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1.
FEMS Microbiol Lett ; 366(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30715321

RESUMO

Oxidosqualene cyclases (OSCs) are remarkable enzymes that catalyze the production of the first sterol, lanosterol, in sterol biosynthetic pathways. These reactions are present in a limited number of bacterial species unlike eukaryotic species where sterol synthesis is ubiquitous. The biological role(s) of OSCs, and the sterols produced by the different sterol biosynthetic pathways in bacteria, are not clearly understood. Here, we show that inhibition of the Gemmata obscuriglobus OSC enzyme resulted in the inability of cells to form colonies on solid medium and resulted in cell death within 24 hr of inactivation for planktonic cells. The inclusion of lanosterol in cell culture medium was able to rescue the cell lethality associated with the OSC inhibitors. We purified active, recombinant bacterial OSC to high levels (> 3 mg L-1 of culture) and demonstrated that the purified enzyme is active and inhibited by common OSC inhibitors. Comparable inhibitor concentrations were used in in vivo lethality experiments and in vitro enzymatic assays. Together, these results show that OSC, and the sterols produced by this enzyme, are essential for G. obscuriglobus viability.


Assuntos
Transferases Intramoleculares/metabolismo , Planctomycetales/enzimologia , Esteróis/metabolismo , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Lanosterol/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Planctomycetales/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Esteróis/biossíntese
2.
AAPS PharmSciTech ; 16(6): 1425-33, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25986597

RESUMO

Unprotected sunlight exposure is a risk factor for a variety of cutaneous cancers. Topically used dihydroxyacetone (DHA) creates, via Maillard reaction, chemically fixed keratin sunscreen in the stratum corneum with significant protection against UVA/Soret radiation. When used in conjunction with naphthoquinones a naphthoquinone-modified DHA Maillard reaction is produced that provides protection across the UVB/UVA/Soret spectra lasting up to 1 week, resisting sweating and contact removal. The aim of this study was to examine a simplified version of this formulation for effect on UV transmission and to determine if penetration levels merit toxicity concerns. Permeability was demonstrated for freshly prepared DHA (30 mg/mL) and lawsone (0.035 mg/mL) alone and in combination using a side-by-side diffusion apparatus at 37°C over 48 h across shed snake skin and dermatomed pig skin. These samples were then examined for effectiveness and safety. Concentrations were determined by HPLC and UPLC monitored from 250-500 nm. Lawsone flux significantly decreased across pig skin (20.8 (± 4.8) and 0.09 (± 0.1) mg/cm(2) h without and with DHA, respectively) but did not change across shed snake skin in the presence of DHA. Significantly reduced lawsone concentration was noted in donor chambers of combined solutions. Damage was not observed in any skins. Darker coloration with greater UV absorbance was observed in skins exposed to the combined solution versus individual solutions. This study confirmed that combined DHA and lawsone provided effective blocking of ultraviolet light through products bound in keratinized tissue. DHA permeation levels in pig skin suggest further in vitro and in vivo study is required to determine the safety of this system.


Assuntos
Di-Hidroxiacetona/administração & dosagem , Naftoquinonas/administração & dosagem , Pele/efeitos dos fármacos , Protetores Solares/administração & dosagem , Animais , Boidae/metabolismo , Difusão , Suínos , Raios Ultravioleta
3.
Synthesis (Stuttg) ; 46(4): 515-521, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29097824

RESUMO

An efficient two-step synthesis of 4(5)-benzyl-L-histidine from L-histidine was developed. A Pictet-Spengler reaction between L-histidine and benzaldehyde in the presence of excess strong base yielded 4-phenylspinacine within one hour. Catalytic transfer hydrogenolysis in methanol at reflux using ammonium formate rapidly converted 4-L-phenylspinacine to 4(5)-benzyl-L-histidine within five minutes. No racemization of the final product 4(5)-benzyl-L-histidine was observed using the Marfey reagent. To show the utility of this methodology, a series of fluorinated benzylhistidines is presented.

