Identification of potential modulators of osteosarcoma metastasis by high-throughput cellular screening of natural products.

A high-throughput screening assay was developed and applied to a large library of natural product extract samples, in order to identify compounds which preferentially inhibited the in vitro 2D growth of a highly metastatic osteosarcoma cell line (MG63.3) compared to a cognate parental cell line (MG63) with low metastatic potential. Evaluation of differentially active natural product extracts with bioassay-guided fractionation led to the identification of lovastatin (IC50  = 11 µm) and the limonoid toosendanin (IC50  = 26 nm). Other statins and limonoids were then tested, and cerivastatin was identified as a particularly potent (IC50  < 0.1 µm) and selective agent. These compounds potently and selectively induced apoptosis in MG63.3 cells, but not MG63. Assays with other cell pairs were used to examine the generality of these results. Statins and limonoids may represent unexplored opportunities for development of modulators of osteosarcoma metastasis. As cerivastatin was previously approved for clinical use, it could be considered for repurposing in osteosarcoma, pending validation in further models.

Development of One Pot Strategy for Hyper Production and In Vivo Evaluation of Lovastatin.

The aim of this project was to improve the Aspergillus terreus strain and pretreatment of sugarcane bagasse as carrier substrate for bulk production of lovastatin, a cholesterol-lowering drug, in solid state fermentation. Sugarcane bagasse was treated with alkali (1-3% NaOH) for the conversion of complex polysaccharides into simple sugars for better utilization of carrier substrate by microorganism for maximum lovastatin production. Ethidium bromide (time of exposure 30-180 min) was used to induce mutation in Aspergillus terreus and the best mutant was selected on the basis of inhibition zone appeared on petri plates. Fermented lovastatin was quantified by high-performance liquid chromatography. The fermented lovastatin, produced by parent and mutant Aspergillus terreus strain, was checked on body weight, blood glucose and serum cholesterol, ALT, AST, HDL-C, LDL-C, TG and TC levels of rats for their cholesterol lowering capacity. Our results indicate that selected strain along with 2% NaOH treated sugar cane bagasse was best suitable for bulk production of lovastatin by fermentation and fermented lovastatin effectively lower the cholesterol level of rats.

Weighted gene co-expression network analysis and connectivity map identifies lovastatin as a treatment option of gastric cancer by inhibiting HDAC2.

OBJECTIVE: This study aimed to identifying and validating therapeutic compounds which might have positive effects on patients with gastric cancer (GC) based on weighted gene co-expression network analysis (WGCNA) and connectivity map (CMap). METHODS: We performed WGCNA to gain insights into the molecular aspects of GC. Raw microarray datasets (including 132 samples) were downloaded from the Gene Expression Omnibus (GEO) website. We utilized the WGCNA to identify the coexpressed genes (modules) and modular hub genes after non-specific filtering. Furthermore, these differentially expressed genes were submitted to CMap analysis to identify candidate therapeutic compounds for GC. In experimental part, cell growth inhibition was evaluated by Cell Counting Kit-8 (CCK-8) and colony formation assays. Tumor growth was assessed using nude mice with xenografts established in vivo. QRT-PCR and western blot were used for determination of HDAC2 expression level and immunohistochemistry was performed to quantify HDAC2 in gastric tumor samples. RESULTS: Through WGCNA and CMap analysis, we found two potential therapeutic compounds, the valproic acid (VPA), which is the histone deacetylase (HDAC) inhibitor and lovastatin. HDAC2 was overexpressed in gastric cancer cell lines including AGS, BGC-823, NCI-N87 and MKN28. Dose-dependent inhibition of gastric cancer cells by VPA and lovastatin was verified in vitro. Apoptosis of GC cells was induced after treatment with VPA and lovastatin through suppressing HDAC2 expression. Furthermore, the inhibition of VPA with cisplatin and lovastatin with cisplatin were also dose-dependent and cisplatin exhibited synergistic effects. In the xenografts, similar results were found. CONCLUSION: WGCNA was able to identify significant groups of genes associated with cancer prognosis. Moreover, analysis of gene expression signature using CMap is a powerful way to explore potential therapeutics for human diseases. For treating GC, lovastatin may be a potential drug.

Uncovering the repertoire of fungal secondary metabolites: From Fleming's laboratory to the International Space Station.

