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Drug Discovery using Cell-Free 'Meta-biosynthesis' of Pyrrole-Imidazole Alkaloids

E. Paige Stout

Appointment Period: 2011-2013, Grant Years: [27,28]

E. Paige StoutPyrrole-aminoimidazole alkaloids (PAIs) are structurally diverse natural products from marine sponges of the genera Agelas, Stylissa, Phakellia, Axinella, Cymbastella and Hymeniacedon. Sceptrin, an intriguing dimeric C2-symmetric cyclobutane, exhibits a broad range of biological activities,2 including antimicrobial and antimuscarinic properties, and inhibition of motility of a variety of cancer cell lines.2c Agelastatins A and B are potent antitumor agents; 2a inhibits cultured KB nasopharyngeal tumor cells (IC50 0.5-1 ug.mL-1), suppresses osteopontin-mediated malignant transformation by beta-catenin inhibition, and inhibits glycogen synthase kinase (GSK-3b). MCC-001, a PAI isolated in our lab, inhibits the wnt-beta-catenin pathway; a validated cancer target. For the past four years, we have been evaluating an antitumor PAI, agelastatin A - obtained in our laboratory from extracts of Cymbastella sp. in murine models of chronic lymphocytic leukemia (CLL, IC50 ~50 nM). 

It is widely accepted that complex PAIs are biosynthesized by sponges through permutations of reactions (cyclization, oxidation, etc.) of three simple building-block PAIs; hymenidin, oroidin and clathrodin; that also occur naturally. We propose to expand an exciting new concept of 'meta-biosynthesis' developed in our labs in the past 4 months, and demonstrated by cell-free synthesis of non-natural chlorinated analog 4b from synthetic dichloroclathrodin. Harnessing the biosynthetic potential of enzymes in Agelasidae would facilitate synthesis of libraries of non-natural agelastatin analogs for evaluation in preclinical antitumor models. Cell free extracts of Agelas conifera, Stylissa caribica and Cymbastella sp. will be used for preparation of non-natural PAI analogs and evaluation in tumor models. Non-natural PAIs prepared by 'meta-biosynthesis' will be assayed against cultured cell lines to select suitable candidates for further evaluation in murine models of CLL. Multiplexed 'meta-biosynthesis' of PAIs will be undertaken with variations of substrate 3 (R=CN, NO2, F, etc) to produce electron-rich and poor agelastatin and MCC-001 analogs. Meta-biosynthesis with Cymbastella sp. enzymes will be investigated in attempts to prepare nonnatural agelastatin A-like PAIs. Lead compounds generated through this highly innovative approach to natural products discovery will be prioritized for evaluation in murine models according to potency and PK properties.

PUBLICATIONS (resulting from this training)

Stout EP, Wang YG, Romo D, Molinski TF. Pyrrole Aminoimidazole Alkaloid Metabiosynthesis with Marine Sponges Agelas conifera and Stylissa caribica. Angew. Chem. Intl. Ed. Engl. 2012, 51(xx), 4877-xxx. PubMed PMID: 22473581.

Stout EP, Morinaka BI, Wang YG, Romo D, Molinski TF. De Novo Synthesis of Benzosceptrin C and Nagelamide H from 7-(15)N-Oroidin: Implications for Pyrrole-Aminoimidazole Alkaloid Biosynthesis. J Nat Prod. 2012, 75(4), 527-530. PubMed PMID: 22455452.

Other publications (prior to Training Grant appointment):

Rasher DB, Stout EP, Engel S, Kubanek J, Hay ME. Macroalgal terpenes function as allelopathic agents against reef corals. Proc Natl Acad Sci USA. (2011) 108:17726-31. PMID: 22006333; PMCID: PMC3203809.

Stout EP, Cervantes S, Prudhomme J, France S, La Clair JJ, Le Roch K, Kubanek J. Bromophycolide A Targets Heme Crystallization in the Human Malaria Parasite Plasmodium falciparum. ChemMedChem. 2011 Jul 5. doi: 10.1002/cmdc.201100252. [Epub ahead of print] PubMed PMID: 21732541.

Stout EP, Prudhomme J, Roch KL, Fairchild CR, Franzblau SG, Aalbersberg W, Hay ME, Kubanek J. Unusual antimalarial meroditerpenes from tropical red macroalgae. Bioorg Med Chem Lett. 2010 Oct 1;20(19):5662-5. Epub 2010 Aug 10. PubMed PMID: 20801038; PubMed Central PMCID: PMC2939151.

Stout EP, La Clair JJ, Snell TW, Shearer TL, Kubanek J. Conservation of progesterone hormone function in invertebrate reproduction. Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):11859-64. Epub 2010 Jun 14. PubMed PMID: 20547846; PubMed Central PMCID: PMC2900687.

Lin AS, Stout EP, Prudhomme J, Le Roch K, Fairchild CR, Franzblau SG, Aalbersberg W, Hay ME, Kubanek J. Bioactive bromophycolides R-U from the Fijian red alga Callophycus serratus. J Nat Prod. 2010 Feb 26;73(2):275-8. PubMed PMID: 20141173.

Lane AL, Nyadong L, Galhena AS, Shearer TL, Stout EP, Parry RM, Kwasnik M, Wang MD, Hay ME, Fernandez FM, Kubanek J. Desorption electrospray ionization mass spectrometry reveals surface-mediated antifungal chemical defense of a tropical seaweed. Proc Natl Acad Sci U S A. 2009 May 5;106(18):7314-9. Epub 2009 Apr 6. PubMed PMID: 19366672; PubMed Central PMCID: PMC2678663.

Lane AL, Stout EP, Lin AS, Prudhomme J, Le Roch K, Fairchild CR, Franzblau SG, Hay ME, Aalbersberg W, Kubanek J. Antimalarial bromophycolides J-Q from the Fijian red alga Callophycus serratus. J Org Chem. 2009 Apr 3;74(7):2736-42. PubMed PMID: 19271727; PubMed Central PMCID: PMC2707147.

Stout EP, Hasemeyer AP, Lane AL, Davenport TM, Engel S, Hay ME, Fairchild CR, Prudhomme J, Le Roch K, Aalbersberg W, Kubanek J. Antibacterial neurymenolides from the Fijian red alga Neurymenia fraxinifolia. Org Lett. 2009 Jan 1;11(1):225-8. PubMed PMID: 19053716; PubMed Central PMCID: PMC2646105.

Lane AL, Stout EP, Hay ME, Prusak AC, Hardcastle K, Fairchild CR, Franzblau SG, Le Roch K, Prudhomme J, Aalbersberg W, Kubanek J. Callophycoic acids and callophycols from the Fijian red alga Callophycus serratus. J Org Chem. 2007 Sep 14;72(19):7343-51. Epub 2007 Aug 23. PubMed PMID: 17715978.

(maiden name is Auten) Alberti MJ, Auten EP, Lackey KE, McDonald OB, Wood ER, Preugschat F, Cutler GJ, Kane-Carson L, Liu W, Jung DK. Discovery and in vitro evaluation of potent kinase inhibitors: Pyrido[1',2':1,5]pyrazolo[3,4-d]pyrimidines. Bioorg Med Chem Lett. 2005 Aug 15;15(16):3778-81. PubMed PMID: 15993060.