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Asma , Humanos , Niño , Asma/epidemiología , Asma/terapia , Instituciones de Atención AmbulatoriaRESUMEN
OBJECTIVE: Caregiver depressive symptoms are prevalent among children with asthma and associated with greater asthma morbidity. Identifying caregivers with depression and connecting them to appropriate treatment may reduce child asthma morbidity. The goal of this project was to implement a workflow for caregiver depression screening and treatment referral in an urban, community-based, asthma clinic serving under-resourced children. METHODS: The Model for Improvement with weekly Plan-Do-Study-Act cycles was utilized. A two-item depression screening tool (Patient Health Questionnaire-2; PHQ-2) and an acceptability question using a 5-point Likert scale were added to an existing social needs screening checklist administered to all caregivers during the child's clinic visit. Caregivers with a positive PHQ-2 score (≥3) received the PHQ-9. Positive screens on the PHQ-9 (≥5) received information and referrals by level of risk. PHQ-9 positive caregivers received a follow-up phone call two weeks post-visit to assess connection to support, improvement in depressive symptoms, and satisfaction with resources provided. RESULTS: The PHQ-2 was completed by 84.4% of caregivers (233/276). Caregivers had a mean age of 33.8 years (SD = 8.3; Range: 18-68) and were predominately female (86.4%), Black (80.4%), and non-Hispanic (78.4%). The majority (72.3%) found the screening acceptable (agree/strongly agree). Nearly one in six caregivers (37/233, 15.9%) reported depressive symptoms (PHQ-2 ≥ 3); 11.6% (27/233) had clinically significant symptoms (PHQ-9 score ≥ 10); and 2.1% (5/233) reported suicidal thoughts. Of those with depressive symptoms, 70.3% (26/37) participated in the follow-up phone call. While 50% (13/26) reported the resources given in clinic were "extremely helpful," no caregivers contacted or used them. CONCLUSIONS: Caregiver depression screening was successfully integrated into a pediatric asthma clinic serving under-resourced children. While caregivers found screening to be acceptable, it did not facilitate short-term connection to treatment among those with depressive symptoms.
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Asma , Humanos , Niño , Femenino , Adulto , Asma/diagnóstico , Asma/terapia , Depresión/diagnóstico , Depresión/epidemiología , Mejoramiento de la Calidad , Cuidadores , Instituciones de Atención AmbulatoriaRESUMEN
Human cancers require fatty acid synthase (FASN)-dependent de novo long-chain fatty acid synthesis for proliferation. FASN is therefore an attractive drug target, but fast technologies for reliable label-free cellular compound profiling are lacking. Recently, MALDI-mass spectrometry (MALDI-MS) has emerged as an effective technology for discovery of recombinant protein target inhibitors. Here we present an automated, mechanistic MALDI-MS cell assay, which monitors accumulation of the FASN substrate, malonyl-coenzyme A (CoA), in whole cells with limited sample preparation. Profiling of inhibitors, including unpublished compounds, identified compound 1 as the most potent FASN inhibitor (1 nM in A549 cells) discovered to date. Moreover, cellular MALDI-MS assays enable parallel profiling of additional pathway metabolites. Surprisingly, several compounds triggered cytidine 5'-diphosphocholine (CDP-choline) but not malonyl-CoA accumulation indicating that they inhibit diacylglycerol generation but not FASN activity. Taken together, our study suggests that MALDI-MS cell assays may become important tools in drug profiling that provide additional mechanistic insights concerning compound action on metabolic pathways.
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Ácido Graso Sintasas/antagonistas & inhibidores , Ácido Graso Sintasas/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Células A549 , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Acido Graso Sintasa Tipo I/antagonistas & inhibidores , Acido Graso Sintasa Tipo I/metabolismo , Humanos , Concentración 50 Inhibidora , Células K562 , Lipogénesis , Malonil Coenzima A/metabolismo , Prueba de Estudio ConceptualRESUMEN
This study investigates the role of physical mobility in street harassment by analyzing a stratified random sample of 334 cases posted to Hollaback!, an online community documenting experiences of street harassment. Findings suggest that harassers utilize means of transportation as weapons to inflict or threaten physical harm, to escape or preserve anonymity, and to pursue targets and as a hunting ground for potential targets. By identifying the mechanisms of street harassment, we theorize how harassers negotiate mobility-particular types of mobility, especially that which is enabled by public transportation and owner vehicles-to gain advantage over targets of harassment.
