Cunninghamella--a microbial model for drug metabolism studies--a review

Drug metabolism studies constitute an important and necessary step in the evaluation of drug efficacy and safety. In vivo drug metabolism studies suffer from many disadvantages. Hence there is a rise in validation of in vitro microbial models. This review describes the transformation studies of drugs by the fungus, Cunninghamella and correlating them with the metabolism/biotransformation in animal systems and providing technical methods to develop microbial models. Emphasis is laid on the potential of Cunninghamella fungus to mimic mammalian drug biotransformations and to use as in vitro model for drug metabolism studies and for further toxicological and pharmacological studies of metabolites.     

Biotransformation of drugs by microbial cultures for predicting mammalian drug metabolism

This review discusses the microbial transformation studies of drugs, correlating them with the corresponding metabolism (biotransformation) in animal systems. Approaches are provided for development of microbial models for mammalian metabolism. Emphasis is placed on the potential of microorganisms to mimic mammalian metabolism and provide ways for structural elucidation and toxicological and pharmacological studies of metabolites. Microorganisms can provide difficult-to-synthesize drugs and assist in identifying metabolic pathways of drugs.     

真菌代谢产物的药物发现——资源、问题和策略

本期菌物学报“真菌生物活性代谢产物的药物发现专刊”刊登了7篇深度论述的综述性文章,19篇研究论文及2篇简报。内容涉及植物内生真菌产紫杉醇/紫杉烷的研究现状及最新进展、虫草菌素产生菌的培养策略及其药理学研究进展、灵芝三萜和胞外多糖生物合成的调控策略、淡水和海水真菌活性代谢产物研究进展等。本期内容还涉及内生真菌的分离和鉴定、海洋真菌的代谢产物分离、大型真菌的培养、代谢产物的分离及其药理学研究、酚类化合物测定方法、蛋白质组学以及生物转化等研究,基本体现了我国真菌代谢产物新药发现研究的最新进展,对真菌代谢产物合成调控、活性代谢产物分离和结构鉴定以及药理学研究等都具有重要的参考价值。     

天然活性先导化合物生物转化研究进展

天然活性先导化合物生物转化是利用生物催化剂(如：酶、微生物、动植物细胞)将加入到生物反应系统中的天然活性先导化合物进行特异性的分子结构修饰以获得高效、低毒新化合物的方法。该方法可以有效地提高已知的天然活性先导化合物的活性、降低毒副作用、改善水溶性和生物利用度,也可以用来生产具有重要应用价值的微量天然活性先导化合物,同时可用于药物代谢机制的研究。国内外学者已经针对甾体、醌类、黄酮类、萜类等化合物开展了天然活性先导化合物生物转化研究,筛选出一批有重要应用价值的生物转化反应类型,但针对天然活性先导化合物生物转化的机制、生物转化过程工程以及生物转化产物活性等方面的研究较少。将现代生命科学技术(如：生物催化剂的定向改造、高通量筛选、组合生物转化、非水相生物转化)引入天然活性先导化合物生物转化研究中,必将推进天然活性先导化合物的快速发展。     

Metabolism of fluoroorganic compounds in microorganisms: impacts for the environment and the production of fine chemicals

Incorporation of fluorine into an organic compound can favourably alter its physicochemical properties with respect to biological activity, stability and lipophilicity. Accordingly, this element is found in many pharmaceutical and industrial chemicals. Organofluorine compounds are accepted as substrates by many enzymes, and the interactions of microorganisms with these compounds are of relevance to the environment and the fine chemicals industry. On the one hand, the microbial transformation of organofluorines can lead to the generation of toxic compounds that are of environmental concern, yet similar biotransformations can yield difficult-to-synthesise products and intermediates, in particular derivatives of biologically active secondary metabolites. In this paper, we review the historical and recent developments of organofluorine biotransformation in microorganisms and highlight the possibility of using microbes as models of fluorinated drug metabolism in mammals.     

