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Microbial models of mammalian metabolism: uses and misuses (clarification of some misconceptions)
Journal of Molecular Catalysis B: Enzymatic 5 Ž1998. 371–377
Microbial models of mammalian metabolism: uses and misuses ž clarification of some misconceptions/ Serge G. Jezequel
)
Department of Drug Metabolism, Pfizer Central Research, Sandwich, Kent CT13 9NJ, UK Received 9 October 1997; accepted 9 February 1998
Abstract Oxidative metabolism is the most commonly encountered metabolic clearance pathway of xenobiotics by mammalian systems. Similar pathways are often encountered in microorganisms and have been used to metabolise xenobiotics. This paper reviews the background to these observations and the potential uses they imply. It proposes that although microbial metabolism can have a useful role in preparative biocatalysis, any predictive value to man is negated by the lack of correlation with mammalian systems and may potentially be misleading. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; CYP; Microbial models; Drug metabolism; Metabolite synthesis
1. Introduction Oxidative metabolism is the most commonly encountered metabolic clearance pathway for drugs in animals and man w1,2x. This metabolism is often carried out by a super-family of enzymes: the cytochrome P450s Ž CYPs. and many CYPs have now been gene sequenced for man Ž) 40. and other species Ž) 200. w3x. Such enzymes are not specific to animals, but are also found in plants and, more relevantly here, in microorganisms. Microorganisms have been extensively investigated as a tool to provide mechanistic insight or for their biocatalytic properties and there are many reports of drugs which are successfully metabolised by microorganisms:
)
Corresponding author. Tel.: q44-1304-616079; fax: q441304-616433; E-mail: [email protected]
warfarin, propranolol, phenacetin, quinidine, carbamazepine, ibuprofen, phenytoin, diazepam, aminopyrene, theophilline, tamoxifen, sulindac, imipramine, etc. The use of microorganisms appeals as a potentially very useful tool in the design of novel drugs. However; two key questions must be asked: can useful and reliable predictions be made from microbial systems and can they be used as reliable biocatalytic reagents?
2. Background 2.1. Metabolism by mammalian systems The mammalian CYPs responsible for the metabolism of xeno- and endobiotics are a super-family of membrane bound, heme-contain-
1381-1177r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 1 - 1 1 7 7 Ž 9 8 . 0 0 0 8 9 - 7
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
372 Table 1 CYPs reaction types
ing enzymes. They are found in many organs in low concentrations but reside mainly in high levels in hepatic tissues and intestinal epithelium. A fundamental property of metabolic reactions carried out by CYPs is that they are all based on electrophilic attack ŽTable 1. w4x. For all CYPs, the different types of ‘oxidative metabolism’ Žhydroxylation, de-alkylation, N-oxidation, epoxidation, etc.. are dependent on substrate structure and physicochemical properties, as well as enzyme active site topology dictating the orientation of the substrate. From a biocatalyst viewpoint, the concept of ‘reaction specific enzyme’ is therefore not appropriate. CYPs have been classified on the basis of their gene sequences w3x. CYPs fall into two broad groups: families I, II and III are responsible for nearly all CYP-dependent oxidative drug metabolism in mammals. In particular, these subfamilies are responsible for most of the oxi-
dation of drugs: CYP2C, CYP2D, CYP2E and CYP3A. These subfamilies are represented in different species, as shown below for rats and humans Ž Table 2.. Substrate structure–activity relationships ŽSSAR. have been characterised for human CYPs. Some prediction of preferredrlikely substrates can therefore be made on the basis of lipophilicity, molecular size, p K a and optimal distances between binding and metabolism sites as summarised in Table 3 w5x. The second group comprises families IV– XXIV. These enzymes are essentially responsible for the metabolism of endobiotic molecules such as hormones and are usually highly substrate specific w5x. They are not commonly involved in the metabolism of drugs. Table 3 SSAR for major human CYPs Some limited SSAR from overlap technique for key CYPs
Table 2 Major rat and human CYPs involved in drug metabolism Rat
Man
CYP2
CYP2C6 CYP2C11 CYP2D1 CYP2E1
CYP2C9 CYP2C19 Žpolymorphic. CYP2D6 Žpolymorphic. CYP2E1
CYP3
CYP2A1 Žinducible. CYP3A2 Žinducible.