5.
Chem Biol Drug Des ; 70(3): 216-26, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17718716

RESUMO

Synthesis of the first irreversible calcitonin gene-related peptide receptor antagonists is described. bis-(2-chloroethyl)amino and fluorosulphonyl groups were incorporated into the 4-position of the N-terminal benzoyl group of a potent competitive antagonist, N-alpha-benzoyl-h-alpha-CGRP(8-37) (analogues 4 and 6). Based on previous structure-activity relationships, a second pair of N-terminally modified analogues was synthesized containing a novel benzylated-His residue in position 10 (analogues 5 and 7). In separate experiments, SK-N-MC cells and mouse thoracic aortas were bathed in solutions containing 5 microM and 1.5 microM of each analogue, respectively. After extensive washing, calcitonin gene-related peptide concentration-response curves were generated for cAMP production in SK-N-MC cells and relaxation of mouse aortas. All analogues caused >20% reductions in maximal calcitonin gene-related peptide efficacy in both assays with analogue 5 containing an N-terminal bis-(2-chloroethyl)amino-benzoyl group and a benzylated-His10 residue completely abolishing cAMP production in SK-N-MC cells. Reductions in maximal responses were dependent on the analogue concentration. Analogue 4 also caused more than 10-fold reductions in the potency of the calcitonin gene-related peptide-mediated effects, whereas analogues 5, 6 and 7 have no significant effect on calcitonin gene-related peptide potency. These data indicate that all analogues bind irreversibly to calcitonin gene-related peptide receptors. The bis-(2-chloroethyl)amino-modified analogues 4 and 5 were more effective than the fluorosulphonyl-modified analogues 6 and 7.


Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/análogos & derivados , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cloro/química , Flúor/química , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Enxofre/química , Aminação , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Linhagem Celular Tumoral , Fenômenos Químicos , Físico-Química , Humanos , Masculino , Camundongos , Estrutura Molecular
6.
J Pharmacol Exp Ther ; 319(2): 749-57, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16873605

RESUMO

Human alpha-calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that produces a variety of cardiovascular and other effects via activation of specific CGRP receptors that produce cAMP. Functional CGRP receptors are a heterodimeric complex composed of the heptahelical calcitonin receptor-like receptor and the single transmembrane receptor activity-modifying protein 1. Based on the known structures of the antagonist CGRP((8-37)) and the human CGRP receptor, we designed novel CGRP receptor peptide antagonists with modifications to promote high affinity and selectivity for human CGRP receptors. Antagonist affinity (K(B)) at CGRP receptors was determined using the mouse thoracic aorta and human SK-N-MC cells. In aorta, CGRP((8-37)), [N-alpha-benzoyl]human alpha-CGRP((8-37)) [bzl-CGRP((8-37))], and [N-alpha-benzoyl-His(10)-benzyl]human alpha-CGRP((8-37)) [bzl-bn-CGRP((8-37))] caused rightward shifts in the concentration-response relaxation curve for CGRP with K(B) values of 1000, 88, and 50 nM, respectively. In human SK-N-MC cells, CGRP((8-37)), bzl-CGRP((8-37)), and bzl-bn-CGRP((8-37)) caused rightward shifts in the concentration-response curve for CGRP-stimulated cAMP production with K(B) values of 797, 15, and 0.63 nM, respectively. Thus, CGRP((8-37)) had the same affinity for human and mouse CGRP receptors, whereas bzl-CGRP((8-37)) and bzl-bn-CGRP((8-37)) displayed 6- and 80-fold higher affinities, respectively, for human CGRP receptors. In addition, the selectivity of the antagonists for human CGRP receptors was highly correlated with the antagonist hydrophobicity index. These relatively high-affinity, species-selective peptide antagonists provide novel tools to differentiate structural and functional features that are unique to the human CGRP receptor. Thus, these analogs may be useful compounds for development of drugs to treat migraine headache and other cardiovascular diseases.


Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Proteína Semelhante a Receptor de Calcitonina , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Humanos , Interações Hidrofóbicas e Hidrofílicas , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas de Membrana/química , Camundongos , Proteínas Modificadoras da Atividade de Receptores , Receptores da Calcitonina/química , Especificidade da Espécie , Relação Estrutura-Atividade
7.
Am J Physiol Regul Integr Comp Physiol ; 286(6): R1005-12, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-14701717

RESUMO

CCK type 1 (CCK1) receptor antagonists differing in blood-brain barrier permeability were used to test the hypothesis that satiety is mediated in part by CCK action at CCK1 receptors on vagal sensory nerves innervating the small intestine. Devazepide penetrates the blood-brain barrier; A-70104, the dicyclohexylammonium salt of N alpha-3-quinolinoyl-D-Glu-N,N-dipentylamide, does not. At dark onset, non-food-deprived control rats and rats with subdiaphragmatic vagotomies received a bolus injection of devazepide (2.5 micromol/kg i.v.) or a 3-h infusion of A-70104 (3 micromol.kg(-1).h(-1) i.v.) either alone or coadministered with a 2-h intragastric infusion of peptone (0.75 or 1 g/h). Food intake was determined from continuous computer recordings of changes in food bowl weight. In control rats both antagonists stimulated food intake and attenuated the anorexic response to intragastric infusion of peptone. In contrast, only devazepide was effective in stimulating food intake in vagotomized rats. Thus endogenous CCK appears to act both at CCK1 receptors beyond the blood-brain barrier and by a CCK1 receptor-mediated mechanism involving abdominal vagal nerves to inhibit food intake.


Assuntos
Colecistocinina/fisiologia , Resposta de Saciedade/fisiologia , Nervo Vago/fisiologia , Abdome/inervação , Animais , Colecistocinina/antagonistas & inibidores , Colecistocinina/farmacologia , Devazepida/administração & dosagem , Devazepida/farmacologia , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/fisiologia , Antagonistas de Hormônios/administração & dosagem , Antagonistas de Hormônios/farmacologia , Infusões Intravenosas , Masculino , Peptonas/administração & dosagem , Peptonas/farmacologia , Quinolinas/administração & dosagem , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/antagonistas & inibidores , Receptores da Colecistocinina/fisiologia , Sincalida/administração & dosagem , Sincalida/farmacologia , Vagotomia
8.
J Med Chem ; 46(12): 2427-35, 2003 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-12773046

RESUMO

Seventeen novel analogues of human calcitonin gene-related peptide(8-37) (hCGRP(8-37)) were synthesized by solid-phase methods and purified to apparent homogeneity by semipreparative cation exchange and/or reversed-phase high-performance liquid chromatography. The C-terminal Phe was replaced by Gly, cyclohexylalanine (Cha), Tyr, all four isomers of beta-methylphenylalanine (beta-MePhe), and l- and d-tetrahydroisoquinoline carboxylic acid (Tic), resulting in analogues 3-11. For the synthesis of the beta-MePhe-containing analogues 6-9, crystallization was used to separate a mixture of all four isomers of beta-MePhe into the erythro pair of enantiomers (2S,3S, 2R,3R) and the threo pair of enantiomers (2S,3R, 2R,3S), which were then converted to Fmoc derivatives and used in two separate syntheses. Two diastereomeric peptides were obtained from each synthesis and were separated by RP-HPLC to yield enantiomerically pure 6-9. Substitution of Tyr for Phe caused no change in binding affinity at CGRP receptors. All other substitutions for Phe resulted in substantial reductions in binding affinity. Indeed, no binding was observed for analogues 7, 9, and 11, all of which contained a d-amino acid residue in the C-terminal position, and the binding affinities of the remaining analogues were >10-fold lower than that of h-alpha-CGRP(8-37). These data suggest that a conformationally flexible phenyl ring in the C-terminal position of h-alpha-CGRP(8-37) is preferred for high-affinity binding to CGRP receptors. Acetylation, benzoylation, and benzylation of the N-termini of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) produced analogues 12-14 and 16-18, respectively. A byproduct was isolated by RP-HPLC from the resin-cleaved crude product of each benzylated analogue, which was characterized as the dibenzylated derivative of h-alpha-CGRP(8-37) and h-beta-CGRP(8-37) (analogues 15 and 19, respectively). Amino acid analysis and (1)H NMR showed that the second benzyl group was located on the C4 carbon of the imidazole ring of His(10). Radioligand binding experiments showed that derivatizing the N-termini substantially increased binding affinities at CGRP receptors. The benzoylated and dibenzylated derivatives had the highest affinities, which were approximately 50-fold greater than those of h-alpha-CGRP(8-37). Functional experiments confirmed that the N-terminally derivatized analogues of h-alpha-CGRP(8-37) are antagonists that are more potent than h-alpha-CGRP(8-37). In conclusion, these studies underscore the importance of Phe(37) of h-alpha-CGRP(8-37) for binding to CGRP receptors and have identified the N-terminus and His(10) as two positions that can be used for the design of antagonists with increased affinity for CGRP receptors.


