Metabolite generation via microbial biotransformations with Actinomycetes: rapid screening for active strains and biosynthesis of important human metabolites of two development-stage compounds, 5-[(5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic acid (BMS-587101) and dasatinib.
The enzymes present in many microbial strains are capable of carrying out a variety of biotransformations when presented with drug-like molecules. Although the enzymes responsible for the biotransformations are not well characterized, microbial strains can often be found that produce metabolites identical to those found in mammalian systems. However, traditional screening for microbial strains that produce metabolites of interest is done with many labor intensive steps that include multiple shake flasks and many manual manipulations, which hinder the application of these techniques in drug metabolite preparation. A 24-well microtiter plate screening system was developed for rapid screening of actinomycetes strains for their ability to selectively produce metabolites of interest. The utility of this system was first demonstrated with the well characterized cytochrome P450 substrate diclofenac. Subsequently, the use of this system allowed the rapid identification of several actinomycetes strains that were capable of converting two drug candidates under development, 5-[(5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic acid and N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, Sprycel, BMS-345825), to mammalian metabolites of interest. Milligram quantities of the metabolites were then prepared by scaling-up the microbial biotransformation reactions. These quantities were sufficient for initial characterization, such as testing for pharmacological activity and use as analytical standards, prior to the availability of authentic chemically synthesized compounds.
Li
Wenying
W
Department of Biotransformation, Research and Development, Bristol-Myers Squibb, Princeton, New Jersey 08543, USA. wenying.li@bms.com
Josephs
Jonathan L
JL
Skiles
Gary L
GL
Humphreys
W Griffith
WG
D039903:Actinobacteria / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001711:Biotransformation / Q000187:drug effects / Q000502:physiology; D000069439:Dasatinib; D015507:Drugs, Investigational / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods / Q000592:standards; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D011743:Pyrimidines / Q000737:chemistry* / Q000493:pharmacokinetics* / Q000494:pharmacology; D051381:Rats; D013141:Spiro Compounds / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D013552:Swine; D013844:Thiazoles / Q000737:chemistry* / Q000493:pharmacokinetics* / Q000494:pharmacology; D013876:Thiophenes / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology
C517055:5-(9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro(4,4)non-7-ylmethyl)-3-thiophenecarboxylic acid; D015507:Drugs, Investigational; D011743:Pyrimidines; D013141:Spiro Compounds; D013844:Thiazoles; D013876:Thiophenes; D000069439:Dasatinib