CYP3A4 Ž20= variable.
CYP2D6 Ž2.5% of CYPs but 19% of metabolism. structural requirements: l basic nitrogen atom l extended hydrophobic region Žnonspecific binding., site for oxidation 5-7A from nitrogen CYP3A4 Ž28% of CYPs and 34% of metabolism. l wide range of substrate-no apparent structural requirements Žfrom ethylene to cyclosporin. l overall lipophilicity probably important CYP2E1 Ž7% of CYPs and 4% of metabolism. l only small molecules ŽMW-100 Da.
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
373
Fig. 1. Structures of typical CYP101 substrate.
2.2. Metabolism by microbial systems Overall, the characterisation of microbial CYPs Žfamilies LI, LII, CI and CII. has been limited and therefore has not clearly identified families of xenobiotic metabolising enzymes. One area of exception is that of the soluble bacterial CYPs. 2.2.1. Soluble bacterial CYPs Soluble CYPs from bacteria such as those from Pseudomonas putida Ž CYP101. and Bacillus megaterium Ž CYP102 . have been extensively characterised over the past 25 years w6x. In particular, CYP101 ŽP450cam in old nomenclature. has provided structure and mechanistic information generic to all CYPs w7x. Knowledge of its 3D crystal structure Ždown to ˚ resolution w8,9x. has allowed some ex1.6 A tremely useful extrapolation to all CYPs. However, there are significant limitations with re-
gard to range of substrates metabolised by CYP101 as shown below ŽFig. 1.. Similarly, other soluble bacterial CYPs also exhibit very narrow substrate specificity: CYP102 w10x is limited to oxidation of fatty acids and CYP108 w11x Žpseudomonad. is limited to a narrow range of terpenes.
Table 4 Selected applications of microbial CYPs l C. elegans phenacetin w15x carbamazepine w16x furosemide w17x amitriptyline w18x chlorpramazine w19x l B. bassiana diazepam w20x l C. banieri propranolol w21x l S. rimosus zolasartan w22x
374
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
Fig. 2. Microbial oxidation of terfenadine.
Consequently, although soluble CYPs have helped to understand generic and mechanistic aspects of CYPs, their substrate limitations makes them poor predictors of mammalian enzymes and, therefore, unsuitable tools for drug design or as biocatalysts.
malian CYPs, no such information is available for eukaryotic CYPs. Some Žprobably all. possess several CYPs w13x, and it is tempting to speculate that some of these enzymes may have a detoxification role.
2.2.2. Eukaryotic CYPs These CYPs are all membrane bound and appear to have similar structural and mechanistic constraints as mammalian CYPs w3x. Little fundamental research is available, mostly due to the difficulties of isolating individual CYPs for characterisation. One notable exception is the steroid C14 demethylase w12x, principally because of its importance in the treatment of fungal infections with azole antifungal drugs. There is, however, no or little SAR available and in spite of being inhibited by xenobiotics such as fluconazole, ketoconazole or itraconazole, this enzyme is probably highly specific to the metabolism of steroidal substrates. Whereas inducibility is a characteristic of several mam-
3. Applications of microbial CYPs Since the early days of Smith and Rosazza w14x when the basic principles of microbial modelling of mammalian metabolism were set out, much research has been undertaken to characterise this approach. Most of this research has led to factual reporting of microbial biocatalysis results, and although their predictive capabilities are occasionally mentioned, it seems always to be as a generic or throw away comment usually in the introduction. There are many literature examples of microorganisms being used in the metabolism of drugs. A few of the more commonly used microorganisms have been selected below ŽTable 4. to serve as examples.
Fig. 3. Microbial oxidation of diclofenac.
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
Fig. 4. Human metabolism of warfarin w24x.
In our own laboratories, several synthesis problems Ž typically difficulties to synthesise metabolites in a regio-specific manner. have been resolved with microorganisms over the last few years. Two examples are summarised below: terfenadine and diclofenac ŽFigs. 2 and 3. w23x.
4. Discussions 4.1. Are microbial biotransformations predictiÕe of human drug metabolism? At first sight, there appear to be a lot of commonality between all CYPs. But does this include reliable commonality of metabolic pathways of xenobiotics? One of the most frequently reported substrate which is metabolised by both mammalian and microbial CYPs is warfarin. This drug has, therefore, been used to examine any overlap of metabolism patterns between mammalian and microbial CYPs. The human metabolism of warfarin is summarised in Fig. 4, with relevant specificity of individual CYPs.