Assuntos
Antagonistas do Receptor do Peptídeo Relacionado ao Gene de Calcitonina , Peptídeo Relacionado com Gene de Calcitonina/síntese química , Fragmentos de Peptídeos/síntese química , Animais , Peptídeo Relacionado com Gene de Calcitonina/química , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Vasos Coronários/metabolismo , Vasos Coronários/fisiologia , Humanos , Técnicas In Vitro , Membranas , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Ensaio Radioligante , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/agonistas , Espectrometria de Massas por Ionização por Electrospray , Estereoisomerismo , Relação Estrutura-Atividade , Suínos
9.
Am J Physiol Regul Integr Comp Physiol ; 285(2): R429-37, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12738611

RESUMO

Type A cholecystokinin receptor (CCKAR) antagonists differing in blood-brain barrier permeability were used to test the hypothesis that satiety is mediated, in part, by CCK action at CCKARs located peripheral to the blood-brain barrier. At dark onset, non-food-deprived rats received a bolus injection of devazepide (2.5 micromol/kg iv), a 3-h infusion of A-70104 (1 or 3 micromol x kg-1 x h-1 iv), or vehicle either alone or coadministered with a 3-h infusion of CCK-8 (10 nmol x kg-1 x h-1 iv) or a 2-h intragastric infusion of peptone (1 g/h). Food intake was determined from continuous computer recordings of changes in food bowl weight. Devazepide penetrates the blood-brain barrier; A-70104, the dicyclohexylammonium salt of Nalpha-3-quinolinoyl-d-Glu-N,N-dipentylamide (A-65186), does not. CCK-8 inhibited 3-h food intake by more than 50% and both A-70104 and devazepide abolished this response. A-70104 and devazepide stimulated food intake and similarly attenuated the anorexic response to intragastric infusion of peptone. Thus endogenous CCK appears to act, in part, at CCKARs peripheral to the blood-brain barrier to inhibit food intake.


Assuntos
Comportamento Alimentar/efeitos dos fármacos , Receptores da Colecistocinina/antagonistas & inibidores , Animais , Apetite/efeitos dos fármacos , Devazepida/farmacologia , Antagonistas de Hormônios/farmacologia , Masculino , Peptonas/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/metabolismo , Sincalida/farmacologia
10.
Am J Physiol Regul Integr Comp Physiol ; 284(1): R66-75, 2003 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12388446

