RESUMO
Oxetane synthase (TmCYP1), a novel cytochrome P450 enzyme from Taxus×media cell cultures, has been functionally characterized to efficiently catalyse the formation of the oxetane ring in tetracyclic taxoids. Transient expression of TmCYP1 in Nicotiana benthamiana using 2α,5α,7ß,9α,10ß,13α-hexaacetoxytaxa-4(20),11(12)-diene (1) as a substrate led to the production of a major oxetane derivative, 1ß-dehydroxybaccatin IV (1 a), and a minor 4ß,20-epoxide derivative, baccatin I (1 b). However, feeding the substrate decinnamoyltaxinine J (2), a 5-deacetylated derivative of 1, yielded only 5α-deacetylbaccatin I (2 b), a 4ß,20-epoxide. A possible reaction mechanism was proposed on the basis of substrate-feeding, 2H and 18O isotope labelling experiments, and density functional theory calculations. This reaction could be an intramolecular oxidation-acetoxyl rearrangement and the construction of the oxetane ring may occur through a concerted process; however, the 4ß,20-epoxide might be a shunt product. In this process, the C5-O-acetyl group in substrate is crucial for the oxetane ring formation but not for the 4(20)-epoxy ring formation by TmCYP1. These findings provide a better understanding of the enzymatic formation of the oxetane ring in paclitaxel biosynthesis.
Assuntos
Sistema Enzimático do Citocromo P-450 , Éteres Cíclicos , Paclitaxel , Sistema Enzimático do Citocromo P-450/metabolismo , Paclitaxel/biossíntese , Paclitaxel/química , Paclitaxel/metabolismo , Éteres Cíclicos/química , Éteres Cíclicos/metabolismo , Taxus/enzimologia , Taxus/metabolismo , Biocatálise , Nicotiana/metabolismo , Nicotiana/enzimologia , Estrutura MolecularRESUMO
Short-chain dehydrogenase/reductases (SDRs) belong to the NAD(P)(H)-dependent oxidoreductase superfamily, which have various functions of catalyzing oxidation/reduction reactions and have been generally used as powerful biocatalysts in the production of pharmaceuticals. In this study, ScSDR1 and ScSDR2, two new SDRs have been identified and characterized from Stachybotrys chartarum 3.5365. Substrate scope investigation revealed that both of the enzymes possessed the ability to oxidize ß-OH to ketone specifically, and exhibited substrate promiscuity and high stereo-selectivity for efficiently catalyzing the structurally different prochiral ketones to chiral alcohols. These findings not only suggest that ScSDR1 and ScSDR2 might be potent synthetic tools in drug research and development, but also provide good examples for further engineered enzymes with higher efficiency and stereo-selectivity.
Assuntos
Redutases-Desidrogenases de Cadeia Curta , Stachybotrys , Oxirredutases , Catálise , Álcoois/químicaRESUMO
CsCTS, a new diterpene synthase from Cephalotaxus sinensis responsible for forming cephalotene, the core skeleton of cephalotane-type diterpenoids with a highly rigid 6/6/5/7 tetracyclic ring system, was functionally characterized. The stepwise cyclization mechanism is proposed mainly based on structural investigation of its derailment products, and further demonstrated through isotopic labeling experiments and density functional theory calculations. Homology modeling and molecular dynamics simulation combined with site-directed mutagenesis revealed the critical amino acid residues for the unique carbocation-driven cascade cyclization mechanism of CsCTS. Altogether, this study reports the discovery of the diterpene synthase that catalyzes the first committed step of cephalotane-type diterpenoid biosynthesis and delineates its cyclization mechanism, laying the foundation to decipher and artificially construct the complete biosynthetic pathway of this type diterpenoids.
Assuntos
Diterpenos , Diterpenos/química , Diterpenos/metabolismo , Ciclização , Catálise , Modelos Moleculares , Mutagênese Sítio-Dirigida , Sítios de LigaçãoRESUMO
With the purpose to improve antiproliferative activity, 26 new betulonic acid-diazine derivatives were designed and synthesized from betulinic acid. The anticancer activity of these semi-synthetic compounds was evaluated by MTT assay in both tumor cell lines and normal cell line. The results indicated that majority of new compounds exhibited improved antitumor activity compared with the parent compound betulonic acid. Compound BoA2C, in particular, had the most significant action with IC50 value of 3.39 µM against MCF-7 cells, while it showed lower cytotoxicity on MDCK cell line than cisplatin. Furthermore, we discovered that BoA2C strongly increased MCF-7 cell damage mostly by influencing arginine and fatty acid metabolism. In addition, the structure-activity relationships were briefly discussed. The results of this study suggested that the introduction of different diazines at C-28 could selectively inhibit different kinds of cancer cells and might be an effective way to synthesize potent anticancer lead compound from betulonic acid.
RESUMO
Hederagenin (He) is a novel triterpene template for the development of new antitumor compounds. In this study, 26 new Heâ»pyrazine derivatives were synthetized in an attempt to develop potent antitumor agents; they were screened for in vitro cytotoxicity against tumor and non-tumor cell lines. The majority of these derivatives showed much stronger cytotoxic activity than He. Remarkably, the most potent was compound 9 (half maximal inhibitory concentration (IC50) was 3.45 ± 0.59 µM), which exhibited similar antitumor activities against A549 (human non-small-cell lung cancer) as the positive drug cisplatin (DDP; IC50 was 3.85 ± 0.63 µM), while it showed lower cytotoxicity on H9c2 (murine heart myoblast; IC50 was 16.69 ± 0.12 µM) cell lines. Compound 9 could induce the early apoptosis and evoke cell-cycle arrest at the synthesis (S) phase of A549 cells. Impressively, we innovatively introduced the method of cluster analysis modeled as partial least squares discriminant analysis (PLS-DA) into the structureâ»activity relationship (SAR) evaluation, and SAR confirmed that pyrazine had a profound effect on the antitumor activity of He. The present studies highlight the importance of pyrazine derivatives of He in the discovery and development of novel antitumor agents.