375
In addition, to allow direct correlation with microbial metabolism of warfarin, comparative data for man and six suitably reported microbial biotransformations are shown in Table 5. Examination of Table 5 reveals that although warfarin is a good substrate for several microorganisms, there is no evidence of correlation with human pathways. The major human metabolite Ž 7-OH. for ŽS. warfarin is only observed as minor with two microorganisms, and the two ‘intermediate’ human metabolites for ŽR. warfarin are not reported for any microorganisms. The 4X-OH metabolite seems to be the most easily obtained compound with microbial systems, being observed in three out of six biotransformations. It is our view that the example of warfarin, one of the most universally characterised probes for CYPs, is probably a fair representation of a general situation. Nonhumanised Ž see below. microorganisms do not possess the necessary attribute of good correlation with human metabolism to be models of human drug metabolism. 4.2. Implications for the use of microbial models of mammalian metabolism Although many microorganisms are very apt at metabolising xenobiotics, the little data available do not support their use as predictive models of human metabolism of drugs and therefore cannot be useful tools for designing drugs with optimum clearance and pharmacokinetics. It is reasonable to speculate that every microorganism will have its own set of CYPs genetically
Table 5 Warfarin: human vs. microbial metabolism X
Man N. corallina w25x B. bassiana w26x C. bainieri w27x C. elegans w28x A. niger w29x S. rimosus w30x
4 -OH
6-OH
7-OH
8-OH
10-OH
minor ŽR. y major major y y minor
intermediate ŽR. y y y y y y
major ŽS. y minor y y y minor
intermediate ŽR. y
minor ŽR. y y y y y y
y y y y
Others 1 metabolism only for S several minor novel X
3 OH oxidative cleavage 12-OH
376
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
encoded, possibly inducible, with a broad variation in specificity, similarly to higher organisms variation in CYPs. Although it is likely that all cytochrome genes have evolved from a common ancestral gene w3x, the basic structural similarities exhibited by all the CYPs is mostly confined to mechanism and do not encompass substrate specificity. After all, even enzymes from the same family Ž e.g., 2C. can have differing substrate specificity across species Že.g., rat CYP2C6 and CYP2C11, human CYP2C9 and CYP2C19 . w4x. In addition, it is our experience that growth conditions can be critical; different metabolising activities may be obtained if microorganisms are inoculated or harvested at different stages of their growth. It is therefore wrong and potentially misleading to assume that because a microorganism metabolises a molecule in a particular way, it has an automatic bearing on its fate in man or any animal species. Any such predictions extended to potential toxicity of xenobiotics or their metabolites would also be highly speculative or even misleading. 4.3. Microorganisms as biocatalytic reagents On a positive note, the area of most potential in the use of microorganisms has to be as a biocatalyst for the synthesis of drug metabolites. This approach has been successfully used in our laboratories, to carry out the synthesis of authenticated mammalian metabolites, when classical methods have failed. The chemical synthesis of regio- or stereospecific hydroxylated compound can be a particularly intractable task for the organic chemist, and biocatalysis is generally under-used in the field of preparative drug metabolism. In practice, the lack of knowledge on specific microbial enzymes implies that there is a need for screening to identify the most suitable microorganismŽs. prior to production. This is not, however, a serious barrier for well equipped laboratories. In this context, it is also critical to remember that such biocatalysts are not ‘reac-
tion specific’, rather the combination of structure of each substrate and enzyme topology at the active site will determine the type and position of oxidative process Ž e.g., hydroxylation vs. epoxidation vs. dealkylation vs. N-oxidation. . 4.4. The future of microbial models of mammalian metabolism? In due course, ‘humanised’ cell-lines may provide a reliable tool for predicting routes and rates of human metabolism as well as biocatalysis. Their usefulness as predictors of human metabolism is presently undergoing characterisation in many laboratories and looks very promising w31x. Little work has yet been reported on their potential as specific biocatalysts, and their robustness, in particular, needs investigating.