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Ambiente , Relaciones Interpersonales , Acoso Sexual/psicología , Adulto , Chicago , Femenino , Humanos , Internet , Masculino , Ciudad de Nueva York , Acoso Sexual/estadística & datos numéricos , Medios de Comunicación Sociales/instrumentación , Medios de Comunicación Sociales/tendenciasRESUMEN
Human fatty acid synthase (hFAS) is a complex, multifunctional enzyme that is solely responsible for the de novo synthesis of long chain fatty acids. hFAS is highly expressed in a number of cancers, with low expression observed in most normal tissues. Although normal tissues tend to obtain fatty acids from the diet, tumor tissues rely on de novo fatty acid synthesis, making hFAS an attractive metabolic target for the treatment of cancer. We describe here the identification of GSK2194069, a potent and specific inhibitor of the ß-ketoacyl reductase (KR) activity of hFAS; the characterization of its enzymatic and cellular mechanism of action; and its inhibition of human tumor cell growth. We also present the design of a new protein construct suitable for crystallography, which resulted in what is to our knowledge the first co-crystal structure of the human KR domain and includes a bound inhibitor.
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3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/metabolismo , Inhibidores Enzimáticos/metabolismo , Ácido Graso Sintasas/antagonistas & inhibidores , Pirrolidinas/metabolismo , Pirrolidinas/farmacología , Triazoles/metabolismo , Triazoles/farmacología , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa/química , Dominio Catalítico , Línea Celular Tumoral , Ácido Graso Sintasas/química , Humanos , Modelos Moleculares , Conformación Proteica , Difracción de Rayos XRESUMEN
Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position.
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Anticoagulantes/síntesis química , Trastornos de la Coagulación Sanguínea/tratamiento farmacológico , Diseño de Fármacos , Pirazinas/síntesis química , Trombina/antagonistas & inhibidores , Administración Oral , Animales , Anticoagulantes/química , Anticoagulantes/farmacología , Sitios de Unión , Disponibilidad Biológica , Perros , Estructura Molecular , Pirazinas/química , Pirazinas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
A novel 1,3,5-trisubstituted benzamide thrombin inhibitor template was designed via hybridization of a known aminopyridinoneacetamide and a known 1,3,5-trisubstituted phenyl ether. Optimization of this lead afforded a novel potent series of biaryl 1,3,5-trisubstituted benzenes with excellent functional anticoagulant potency.
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Antitrombinas/síntesis química , Benceno/síntesis química , Diseño de Fármacos , Trombina/antagonistas & inhibidores , Antitrombinas/química , Antitrombinas/farmacología , Benceno/química , Benceno/farmacología , Humanos , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Gap junction uncoupling can alter conduction pathways and promote cardiac re-entry mechanisms that potentiate many supraventricular arrhythmias, such as atrial fibrillation (AF) and atrial flutter (AFL). Our objective was to determine whether GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], a small dipeptide gap junction modifier, can improve conduction and ultimately prevent AF/AFL. In rat atrial strips subjected to metabolic stress, GAP-134 prevented significantly conduction velocity slowing at 10 nM compared with vehicle (p < 0.01). In the canine sterile pericarditis model, conduction time (CT; n = 5), atrial effective refractory period (AERP; n = 3), and AF/AFL duration/inducibility (n = 16) were measured 2 to 3 days postoperatively in conscious dogs. CT was significantly faster after GAP-134 infusion (average plasma concentration, 250 nM) at cycle lengths of 300 ms (66.2 +/- 1.0 versus 62.0 +/- 1.0 ms; p < 0.001) and 200 ms (64.4 +/- 0.9 versus 61.0 +/- 1.3 ms; p < 0.001). No significant changes in AERP were noted after GAP-134 infusion. The mean number of AF/AFL inductions per animal was significantly decreased after GAP-134 infusion (2.7 +/- 0.6 versus 1.6 +/- 0.8; p < 0.01), with total AF/AFL burden being decreased from 12,280 to 6063 s. Western blot experiments showed no change in connexin 43 expression. At concentrations exceeding those described in the AF/AFL experiments, GAP-134 had no effect on heart rate, blood pressure, or any electrocardiogram parameters. In conclusion, GAP-134 shows consistent efficacy on measures of conduction and AF/AFL inducibility in the canine sterile pericarditis model. These findings, along with its oral bioavailability, underscore its potential antiarrhythmic efficacy.
