Mechanism of azole antifungal activity as determined by liquid chromatographic/mass spectrometric monitoring of ergosterol biosynthesis

A liquid chromatography/mass spectrometry (LC/MS) method for separation and characterization of ergosterol biosynthetic precursors was developed to study the effect of Posaconazole on sterol biosynthesis in fungi. Ergosterol biosynthetic precursors were characterized from their electron ionization mass spectra acquired by a normal-phase chromatography, particle beam LC/MS method. Fragment ions resulting from cleavage across the D-ring and an abundant M - 15 fragment ion were diagnostic for methyl substitution at C-4 and C-14. Comparison of the sterol profile in control and treated Candida albicans incubations showed depletion of ergosterol and accumulation of C-4 and C-14 methyl-substituted sterols following treatment with Posaconazole. These C-4 and C-14 methyl sterols are known to be incapable of sustaining cell growth. The results demonstrate that Posaconazole exerts its antifungal activity by inhibition of ergosterol biosynthesis. Furthermore, Posaconazole appears to disrupt ergosterol biosynthesis by inhibition of lanosterol 14alpha-demethylase.     

Separation and purification of lanosterol,dihydrolanosterol, and cholesterol from lanolin by high‐performance counter‐current chromatography dual‐mode elution method

Lanosterol is a potential drug for cataracts treatment, which can reverse the aggregation of intracrystalline proteins. The low concentration in lanolin calls for high‐performance separation methods. In this study, a counter‐current chromatography dual‐mode elution method was developed for the first time to separate and purify lanosterol from hexane extract of lanolin after saponification, in which the column was first eluted with the lower phase as mobile phase in head‐to‐tail mode, followed by the upper phase in the tail‐to‐head mode. High purity of lanosterol, dihydrolanosterol, and cholesterol can be obtained simultaneously. A solvent system composed of n‐heptane/acetonitrile/ethyl acetate (5:5:1, v/v/v) was selected and optimized via partition coefficient determination. Compounds such as 111 mg lanosterol, 84 mg dihydrolanosterol, and 183 mg cholesterol with high purity of 99.77, 95.71, and 91.43%, respectively, analyzed by high‐performance liquid chromatography were obtained within 80 min from 700 mg crude extract from 1.78 g lanolin. The method was also used to improve the purity of commercial lanosterol product from 66.97 to above 99%. Counter‐current chromatography could serve as a potential and powerful technique for commercial production of highly pure lanosterol.     

Human Lanosterol 14-Alpha Demethylase (CYP51A1) Is a Putative Target for Natural Flavonoid Luteolin 7,3′-Disulfate

Widespread pathologies such as atherosclerosis, metabolic syndrome and cancer are associated with dysregulation of sterol biosynthesis and metabolism. Cholesterol modulates the signaling pathways of neoplastic transformation and tumor progression. Lanosterol 14-alpha demethylase (cytochrome P450(51), CYP51A1) catalyzes one of the key steps in cholesterol biosynthesis. The fairly low somatic mutation frequency of CYP51A1, its druggability, as well as the possibility of interfering with cholesterol metabolism in cancer cells collectively suggest the clinical importance of CYP51A1. Here, we show that the natural flavonoid, luteolin 7,3′-disulfate, inhibits CYP51A1 activity. We also screened baicalein and luteolin, known to have antitumor activities and low toxicity, for their ability to interact with CYP51A1. The K_d values were estimated using both a surface plasmon resonance optical biosensor and spectral titration assays. Unexpectedly, in the enzymatic activity assays, only the water-soluble form of luteolin—luteolin 7,3′-disulfate—showed the ability to potently inhibit CYP51A1. Based on molecular docking, luteolin 7,3′-disulfate binding suggests blocking of the substrate access channel. However, an alternative site on the proximal surface where the redox partner binds cannot be excluded. Overall, flavonoids have the potential to inhibit the activity of human CYP51A1 and should be further explored for their cholesterol-lowering and anti-cancer activity.     

A molecular mechanics force field for biologically important sterols

A parameterization has been performed of the biologically important sterols cholesterol, ergosterol, and lanosterol for the CHARMM27 all-atom molecular mechanics force field. An automated parameterization method was used that involves fitting the potential to vibrational frequencies and eigenvectors derived from quantum-chemical calculations. The partial charges were derived by fitting point charges to quantum-chemically calculated electrostatic potentials. To model the dynamics of the hydroxyl groups of the sterols correctly, the parameter set was refined to reproduce the energy barrier for the rotation of the hydroxyl group around the carbon connected to the hydroxyl of each sterol. The frequency-matching plots show good agreement between the CHARMM and quantum chemical normal modes. The parameters are tested in a molecular dynamics simulation of the cholesterol crystal structure. The experimental geometry and cell dimensions are well reproduced. The force field derived here is also useful for simulating other sterols such as the phytosterols sigmasterol, and campesterol, and a variety of steroids.     

