Point of
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Cryptophycin Anticancer Drugs Revisited Jürgen Rohr*
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, 725 Rose Street, Lexington, Kentucky 40536-0082
I
magine a new biotechnologically producible anticancer drug of marine origin with a tubulin-destabilizing mechanism of action that is not negatively affected by P-glycoprotein, a commonly used efflux system of resistant cancer cell lines. This would add another unique natural product into the armamentarium of cancer chemotherapeutics and justify the tremendous efforts that have been invested into marine drug discovery efforts over the past decade or so. The cryptophycins (e.g., cryptophycin 1; Figure 1, panel a) are among the most promising candidates for such a new drug. Like many other natural products, several of marine origin, the cryptophycins interfere with the dynamics of tubulin polymerization and depolymerization. This activity has a long history. Microtubules are dynamic cell structures that play an important role in cell division. Various natural products, such as colchicine, combretastatins, vinca alkaloids, taxanes, epothilones, discodermolides, and dolastatins, bind to tubulin. They eventually cause metaphasic mitotic arrest and consequently apoptotic cell death (1–9). The oldest drug in this context is colchicine. Its damaging effect on tumor vasculature was known in the 1930s, but it was too toxic as an anticancer agent and is now used mostly to treat severe inflammation from gout (10). However, derivatives of the structurally closely related combretastatins A-1 and A-4P were developed and are currently in phase 2 clinical trials as anticancer agents (10). Other tubulin binders, especially several natural (e.g., vincristine and vinblastine) and semisynthetic (e.g., vin-
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desine and vinorelbine) vinca alkaloids and some taxanes (e.g., paclitaxel and docetaxel), have a solid place within regimens of anticancer chemotherapeutics. Various epothilones and their derivatives are in clinical trials as well. Epothilones are easily produced by fermentation and are not affected by P-glycoprotein, so they are favorable over the well-established drug Taxol (paclitaxel) (1, 2). Colchicine, combretastatins, vinca alkaloids, and cryptophycins inhibit the polymerization of tubulin, destabilize the microtubules, and eventually cause metaphasic arrest, whereas taxanes, epothilones, and discodermolides follow a different mechanism of action: they stabilize the microtubules and thus cause misarrangements. This eventually leads to the same overall effect: mitotic arrest and cell death. Interestingly, some recently developed colchicine glycosides obtained through a glycorandomization study (e.g., Col19) seemed to act as microtubule stabilizers; this shows that chemical derivatization can change the principal mechanism of action of a tubulin-binding drug (11). Figure 1 shows examples of tubulin-binding natural products and semisynthetic derivatives. When the cryptophycins, a group of ⬎25 cyanobacterial metabolites with strong tubulin-destabilizing activities (12–14), were discovered, hopes were great that one of these natural products could be developed into a useful anticancer drug. In fact, the prototype cryptophycin 1, the major representative of this class of natural anticancer drugs from the cyanobacterial symbiont
A B S T R A C T A recent publication reveals the biosynthetic building blocks, genetic code, and broad substrate tolerance of the enzymes of the cryptophycin biosynthetic pathway. This work lays the foundation for the production of poorly accessible yet very promising members of this family of anticancer compounds from lichen cyanobacterial symbionts. Chemoenzymatic production or precursor-directed biosynthesis might bring candidates from this family of natural products back to clinical trials.
*Corresponding author,
[email protected].
Published online December 15, 2006 10.1021/cb6004678 CCC: $33.50 © 2006 by American Chemical Society
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The scientific results might provide the basis for the biotechnological development of promising cryptophycin analogues.