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Fibrilación Atrial/tratamiento farmacológico , Aleteo Atrial/tratamiento farmacológico , Benzamidas/uso terapéutico , Dipéptidos/uso terapéutico , Uniones Comunicantes/efectos de los fármacos , Sistema de Conducción Cardíaco/efectos de los fármacos , Pericarditis/tratamiento farmacológico , Prolina/análogos & derivados , Animales , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Fibrilación Atrial/fisiopatología , Aleteo Atrial/fisiopatología , Benzamidas/farmacología , Conexina 43/metabolismo , Dipéptidos/efectos adversos , Dipéptidos/farmacología , Modelos Animales de Enfermedad , Perros , Conductividad Eléctrica , Femenino , Uniones Comunicantes/fisiología , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/metabolismo , Atrios Cardíacos/fisiopatología , Sistema de Conducción Cardíaco/fisiología , Masculino , Estructura Molecular , Oligopéptidos/farmacología , Oligopéptidos/uso terapéutico , Pericarditis/fisiopatología , Complicaciones Posoperatorias/tratamiento farmacológico , Complicaciones Posoperatorias/fisiopatología , Prolina/farmacología , Prolina/uso terapéutico , Ratas , Ratas Sprague-Dawley , Periodo Refractario Electrofisiológico/efectos de los fármacosRESUMEN
The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR(-/-)) or apolipoprotein E (apoE(-/-)) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7alpha-hydroxylase (CYP7A1) and sterol 12 alpha-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR(-/-)SHP(-/-) and apoE(-/-)SHP(-/-) mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR(-/-)SHP(-/-) or apoE(-/-)SHP(-/-) mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR(-/-) or apoE(-/-) mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.
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Apolipoproteínas E/deficiencia , Aterosclerosis/prevención & control , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores de LDL/deficiencia , Animales , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Azepinas/farmacología , Ácidos y Sales Biliares/metabolismo , Colesterol 7-alfa-Hidroxilasa/antagonistas & inhibidores , Colesterol 7-alfa-Hidroxilasa/genética , Dislipidemias/genética , Dislipidemias/metabolismo , Dislipidemias/prevención & control , Femenino , Expresión Génica/efectos de los fármacos , Indoles/farmacología , Lípidos/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de LDL/genética , Esteroide 12-alfa-Hidroxilasa/antagonistas & inhibidores , Esteroide 12-alfa-Hidroxilasa/genéticaRESUMEN
The nuclear hormone receptor farnesoid X receptor (FXR) plays a critical role in the regulation of bile acid, triglyceride (TG), and cholesterol homeostasis. WAY-362450 (FXR-450/XL335) is a potent synthetic FXR agonist as characterized in luciferase reporter assays and in mediating FXR target gene regulation in primary human and immortalized mouse hepatocytes. In vivo, WAY-362450 dose dependently decreased serum TG levels after 7 days of oral dosing in western diet-fed low-density lipoprotein receptor-/- mice and in the diabetic mouse strains KK-Ay and db/db comparable to that achieved with the peroxisome proliferator activated receptor-alpha agonist, fenofibrate. WAY-362450 treatment also reduced serum cholesterol levels via reductions in LDLc, VLDLc, and HDLc lipoprotein fractions that were not accompanied by hepatic cholesterol accumulation. This cholesterol lowering was dependent on FXR as demonstrated in a hypothyroid-induced hypercholesterolemia setting in FXR-/- mice. In fructose-fed models, WAY-362450 also decreased TG and VLDLc levels in rats and hamsters but significantly increased HDLc levels in rats while reducing HDLc levels in hamsters. The differential effect of WAY-362450 on HDLc is likely due to a murine-specific induction of endothelial lipase and scavenger receptor-BI that does not occur in rats. These studies demonstrate a consistent ability of WAY-362450 to lower both serum TG and cholesterol levels and suggest that synthetic FXR agonists may have clinical utility in the treatment of mixed dyslipidemia.
Asunto(s)
Azepinas/farmacología , Colesterol/sangre , Proteínas de Unión al ADN/agonistas , Dislipidemias/tratamiento farmacológico , Dislipidemias/metabolismo , Indoles/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Factores de Transcripción/agonistas , Animales , Apolipoproteína A-I/sangre , Apolipoproteína A-I/genética , Azepinas/química , Células Cultivadas , Colesterol/farmacología , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Dislipidemias/complicaciones , Femenino , Fructosa/farmacología , Humanos , Hiperglucemia/complicaciones , Hiperglucemia/genética , Hiperinsulinismo/complicaciones , Hiperinsulinismo/genética , Indoles/química , Riñón/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de LDL/genética , Receptores de Leptina/genética , Factores de Transcripción/metabolismo , Triglicéridos/sangreRESUMEN
Guided by X-ray crystallography of thrombin-inhibitor complexes and molecular modeling, alkylation of the N1 nitrogen of the imidazole P1 ligand of the pyridinoneacetamide thrombin inhibitor 1 with various acetamide moieties furnished inhibitors with significantly improved thrombin potency, trypsin selectivity, functional in vitro anticoagulant potency and in vivo antithrombotic efficacy. In the pyrazinoneacetamide series, oral bioavailability was also improved.