References w1x S. Rendic, F.J. di Carlo, Drug Metab. Rev. 29 Ž1&2. Ž1997. 413. w2x D.A. Smith, B. Jones, Biochem. Pharmacol. 44 Ž11. Ž1992. 2089. w3x D.R. Nelson, T. Kamatala, D.J. Waxman, F.P. Guengerich, R.W. Estabrook, D.W. Nebert, DNA Cell Biol. 12 Ž1. Ž1993. 1. w4x F.P. Guengerich, Prog. Drug Metab. 10 Ž1987. 1. w5x D.A. Smith, B.C. Jones, D.K. Walker, Med. Res. Dev. 16 Ž3. Ž1996. 243. w6x S.P. Crammer, J.H. Dawson, K.O. Hodgson, L.P. Hages, J. Am. Chem. Soc. 100 Ž1978. 7282. w7x J.A. Peterson, S.E. Graham-Lorence, in: P. Ortiz de Montellano ŽEd.., Cytochrome P450, Plenum, 1995, p. 175. w8x T.L. Poulos, B.C. Finzel, I.C. Gonzalus, J. Biol. Chem. 260 Ž1985. 16122. w9x T.L. Poulos, B.C. Finzel, A.F. Howard, J. Mol. Biol. 195 Ž1987. 687. w10x A.J. Fulco, Annu. Rev. Pharmacol. Toxicol. 31 Ž1991. 177. w11x J.A. Peterson, J.Y. Lu, J. Geisselsoder, M.C. Lorence, J. Biol. Chem. 267 Ž1992. 14193. w12x C.A. Hitchcok, K. Dickinson, S.B. Brown, D.J. Adams, Biochem J. 263 Ž1989. 573. w13x W.H. Schunck, F. Vogel, B. Gross, E. Kangel, K. Kopke, H.G. Muller, Eur. J. Cell. Biol. 55 Ž1991. 336. w14x R.V. Smith, J.P. Rosazza, Arch. Biochem. Biophys. 161 Ž1974. 551. w15x C.S. Reddy, D. Accosta, P.J. Davies, Xenobiotica 20 Ž12. Ž1990. 1281.
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377 w16x D. Davis, P.J. Davis, Pharm. Res. 9 Ž1992. S107, ŽSuppl. 10.. w17x M. Hexari, P.J. Davis, Drug Metab. Dispos. 20 Ž6. Ž1992. 882. w18x D. Zhang, F.E. Evans, J.P. Freeman, B. Duhart, C.E. Cerniglia, Drug Metab. Dispos. 23 Ž12. Ž1995. 1417. w19x D. Zhang, J.P. Freeman, J.B. Sutherland, A.E. Walker, Appl. Environ. Microbiol. 62 Ž3. Ž1996. 798. w20x D.A. Griffiths, D.J. Best, S.G. Jezequel, Appl. Microbiol. Biotechnol. 35 Ž1991. 373. w21x B.C. Foster, H.S. Button, S.A. Qureshi, I.J. McGilveray, Xenobiotica 19 Ž5. Ž1989. 539. w22x R.J.P. Cannell, A.R. Knaggs, G.R. Manchee, P.J. Eddershaw, I. Waterhouse, Drug Metab. Dispos. 23 Ž7. Ž1995. 724.
377
w23x R. Webster, M. Pacey, T. Winchester, P. Johnsonm, S.G. Jezequel, Applied Microbiol and Biotechnol, 1998, in press. w24x J.J.R. Hermans, H.H.W. Thijssen, Br. J. Pharmacol. 110 Ž1993. 482. w25x J.J. McBride, G.W. Hanlon, A.J. Hutt, C.J. Olliff, J. Pharm. Pharmacol. 45 Ž1993. 1106, ŽSuppl. 2.. w26x D.A. Griffiths, D.E. Brown, S.G. Jezequel, Appl. Microbiol Biotechnol. 37 Ž1992. 169. w27x J.D. Rizzo, J.D. Davis, J. Pharm. Sci. 78 Ž3. Ž1989. 183. w28x Y.W. Wong, P.J. Davies, J. Pharm. Sci. 80 Ž4. Ž1991. 305. w29x J.D. Rizzo, P.J. Davies, Xenobiotica 18 Ž1988. 1425. w30x R.J.P. Cannell, T. Rashid, I.M. Ismail, P.J. Sidebottom, A.R. Knaggs, P.S. Marshall, Xenobiotica 27 Ž2. Ž1997. 147. w31x D. Mansuy, J. Mol. Catal. B: Enzymic, these proceedings.
