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George B. Kauffman California State University Fresno, CA 93740
Developing New Antibiotics with Combinatorial Biosynthesis Nicola L. Pohl Department of Chemistry and the Plant Sciences Institute, Iowa State University, Ames, IA 50011;
[email protected] The rapid and extensive emergence of antibiotic-resistant bacteria has resulted in a clear need to discover new compounds for human medicine (1). Many of the old “wonder” drugs, such as penicillin and erythromycin, originate from natural sources such as bacteria and fungi; the pharmaceutical industry learned to cultivate these organisms on a large scale and then harvest their valuable products to treat a wide variety of formerly lethal infections. Erythromycin (Fig. 1), isolated from soil bacteria in 1952 (2), now is produced in multikiloton quantities annually (3). Ironically, the very ubiquity of antibiotics has also led to the explosion of microorganisms that have evolved ways to evade the toxicity of these drugs. Fear of another pre-antibiotic era therefore fuels a quest for new antibacterial drugs. Antibiotics, compounds that are produced by one microorganism to kill another microorganism, are not necessarily the ideal therapeutics for human use. Erythromycin suffered from marginal chemical stability in the acidic environment of the human gut, but synthetic chemists could selectively modify this natural product by methylation of a tertiary hydroxyl group, thereby creating the more stable clarithromycin (4) (Fig. 1). Despite monumental efforts (5), chemical synthesis of the entire erythromycin structure is cost-prohibitive for commercial purposes; moreover, the complex structure makes site-selective modification difficult. Because each new structure often correlates with unique functions, the generation of a large array of structures is key to discovering molecules that have new functions in the war against infectious bacteria. It is reasonable to propose that a large array of novel compounds could be produced by manipulating the biosynthetic pathways of antibiotic-producing bacteria. This hope drives the emerging field of combinatorial biosynthesis (6–8), which allows further exploitation of a historically rich fount of antibiotics in addition to providing an avenue for the engineering of chemical handles on complex molecules for the synthesis of an even greater diversity of structures and functions.
tions of this chain, such as reduction of a ketone to an alcohol, serve to variegate the backbone, thereby creating a plethora of structures. This great structural complexity has made polyketides a prime source for therapeutically active compounds that include not only antibiotics such as erythromycin, but also antitumor compounds such as doxorubicin (Fig. 1). Inspection of the final products of these biosynthetic pathways provides valuable clues as to how polyketides are made; however, an understanding of the actual machinery that executes these reactions has surfaced only recently. Sequencing
O O HO
O OH
OR
OH
N(CH3)2 HO O
O O
OH
O OCH3 O
OH
OCH3
O
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O NH2 OH
OH
O
Erythromycin R=H Clarithromycin R=CH3 (antibiotics)
Doxorubicin (anticancer agent)
Figure 1. Polyketides comprise a large, structurally diverse class that is the source of many therapeutically relevant drugs.
O _
O O
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Condensation O S
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OH
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Reduction
Polyketide Antibiotics Erythromycin, in the absence of its two crucial sugar appendages, belongs to a structurally diverse class of compounds called polyketides (9). Polyketides are named for their apparent biosynthetic origins as a linear sequence of ketones separated by single carbons. Collie hypothesized in 1893 that these compounds were synthesized biologically from ketenes (CH2=C=O) or their equivalents (10). Later, Birch proposed that a condensation reaction between acetate groups could slowly build the carbon backbone of polyketides (11) (Fig. 2). In fact, malonic acid derivatives are used by bacteria as a source of an anion equivalent by decarboxylation and concomitant attack at the carbonyl carbon of a reactive acetic acid derivative. The result is a long chain of ketone functionalities. Modifica-
OH
"H_" OH
O
O Dehydration
"H_"
O
O Reduction
Figure 2. The assembly of a polyketide chain by classic organic reactions.
