RESUMEN
By screening directed libraries of serine hydrolase inhibitors using the cell surface form of endothelial lipase (EL), we identified a series of carbamate-derived (EL) inhibitors. Compound 3 raised plasma HDL-C levels in the mouse, and a correlation was found between HDL-C and plasma compound levels. Spectroscopic and kinetic studies support a covalent mechanism of inhibition. Our findings represent the first report of EL inhibition as an effective means for increasing HDL-C in an in vivo model.
Asunto(s)
HDL-Colesterol/sangre , Endotelio Vascular/enzimología , Inhibidores Enzimáticos/química , Lipasa/antagonistas & inhibidores , Tiocarbamatos/química , Animales , Endotelio Vascular/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Lipasa/metabolismo , Lipoproteínas HDL/deficiencia , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Tiocarbamatos/síntesis química , Tiocarbamatos/farmacologíaRESUMEN
STUDY DESIGN: Retrospective review. OBJECTIVES: To identify relationships between lower extremity innervation and level of injury, mechanism of injury, and age at injury in a pediatric population with spinal cord injury (SCI). Secondarily, relationships between innervation and completeness of injury, time since injury, race, and sex were evaluated. SETTING: Pediatric orthopedic referral hospital, Philadelphia, Pennsylvania. METHODS: Records of 190 subjects, ages 1-21 years, were reviewed. Data collected from the medical record included lower extremity muscle innervation, American Spinal Injury Association (ASIA) level and class, mechanism of injury, age at injury, time since injury, race, and sex. To determine innervation, lower extremity muscles had been tested using surface electrical stimulation and identified as being innervated or denervated. If a muscle responded weakly, strength duration testing was performed. For analysis via logistic regression, subjects were grouped based upon level and mechanism of injury. RESULTS: A relationship (P<0.0001) was found between ASIA level and lower extremity innervation of all muscles and between length of time since injury and lower extremity innervation for some muscles. Following multiple logistic regression, only ASIA level remained as an independent predictor of lower extremity innervation status. CONCLUSION: Our results show that lower extremity innervation does differ based on the level of the injury. Denervation began to be seen with injuries in the lower thoracic region and more predominantly with injuries in the lumbar region. This supports our hypothesis that the incidence of lower motor neuron injuries would increase as injuries became more caudal. Our hypotheses of a relationship between innervation status and mechanism of injury and age at injury were not supported. This information is important in determining treatment strategies, eligibility for electrical stimulation techniques, and potential regenerative strategies. SPONSORSHIP: This study was funded by Shriners Hospitals for Children, Grant #8530.
Asunto(s)
Extremidad Inferior/inervación , Músculo Esquelético/inervación , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/fisiopatología , Adolescente , Adulto , Distribución por Edad , Niño , Preescolar , Demografía , Femenino , Lateralidad Funcional/fisiología , Humanos , Lactante , Modelos Logísticos , Extremidad Inferior/fisiopatología , Masculino , Análisis Multivariante , Músculo Esquelético/fisiopatología , Estudios Retrospectivos , Factores Sexuales , Traumatismos de la Médula Espinal/etiología , Factores de TiempoRESUMEN
We have explored the structure-activity relationship (SAR) surrounding the clinically efficacious antiepileptic drug topiramate (1), a unique sugar sulfamate anticonvulsant that was discovered in our laboratories. Systematic structural modification of the parent compound was directed to identifying potent anticonvulsants with a long duration of action and a favorable neurotoxicity index. In this context, we have probed the pharmacological importance of several molecular features: (1) the sulfamate group (6-8, 22-25, 27, 84), (2) the linker between the sulfamate group and the pyran ring (9, 10, 21a,b), (3) the substituents on the 2,3- (58-60, 85, 86) and 4, 5-fused (30-38, 43, 45-47, 52, 53) 1,3-dioxolane rings, (4) the constitution of the 4,5-fused 1,3-dioxolane ring (2, 54, 55, 63-68, 76, 77, 80, 83a-r, 84-87, 90a, 91a, 93a), (5) the ring oxygen atoms (95, 96, 100-102, 104, 105), and (6) the absolute stereochemistry (106 and 107). We established the C1 configuration as R for the predominant alcohol diastereomer from the highly selective addition of methylmagnesium bromide to aldehyde 15 (16:1 ratio) by single-crystal X-ray analysis of the major diastereomer of sulfamate 21a. Details for the stereoselective syntheses of the hydrindane carbocyclic analogues 95, 96, 100, and 104 are presented. We also report the synthesis of cyclic imidosulfites 90a and 93a, and imidosulfate 91a, which are rare examples in the class of such five-membered-ring sulfur species. Imidosulfite 93a required the preparation and use of the novel sulfur dichloride reagent, BocN=SCl2. Our SAR investigation led to the impressive 4,5-cyclic sulfate analogue 2 (RWJ-37947), which exhibits potent anticonvulsant activity in the maximal electroshock seizure (MES) test (ca. 8 times greater than 1 in mice at 4 h, ED50 = 6.3 mg/kg; ca. 15 times greater than 1 in rats at 8 h, ED50 = 1.0 mg/kg) with a long duration of action (>24 h in mice and rats, po) and very low neurotoxicity (TD50 value of >1000 mg/kg at 2 h, po in mice). Cyclic sulfate 2, like topiramate and phenytoin, did not interfere with seizures induced by pentylenetetrazole, bicucculine, picrotoxin, and strychnine; also, 2 was not active in diverse in vitro receptor binding and uptake assays. However, 2 turned