Asunto(s)
Anticoagulantes/farmacología , Antitrombinas/farmacología , Diseño de Fármacos , Imidazoles/síntesis química , Imidazoles/farmacología , Trombina/antagonistas & inhibidores , Administración Oral , Animales , Anticoagulantes/síntesis química , Anticoagulantes/química , Anticoagulantes/farmacocinética , Antitrombinas/síntesis química , Antitrombinas/química , Antitrombinas/farmacocinética , Disponibilidad Biológica , Cloruros , Cristalografía por Rayos X , Perros , Compuestos Férricos/farmacología , Imidazoles/química , Imidazoles/farmacocinética , Macaca mulatta , Modelos Moleculares , Estructura Molecular , Tiempo de Tromboplastina Parcial , Ratas , Relación Estructura-Actividad , Trombina/química , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.
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Indoles/farmacología , Inhibidor 1 de Activador Plasminogénico/metabolismo , Tetrazoles/farmacología , Biopolímeros/metabolismo , Electroforesis en Gel de Poliacrilamida , Fluorescencia , Humanos , Hidrólisis , Micelas , Vitronectina/metabolismo , Vitronectina/farmacologíaRESUMEN
Previous reports from our laboratories described potent tripeptide thrombin inhibitors which incorporate heterocycle-substituted chlorophenyl groups in the P1 position. Using these as lead compounds for further optimization, we identified sites of metabolism and designed analogs with 4-fluoroproline in P2 and cyclopropane-containing side chains in P3 as an approach to reducing metabolism and improving their oral pharmacokinetic performance. The large (300-fold) difference in potency between analogs containing (4R)- and (4S)-4-fluoroproline was rationalized by analyzing inhibitor-enzyme interactions in crystal structures of related compounds and by molecular modeling which indicated that the more potent (4R)-4-fluoroproline isomer stabilizes a proline ring conformation that is preferred for binding to the enzyme. An optimal compound from this work, 41, exhibits high potency in a coagulation assay in human plasma (2xAPTT=190 nM), excellent selectivity versus the digestive enzyme trypsin (K(i)=3300 nM), and excellent oral bioavailability in dogs with moderate clearance (F=100%, CL=12 mL/min/kg).
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Inhibidores Enzimáticos/farmacología , Prolina/análogos & derivados , Trombina/antagonistas & inhibidores , Sitios de Unión , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Humanos , Técnicas In Vitro , Modelos Moleculares , Conformación Molecular , Prolina/química , Conformación Proteica , Estructura Terciaria de Proteína , Estereoisomerismo , Relación Estructura-Actividad , Trombina/metabolismo , Tripsina/efectos de los fármacos , Tripsina/metabolismoRESUMEN
Antagonism of the bradykinin B1 receptor represents a potential treatment for chronic pain and inflammation. Novel antagonists were designed that display low-nanomolar affinity for the human bradykinin B1 receptor and good bioavailability in the rat.
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Amidas/síntesis química , Analgésicos/síntesis química , Antiinflamatorios no Esteroideos/síntesis química , Antagonistas del Receptor de Bradiquinina B1 , Ciclopropanos/síntesis química , Piridinas/síntesis química , Amidas/química , Amidas/farmacología , Analgésicos/química , Analgésicos/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Disponibilidad Biológica , Ciclopropanos/química , Ciclopropanos/farmacología , Diseño de Fármacos , Humanos , Conformación Molecular , Piridinas/química , Piridinas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Despite their relatively weak basicity, simple azoles, specifically imidazoles and aminothiazoles, can function as potent surrogates for the more basic amines (e.g., alkyl amines, amidines, guanidines, etc.) which are most often employed as the P1 ligand in the design of noncovalent small molecule inhibitors of thrombin.
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Azoles/farmacología , Inhibidores Enzimáticos/farmacología , Trombina/antagonistas & inhibidores , Azoles/química , Diseño de Fármacos , Ligandos , Estructura Molecular , Relación Estructura-Actividad , Tripsina/efectos de los fármacosRESUMEN
Thrombin-inhibitor X-ray crystal structures, in combination with the installation of binding elements optimized within the pyrazinone series of thrombin inhibitors, were utilized to transform a weak triazolopyrimidine lead into a series of potent oxazolopyridines. A modification intended to attenuate plasma protein binding (i.e., conversion of the P3 pyridine to a piperidine) conferred significant factor Xa activity to this series. Ultimately, these dual thrombin/factor Xa inhibitors demonstrated excellent in vitro and in vivo anticoagulant efficacy.