JChemEd.chem.wisc.edu • Vol. 77 No. 11 November 2000 • Journal of Chemical Education
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DEBS1
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DEBS3
module 3
module 1
module 5 module 4
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O
AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP KS AT DH ER KR ACP KS AT KR ACP KS AT KR ACP TE HO
S
S O
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HO
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HO
HO
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HO HO
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S
S O
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OH OH
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HO O HO
O
HO
HO HO
Figure 3. The machinery that assembles the erythromycin backbone comprises three large separate proteins (DEBS1–3), each of which contains two modules that perform one condensation cycle each. The separate domains of each module are abbreviated as follows: AT = acyltransferase, ACP = acyl carrier protein, KS = ketosynthase, KR = ketoreductase, DH = dehydratase, ER = enoyl reductase, TE = thioesterase.
of a portion of the genetic code of the erythromycin-producing soil bacteria suddenly unveiled an assembly line of protein domains whose successive functions mimicked the successive chemical reactions required to form the long chain of erythromycin (12, 13). An enzyme domain dubbed a ketosynthase (KS) is responsible for the condensation reaction between units provided by an acyltransferase (AT), followed by a ketoreductase (KR), which when present reduces this ketone to an alcohol. A dehydratase eliminates the resulting hydroxyl to form an alkene. Finally an enoylreductase (ER) domain reduces the ketone-conjugated alkene to ultimately form a fully saturated carbon where a carbonyl once was (Fig. 3). Successive methylenes constitute the alkyl chain of a fatty acid. The polyketide-producing proteins are actually close relatives of the fatty-acid-producing proteins (14). Both the fatty-acid- and the polyketide-synthesizing proteins perform a succession of reactions that are ideal examples in the natural world of reactions taught in basic organic chemistry. Harnessing Nature The wide variety of biologically potent polyketides sparks interest in the manipulation of these biosynthetic pathways to new ends. Imagining the biosynthetic machinery of erythromycin as an assembly line leads to the possibility of altering the product by redesigning the assembly line. Can the elimination of one function along the line produce a new polyketide chain that is capable of undergoing further elaboration into a novel compound? Several experiments conclusively show that the answer is yes. If the ketoreductase function is excised, for example, a ketone rather than an alcohol ends up in the final product (13). With this information, a method of generating novel structures that can be tested for antibiotic or other activity emerges. The field of chemistry has already embraced the possibility of great chemical diversity by the use of various combinations of reagents in multistep syntheses. This approach has been called combinatorial chemistry (15). A simple example of this concept is the combination of three acid chlorides (1–3) with three amines (4–6) in solution (Fig. 4). Nine new amides can 1422
be formed this way. If each set of reactions involved in one round of condensation in polyketide formation is seen as a separate module (1–6, Fig. 3), the same variety of products should be available by combining these modules in different orders. This paradigm is the core of combinatorial biosynthesis of polyketides (6, 16 ) and has already generated an array of new compounds, some of which show antibacterial activity (17, 18). In essence, combinatorial biosynthesis exploits nature’s toolbox of basic organic reactions to do combinatorial chemistry. Future Directions Combinatorial biosynthesis holds great promise for the synthesis of novel chemical entities. Other classes of natural products such as the penicillins also have biosynthetic pathways that are currently being elucidated and are amenable to reengineering (19). Erythromycin requires its sugar appendages for bioactivity; those carbohydrate biosynthetic pathways are likewise under investigation (20). Terpene-
A Combinatorial Array R 1
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+ R
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N H
Figure 4. The principle behind combinatorial chemistry and combinatorial biosynthesis relies on various combinations of sets to create arrays, or libraries, of products. These sets could contain reactive acyl chlorides (1–3) and amines (4–6) or different modules (modules 1–6, Fig. 3) of a polyketide-synthesizing protein.