Assuntos
Desenho de Fármacos , Ácido Oleanólico/análogos & derivados , Pirazinas/síntese química , Pirazinas/toxicidade , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Morte Celular , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Análise por Conglomerados , Análise Discriminante , Humanos , Concentração Inibidora 50 , Análise dos Mínimos Quadrados , Ácido Oleanólico/síntese química , Ácido Oleanólico/química , Ácido Oleanólico/toxicidade , Análise de Componente Principal , Pirazinas/química , Coloração e Rotulagem , Relação Estrutura-AtividadeRESUMO
A new series of ligustrazine-cinnamon acid derivatives had been designed and synthesized as potential neuro-protective agents. Among the derivatives, 3a exhibited the promising neuroprotective activity (EC50â¯=â¯3.68⯵M). Moreover, with the deep research of the drug pathway, it (the further mechanism researches) suggested compound 3a could inhibit the apoptosis of injured PC12 cells via blocking the mitochondria apoptosis pathway including up-regulation the ratio of Bcl-2/Bax, down-regulation the expression of cytochrome-c (Cyt-c) and inhibition of the activity of caspase-9 and -3. In addition, the structure-activity relationships (SARs) of novel compounds were also discussed.
Assuntos
Apoptose/efeitos dos fármacos , Cinamatos/farmacologia , Fármacos Neuroprotetores/farmacologia , Pirazinas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cinamatos/química , Cobalto/farmacologia , Relação Dose-Resposta a Droga , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Células PC12 , Pirazinas/química , Ratos , Relação Estrutura-AtividadeRESUMO
A novel hepatoprotective oleanolic acid derivative, 3-oxours-oleana-9(11), 12-dien-28-oic acid (Oxy-Di-OA), has been reported. In previous studies, we found that Oxy-Di-OA presented the anti-HBV (Hepatitis B Virus) activity (IC50 = 3.13 µg/mL). Remarkably, it is superior to lamivudine in the inhibition of the rebound of the viral replication rate. Furthermore, Oxy-Di-OA showed good performance of anti-HBV activity in vivo. Some studies showed that liver fibrosis may affiliate with HBV gene mutations. In addition, the anti-hepatic fibrosis activity of Oxy-Di-OA has not been studied. Therefore, we evaluated the protective effect of Oxy-Di-OA against carbon tetrachloride (CCl4)-induced liver injury in rats. Daily intraperitoneally administration of Oxy-Di-OA prevented the development of CCl4-induced liver fibrosis, which was evidenced by histological study and immunohistochemical analysis. The entire experimental protocol lasted nine weeks. Oxy-Di-OA significantly suppressed the increases of plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels (p < 0.05). Furthermore, Oxy-Di-OA could prevent expression of transforming growth factor ß1 (TGF-ß1). It is worth noting that the high-dose group Oxy-Di-OA is superior to bifendate in elevating hepatic function. Compared to the model group, Oxy-Di-OA in the high-dose group and low-dose group can significantly reduce the liver and spleen indices (p < 0.05). The acute toxicity test showed that LD50 and a 95% confidence interval (CIs) value of Oxy-Di-OA were 714.83 mg/kg and 639.73-798.73 mg/kg via intraperitoneal injection in mice, respectively. The LD50 value of Oxy-Di-OA exceeded 2000 mg/kg via gavage in mice. In addition, a simple and rapid high performance liquid chromatography-ultraviolet (HPLC-UV) method was developed and validated to study the pharmacokinetic characteristics of the compound. After single-dose oral administration, time to reach peak concentration of Oxy-Di-OA (Cmax = 8.18 ± 0.66 µg/mL) was 10 ± 2.19 h; the elimination half-life and area under the concentration-time curve from t = 0 to the last time of Oxy-Di-OA was 2.19 h and 90.21 µg·h/mL, respectively.
Assuntos
Cirrose Hepática/tratamento farmacológico , Ácido Oleanólico/análogos & derivados , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Tetracloreto de Carbono/toxicidade , Feminino , Cirrose Hepática/etiologia , Masculino , Camundongos , Ácido Oleanólico/administração & dosagem , Ácido Oleanólico/efeitos adversos , Ácido Oleanólico/química , Ácido Oleanólico/farmacologia , Ácido Oleanólico/uso terapêutico , Ratos , Ratos Sprague-Dawley , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The lead compound TBA, 3ß-Hydroxy-lup-20(29)-ene-28-oic acid-3, 5, 6-trimethylpyrazin-2-methyl ester, which exhibited promising antitumor activity and induced tumor cell apoptosis in various cancer cell lines, had previously been reported. Moreover, reports have revealed that the introduction of amino acid to betulinic acid could improve selective cytotoxicity as well as water solubility. Thus, a series of novel TBA amino acid and dipeptide derivatives were designed, synthesized and screened for selective cytotoxic activity against five cancer cell lines (HepG2, HT-29, Hela, BCG-823 and A549) and the not malignant cell line MDCK by standard MTT assay. Most of the tested TBA-amino acid and dipeptide analogues showed stronger anti-proliferative activity against all tested tumor cell lines than TBA. Among them, BA-25 exhibited the greatest cytotoxic activity on tumor cell lines (mean IC50 = 2.31 ± 0.78 µM), that was twofold than the positive drug cisplatin (DDP), while it showed lower cytotoxicity on MDCK cell line than DDP. Further cell apoptosis analyses indicated BA-25-induced apoptosis was associated with loss of mitochondrial membrane potential and increase of intracellular free Ca2+ concentration.