A commentary on methodological aspects of hydrolysable tannins metabolism in ruminant: a perspective view

Although, the application of tannic acid (TA), gallic acid (GA), natural hydrolysable tannins (HT)-rich ingredients, and HT-rich feeds in ruminant feeding have been explored in order to modify or manipulate microbial activities of digestive tract of animals, the interaction between HT and gastrointestinal microbiota and the fate of HT metabolites (GA, ellagic acid, pyrogallol, resorcinol, phloroglucinol, catechol and urolithin) derived from gastrointestinal microbial HT metabolism in the animal as a whole and animal products are missing. Incomplete biotransformation of HT and TA to GA, pyrogallol, resorcinol, phloroglucinol and other phenolic metabolites is a prevalent phenomenon discovered by researchers who examine the fate of HT metabolites in ruminant. While the rest of fellow researchers do not even examine the fate of HT metabolites and assume the complete biotransformation and fermentation of HT metabolites to volatile fatty acids (VFA). Only three studies have successfully identified the complete biotransformation and fermentation of HT metabolites to VFA in ruminant. The HT metabolites, mostly pyrogallol, produced through incomplete biotransformation of HT have adverse effects on gastrointestinal microbiota and host animal. Lack of awareness regarding the metabolism of HT metabolites and its consequences would compromise ruminant gastrointestinal microbiota, animal welfare, our environment and the power of research papers’ findings. In this perspective paper, I will bring to attention a new angle on the biotransformation and fermentation of HT metabolites in gastrointestinal tract, the role of gastrointestinal microbiota and deficiency of current approach in isolating tannin-degrading bacteria from rumen. Also, suggestions for better monitoring and understanding HT metabolisms in ruminant are presented.     

Use of a semicontinuous culture system as a model for determining the role of human intestinal microflora in the metabolism of xenobiotics

The use of semicontinuous culture system for determining the role of the human intestinal microflora int he biotransformation of xenobiotics is discussed. This model system, which stimulates the lumen of the large intestine, has been used to investigate human intestinal microflora metabolism of compounds representative of three different classes of chemicals; a benzidine-based azo dye, Direct Black 38, a nitropolycyclic aromatic hydrocarbon, 1-nitropyrene and a substituted pyrimidine, 5-fluorocytosine. Metabolites of each of the test compounds were identified, and the kinetics of production and biological activity of the metabolites determined. Metabolic adaptation was observed with 1-nitropyrene and 5-fluorocytosine incubations. This microbial culture system could be quite useful, especially in concert with other in vitro models and animal studies, for determining the pharmacological and toxicological role of the human intestinal microflora in the transformation of xenobiotics. Particular emphasis on the application of this method for toxicological studies in elucidating the role of the intestinal microflora in the etiology of cancer is described.     

微生物转化技术在中药开发中的应用进展

中药现代化是目前中药研究最迫切的需要,微生物转化技术可以利用微生物的特性解决中药现代化研究中难以解决的诸多问题,我们总结了微生物转化中药的特点,转化酶系统及反应类型,分析了微生物生物转化对中药的影响,认为微生物转化技术酶系统广泛,选择性强,反应条件温和可控,反应类型广泛,适用于所有类型的中药有效成分的生物转化,经过转化后,可以提高中药药效,降低毒性,去除杂质,帮助有效成分的体内代谢及产生新的药物成分。中药微生物转化技术必定成为中药学与微生物学完美契合的典范,推进中药现代化的进程。     

Production of natural value-added compounds: an insight into the eugenol biotransformation pathway

During the past few years, the production of natural value-added compounds from microbial sources has gained tremendous importance. Due to an increase in consumer demand for natural products, various food and pharmaceutical industries are continuously in search of novel metabolites obtained from microbial biotransformation. The exploitation of microbial biosynthetic pathways is both feasible and cost effective in the production of natural compounds. The environmentally compatible nature of these products is one major reason for their increasing demand. Novel approaches for natural product biogeneration will take advantage of the current studies on biotechnology, biochemical pathways and microbiology. The interest of the scientific community has shifted toward the use of microbial bioconversion for the production of valuable compounds from natural substrates. The present review focuses on eugenol biotransformation by microorganisms resulting in the formation of various value-added products such as ferulic acid, coniferyl alcohol, vanillin and vanillic acid.     