RESUMO

Type A CCK receptor (CCKAR) antagonists differing in blood-brain barrier permeability [devazepide penetrates; the dicyclohexylammonium salt of Nalpha-3-quinolinoyl-d-Glu-N,N-dipentylamide (A-70104) does not] were used to test the hypothesis that duodenal nutrient-induced inhibition of gastric emptying is mediated by CCKARs located peripheral to the blood-brain barrier. Rats received A-70104 (700 or 3,000 nmol. kg(-1). h(-1) iv) or devazepide (2.5 micromol/kg iv) and either a 15-min intravenous infusion of CCK-8 (3 nmol. kg(-1). h(-1)) or duodenal infusion of casein, peptone, Intralipid, or maltose. Gastric emptying of saline was measured during the last 5 min of each infusion. A-70104 and devazepide abolished the gastric emptying response to a maximal inhibitory dose of CCK-8. Each of the macronutrients inhibited gastric emptying. A-70104 and devazepide attenuated inhibitory responses to each macronutrient. Intravenous injection of a CCK antibody to immunoneutralize circulating CCK had no effect on peptone or Intralipid-induced responses. Thus endogenous CCK appears to act in part by a paracrine or neurocrine mechanism at CCKARs peripheral to the blood-brain barrier to inhibit gastric emptying.


Assuntos
Colecistocinina/farmacologia , Esvaziamento Gástrico/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Receptores da Colecistocinina/antagonistas & inibidores , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Caseínas/administração & dosagem , Caseínas/farmacologia , Colecistocinina/antagonistas & inibidores , Colecistocinina/imunologia , Colecistocinina/metabolismo , Devazepida/farmacologia , Vias de Administração de Medicamentos , Emulsões Gordurosas Intravenosas/administração & dosagem , Emulsões Gordurosas Intravenosas/farmacologia , Antagonistas de Hormônios/farmacologia , Masculino , Maltose/administração & dosagem , Maltose/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Peptonas/administração & dosagem , Peptonas/farmacologia , Quinolinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/metabolismo
11.
Am J Physiol Regul Integr Comp Physiol ; 284(2): R389-98, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12414437

RESUMO

Type A cholecystokinin receptor (CCKAR) antagonists differing in blood-brain barrier permeability were used to test the hypothesis that duodenal delivery of protein, carbohydrate, and fat produces satiety in part by an essential CCK action at CCKARs located peripheral to the blood-brain barrier. Fasted rats with open gastric fistulas received devazepide (1 mg/kg iv) or A-70104 (700 nmol. kg(-1). h(-1) iv) and either a 30-min intravenous infusion of CCK-8 (10 nmol. kg(-1). h(-1)) or duodenal infusion of peptone, maltose, or Intralipid beginning 10 min before 30-min access to 15% sucrose. Devazepide penetrates the blood-brain barrier; A-70104, the dicyclohexylammonium salt of Nalpha-3-quinolinoyl-d-Glu-N,N-dipentylamide, does not. CCK-8 inhibited sham feeding by approximately 50%, and both A-70104 and devazepide abolished this response. Duodenal infusion of each of the macronutrients dose dependently inhibited sham feeding. A-70104 and devazepide attenuated inhibitory responses to each macronutrient. Thus endogenous CCK appears to act in part at CCKARs peripheral to the blood-brain barrier to inhibit food intake.


Assuntos
Regulação do Apetite/efeitos dos fármacos , Devazepida/farmacologia , Duodeno/efeitos dos fármacos , Duodeno/metabolismo , Quinolinas/farmacologia , Receptores da Colecistocinina/antagonistas & inibidores , Animais , Barreira Hematoencefálica , Devazepida/administração & dosagem , Relação Dose-Resposta a Droga , Vias de Administração de Medicamentos , Ingestão de Alimentos , Emulsões Gordurosas Intravenosas/administração & dosagem , Emulsões Gordurosas Intravenosas/farmacologia , Masculino , Maltose/administração & dosagem , Maltose/farmacologia , Peptonas/administração & dosagem , Peptonas/farmacologia , Quinolinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/metabolismo , Sincalida/antagonistas & inibidores , Sincalida/farmacologia
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