Asunto(s)
Antitrombinas/química , Antitrombinas/farmacología , Inhibidores del Factor Xa , Piridinas/química , Piridinas/farmacología , Cristalografía por Rayos X , Modelos Moleculares , Estructura MolecularRESUMEN
In this study, we have demonstrated that the critical hydrogen bonding motif of the established 3-aminopyrazinone thrombin inhibitors can be effectively mimicked by a 2-aminopyridine N-oxide. As this peptidomimetic core is more resistant toward oxidative metabolism, it also overcomes the metabolic liability associated with the pyrazinones. An optimization study of the P(1) benzylamide delivered the potent thrombin inhibitor 21 (K(i) = 3.2 nM, 2xaPTT = 360 nM), which exhibited good plasma levels and half-life after oral dosing in the dog (C(max) = 2.6 microM, t(1/2) = 4.5 h).
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Antitrombinas/química , Pirimidinas/química , Enlace de Hidrógeno , Modelos Moleculares , Imitación MolecularRESUMEN
Optimization of a previously reported thrombin inhibitor, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-trans-4-aminocyclohexylmethylamide (1), by replacing the aminocyclohexyl P1 group provided a new lead structure, 9-hydroxy-9-fluorenylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (2), with improved potency (K(i) = 0.49 nM for human thrombin, 2x APTT = 0.37 microM in human plasma) and pharmacokinetic properties (F = 39%, iv T(1/2) = 13 h in dogs). An effective strategy for reducing plasma protein binding of 2 and improving efficacy in an in vivo thrombosis model in rats was to replace the lipophilic fluorenyl group in P3 with an azafluorenyl group. Systematic investigation of all possible azafluorenyl P3 isomers and azafluorenyl-N-oxide analogues of 2 led to the identification of an optimal compound, 3-aza-9-hydroxyfluoren-9(R)-ylcarbonyl-l-prolyl-2-aminomethyl-5-chlorobenzylamide (19b), with high potency (K(i) = 0.40 nM, 2x APTT = 0.18 microM), excellent pharmacokinetic properties (F = 55%, T(1/2) = 14 h in dogs), and complete efficacy in the in vivo thrombosis model in rats (inhibition of FeCl(3)-induced vessel occlusions in six of six rats receiving an intravenous infusion of 10 microg/kg/min of 19b). The stereochemistry of the azafluorenyl group in 19b was determined by X-ray crystallographic analysis of its N-oxide derivative (23b) bound in the active site of human thrombin.
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Fluorenos/síntesis química , Prolina/análogos & derivados , Prolina/síntesis química , Trombina/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Proteínas Sanguíneas/metabolismo , Cristalografía por Rayos X , Perros , Fluorenos/química , Fluorenos/farmacología , Semivida , Humanos , Técnicas In Vitro , Macaca mulatta , Masculino , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Prolina/química , Prolina/farmacología , Unión Proteica , Ratas , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Although HIV-1 reverse transcriptase (RT) DNA polymerase and ribonuclease H (RNase H) activities reside in spatially distinct domains of the enzyme, inhibitors that bind in the RT polymerase domain can affect RNase H activity. We used both gel assays and a real-time FRET assay to analyze the impact of three mechanistically distinct RT polymerase inhibitors on RNase H activity in vitro. The nucleoside analogue 3'-azido-3'-deoxythymidine triphosphate (AZT-TP) had no effect, whereas the pyrophosphate analogue phosphonoformate (PFA) inhibited RNase H activity in a concentration-dependent manner. Nonnucleoside RT inhibitors (NNRTIs) enhanced RNase H catalysis, but the cleavage products differed substantially for RNA/DNA hybrid substrates of different lengths. A comparison of 61 different RT crystal structures revealed that NNRTI binding opened the angle between the polymerase and RNase H domains of the p66 subunit and reduced the relative motion of the thumb and RNase H regions, suggesting that NNRTI enhancement of RNase H cleavage may result from increased accessibility of the RNase H active site to the RNA/DNA hybrid duplex. We also examined the effects of combining a diketo acid (DKA) RNase H inhibitor with various RT polymerase inhibitors on polymerase-independent RNase H cleavage, RNA-dependent DNA polymerization, and in reverse-transcription assays. Interestingly, although the NNRTI decreased DKA potency in polymerase-independent RNase H assays, NNRTI/DKA combinations were synergistic in inhibiting reverse transcription overall, indicating that regimens incorporating both NNRTI and RNase H inhibitors may be therapeutically beneficial.