Microbial models of mammalian metabolism: uses and misuses ž clarification of some misconceptions/ Serge G. Jezequel
)
Department of Drug Metabolism, Pfizer Central Research, Sandwich, Kent CT13 9NJ, UK Received 9 October 1997; accepted 9 February 1998
Abstract Oxidative metabolism is the most commonly encountered metabolic clearance pathway of xenobiotics by mammalian systems. Similar pathways are often encountered in microorganisms and have been used to metabolise xenobiotics. This paper reviews the background to these observations and the potential uses they imply. It proposes that although microbial metabolism can have a useful role in preparative biocatalysis, any predictive value to man is negated by the lack of correlation with mammalian systems and may potentially be misleading. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome P450; CYP; Microbial models; Drug metabolism; Metabolite synthesis
1. Introduction Oxidative metabolism is the most commonly encountered metabolic clearance pathway for drugs in animals and man w1,2x. This metabolism is often carried out by a super-family of enzymes: the cytochrome P450s Ž CYPs. and many CYPs have now been gene sequenced for man Ž) 40. and other species Ž) 200. w3x. Such enzymes are not specific to animals, but are also found in plants and, more relevantly here, in microorganisms. Microorganisms have been extensively investigated as a tool to provide mechanistic insight or for their biocatalytic properties and there are many reports of drugs which are successfully metabolised by microorganisms:
)
Corresponding author. Tel.: q44-1304-616079; fax: q441304-616433; E-mail: [email protected]
warfarin, propranolol, phenacetin, quinidine, carbamazepine, ibuprofen, phenytoin, diazepam, aminopyrene, theophilline, tamoxifen, sulindac, imipramine, etc. The use of microorganisms appeals as a potentially very useful tool in the design of novel drugs. However; two key questions must be asked: can useful and reliable predictions be made from microbial systems and can they be used as reliable biocatalytic reagents?
2. Background 2.1. Metabolism by mammalian systems The mammalian CYPs responsible for the metabolism of xeno- and endobiotics are a super-family of membrane bound, heme-contain-
1381-1177r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 1 - 1 1 7 7 Ž 9 8 . 0 0 0 8 9 - 7
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
372 Table 1 CYPs reaction types
ing enzymes. They are found in many organs in low concentrations but reside mainly in high levels in hepatic tissues and intestinal epithelium. A fundamental property of metabolic reactions carried out by CYPs is that they are all based on electrophilic attack ŽTable 1. w4x. For all CYPs, the different types of ‘oxidative metabolism’ Žhydroxylation, de-alkylation, N-oxidation, epoxidation, etc.. are dependent on substrate structure and physicochemical properties, as well as enzyme active site topology dictating the orientation of the substrate. From a biocatalyst viewpoint, the concept of ‘reaction specific enzyme’ is therefore not appropriate. CYPs have been classified on the basis of their gene sequences w3x. CYPs fall into two broad groups: families I, II and III are responsible for nearly all CYP-dependent oxidative drug metabolism in mammals. In particular, these subfamilies are responsible for most of the oxi-
dation of drugs: CYP2C, CYP2D, CYP2E and CYP3A. These subfamilies are represented in different species, as shown below for rats and humans Ž Table 2.. Substrate structure–activity relationships ŽSSAR. have been characterised for human CYPs. Some prediction of preferredrlikely substrates can therefore be made on the basis of lipophilicity, molecular size, p K a and optimal distances between binding and metabolism sites as summarised in Table 3 w5x. The second group comprises families IV– XXIV. These enzymes are essentially responsible for the metabolism of endobiotic molecules such as hormones and are usually highly substrate specific w5x. They are not commonly involved in the metabolism of drugs. Table 3 SSAR for major human CYPs Some limited SSAR from overlap technique for key CYPs
Table 2 Major rat and human CYPs involved in drug metabolism Rat
Man
CYP2
CYP2C6 CYP2C11 CYP2D1 CYP2E1
CYP2C9 CYP2C19 Žpolymorphic. CYP2D6 Žpolymorphic. CYP2E1
CYP3
CYP2A1 Žinducible. CYP3A2 Žinducible.