Small bugs, big business: the economic power of the microbe

The versatility of microbial biosynthesis is enormous. The most industrially important primary metabolites are the amino acids, nucleotides, vitamins, solvents, and organic acids. Millions of tons of amino acids are produced each year with a total multibillion dollar market. Many synthetic vitamin production processes are being replaced by microbial fermentations. In addition to the multiple reaction sequences of fermentations, microorganisms are extremely useful in carrying out biotransformation processes. These are becoming essential to the fine chemical industry in the production of single-isomer intermediates. Microbially produced secondary metabolites are extremely important to our health and nutrition. As a group, they have tremendous economic importance. The antibiotic market amounts to almost 30 billion dollars and includes about 160 antibiotics and derivatives such as the beta-lactam peptide antibiotics, the macrolide polyketide erythromycin, tetracyclines, aminoglycosides and others. Other important pharmaceutical products produced by microrganisms are hypocholesterolemic agents, enzyme inhibitors, immunosuppressants and antitumor compounds, some having markets of over 1 billion dollars per year. Agriculturally important secondary metabolites include coccidiostats, animal growth promotants, antihelmintics and biopesticides. The modern biotechnology industry has made a major impact in the business world, biopharmaceuticals (recombinant protein drugs, vaccines and monoclonal antibodies) having a market of 15 billion dollars. Recombinant DNA technology has also produced a revolution in agriculture and has markedly increased markets for microbial enzymes. Molecular manipulations have been added to mutational techniques as means of increasing titers and yields of microbial procresses and in discovery of new drugs. Today, microbiology is a major participant in global industry. The best is yet to come as microbes move into the environmental and energy sectors.     

Biotechnological applications of microbial bioconversions

Modern research has focused on the microbial transformation of a huge variety of organic compounds to obtain compounds of therapeutic and/or industrial interest. Microbial transformation is a useful tool for producing new compounds, as a consequence of the variety of reactions for natural products. This article describes the production of many important compounds by biotransformation. Emphasis is placed on reporting the metabolites that may be of special interest to the pharmaceutical and biotechnological industries, as well as the practical aspects of this work in the field of microbial transformations.     

Preliminary studies about novel strategies to reverse chemoresistance to adriamycin regarding glutathione metabolism,peroxisomal and extraperoxisomal hydroperoxide and valproic acid metabolic pathways

Summary— The present work was aimed at defining novel strategies to reverse chemoresistance to anticancer drugs, especially by interfering with cellular glutathione metabolism, peroxisomal and/or extraperoxisomal hydroperoxide metabolic pathways. Preliminary results are presented about molecules we demonstrated to be capable of interfering with hydrogen peroxide metabolism in cells. Prior to describing these molecules, a short overview of glutathione and free radical metabolic pathways is presented as well as a rapid presentation of the characteristics of chemo-sensitivity and -resistance towards the anticancer drug adriamycin, with special emphasis on hydrogen peroxide metabolism. The strategies currently developed to reverse chemoresistance are further presented. in subsequent sections, our own strategy to achieve inhibition of hydrogen peroxide breakdown and stimulation of peroxisomal hydrogen peroxide production is illustrated on the basis of molecular modelling studies and biochemical investigations on extraperoxisomal and peroxisomal metabolic pathways. Preliminary studies on cultured cells have been initiated. The perspective for future studies is presented as well as other possible models of chemoresistance as target for the design of hydrogen peroxide metabolism-interfering pharmacomolecules.     

The relations between metabolic variations and genetic evolution of different species

Metabonomics has been applied in many bio-related scientific fields. Nevertheless, some animal research works are shown to fail when they are extended to humans. Therefore, it is essential to figure out suitable animal modeling to mimic human metabolism so that animal findings can serve humans. In this study, two kinds of commonly selected body fluids, serum and urine, from humans and various experimental animals were characterized by integration of nuclear magnetic resonance (NMR) spectroscopy with multivariate statistical analysis to identify the interspecies metabolic differences and similarities at a baseline physiological status. Our results highlight that the dairy cow and pig may be an optimal choice for transportation and biodistribution studies of drugs and that the Kunming (KM) mouse model may be the most effective for excretion studies of drugs, whereas the Sprague–Dawley (SD) rat could be the most suitable candidate for animal modeling under overall considerations. The biochemical pathways analyses further provide an interconnection between genetic evolution and metabolic variations, where species evolution most strongly affects microbial biodiversity and, consequently, has effects on the species-specific biological substances of biosynthesis and corresponding biological activities. Knowledge of the metabolic effects from species difference will enable the construction of better models for disease diagnosis, drug metabolism, and toxicology research.     

