Advances in exploring alternative Taxol sources

Acta Pharmaceutica Sinica B

Wang

et al. 2019

Taxol is a “blockbuster” antitumor drug produced by Taxus species with extremely low amount, while its analogue 7- β -xylosyl-10-deacetyltaxol is generally much higher in the plants. Both the fungal enzymes LXYL-P1−1 and LXYL-P1−2 can convert 7- β -xylosyl-10-deacetyltaxol into 10-deacetyltaxol for Taxol semi-synthesis. Of them, LXYL-P1−2 is twice more active than LXYL-P1−1, but there are only 11 significantly different amino acids in terms of the polarity and acidic-basic properties between them. In this study, single and multiple site-directed mutations at the 11 sites from LXYL-P1−1 to LXYL-P1−2 were performed to define the amino acids with upward bias in activities and to acquire variants with improved catalytic properties. Among all the 17 mutants, E12 (A72T/V91S) was the most active and even displayed 2.8- and 3-fold higher than LXYL-P1−2 on β -xylosidase and β -glucosidase activities. The possible mechanism for such improvement was proposed by homology modeling and molecular docking between E12 and 7- β -xylosyl-10-deacetyltaxol. The recombinant yeast GS115-P1E12-7 was constructed by introducing variant E12 , the molecular chaperone gene pdi and the bacterial hemoglobin gene vhb . This engineered yeast rendered 4 times higher biomass enzyme activity than GS115-3.5K-P1−2 that had been used for demo-scale fermentation. Thus, GS115-P1E12-7 becomes a promising candidate to replace GS115-3.5K-P1−2 for industrial purpose.

Section: Introductionmentioning

confidence: 99%

Combinatorial mutation on the β-glycosidase specific to 7-β-xylosyltaxanes and increasing the mutated enzyme production by engineering the recombinant yeast

Chen

Acta Pharmaceutica Sinica B

Wang

et al. 2019

“…With the development of its new indications and formulations, its clinical application has also expanded. Both the domestic and overseas market demand for paclitaxel is still increasing, although the natural concentrations in yew plants is extremely low [ 4 , 5 ]. 7-β-Xylosyltaxanes, such as 7-β-xylosyl-10-deacetyltaxol (XDT), are the analogues of paclitaxel produced by yew plants ( Taxus species), which are generally discarded during paclitaxel extraction, leading to both resource waste and potential environmental pollution [ 4 , 6 ].…”

Section: Introductionmentioning

confidence: 99%

Improving the Catalytic Property of the Glycoside Hydrolase LXYL-P1–2 by Directed Evolution

Chen

et al. 2017

Molecules

The glycoside hydrolase LXYL-P1–2 from Lentinula edodes can specifically hydrolyze 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes for the semi-synthesis of paclitaxel. In order to improve the catalytic properties of the enzyme, we performed error-prone PCR to construct the random mutant library of LXYL-P1–2 and used the methanol-induced plate method to screen the mutants with improved catalytic properties. Two variants, LXYL-P1–2-EP1 (EP1, S91D mutation) and LXYL-P1–2-EP2 (EP2, T368E mutation), were obtained from the library and exhibited 17% and 47% increases in their catalytic efficiencies on 7-β-xylosyl-10-deacetyltaxol. Meanwhile, compared with LXYL-P1–2, EP1 and EP2 showed elevated stabilities in the range of pH ≥ 6 conditions. After treatment at pH 12 for 48 h, EP1 and EP2 retained 77% and 63% activities, respectively, while the wild-type only retained 33% activity under the same condition. Molecular docking results revealed that the S91D mutation led to a shorter distance between the R-chain and the substrate, while the T368E mutation increased negative charge at the surface of the enzyme, and may introduce alterations of the loop near the active pocket, both of which may result in improved stabilities and catalytic activities of enzymes. This study provides a practical directed evolution method for exploring catalytically improved glycoside hydrolase.

“…Larotaxel has a much lower affinity for P-glycoprotein 1 than docetaxel and is shown to be active in cell lines resistant to doxorubicin, vinblastine, paclitaxel and docetaxel [7] . The antitumor activity in non-small cell lung cancer, the reproducible profile of toxicity and the ability to cross the blood-brain barrier make larotaxel worthy of further disease-oriented clinical development [8] , [9] , [10] . Since new molecular entities are evaluated in clinical development, the understanding, identification and control of degradation products or impurities in drug substances are essential, which can also assist the development of proper formulations and storage conditions of the drugs [11] , [12] .…”

Section: Introductionmentioning

confidence: 99%

Degradation kinetics of larotaxel and identification of its degradation products in alkaline condition

Journal of Pharmaceutical Analysis

Liu

Shi

et al. 2017

Larotaxel, a new taxane compound prepared by partial synthesis from 10-deacetyl baccatin III, is active against tumors. In this research, a selective LC–MS method was developed and validated for the study of degradation kinetics of larotaxel, which was carried out in aqueous solutions with different pH (1.5, 3.0, 5.0, 6.5, 7.4, 9.0, 10 and 11.0) and temperature (0, 25, 37 and 45 °C). The linear range was 0.5–25 μg/mL, the intra- and inter-day precisions were less than 7.0%, and accuracy ranged from 97.4–104.5% for each analyte. The observed rate obtained by measuring the remaining intact larotaxel was shown to follow first-order kinetics. The activation energies for degradation were 126.7 and 87.01 kJ/mol at pH 1.5 and 11, respectively. Although larotaxel was stable in pH 5, 6.5 and 7.4 buffers at 37 °C for 24 h during our study, increasing or decreasing the pH of the solutions would decrease its stabilities. Moreover, three main degradation products in alkaline condition were separated by HPLC and identified by Q–TOF–MS. The three degradation products were confirmed as 10-deacetyl larotaxel, 7, 8-cyclopropyl baccatin Ⅲ and 10-deacetyl-7, 8-cyclopropyl baccatin Ⅲ.