Toxicological investigation of drug metabolites has gained huge importance in the last years especially since the FDA has issued their guidelines for metabolites in safety testing (MIST). The metabolic steps that lead to drug clearance in the human body are divided into two distinct parts. First, the parent compound is chemically modified (phase I) and in a second step (phase II) conjugated with endogenous molecules facilitating the final clearance e.g. by increasing the solubility of the compound. The most important enzymes involved in Phase I metabolism are cytochrome P450s and UDP glycosyltransferases (UGTs) for Phase II reactions. In order to investigate and characterize such metabolites the synthetic or biosynthetic accessibility of drug metabolites is a prerequisite. Unfortunately, assessing some drug metabolites during the drug development phase e.g. for toxicological assays can be very difficult. For instance chemical synthesis is often cumbersome, time consuming or even fails due to the difficulties in generating metabolites containing e.g. regio-and stereospecific modifications.
In this context, the biotechnological generation of metabolites using genetically modified organisms expressing the enzymes of interest is gaining more and more importance. Especially whole-cell biotransformation using recombinant organisms is becoming the method of choice for many pharmaceutical companies. Different microorganisms have been used for the production of metabolites. One organism that has turned out to be very well suited for performing such biotransformation reactions is the fission yeast Schizosaccharomyces pombe. This yeast was first isolated by the German botanist Lindner from the millet of an east African beer called pombe by the locals. From an evolutionary point of view S. pombe and the more commonly known baker's yeast Saccharomyces cerevisiae have a long evolutionary distance. Our company, PomBioTech, has developed; a whole-cell biotransformation technique using recombinant fission yeasts that functionally express human UGTs and P450s. The recombinant fission yeast cells are cultivated together with the substrate of interest, which is taken up by the cells, converted into the product of interest by the action of the expressed recombinant enzyme and is then secreted into media. One of the mayor advantages of this system is that no expensive co-substrates are needed since the yeasts can produce these co-factors by themselves. In the case of UDP-glucuronic acid, the co-factor by UGTs, we have co-expressed another enzyme, human UDP-glucose-6-dehydrogenase, that catalysis the synthesis of UDP-glucuronic acid from yeast endogenous UDP-glucose. Moreover, using our approach we can also obtain stable isotope-labeled glucuronids and P450 metabolites by using a labeled aglycon or P450 substrate. In the case of glucuronide production 13C labeled glucose can also be used as a metabolic precursor of isotope-labeled UDP-glucuronic acid resulting in the formation of 13C labeled glucuronides. The possibility of producing labeled metabolites enables the use of these compounds as reference standards for LC-MS and LC-MS/MS studies. Moreover, the amount of metabolites that can be produced using this system goes up to 100 g which enables the possibility to carry out different toxicological assays in the context of MIST.
Summarizing, we have now a unique biotechnological tool at hands that allows the relatively easy production of human Phase I and Phase II metabolites up to 100 g. The door is now wide open for investigating the effects of numerous metabolites, which in some cases can exert improved or different mode of actions compared to the parental substances.
About Author / Additional Info:
Dr. Andy Zöllner studied biology at the Saarland University, Saarbrücken, Germany. After his Ph.D. thesis and a first PostDoc time, he decided to join PomBioTech (www.pombiotech.com) and exploit his knowledge on P450s for commercial purposes. Contact: firstname.lastname@example.org.