Manipulation of fungal development as source of novel secondary metabolites for biotechnology

Fungal genomics revealed a large potential of yet-unexplored secondary metabolites, which are not produced during vegetative growth. The discovery of novel bioactive compounds is increasingly gaining importance. The high number of resistances against established antibiotics requires novel drugs to counteract increasing human and animal mortality rates. In addition, growth of plant pathogens has to be controlled to minimize harvest losses. An additional critical issue is the post-harvest production of deleterious mycotoxins. Fungal development and secondary metabolite production are linked processes. Therefore, molecular regulators of development might be suitable to discover new bioactive fungal molecules or to serve as targets to control fungal growth, development, or secondary metabolite production. The fungal impact is relevant as well for our healthcare systems as for agriculture. We propose here to use the knowledge about mutant strains discovered in fungal model systems for a broader application to detect and explore new fungal drugs or toxins. As examples, mutant strains impaired in two conserved eukaryotic regulatory complexes are discussed. The COP9 signalosome (CSN) and the velvet complex act at the interface between development and secondary metabolism. The CSN is a multi-protein complex of up to eight subunits and controls the activation of CULLIN-RING E3 ubiquitin ligases, which mark substrates with ubiquitin chains for protein degradation by the proteasome. The nuclear velvet complex consists of the velvet-domain proteins VeA and VelB and the putative methyltransferase LaeA acting as a global regulator for secondary metabolism. Defects in both complexes disturb fungal development, light perception, and the control of secondary metabolism. The potential biotechnological relevance of these developmental fungal mutant strains for drug discovery, agriculture, food safety, and human healthcare is discussed.     

Arsenic metabolism and thioarsenicals

Arsenic has received considerable attention in the world, since it can lead to a multitude of toxic effects and has been recognized as a human carcinogen causing cancers. Here, we focus on the current state of knowledge regarding the proposed mechanisms of arsenic biotransformation, with a little about cellular uptake, toxicity and clinical utilization of arsenicals. Since pentavalent methylated metabolites were found in animal urine after exposure to iAs(III), methylation was considered to be a detoxification process, but the discovery of methylated trivalent intermediates and thioarsenicals in urine has diverted the view and gained much interest regarding arsenic biotransformation. To further investigate the partially understood phenomena relating to arsenic toxicity and the uses of arsenic as a drug, it is important to elucidate the exact pathways involved in metabolism of this metalloid, as the toxicity and the clinical uses of arsenic can be best recognized in context of its biotransformation. Thereby, in this perspective, we have focused on arsenic metabolic pathways including three proposed mechanisms: a classic pathway by Challenger in 1945, followed by a new metabolic pathway proposed by Hayakawa in 2005 involving arsenic-glutathione complexes, while the third is a new reductive methylation pathway that is proposed by our group involving As-protein complexes. According to previous and present in vivo and in vitro experiments, we conclude that the methylation reaction takes place with simultaneous reductive rather than stepwise oxidative methylation. In addition, production of pentavalent methylated arsenic metabolites are suggested to be as the end product of metabolism, rather than intermediates.     

The use of transgenic systems in pharmaceutical research.

Those pharmaceutical companies whose goal is to generate novel innovative drugs are faced with the challenge that only a fraction of the compounds tested in clinical trials eventually become a registered drug. This problem of attrition is compounded by the fact that the clinical trial or development stage is by far the most costly phase of bringing a new drug to market, consuming around 80 per cent of the total spend. Transgenic technology represents an attractive approach to reducing the attrition rate of compounds entering clinical trials by increasing the quality of the target and compound combinations making the transition from discovery into development. Transgenic technology can impact at many points in the discovery process, including target identification and target validation, and provides models designed to alert researchers early to potential problems with drug metabolism and toxicity, as well as providing better models for human diseases. In target identification, transgenic animals harbouring large DNA fragments can be used to narrow down genetic regions. Genetic studies often result in the identification of large genomic regions and one way to decrease the region size is to do complementation studies in transgenic animals using, for example, inserts from bacterial artificial chromosome (BAC) clones. In target validation, transgenic animals can be used for in vivo validation of a specific target. Considerable efforts are being made to establish new, rapid and robust tools with general utility for in vivo validation, but, so far, only transgenic animals work reliably on a wide range of targets. Transgenic animals can also be used to generate better disease models. Predictive animal models to test new compounds and targets will significantly speed up the drug discovery process and, more importantly, increase the quality of the compounds taken further in the research and development process. Humanised transgenic animals harbouring the human target molecule can be used to understand the effect of a compound acting on the human target in vivo. Also, models mimicking human drug metabolism will provide a means of assessing the effect of human-specific metabolites and of understanding the pharmacokinetic properties of potential drugs. In toxicology studies, transgenic animals are providing more predictive models. A good example of this are those models routinely used to look for carcinogenicity associated with new compounds.     