Journal of Chemical Education • Vol. 77 No. 11 November 2000 • JChemEd.chem.wisc.edu
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derived natural products pathways could similarly be harnessed. Of course, newly generated compounds that display antibacterial action by a mechanism unrelated to the original compound will kill the host bacteria or be chemically inactivated by the host. In these cases, alternative host organisms are sought. Combinatorial biosynthesis will allow the generation of many presently unknown complex structures that can be tested for antibacterial activity, in addition to other therapeutic bioactivities, thereby successfully contributing to the race against antibiotic-resistant infectious bacteria. Acknowledgments I thank Chaitan Khosla and anonymous referees for reviewing this manuscript and the National Institutes of Health for a postdoctoral fellowship (1 F32 GM19540-01). Literature Cited
6. 7. 8. 9. 10. 11. 12. 13.
1. Levy, S. B. Sci. Am. 1998, 278 (3), 46. 2. McGuire, J. M.; Bunch, R. L.; Anderson, R. C.; Boaz, H. E.; Flynn, E. H.; Powell, M.; Smith, J. W. Antibiot. Chemother. 1952, 2, 281. 3. Minas, W; Bruenker, P.; Kallio, P. T.; Bailey, J. E. Biotechnol. Prog. 1998, 14, 561. 4. Morimoto, S.; Takahashi, Y.; Watanabe, Y.; Omura, S. J. Antibiot. 1984, 37, 187. 5. Woodward, R. B.; Logusch, E.; Nambiar, K. P.; Sakan, K.; Ward, D. E.; Au-Yeung, B.-W.; Balaram, P.; Browne, L. J.; Gard, P. J.; Chen, C. H.; Chenevert, R. B.; Fliri, A.; Frobel, K; Gais, H.-J.; Garrat, D. G.; Hayakawa, K.; Heggie, W.; Hesson, D.
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P.; Hoppe, D.; Hoppe, I.; Hyatt, J. A.; Ikeda, J.; Jacobi, P. A.; Kim, K. S.; Kobuke, Y.; Kojima, K.; Krowicki, K.; Lee, V. J.; Leutert, T.; Malchenko, S.; Martins, J.; Matthews, R. S.; Ong, B. S.; Press, J. B.; Rajan Babu, T. V.; Rosseau, G.; Sauter, H. M.; Suzuki, M.; Tatsuta, K.; Tolbert, L. M.; Truesdale, E. A.; Uchida, I.; Udea, Y.; Uyehara, T.; Vasella, A. T.; Vladuchick, W. C.; Wade, P. A.; Williams, R. M.; Wong, H. N.-C. J. Am. Chem. Soc. 1981, 103, 3210, 3213, 3215. Tsoi, C. J.; Khosla, C. Chem. Biol. 1995, 2, 355. Khosla, C. Chem. Rev. 1997, 97, 2577. Cane, D. E.; Walsh, C. T.; Khosla, C. K. Science 1998, 282, 63. Mann, J. Chemical Aspects of Biosynthesis; Oxford University Press: Oxford, 1994; p 19. Collie, J. N. J. Chem. Soc. 1893, 63, 329. Birch, A. J. Science 1967, 156, 202. Cortes, J.; Haydock, S. F.; Roberts, G. A.; Bevitt, D. J.; Leadley, P. F. Nature 1990, 348, 176. Donadio, S.; Staver, M. J.; McAlpine, J. B.; Swanson, S. J.; Katz, L. Science 1991, 252, 675. Carreras, C. W.; Pieper, R.; Khosla, C. Top. Curr. Chem. 1997, 188, 85. Frobel, K. Sci. Spectra 1999, (15), 52. Katz, L. Chem. Rev. 1997, 97, 2557. Jacobsen, J. R.; Hutchinson, C. R.; Cane, D. E.; Khosla, C. Science 1997, 277, 367. Marsden, A. F. A.; Wilkinson, B.; Cortes, J.; Dunster, N. J.; Staunton, J.; Leadley, P. F. Science 1998, 279, 199. Byford, M. F.; Baldwin, J. E.; Shiau, C.-Y.; Schofield, C. J. Chem. Rev. 1997, 97, 2631. Hallis, T. L.; Lui, H.-W. Acc. Chem. Res. 1999, 32, 579.
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