Theoretical study of structural features of endo,exo double bonds and side chain in 14α‐demethylation of lanosterol

The 14α‐demethylation of biosynthetic reactions is carried out exclusively by lanosta‐8,24‐dien‐3β‐ol (lanosterol). Theoretical calculations were done to determine the importance of this structure with the Hartree–Fock scheme. It was modeled on structures similar to lanosterol with variations on the endo between C8? C9 (lanosta‐7,24‐dien‐3β‐ol and lanosta‐6,24‐dien‐3β‐ol) as compared with the exo between C24? C25 (Lanosta‐8‐en‐3β‐ol) and the size of the side chain (27‐norlanosta‐8‐en‐3β‐ol, 26,27‐dinorlanosta‐8‐en‐3β‐ol, 25,26,27‐trinorlanosta‐8‐en‐3β‐ol, 24,25,26,27‐tetranorlanosta‐8‐en‐3β‐ol, 23,24,25,26,27‐pentanorlanosta‐8‐en‐3β‐ol, 22,23,24,25,26,27‐hexanorlanosta‐8‐en‐3β‐ol). Energetic, electronic, and orbital evidence was found. The theoretical data were analyzed and were found to be important for the 14α‐demethylation, the presence of HOMO on the region C8? C9 and C14? C14¹, a negative charge on the outgoing methyl C14¹. This atomic charge varies with the size of the side chain, and with the modification of the endo and exo double bonds. The conformation of the structure of the steroid is also related to its reactivity. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2006     

Biosynthetic Mechanism of Lanosterol: Cyclization

The remarkable cyclization mechanism of the formation of the 6‐6‐6‐5 tetracyclic lanosterol (a key triterpenoid intermediate in the biosynthesis of cholesterol) from the acyclic 2,3‐oxidosqualene catalyzed by oxidosqualene cyclase (OSC) has stimulated the interest of chemists and biologists for over a half century. Herein, the elaborate, state‐of‐the‐art two‐dimensional (2D) QM/MM MD simulations have clearly shown that the cyclization of the A–C rings involves a nearly concerted, but highly asynchronous cyclization, to yield a stable intermediate with “6‐6‐5” rings followed by the ring expansion of the C‐ring concomitant with the formation of the D‐ring to yield the “6‐6‐6‐5” protosterol cation. The calculated reaction barrier of the rate‐limiting step (≈22 kcal mol^?1) is comparable to the experimental kinetic results. Furthermore all previous experimental mutagenic evidence is highly consistent with the identified reaction mechanism.     

反相高效液相色谱法分析禾谷丝核菌菌丝中麦角甾醇和羊毛甾醇的含量

采用反相高效液相色谱法,建立了检测禾谷丝核菌中麦角甾醇和羊毛甾醇含量的方法.样品经过破碎、离心、皂化后定量测定.采用DiamonsilTMC18色谱柱(250 mm×4.6 mm i.d, 5 μm i.d.),流动相为100%甲醇,流速为1 mL/min,紫外检测波长为210 nm,保留时间定性,外标法定量.麦角甾醇和羊毛甾醇的保留时间分别为21 min和27min.麦角甾醇在0.125～12.5 mg/L、羊毛甾醇在0.1～10.0 mg/L 范围内,两者峰面积与浓度均呈线性关系,相关系数分别为0.9993和0.9983.添加回收率实验表明,2种甾醇的平均添加回收率为91.8%～99.7%;相对标准偏差为2.8%～7.8%;麦角甾醇和羊毛甾醇的最小检出量分别为1.0和0.8 ng,最低检出浓度分别为0.05和0.04 mg/L.本方法准确、简便、重现性好.     

基于真菌14α-去甲基化酶的非氮唑类抗真菌先导化合物的设计、合成及其体外抗真菌活性研究

基于真菌羊毛甾醇14α-去甲基化酶(CYP51)活性位点的三维结构设计并合成了新型四氢异喹啉类抗真菌先导化合物. 体外抗真菌活性研究显示: 设计的先导化合物具有较好的抗真菌活性. 其中化合物5f和5g对于5种测试菌的抗真菌活性强于或相当于对照药物氟康唑. 先导分子通过与靶酶活性腔氨基酸残基的非共价键结合产生抗真菌作用, 避免与血红素辅基Fe原子发生配位结合, 为一类具有新作用机理的非氮唑类抗真菌先导化合物. 本研究为抗真菌药物研究提供了新的结合方式及结构类型.     

Mechanistic insights into oxidosqualene cyclizations through homology modeling

2,3-Oxidosqualene cyclases (OSC) are key enzymes in sterol biosynthesis. They catalyze the stereoselective cyclization and skeletal rearrangement of (3S)-2,3-oxidosqualene to lanosterol in mammals and fungi and to cycloartenol in algae and higher plants. Sequence information and proposed mechanism of 2,3-oxidosqualene cyclases are closely related to those of squalene-hopene cyclases (SHC), which represent functional analogs of OSCs in bacteria. SHCs catalyze the cationic cyclization cascade converting the linear triterpene squalene to fused ring compounds called hopanoids. High stereoselectivity and precision of the skeletal rearrangements has aroused the interest of researchers for nearly half a century, and valuable data on studying mechanistic details in the complex enzyme-catalyzed cyclization cascade has been collected. Today, interest in cyclases is still unbroken, because OSCs became targets for the development of antifungal and hypocholesterolemic drugs. However, due to the large size and membrane-bound nature of OSCs, three-dimensional structural information is still not available, thus preventing a complete understanding of the atomic details of the catalytic mechanism. In this work, we discuss results gained from homology modeling of human OSC based on structural information of SHC from Alicyclobacillus acidocaldarius and propose a structural model of human OSC. The model is in accordance with previously performed experimental studies with mechanism-based suicide inhibitors and mutagenesis experiments with altered activity and product specificity. Structural insight should strongly stimulate structure-based design of antifungal or cholesterol-lowering drugs.     

新型四氢萘类化合物合成及体外抗真菌活性研究

根据真菌羊毛甾醇14α-去甲基化酶的三维结构模型设计、合成了48个新型四氢萘类化合物, 所有化合物的结构经过IR, ¹H NMR和MS确证. 体外抗真菌活性研究表明所有化合物对7种临床致病真菌都有抗真菌活性, 特别是化合物18, 21, 22, 24的抗真菌活性最强. 对接研究显示设计的先导化合物与靶酶活性腔中氨基酸功能残基结合, 作用模式不同于氮唑类化合物. 结果表明新型四氢萘类化合物是一类全新结构类型的抗真菌化合物.