Regulation of secondary metabolite production in filamentous ascomycetes

Fungi are renowned for their ability to produce bioactive small molecules otherwise known as secondary metabolites. These molecules have attracted much attention due to both detrimental (e.g. toxins) and beneficial (e.g. pharmaceuticals) effects on human endeavors. Once the topic only of chemical and biochemical studies, secondary metabolism research has reached a sophisticated level in the realm of genetic regulation. This review covers the latest insights into the processes regulating secondary metabolite production in filamentous fungi.     

Human hepatic cell cultures: in vitro and in vivo drug metabolism

Drug metabolism is the major determinant of drug clearance, and the factor most frequently responsible for inter-individual differences in drug pharmacokinetics. The expression of drug metabolising enzymes shows significant interspecies differences, and variability among human individuals (polymorphic or inducible enzymes) makes the accurate prediction of the metabolism of a new compound in humans difficult. Several key issues need to be addressed at the early stages of drug development to improve drug candidate selection: a) how fast the compound will be metabolised; b) what metabolites will be formed (metabolic profile); c) which enzymes are involved and to what extent; and d) whether drug metabolism will be affected directly (drug-drug interactions) or indirectly (enzyme induction) by the administered compound. Drug metabolism studies are routinely performed in laboratory animals, but they are not sufficiently accurate to predict the metabolic profiles of drugs in humans. Many of these issues can now be addressed by the use of relevant human in vitro models, which speed up the selection of new candidate drugs. Human hepatocytes are the closest in vitro model to the human liver, and they are the only model which can produce a metabolic profile of a drug which is very similar to that found in vivo. However, the use of human hepatocytes is restricted, because limited access to suitable tissue samples prevents their use in high throughput screening systems. The pharmaceutical industry has made great efforts to develop fast and reliable in vitro models to overcome these drawbacks. Comparative studies on liver microsomes and cells from animal species, including humans, are very useful for demonstrating species differences in the metabolic profile of given drug candidates, and are of great value in the judicious and justifiable selection of animal species for later pharmacokinetic and toxicological studies. Cytochrome P450 (CYP)-engineered cells (or microsomes from CYP-engineered cells, for example, Supersomes) have made the identification of the CYPs involved in the metabolism of a drug candidate more straightforward and much easier. However, the screening of compounds acting as potential CYP inducers can only be conducted in cellular systems fully capable of transcribing and translating CYP genes.     

Delivery of retinoid-based therapies to target tissues

Through its various metabolites, vitamin A controls essential physiological functions. Both naturally occurring metabolites and novel retinoid analogues have shown effectiveness in many clinical settings that include skin diseases and cancer, and in animal models of human conditions affecting vision. In this review, we analyze several potential retinoid-based therapies from the point of view of drug metabolism and transport to target tissues. We focus on the endogenous factors that affect the absorption, transport, and metabolism of retinoids by taking into account data obtained from the analysis of animal models that lack the enzymes or proteins involved in the storage and absorption of retinoids. We also discuss findings of toxicity associated with retinoids in an effort to improve the outcome of retinoid-based therapies. In this context, we review evidence that esterification of retinol and retinol-based drugs within target tissues provides one of the most efficient means to improve the absorption and to reduce the toxicity associated with pharmacological doses of retinoids. Future retinoid-based therapeutic strategies could involve targeted delivery mechanisms leading to lower toxicity and improved effectiveness of retinoids.     

Lapachol biotransformation by filamentous fungi yields bioactive quinone derivatives and lapachol-stimulated secondary metabolites

Abstract

Lapachol is a natural naphthoquinone with a range of biological effects, including anticancer activity. Microbial transformations of lapachol can lead to the formation of new biologically active compounds. In addition, fungi can produce secondary metabolites that are also important for drug discovery. The goal of this study was to evaluate the ability of filamentous fungi to biotransform lapachol into biologically active compounds and identify secondary metabolites produced in the presence of lapachol. Seven out of nine strains of filamentous fungi tested exhibited the ability to biotransform or biodegrade lapachol. The bioactive derivatives norlapachol and isolapachol were identified among biotransformation products. Moreover, lapachol stimulated the production of pyrrolo-[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) and phenol-2,4-bis-(1,1-dimethylethyl), secondary metabolites already known to have antimicrobial and antioxidant activities. These results open the perspective of using these strains of filamentous fungi for lapachol biotransformation and efficient production of several biologically active compounds.