RESUMO
Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.
RESUMO
Novel O-acylated (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-one oximes were designed as potential HSP90 inhibitors. A series of the compounds was synthesized by oximation of (E)-3-aryl-6,7-dihydrobenzisoxazol-4(5H)-ones followed by O-acylation with acylamidobenzoic acids. The obtained compounds showed an antiproliferative effect on three breast cancer cell lines (MCF7, MDA-MB-231 and HCC1954). Compound 16s exhibited high antiproliferative potency against HCC1954 breast cancer cells with the IC50 value of 6 µM was selected for in-depth evaluation. Compound 16s did not inhibit the growth of normal epithelial cells. We have demonstrated that the compound 16s can induce apoptosis in cancer cells via inhibition of HSP90 "client" proteins including a key oncogenic receptor, HER2/neu. Described here compounds can be considered for further basic and preclinical investigation as a part of HSP90/HER2-targeted therapies.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Oxazóis/farmacologia , Oximas/farmacologia , Acilação , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Modelos Moleculares , Estrutura Molecular , Oxazóis/síntese química , Oxazóis/química , Oximas/síntese química , Oximas/química , Relação Estrutura-AtividadeRESUMO
A series of novel fluorine-containing lupane triterpenoid acid derivatives with fluoroaromatic amide moieties at the C-28 position (1-8) or with 2-(fluoroacyl)cyclopentane-1,3-dione fragments at the C-3 position (9-18) of lupane skeleton was synthesized. A simple synthesis of novel lupane triterpenoid hybrids with 2-(fluoroacyl)-2-cyclopenten-1-one moieties was developed. An interaction of 2-acyl-3-chlorocyclopent-2-en-1-ones, obtained from corresponding cyclic ß-triketones, with methyl 3-amino-3-deoxybetulinate gave 3ß-isomers (9-13) and 3α-isomers (14-18) of target hybrids, which were isolated as individual compounds. Anti-inflammatory properties of selected synthesized compounds were studied in vivo using the histamine-, concanavalin A- and sheep erythrocytes immunization-induced mouse paw edema models. The antioxidant activity was investigated in vivo on the model of tetracycline-induced hepatitis. Majority of synthesized fluorine-containing lupane triterpenoid acid derivatives exhibited significant anti-inflammatory and antioxidant effects. Among studied compounds, 3ß-hybrid 11 with 2-perfluorobutanoyl-2-cyclopenten-1-one moiety was the most potent bioactive compound.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Edema/tratamento farmacológico , Hepatite/tratamento farmacológico , Triterpenos Pentacíclicos/farmacologia , Animais , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Antioxidantes/síntese química , Antioxidantes/química , Concanavalina A , Modelos Animais de Doenças , Desenho de Fármacos , Edema/induzido quimicamente , Feminino , Flúor/química , Flúor/farmacologia , Histamina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Conformação Molecular , Triterpenos Pentacíclicos/síntese química , Triterpenos Pentacíclicos/química , Tetraciclina/administração & dosagem , Triterpenos/síntese química , Triterpenos/química , Triterpenos/farmacologia , Ácido BetulínicoRESUMO
An efficient protocol for the synthesis of novel lupane triterpenoid-indazolone hybrids with oxime ester linkage has been developed from naturally accessible precursor betulin. For the first time a series of betulonic acid-indazolone hybrids have been synthesized via an acylation of corresponding 6,7-dihydro-1H-indazol-4(5H)-one oximes with betulonic acid chloride. Diastereoselective reduction of the obtained betulonic acid conjugates with NaBH4 resulted in a formation of betulinic acid-indazolone hybrids in excellent yields. The configuration of the key compounds has been fully established by X-ray and 2D NMR analysis.
Assuntos
Triterpenos/química , Estrutura Molecular , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/química , Triterpenos Pentacíclicos , Relação Estrutura-Atividade , Ácido BetulínicoRESUMO
The ethanol solvolysis of 3-methoxy-14,17-etheno-16α-nitroestra-1,3,5(10)-trien-17ß-yl acetate in the presence of NaHCO3 was studied by means of real-time NMR experiments, LC-SPE-NMR, and LC-MS. The pathway to form 3-methoxy-2'-oxopyrrolidino-[4',5':14ß,15ß]-estra-1,3,5(10)-trien-17-one was disclosed. The intermediacy of nitrile oxide and alkoxynitrone was postulated based on the analysis of the reaction products. The proposed mechanism of cleaving the bridge in the nitro compound is legal for the formation of N-acetoxylactams, nitriles, isoxazoles and isoxazolines.
Assuntos
Estradiol/análogos & derivados , Lactamas/síntese química , Pirrolidinonas/síntese química , Cromatografia Líquida , Estradiol/química , Lactamas/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Conformação Molecular , Pirrolidinonas/química , Bicarbonato de Sódio/química , SolubilidadeRESUMO
Starting from (22R,23R)-2α,3α,22,23,26-pentahydroxy-5α-cholestan-6-one 26-hemisuccinate, conjugates of 28-norcastasterone with horse radish peroxidase and bovine serum albumin were prepared. The latter conjugate was injected into rabbits; produced polyclonal antibodies were used to quantitate 6-keto-brassinosteroids. The newly developed analytical system was used in combination with two other immunoenzymatic assays for brassinosteroids to determine individual compounds of this series. In addition, a direct method of brassinosteroid analysis was proposed. It has the advantage of requiring no sample pretreatment steps such as extraction with organic solvents and chromatography.