Fungi produce a variety of secondary metabolites (SMs), low-molecular weight compounds associated with many potentially useful biologic activities. The examples of biotechnologically relevant fungal metabolites include penicillin, a ß-lactam antibiotic, and lovastatin, a cholesterol-lowering drug. The discovery of pharmaceutical lead compounds within the microbial metabolic pools relies on the selection and biochemical characterization of promising strains. Not all SMs are produced under standard cultivation conditions, hence the uncovering of chemical potential of investigated strains often requires the use of induction strategies to awake the associated biosynthetic genes. Triggering the secondary metabolic pathways can be achieved through the variation of cultivation conditions and growth media composition. The alternative strategy is to use genetic engineering to activate the respective genomic segments, e.g. by the manipulation of regulators or chromatin-modifying enzymes. Recently, whole-genome sequencing of several fungi isolated from the Chernobyl accident area was reported by Singh et al. (Genome Announc 2017; 5:e01602-16). These strains were selected for exposure to microgravity at the International Space Station. Biochemical characterization of fungi cultivated under extreme conditions is likely to provide valuable insights into the adaptation mechanism associated with metabolism and, possibly, a catalog of novel molecules of potential pharmaceutical importance.

Comparison of the elution characteristics of individual forms of lovastatin in both isocratic and gradient modes and HPLC-PDA method development for pure and fermentation-derived lovastatin.

The elution characteristics of lovastatin were studied by varying the composition of mobile phase in both isocratic and gradient elution modes to comprehend the role of organic modifier and acidifier on the overall analysis time and retention time of individual forms of lovastatin. Acetonitrile has influenced on the overall analysis time, whereas the acidifier determines the retention time of hydroxy acid form of lovastatin and the retention time gap between the individual forms. A combination of acetonitrile and 0.1% trifluoroacetic acid (TFA) (60:40, v/v) in isocratic elution mode eluted both hydroxy acid and lactone forms of lovastatin at 4.5 and 5.4 min, respectively. This appears to be a better approach for the separation of pharmaceutical and clinical lovastatin samples. At isocratic elution mode, a mixture of acetonitrile and either 0.05% TFA or 0.1% H3PO4 of 60:40 (v/v) has eluted both hydroxy acid and lactone forms of lovastatin at 10 ± 0.5 and 17 ± 0.5 min, respectively. This is suitable for the fermentation-derived samples or for the complex mixtures of structural analogs. The fermentation broth (pH not adjusted) extracted with ethyl acetate at a ratio of 1:1 (v/v) at 60°C for 30 min was the optimal extraction condition for lovastatin.

Lovastatin Analogues from the Soil-Derived Fungus Aspergillus sclerotiorum PSU-RSPG178.

Three new lovastatin analogues (1, 4, and 5) together with four known lovastatin derivatives, namely, lovastatin (2), α,ß-dehydrolovastatin (3), α,ß-dehydrodihydromonacolin K (6), and α,ß-dehydro-4a,5-dihydromonacolin L (7), were isolated from the soil-derived fungus Aspergillus sclerotiorum PSU-RSPG178. Their structures were established using spectroscopic evidence. Compound 5 exhibited the most potent activity against HMG-CoA reductase, with an IC50 value of 387 µM. In addition, the present study indicated the direct interaction of compound 5 with HMG-CoA reductase. Compound 5 was considered to be noncytotoxic against noncancerous Vero cells, with an IC50 value of 40.0 µM, whereas compound 2 displayed much stronger activity, with an IC50 value of 2.2 µM.

Lovastatin production: From molecular basis to industrial process optimization.

Lovastatin, composed of secondary metabolites produced by filamentous fungi, is the most frequently used drug for hypercholesterolemia treatment due to the fact that lovastatin is a competitive inhibitor of HMG-CoA reductase. Moreover, recent studies have shown several important applications for lovastatin including antimicrobial agents and treatments for cancers and bone diseases. Studies regarding the lovastatin biosynthetic pathway have also demonstrated that lovastatin is synthesized from two-chain reactions using acetate and malonyl-CoA as a substrate. It is also known that there are two key enzymes involved in the biosynthetic pathway called polyketide synthases (PKS). Those are characterized as multifunctional enzymes and are encoded by specific genes organized in clusters on the fungal genome. Since it is a secondary metabolite, cultivation process optimization for lovastatin biosynthesis has included nitrogen limitation and non-fermentable carbon sources such as lactose and glycerol. Additionally, the influences of temperature, pH, agitation/aeration, and particle and inoculum size on lovastatin production have been also described. Although many reviews have been published covering different aspects of lovastatin production, this review brings, for the first time, complete information about the genetic basis for lovastatin production, detection and quantification, strain screening and cultivation process optimization. Moreover, this review covers all the information available from patent databases covering each protected aspect during lovastatin bio-production.

A dispersive liquid-liquid microextraction method based on the solidification of a floating organic drop combined with HPLC for the determination of lovastatin and simvastatin in rat urine.