CYP3A4 Ž20= variable.
CYP2D6 Ž2.5% of CYPs but 19% of metabolism. structural requirements: l basic nitrogen atom l extended hydrophobic region Žnonspecific binding., site for oxidation 5-7A from nitrogen CYP3A4 Ž28% of CYPs and 34% of metabolism. l wide range of substrate-no apparent structural requirements Žfrom ethylene to cyclosporin. l overall lipophilicity probably important CYP2E1 Ž7% of CYPs and 4% of metabolism. l only small molecules ŽMW-100 Da.
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
373
Fig. 1. Structures of typical CYP101 substrate.
2.2. Metabolism by microbial systems Overall, the characterisation of microbial CYPs Žfamilies LI, LII, CI and CII. has been limited and therefore has not clearly identified families of xenobiotic metabolising enzymes. One area of exception is that of the soluble bacterial CYPs. 2.2.1. Soluble bacterial CYPs Soluble CYPs from bacteria such as those from Pseudomonas putida Ž CYP101. and Bacillus megaterium Ž CYP102 . have been extensively characterised over the past 25 years w6x. In particular, CYP101 ŽP450cam in old nomenclature. has provided structure and mechanistic information generic to all CYPs w7x. Knowledge of its 3D crystal structure Ždown to ˚ resolution w8,9x. has allowed some ex1.6 A tremely useful extrapolation to all CYPs. However, there are significant limitations with re-
gard to range of substrates metabolised by CYP101 as shown below ŽFig. 1.. Similarly, other soluble bacterial CYPs also exhibit very narrow substrate specificity: CYP102 w10x is limited to oxidation of fatty acids and CYP108 w11x Žpseudomonad. is limited to a narrow range of terpenes.
Table 4 Selected applications of microbial CYPs l C. elegans phenacetin w15x carbamazepine w16x furosemide w17x amitriptyline w18x chlorpramazine w19x l B. bassiana diazepam w20x l C. banieri propranolol w21x l S. rimosus zolasartan w22x
374
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
Fig. 2. Microbial oxidation of terfenadine.
Consequently, although soluble CYPs have helped to understand generic and mechanistic aspects of CYPs, their substrate limitations makes them poor predictors of mammalian enzymes and, therefore, unsuitable tools for drug design or as biocatalysts.
malian CYPs, no such information is available for eukaryotic CYPs. Some Žprobably all. possess several CYPs w13x, and it is tempting to speculate that some of these enzymes may have a detoxification role.
2.2.2. Eukaryotic CYPs These CYPs are all membrane bound and appear to have similar structural and mechanistic constraints as mammalian CYPs w3x. Little fundamental research is available, mostly due to the difficulties of isolating individual CYPs for characterisation. One notable exception is the steroid C14 demethylase w12x, principally because of its importance in the treatment of fungal infections with azole antifungal drugs. There is, however, no or little SAR available and in spite of being inhibited by xenobiotics such as fluconazole, ketoconazole or itraconazole, this enzyme is probably highly specific to the metabolism of steroidal substrates. Whereas inducibility is a characteristic of several mam-
3. Applications of microbial CYPs Since the early days of Smith and Rosazza w14x when the basic principles of microbial modelling of mammalian metabolism were set out, much research has been undertaken to characterise this approach. Most of this research has led to factual reporting of microbial biocatalysis results, and although their predictive capabilities are occasionally mentioned, it seems always to be as a generic or throw away comment usually in the introduction. There are many literature examples of microorganisms being used in the metabolism of drugs. A few of the more commonly used microorganisms have been selected below ŽTable 4. to serve as examples.
Fig. 3. Microbial oxidation of diclofenac.
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
Fig. 4. Human metabolism of warfarin w24x.
In our own laboratories, several synthesis problems Ž typically difficulties to synthesise metabolites in a regio-specific manner. have been resolved with microorganisms over the last few years. Two examples are summarised below: terfenadine and diclofenac ŽFigs. 2 and 3. w23x.
4. Discussions 4.1. Are microbial biotransformations predictiÕe of human drug metabolism? At first sight, there appear to be a lot of commonality between all CYPs. But does this include reliable commonality of metabolic pathways of xenobiotics? One of the most frequently reported substrate which is metabolised by both mammalian and microbial CYPs is warfarin. This drug has, therefore, been used to examine any overlap of metabolism patterns between mammalian and microbial CYPs. The human metabolism of warfarin is summarised in Fig. 4, with relevant specificity of individual CYPs.