Assuntos
Brassinosteroides/análise , Camomila/química , Ensaio de Imunoadsorção Enzimática , Animais , Bovinos , Peroxidase do Rábano Silvestre/química , Peroxidase do Rábano Silvestre/metabolismo , Cavalos , Conformação Molecular , Coelhos , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismoRESUMO
Azide and phosphoramidite functions were found to be compatible within one molecule and stable for months in solution kept frozen at -20 °C. An azide-carrying phosphoramidite was used for direct introduction of multiple azide modifications into synthetic oligonucleotides. A series of azide-containing oligonucleotides were modified further using click reactions with alkynes.
Assuntos
Azidas/síntese química , Oligonucleotídeos/síntese química , Compostos Organofosforados/síntese química , Alcinos/química , Azidas/química , Química Click , Estrutura Molecular , Oligonucleotídeos/química , Compostos Organofosforados/químicaRESUMO
Oxysterols and neurosteroids are important signaling molecules produced by monooxygenases of the cytochrome P450 family that realize their effect through nuclear receptors. CYP7B1 catalyzes the 6- or 7-hydroxylation of both steroids and oxysterols and thus is involved in the metabolism of neurosteroids and bile acid synthesis, respectively. The dual physiological role of CYP7B1 is evidenced from different diseases, liver failure and progressive neuropathy, caused by enzyme malfunction. Here we present biochemical characterization of CYP7B1 at the molecular level to understand substrate specificity and susceptibility to azole drugs. Based on our experiments with purified enzyme, the requirements for CYP7B1 hydroxylation of steroid molecules are as follows: C5 hydrogen in the α-configuration (or double bond at C5), a polar group at C17, a hydroxyl group at C3, and the absence of the hydroxyl group at C20-C24 in the C27-sterol side chain. 21-hydroxy-pregnenolone was identified as a new substrate, and overall low activity toward pregnanes could be related to the increased potency of 7-hydroxy derivatives produced by CYP7B1. Metabolic conversion (deactivation) of oxysterols by CYP7B1 in a reconstituted system proceeds via two sequential hydroxylations. Two mutations that are found in patients with diseases, Gly57Arg and Phe216Ser, result in apo-P450 (devoid of heme) protein formation. Our CYP7B1 homology model provides a rationale for understanding clinical mutations and relatively broad substrate specificity for steroid hydroxylase.
Assuntos
Esteroide Hidroxilases/química , Esteroide Hidroxilases/metabolismo , 17-alfa-Hidroxipregnenolona/metabolismo , Substituição de Aminoácidos , Azóis/metabolismo , Domínio Catalítico , Família 7 do Citocromo P450 , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espectrofotometria , Esteroide Hidroxilases/genética , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
The Diels-Alder cycloaddition of nitroethylene to some androsta-14,16-dien-17-yl acetates has been studied. The addition occurs stereoselectively, giving predominantly head-to-head-adduct. 14ß-Cyanomethyl steroids were obtained via the reductive cleavage reaction of bridged nitro compounds. The structures of the new compounds have been fully characterized by 2D NMR and tandem mass-spectrometry methods.
Assuntos
Androstanos/química , Reação de Cicloadição , Etilenos/química , Indicadores e Reagentes , Espectroscopia de Ressonância MagnéticaRESUMO
Hydrolysis of 3-methoxy-16alpha-nitro-14,17-ethenoestra-1,3,5(10)-trien-17beta-yl acetate under weakly basic conditions leads to formation of 3-methoxy-2'-oxopyrrolidino-[4',5':14beta,15beta]-estra-1,3,5 (10)-trien-17-one, the structure of which has been confirmed by X-ray analysis and some chemical transformations. The reactivity of 3-methoxy-16alpha-nitro-14,17-ethanoestra-1,3,5(10)-trien-17beta-yl acetate under various conditions of basic hydrolysis has been investigated. The derived compounds have been identified by means of NMR spectroscopy and X-ray analysis.
Assuntos
Estrona/análogos & derivados , Pirrolidinas/química , Estrona/síntese química , Estrona/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The ability of a number of hemeproteins to oxidize the flavonoid quercetin has been shown. It was found that quercetin undergoes chemical modification in the presence of cytochrome c, myoglobin, and hemoglobin but not cytochrome b(5). In the range of investigated proteins the most effective oxidant appears to be cytochrome c. Chromatographic analysis of the reaction mixture revealed a number of quercetin oxidation products. The main oxidation product was purified and characterized by means of LC-MS and NMR analyses. It has a dimeric structure similar to the product of quercetin oxidation by horseradish peroxidase and is formed during radical-driven reactions. Our results indicate that a number of hemeproteins can react and modify biologically active flavonoids. However, these reactions might also lead to the generation of active species with deleterious consequences for the cellular macromolecules.