A dispersive liquid-liquid microextraction method based on solidification of floating organic drop combined with HPLC was developed for the determination of lovastatin and simvastatin in rat urine for the first time. 1-Dodecanol and methanol were used as the extraction and disperser solvents, respectively. Several important parameters influencing the micro-extraction efficiency were studied and systematically optimized, including the type and volume of extraction solvent and disperser solvent, extraction time, pH and salt concentration. The analytes were separated on a Kromasil C18 column at 30°C with a mobile phase of methanol and 0.2% acetic acid in water (83:17, v/v) and detected at 238 nm. Under the optimal conditions, the maximum number of enrichment factors for both analytes was 27. The linear ranges were 20.08-1004 and 20.00-1000 µg/L with the correlation coefficients ranging from 0.9990 to 0.9994 for lovastatin and simvastatin, respectively. The volume of organic solvent consumed in extraction was <0.3 mL, and the extraction time was 10 min. The newly developed environment-friendly sample pretreatment method will be a good alternative to conventional techniques, such as solid-phase extraction, liquid-liquid extraction and protein precipitation, for the HPLC determination of lovastatin and simvastatin in biological samples.

Upstream and downstream processing of lovastatin by Aspergillus terreus.

The present study describes the enhanced production and purification of lovastatin by Aspergillus terreus in submerged batch fermentation. The enhancement of lovastatin production from A. terreus was attempted by random mutagenesis using ultraviolet radiations and nitrous acid. UV mutants exhibited increased efficiency for lovastatin production as compared with nitrous acid mutants. Among all the mutants developed, A. terreus UV-4 was found to be the hyper producer of lovastatin. This mutant gave 3.5-fold higher lovastatin production than the wild culture of A. terreus NRRL 265. Various cultural conditions were also optimized for hyper-producing mutant strain. 5 % glucose as carbon source, 1.5 % corn steep liquor as nitrogen source, initial pH value of 6, 120 h of incubation period, and 28 °C of incubation temperature were found as best parameters for higher lovastatin production in shake flasks. Production of lovastatin by wild and mutant strains of A. terreus was also scaled up to laboratory scale fermentor. The fermentation process was conducted at 28 °C, 200 rpm agitation, and 1vvm air flow rate without pH control. After the optimization of cultural conditions in 250 ml Erlenmeyer flasks and scaling up to laboratory scale fermentor, the mutant A. terreus UV-4 gave eightfold higher lovastatin production (3249.95 µg/ml) than its production by wild strain in shake flasks. Purification of lovastatin was carried out by solvent extraction method which yielded 977.1 mg/l of lovastatin with 98.99 % chromatographic purity and 26.76 % recovery. The crystal structure of lovastatin was determined using X-ray diffraction analysis which is first ever reported.

Bacterial production of the fungus-derived cholesterol-lowering agent mevinolin.

Forty-five strains from two different species (Salinispora arenicola and Salinispora pacifica) were isolated from three different marine sponge species in the Great Barrier Reef region of Australia. We found that two of the strains of Salinispora arenicola (MV0335 and MV0029) produced mevinolin, a fungus-derived cholesterol-lowering agent. Compound structure was determined using an integrated approach: (a) high performance liquid chromatography-quadrupole time-of-flight-mass spectrometric analysis with multimode ionization (electrospray ionization and atmospheric pressure chemical ionization) and fast polarity switching; and (b) database searching and matching of monoisotopic masses, retention times and mass spectra of the precursor and product ions of the compounds of interest and the authentic reference standards thereof.

Evaluation of a method using high performance liquid chromatography with ultraviolet detection for the determination of statins in macromycetes of the genus Pleurotus cultivated by fermentation processes.

The applicability of high-performance liquid chromatography with ultraviolet light (HPLC-UV) for the determination of the presence of statins in macromycetes of the genus Pleurotus was analyzed. The fungi were obtained by liquid-state fermentation (LSF) using unconventional sources of carbon as substrates and solid-state fermentation (SSF) employing agro industrial wastes. Five statins were used as standards: lovastatin and simvastatin in the lactone form (LOVL and SIML), their corresponding hydro-acidic forms (LOVH and SIMH) and pravastatin (PRA). The following measures were evaluated: the linearity, accuracy and precision, detection limit (DL) and quantification limit (QL). The results demonstrated HPLC-UV to be an effective tool for detecting the presence of statins in extracts of LSF and SSF products. Likewise, it was hypothesized that the strains that were used for the study do not produce statins. This finding highlights the importance of continuing to evaluate other strains of the same genus by using techniques such as HPLC to first separate sufficient quantities of the compounds that were detected using the standard technique but that did not match the retention time (tR) of any of the standards used.

Simultaneous enrichment of deglycosylated ginsenosides and monacolin K in red ginseng by fermentation with Monascus pilosus.