375
In addition, to allow direct correlation with microbial metabolism of warfarin, comparative data for man and six suitably reported microbial biotransformations are shown in Table 5. Examination of Table 5 reveals that although warfarin is a good substrate for several microorganisms, there is no evidence of correlation with human pathways. The major human metabolite Ž 7-OH. for ŽS. warfarin is only observed as minor with two microorganisms, and the two ‘intermediate’ human metabolites for ŽR. warfarin are not reported for any microorganisms. The 4X-OH metabolite seems to be the most easily obtained compound with microbial systems, being observed in three out of six biotransformations. It is our view that the example of warfarin, one of the most universally characterised probes for CYPs, is probably a fair representation of a general situation. Nonhumanised Ž see below. microorganisms do not possess the necessary attribute of good correlation with human metabolism to be models of human drug metabolism. 4.2. Implications for the use of microbial models of mammalian metabolism Although many microorganisms are very apt at metabolising xenobiotics, the little data available do not support their use as predictive models of human metabolism of drugs and therefore cannot be useful tools for designing drugs with optimum clearance and pharmacokinetics. It is reasonable to speculate that every microorganism will have its own set of CYPs genetically
Table 5 Warfarin: human vs. microbial metabolism X
Man N. corallina w25x B. bassiana w26x C. bainieri w27x C. elegans w28x A. niger w29x S. rimosus w30x
4 -OH
6-OH
7-OH
8-OH
10-OH
minor ŽR. y major major y y minor
intermediate ŽR. y y y y y y
major ŽS. y minor y y y minor
intermediate ŽR. y
minor ŽR. y y y y y y
y y y y
Others 1 metabolism only for S several minor novel X
3 OH oxidative cleavage 12-OH
376
S.G. Jezequelr Journal of Molecular Catalysis B: Enzymatic 5 (1998) 371–377
encoded, possibly inducible, with a broad variation in specificity, similarly to higher organisms variation in CYPs. Although it is likely that all cytochrome genes have evolved from a common ancestral gene w3x, the basic structural similarities exhibited by all the CYPs is mostly confined to mechanism and do not encompass substrate specificity. After all, even enzymes from the same family Ž e.g., 2C. can have differing substrate specificity across species Že.g., rat CYP2C6 and CYP2C11, human CYP2C9 and CYP2C19 . w4x. In addition, it is our experience that growth conditions can be critical; different metabolising activities may be obtained if microorganisms are inoculated or harvested at different stages of their growth. It is therefore wrong and potentially misleading to assume that because a microorganism metabolises a molecule in a particular way, it has an automatic bearing on its fate in man or any animal species. Any such predictions extended to potential toxicity of xenobiotics or their metabolites would also be highly speculative or even misleading. 4.3. Microorganisms as biocatalytic reagents On a positive note, the area of most potential in the use of microorganisms has to be as a biocatalyst for the synthesis of drug metabolites. This approach has been successfully used in our laboratories, to carry out the synthesis of authenticated mammalian metabolites, when classical methods have failed. The chemical synthesis of regio- or stereospecific hydroxylated compound can be a particularly intractable task for the organic chemist, and biocatalysis is generally under-used in the field of preparative drug metabolism. In practice, the lack of knowledge on specific microbial enzymes implies that there is a need for screening to identify the most suitable microorganismŽs. prior to production. This is not, however, a serious barrier for well equipped laboratories. In this context, it is also critical to remember that such biocatalysts are not ‘reac-
tion specific’, rather the combination of structure of each substrate and enzyme topology at the active site will determine the type and position of oxidative process Ž e.g., hydroxylation vs. epoxidation vs. dealkylation vs. N-oxidation. . 4.4. The future of microbial models of mammalian metabolism? In due course, ‘humanised’ cell-lines may provide a reliable tool for predicting routes and rates of human metabolism as well as biocatalysis. Their usefulness as predictors of human metabolism is presently undergoing characterisation in many laboratories and looks very promising w31x. Little work has yet been reported on their potential as specific biocatalysts, and their robustness, in particular, needs investigating.
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