humanos , Técnicas In Vitro , Infusões Intravenosas , Jejuno/efeitos dos fármacos , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Peptídeos/química , Ratos , Sincalida/farmacologia , Relação Estrutura-AtividadeRESUMO
Gastric inhibitory polypeptide (GIP) strongly stimulates insulin secretion in the presence of glucose and also stimulates somatostatin release from gastric mucosa. It was reported recently that both stimulatory activities can be dissociated by removing the C-terminal 12 amino acid residues. Since insulin and somatostatin are involved in regulation of exocrine pancreatic and gastric secretion in rats, we compared the inhibitory effects of pGIP and the pGIP(1-30)NH2 fragment on pancreatic amylase and gastric acid secretion. pGIP(1-30)NH2 displayed full activity on inhibition of bombesin (BN)-stimulated amylase release relative to GIP itself, but was about 10-fold less potent in inhibiting gastric acid secretion. These results suggest that the receptors involved in these two events have quite different ligand binding requirements and that more specific analogues of GIP can be designed which should be of value in elucidating the physiological roles of this hormone.
Assuntos
Amilases/metabolismo , Ácido Gástrico/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Pâncreas/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Animais , Bombesina/farmacologia , Depressão Química , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Masculino , Pâncreas/enzimologia , Perfusão , RatosRESUMO
Following the recent discovery of a new substance P (SP) competitive pancreatic acini cell receptor antagonist containing a reduced peptide bond in place of the C-terminal peptide bond, a new series of full chain and short chain (heptapeptide and hexapeptide) substance P analogues have been prepared in which one of the C-terminal-region peptide bonds has been replaced by CH2NH or CH2O groups. They were compared for their ability to recognize NK1 and/or NK2 tachykinin receptor binding sites on guinea pig ileum and rat duodenum smooth muscle preparations, respectively. It was found that all full sequence SP pseudopeptides were agonists with much reduced bioactivity in both tested systems and, in addition, [Gly9 psi(CH2NH)Leu10,Leu11]SP was found to be a relatively selective agonist for NK1 binding sites. Substitution of leucine at position 11 of SP heptapseudopeptides with phenylalanine generated a pseudopeptide with weak agonist activity when Gln at position 5 was replaced by D-Phe, or antagonists when this residue was replaced by D-Nal or D-Cpa. [Leu10 psi(CH2NH)Leu11]SP-(6-11) with Gln at position 6 substituted by D-Phe was a relatively stronger antagonist in both assay systems. These results suggest that, as with several other peptide systems of late, manipulation of the peptide bonds in SP can produce receptor antagonists which in some cases approach the potency of the classic spantide series and, furthermore, that the approach might be used to induce NK receptor specificity in both agonist and antagonist analogs.
Assuntos
Músculo Liso/efeitos dos fármacos , Substância P/análogos & derivados , Substância P/farmacologia , Sequência de Aminoácidos , Animais , Cobaias , Técnicas In Vitro , Isomerismo , Masculino , Dados de Sequência Molecular , Contração Muscular/efeitos dos fármacos , Ratos , Receptores de Neurotransmissores/antagonistas & inibidores , Receptores de Taquicininas , Substância P/antagonistas & inibidoresRESUMO
The potent inhibitory effect of galanin on basal and pentagastrin-stimulated gastric acid secretion in vivo, and the presence of galanin-containing nerves in gastrointestinal tract and pancreas, suggested that this peptide may regulate the exocrine secretion of the GI system. Male rats were anesthetized with pentobarbital and the dose-dependent inhibitory effects of galanin on basal and stimulated pancreatic protein and amylase secretions were investigated in separate experiments. Galanin was administered intravenously in the following doses: 3, 6, 10, 15 and 20 micrograms/kg/h (0.93, 1.86, 3.1, 4.65 and 6.2 nmol/kg/h), and pancreatic secretions measured. The maximal effective dose of galanin (3.1 nmol/kg/h) on basal pancreatic secretions was found, and was used for evaluating the inhibitory effect of galanin on pancreatic protein and amylase secretions stimulated by bombesin, secretin and cholecystokinin. Galanin potently inhibited basal, bombesin-, secretin- and cholecystokinin-stimulated pancreatic protein and amylase secretion. Inhibitory effect of galanin was dose-dependent and biphasic.
Assuntos
Amilases/antagonistas & inibidores , Neuropeptídeos/farmacologia , Pâncreas/enzimologia , Peptídeos/farmacologia , Amilases/metabolismo , Animais , Bombesina/farmacologia , Relação Dose-Resposta a Droga , Galanina , Cinética , Masculino , Pâncreas/metabolismo , Pentobarbital/farmacologia , Ratos , Secretina/farmacologia , Sincalida/farmacologiaRESUMO
Receptors for galanin in membranes from the rat gastric and jejunal smooth muscle were studied using [125I] radioiodinated synthetic porcine galanin. Specific binding was time and temperature dependent. At 32 degrees C radioligand was degraded in the presence of smooth muscle membranes in a time-dependent manner. At optimal experimental conditions, the equilibrium binding analyses showed the presence of a single population of high affinity binding sites in both the rat stomach and jejunum (Kd value of 2.77 +/- 0.78 nM and 4.93 +/- 1.74 nM for stomach and jejunal smooth muscle membranes, respectively). The concentration of the high affinity binding sites was 58.19 +/- 11.04 and 32.36 +/- 5.68 fmol/mg protein, for gastric and jejunal preparations, respectively. Specific binding was completely inhibited by 10(-6) M of nonradioactive galanin; was 75% blocked by 1 microM of galanin(9-29); it was 10% blocked by 1 microM of galanin(15-29). Galanin(1-15) at a concentration of 1 microM was ineffective for inhibiting [125I]galanin binding. Deletion of four C-terminal amino acid residues from galanin(9-29) to give galanin(9-25) also resulted in almost complete loss of affinity. Radioiodinated galanin and N-terminally deleted fragments had receptor binding potency in the following order: galanin(1-29) greater than galanin(9-29) greater than galanin(15-29). We conclude that the C-terminal part of the galanin chain is important for the rat gastric and jejunal smooth muscle membrane receptor recognition and binding and that N-terminal amino acid sequences are probably not so important, since galanin(1-15) was not active but galanin(9-29) retained most of the receptor binding activity.