tophycin analogues or to develop semisynthetic/biotechnological N O methods for the genO N HN Cl O O O eration of promising O R3 N CO CH cryptophycin anaOCH O N O H OH H O N H logues continued H CO R2 R1 (18–21). Cryptophycin 1 Vincristine: R = CHO, R = OCH , R = COCH Vinblastine: R = CH , R = OCH , R = COCH Now, the research Vindesine: R = CH , R = NH , R = H group of David H. Sherman of the UniverO H CO H CO N sity of Michigan in colN H N laboration with Richard H CO H CO O H CO E. Moore of the UniverOCH O O N CO CH O P O−Na+ H OH O sity of Hawaii (22) has − + O Na OCH O N H OCH H CO studied in a very comOCH HC prehensive way the bioVinorelbine Combretastatin A-4P Colchicine synthesis of the cryptob phycins (see p 766 in OH OH R′ R this issue). The studies OH O O O O OH O 10 O NH O cover a wide range of O O 1 H CO 3′ 13 topics, including incorO N O OCH N OH 15 H OH poration experiments, 2 H S O O HO O H CO 13 N cloning of the gene 12 H CO O O cluster, important bioO OCH synthetic enzymes, and Col19 Epothilone B the production of new cryptophycin anaHO Paclitaxel (taxol): R = , R′ = CH CO logues by precursorO OH O NH O ] directed biosynthesis. OH O Docetaxel (taxotere): R = , R′ = H The scientific results O might provide the basis OH ] Discodermolide for the biotechnological development of promFigure 1. Chemical structures of selected examples of natural product drugs or their semisynthetic derivatives that ising cryptophycin anainterfere with the dynamics of tubulin polymerization and depolymerization. a) Tubulin-destabilizing drugs. b) Tubulin stabilizers. Drugs that are currently used as clinical anticancer drugs or that are in clinical trials are depicted in blue. logues and thus might reopen the door to their Nostoc sp. ATCC 53789, is one of the most was chosen because no large-scale biotech- clinical development. Magarvey et al. (22) nological production method existed for the used a comparative secondary metabolomic potent tubulin-destabilizing agents ever analysis to identify the cryptophycin biosyncryptophycins. Eventually, the high producfound (12). In addition, the cryptophycins, thetic genes (crp). This study is unique in like the epothilones, were not substrates of tion costs and toxic side effects of cryptophycin 52 stopped its development and that that it marks the first time a gene cluster of a P-glycoprotein, an efflux pump that makes natural product has been identified in this multidrug-resistant cancer cell lines immune of any other analogue of the cryptophycin way. The cryptophycin gene cluster (Figure 3) family. Nobody wanted to restart all of the against a multitude of anticancer drugs (4, covers ⬎40 kb of the Nostoc genome. The trials with a different analogue, although 5, 12). Consequently, cryptophycin 52 core of the crp gene cluster consists of two preclinical studies showed that other ana(Figure 2, panel b), a synthetic analogue, modular type-1 polyketide synthase (PKS) was developed and reached phase 2 clinical logues would have been a better choice (18). Nonetheless, studies to find new cryp- genes (crpA and crpB) and two nonribosotrials (15–17). The synthetic analogue 52
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Figure 2. Building blocks of cryptophycins. a) Biosynthetic building blocks of subunits A–D of the cryptophycins. b) Examples of new cryptophycin analogues obtained by precursor-directed biosynthesis via synthetic or natural analogues of the typical building blocks. SAM ⴝ Sadenosyl-methionine
mal peptide synthetase (NRPS) genes (crpC and crpD), as one could expect from the chemical structure of the molecule. These four large genes encode multienzyme complexes, which cover six modules of chain elongation and are followed downstream by four small genes encoding post-PKS/NRPS tailoring enzymes (crpE to H). Interestingly, all genes are assembled with the same reading direction and mirror exactly the biosynthetic assembly-line sequence of events. The most important tailoring enzyme is CrpE, a P450 oxygenase responsible for the epoxide formation found in several cryptophycins, such as cryptophycins 1, 2, and 52. Establishing this epoxide in the desired -stereochemistry was a challenge and required several synthetic steps (23, 24). The characterization of this enzyme allowed the first stereospecific synthesis of cryptophycin 2 through a chemoenzymatic synthewww.acschemicalbiology.org
sis via seco-cryptophycin 4 involving two enzymes, the thioesterase from CrpD and CrpE. Furthermore, through incorporation of stable-isotope-labeled precursors, the biosynthetic origin of all four subunits (A–D) of the cryptophycins could be determined (Figure 2, panel a). The substrate flexibility of the biosynthetic enzymes within the assembly line of the PKS/NRPS multienzyme complex of the cryptophycin pathway was proven by precursor-directed biosynthesis that used unnatural starter units, such as various phenylalanine derivatives and analogues. Surprisingly, these artificial amino acids were incorporated not only into the starter unit but also sometimes into unit B. In addition, halogenase CrpH was tested with bromide and iodide ions, and interestingly, it could also incorporate bromide and iodide atoms into unit B. This result is