out to be a potent inhibitor of carbonic anhydrase from different rat tissue sources (e. g., IC50 of 84 nM for the blood enzyme and 21 nM for the brain enzyme). An examination of several analogues of 2 (83a-r, 85-87, 90a, 91a, 93a) indicated that potent anticonvulsant activity is associated with relatively small alkyl substituents on nitrogen (Me/H, 83a; Me/Me, 83m; Et/H, 83b; allyl/H, 83e; c-Pr/H, 83j; c-Bu/H, 83k) and with limited changes in the cyclic sulfate group, such as 4,5-cyclic sulfite 87a/b. The potent anticonvulsants 83a and 83j had greatly diminished carbonic anhydrase inhibitory activity; thus, inhibition of this enzyme may not be a significant factor in the anticonvulsant activity. The alpha-L-sorbopyranoses 67, 68, and 80, which mainly possess a skew conformation (ref 29), were nearly twice as potent as topiramate (1). The L-fructose enantiomers of 1 (106) and 2 (107), synthesized from L-sorbose, were found to have moderate anticonvulsant activity, with eudysmic ratios (MES ED50 in mice at 4 h, po) of 1:106 = 1.5 and 2:107 = 3.5. The log P values for 1 and 2 were determined experimentally to be 0.53 and 0.42, respectively, which are less than the optimal 2.0 for CNS active agents. However, analogues with more favorable calculated log P (clogP) values, in conjunction with just minor steric perturbation according to the developed SAR profile, such as 47 (clogP = 2.09), 83m (1.93), and 86 (1.50), did not display improved potency: 47 is less potent than 1, 83m is equipotent with 2, and 86 is less potent than 2. Although the measured log P value for diethyl analogue 31 is 1.52, this did not translate into enhanced potency relative to 1. (ABSTRACT TRUNCATED)
Asunto(s)
Anticonvulsivantes , Fructosa/análogos & derivados , Ácidos Sulfónicos , Animales , Anticonvulsivantes/química , Anticonvulsivantes/farmacología , Anticonvulsivantes/toxicidad , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/toxicidad , Cristalografía por Rayos X , Electrochoque , Fructosa/química , Fructosa/farmacología , Fructosa/toxicidad , Ratones , Conformación Molecular , Ratas , Convulsiones/prevención & control , Estereoisomerismo , Relación Estructura-Actividad , Ácidos Sulfónicos/química , Ácidos Sulfónicos/farmacología , Ácidos Sulfónicos/toxicidad , TopiramatoRESUMEN
Macrocyclic pentapeptide analogues (5-9) of the sponge natural product cyclotheonamide A (CtA, 3) were prepared by means of our convergent [3 + 2] synthetic protocol, in which a late-stage primary amine group is available for substitution (Maryanoff et al. Proc. Natl Acad. Sci. U.S.A. 1993, 90, 8048). These analogues, as well as CtA and cyclotheonamide B (CtB, 4), were examined for their ability to inhibit the serine protease alpha-thrombin, in comparison with suitable reference standards. We characterized Michaelis-Menten and slow-binding kinetics for the cyclotheonamide derivatives. An attempt was made to utilize the unoccupied hydrophobic S3 subsite of thrombin via analogues 5 and 6. Also, removal of the hydroxyphenyl group, which is thought to be involved in an aromatic stacking interaction with Trp60D of thrombin, was explored via analogue 9. The importance of the alpha-keto and olefin groups was examined via 7 and 8, respectively. The relationship of structure and function with the analogues proved to be less predictable than anticipated.
Asunto(s)
Antitrombinas/síntesis química , Oligopéptidos/síntesis química , Péptidos Cíclicos/síntesis química , Trombina/antagonistas & inhibidores , Secuencia de Aminoácidos , Antitrombinas/farmacología , Compuestos de Boro/farmacología , Humanos , Indicadores y Reactivos , Cinética , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Estructura Molecular , Oligopéptidos/farmacología , Péptidos Cíclicos/farmacología , Espectrometría de Masa Bombardeada por Átomos Veloces , Relación Estructura-ActividadRESUMEN
The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human alpha-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-A resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the S1 specificity pocket; formation of a hydrogen-bonded two-strand antiparallel beta-sheet with Ser214-Gly216) and the alpha-keto amide group of CtA are primarily responsible for binding to thrombin, with the alpha-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr60A-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp60D as part of an "aromatic stacking chain." Biochemical inhibition data (Ki values at 37 degrees C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human alpha-thrombin (Ki = 0.18 microM) but a potent inhibitor of trypsin (Ki = 0.023 microM) and streptokinase (Ki = 0.035 microM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent [2 + 3] fragment-condensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.
Asunto(s)
Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Inhibidores de Serina Proteinasa/química , Trombina/antagonistas & inhibidores , Trombina/química , Secuencia de Aminoácidos , Humanos , Enlace de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Estructura Secundaria de Proteína , Especificidad por Sustrato , Difracción de Rayos XRESUMEN
A novel series of N-[(2-benzothiazolylthio)alkyl]-N'-hydroxyurea derivatives (9-25) was synthesized and evaluated for biological activity as inhibitors of 5-lipoxygenase both in vivo (mouse zymosan peritonitis assay) and in vitro (Ca2+ ionophore-stimulated human peripheral blood leukocyte model). The compounds of this series were based on the corresponding hydroxamic acid derivatives (1, 3, 4, and 5) which were moderately active in vitro but inactive in vivo. A number of compounds in the hydroxyurea series exhibited oral activity for 5-lipoxygenase inhibition. Results of studies relating structure to in vivo and in vitro 5-lipoxygenase activity are reported.