To improve its bioavailability and pharmacological effects in humans, red ginseng was fermented with a newly isolated fungus, Monascus pilosus KMU103. Most of the ginsenosides were converted to deglycosylated ginsenocides, such as Rh(1), Rh(2), and Rg(3). The total amount of ginsenosides Rh(1), Rh(2), and Rg(3) was 838.7 mg/kg in the red ginseng, and increased to 4,117 mg/kg after 50 L fermentation in 13% red ginseng and 2% glucose. In addition, the Monascus-fermented red ginseng contained 3,089 mg/kg of monacolin K, one of the metabolites produced by Monascus known to reduce cholesterol in the blood. This newly developed Monascus-fermented red ginseng should result in improved health effects, not only by biotransforming gisenosides to deglycosylated ones but also by creating additional bioactive compounds.

Accelerated solvent extraction of monacolin K from red yeast rice and purification by high-speed counter-current chromatography.

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L(9) (3)(3). The optimum extraction conditions were determined as follows: extraction temperature 120°C, static extraction time 7min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (8:2:5:5, v/v/v/v). From 300mg of crude extract, 51.2mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and (1)H NMR.

Compactin production studies using Penicillium brevicompactum under solid-state fermentation conditions.

In the present study, compactin production by Penicillium brevicompactum WA 2315 was optimized using solid-state fermentation. The initial one factor at a time approach resulted in improved compactin production of 905 microg gds(-1) compared to initial 450 microg gds(-1). Subsequently, nutritional, physiological, and biological parameters were screened using fractional factorial and Box-Behnken design. The fractional factorial design studied inoculum age, inoculum volume, pH, NaCl, NH(4)NO(3), MgSO(4), and KH(2)PO(4). All parameters were found to be significant except pH and KH(2)PO(4). The Box-Behnken design studied inoculum volume, inoculum age, glycerol, and NH(4)NO(3) at three different levels. Inoculum volume (p = 0.0013) and glycerol (p = 0.0001) were significant factors with greater effect on response. The interaction effects were not significant. The validation study using model-defined conditions resulted in an improved yield of 1,250 microg gds(-1) compactin. Further improvement in yield was obtained using fed batch mode of carbon supplementation. The feeding of glycerol (20% v/v) on day 3 resulted in further improved compactin yield of 1,406 microg gds(-1). The present study demonstrates that agro-industrial residues can be successfully used for compactin production, and statistical experiment designs provide an easy tool to improve the process conditions for secondary metabolite production.

Production and purification of statins from Pleurotus ostreatus (Basidiomycetes) strains.

Pleurotus ostreatus strains were cultured in liquid medium and on wheat straw. The yields of lovastatin were compared.

[Concentration on Diapak C 16 capsules of lovastatin, mevinolinic acid and other inhibitors of biosynthesis of sterins produced by Penicillium citrinum 89]. / Kontsentrirovanie lovastatina, mevinolinovoi kisloty i drugikh ingibitorov biosinteza sterinov, obrazuemykh Penicillum citrinum 89, na patronakh Diapak C 16.

The method of lovastatine and mevinolinic acid known as competitive inhibitors of HMG-CoA-reductase and produced by micromycetes was elaborated. The inhibitors from diluted water solutions were fully absorbed on Diapak C16 patrons. The rate of inhibitors elution from the patrones was more than 95 per cent. Patrons may be used for concentration of lovastatine group inhibitors from the culture media. Inhibitors synthesis by the Penicillium citrinum 89 was investigated in dynamics with the use of Diapak C16 patrones. It was shown that UV-spectrum of inhibitor produced by P. citrinum 89 was identical with compactin spectrum and had absorbance maximum at 230, 237 and 247 nm.

Production of lovastatin examined by an integrated approach based on chemometrics and DOSY-NMR.

Microbial secondary metabolites are one of the sources of therapeutic molecules in the pharmaceutical industry. Product quality and high yields of secondary metabolites are the main goals for the commercial success of a fermentation process. Our novel approach was based on the decision-tree algorithm to determine the key variables correlated with the process outcome and on DOSY-NMR to identify both co-metabolites and impurities, and it improves fermentation systems and speeds up bioprocess development. The approach has been validated in the case of lovastatin production from Aspergillus terreus.

Chromatographic purification of some 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors.

The purification of pravastatin, simvastatin and lovastatin in the sodium salt or lactone form and of mevastatin in the lactone form by reversed-phase displacement chromatography is presented. The mobile phases consisted of water or mixtures of water-methanol and water-acetonitrile. Six different displacers were successfully used. Up to 0.14 g of raw sample per gram of stationary phase was loaded on a column packed with silica-based octadecyl phase. Crude substances from 85 to 88% chromatographic purity were purified and at least 99.5% purity was achieved.