typical for marine halogenases, because brominated and iodinated natural products are common in the ocean. Finally, unit C could also be altered through the use of suitable -amino acid precursors. This way, even cryptophycin 52 could be produced, a drug that was previously accessible only through total synthesis and that was chosen for the above-mentioned clinical trials. Overall, the efforts yielded 30 new cryptophycin analogues (for four typical examples, see Figure 2, panel b). In summary, the work of Magarvey et al. (22) applied both established and novel methods and yielded a plethora of very interesting and relevant scientific results for cryptophycin biosynthesis. One could imagine that they have presented a solid base from which cryptophycins can be revisted for clinical use as anticancer agents. First, any of the new analogues produced by precursor-directed biosynthesis may have advantageous properties as clinical drugs. Second, epoxide-containing cryptophycins may be more easily accessible now through a chemoenzymatic approach. Finally, cryptophycin 52 is now biotechnologically producible, which may make this drug more easily accessible and less costly to produce if further pursued. REFERENCES 1. Bergstralh, D. T., and Ting, J. P. (2006) Microtubule stabilizing agents: their molecular signaling consequences and the potential for enhancement by drug combination, Cancer Treat. Rev. 32, 166–179. 2. Attard, G., Greystoke, A., Kaye, S., and De Bono, J. (2006) Update on tubulin-binding agents, Pathol. Biol. (Paris) 54, 72–84. 3. Geney, R., Chen, J., and Ojima, I. (2005) Recent advances in the new generation taxane anticancer agents, Med. Chem. 1, 125–39. 4. Fojo, A. T., and Menefee, M. (2005) Microtubule targeting agents: basic mechanisms of multidrug resistance (MDR), Semin. Oncol. 32, S3–S8. 5. Breier, A., Barancik, M., Sulova, Z., and Uhrik, B. (2005) P-Glycoprotein–implications of metabolism of neoplastic cells and cancer therapy, Curr. Cancer Drug Targets 5, 457–468. 6. Pellegrini, F., and Budman, D. R. (2005) Review: tubulin function, action of antitubulin drugs, and new drug development, Cancer Invest. 23, 264–273. VOL.1 NO.12 • 747–750 • 2006
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Figure 3. The cryptophycin gene cluster and the biosynthetic assembly line process. Domains of the PKS and NRPS multienzyme complexes: A ⴝ adenylation domain, ACP ⴝ acyl carrier protein, KS ⴝ -keto synthase, AT ⴝ acyl transferase, DH ⴝ dehydrase, CM ⴝ C-methyl transferase, KR ⴝ ketoreductase, C ⴝ condensation domain, OM ⴝ O-methyl transferase, PCP ⴝ peptidyl carrier protein, and TE ⴝ thioesterase. 7. Verrills, N. M., and Kavallaris, M. (2005) Improving the targeting of tubulin-binding agents: lessons from drug resistance studies, Curr. Pharm. Des. 11, 1719–1733. 8. Zhou, J., and Giannakakou, P. (2005) Targeting microtubules for cancer chemotherapy, Curr. Med. Chem. Anticancer Agents 5, 65–71. 9. Kavallaris, M., Verrills, N. M., and Hill, B. T. (2001) Anticancer therapy with novel tubulin-interacting drugs, Drug Resist. Updates 4, 392–401. 10. Tozer, G. M., Kanthou, C., and Baguley, B. C. (2005) Disrupting tumour blood vessels, Nat. Rev. Cancer 5, 423–435. 11. Ahmed, A., Peters, N. R., Fitzgerald, M. K., Watson, J. A., Jr., Hoffmann, F. M., and Thorson, J. S. (2006) Colchicine glycorandomization influences cytotoxicity and mechanism of action, J. Am. Chem. Soc. 128, 14224–14225. 12. Smith, C. D., Zhang, X., Mooberry, S. L., Patterson, G. M., and Moore, R. E. (1994) Cryptophycin: a new antimicrotubule agent active against drugresistant cells, Cancer Res. 54, 3779–3784. 13. Panda, D., Himes, R. H., Moore, R. E., Wilson, L., and Jordan, M. A. (1997) Mechanism of action of the unusually potent microtubule inhibitor cryptophycin 1, Biochemistry 36, 12948–12953. 14. Corbett, T. H., Valeriote, F. A., Demchik, L., Lowichik, N., Polin, L., Panchapor, C., Pugh, S., White, K., Kushner, J., Rake, J., Wentland, M., Golakoti, T., Heltzel, C., Ogino, J., Patterson, G., and Moore, R. (1997) Discovery of cryptophycin-1 and BCN-183577: examples of strategies and problems in the detection of antitumor activity in mice, Invest. New Drugs 15, 207–218.
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20. Chaganty, S., Golakoti, T., Heltzel, C., Moore, R. E., and Yoshida, W. Y. (2004) Isolation and structure determination of cryptophycins 38, 326, and 327 from the terrestrial cyanobacterium Nostoc sp. GSV 224, J. Nat. Prod. 67, 1403–1406. 21. Shih, C., and Teicher, B. A. (2001) Cryptophycins: a novel class of potent antimitotic antitumor depsipeptides, Curr. Pharm. Des. 7, 1259–1276. 22. Magarvey, N. A., Beck, Z. Q., Golakott, T., Ding, Y., Huber, U., Hemscheidt, T. K., Abelson, D., Moore, R.E., and Sherman, D.H. (2006) Biosynthetic characterization and chemoenzymatic assembly of the cryptophycins, potent anti-cancer agents from Nostoc cyanobionts, ACS Chem. Biol. 1, 766–779. 23. Liang, J., Moher, E. D., Moore, R. E., and Hoard, D. W. (2000) Synthesis of cryptophycin 52 using the Sharpless asymmetric dihydroxylation: diol to epoxide transformation optimized for a base-sensitive substrate, J. Org. Chem. 65, 3143–3147. 24. Eggen, M., and Georg, G. I. The cryptophycins: their synthesis and anticancer activity, Med. Res. Rev. 22, 85–101.
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