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Current Activities

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The research interests of my group are broadly defined in the area of biosynthesis and mode of action of natural products and is supported by several project directions:

• Biosynthesis of the anti-tumor agent azinomycin and its applications in gene therapy
• Biosynthesis of cycloterpenals and its relatives, natural products produced by the milk protein betalactoglobulin, including cycloretinal and A2E linked to causing age-related macular degeneration
• Developing biochemical and genetic methods to activate biosynthetic pathways that are silent within their respective genomes

Nature greatly influences our research interests. Secondary metabolites (natural products), for example, are compounds that are found only in specific organisms and are an expression of the individuality of species. Indeed, at least 60% of all small molecule drugs on the market today are either natural products or have been derived from natural product leads.

The azinomycins are anti-tumor antibiotics isolated from the culture broths of the terrestrial microorganism, Streptomyces sahachiroi. Their biological activity appears to reside in their ability to covalently alkylate and subsequently cross-link double stranded DNA (binding of azinomycin to DNA shown below).

Our research group is actively characterizing the biosynthetic genes of this pathway, which involves a variety of techniques and strategies including: cloning and overexpression of genes, disruption/knockout of genes, enzymology, as well as chemical synthesis/isotopic labeling studies. Functional characterization of the genes of the pathway will not only shed light on the mechanism of azabicycle formation but will also pave the way for genetic engineering of the pathway and the development of new therapeutic methodologies.

We have also been investigating the biosynthesis and cellular effects of cycloterpenals and their derivatives. Cycloretinal (all-trans retinal dimer), a representative member of this family of natural products is attributed to causing age-related macular degeneration (AMD). AMD is the leading cause of blindness in adults over the age of 50 that can lead to the loss of central vision. One of the most common early characteristic features of AMD (the dry form) is the accumulation of yellow deposits in the eye called drusen. A more severe form of the disease, the wet form, is characterized by neovascularization (abnormal blood vessel formation).

Cycloretinal is a non-degradable, toxic byproduct of the visual cycle. Photochemical activation of the metabolite leads to oxidative damage of the retinal pigment epithelium and adjacent photoreceptor cells. Biochemical evidence from our lab has recently revealed that beta-lactoglobulin (BLG), the principal whey protein in milk, biosynthesizes the lipofuscin cycloretinal (all-trans retinal dimer). The protein survives the pasteurization process, is acid stable and a large fraction of the protein is transported intact through the blood stream (as supported by rabbit studies). Proteomic studies performed by Crabb and co-workers have shown that the protein is present in drusen.

Watanabe Group Journal Cover Feature

Our research group aims to study the role of beta-lactoglobulin in cycloretinal synthesis in the eye as an environmental (dietary), non-genetic contributor of AMD. This involves tracking BLG in the eye, monitoring the formation of cycloretinal, and elucidating the mechanism of cycloretinal formation. Research strategies include: chemical synthesis, enzymology, fluorescence/confocal microscopy, PET imaging, dual modality OCT/fluorescence lifetime imaging.

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Selected Publications

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Foulke-Abel, J.; Watanabe, C. M. H. "Characterization of AznR, an Immunity Protein of the DNA-Crosslinking Agent Azinomycin B", 2011, submitted.

Watanabe, C. M. H. "Biosynthesis and Mode of Action of the Azabicycle Containing Natural Products Azinomycin and Ficellomycin", Natural Product Reports 2010, in press (DOI:1039/C0NP0049C).

Bench, B. J.; Foulke-Abel, J.; Watanabe, C. M. H. "Milk, Revealed 'Silent' Chemistry: New Mode of Cycloretinal Synthesis" 2010 published online Nov. (Cover Feature: Jan 2011, 7, 1-268).

Sharma, V.; Kelly, G. T.; Foulke-Abel, J.; Watanabe, C. M. H. "Aminoacetone as the Penultimate Precursor to the Anti-Tumor Agent Azinomycin A," Org. Lett. 2009, 11, 4006-4009.

Sharma, V.; Kelly, G. T.; Watanabe, C. M. H. "Exploration of the Molecular Origin of the Azinomycin Epoxide: Timing of the Biosynthesis Revealed", Org. Lett. 2008, 10, 4815-4818.

Bench, B. J.; Tichy, S. E.; Perez, L. M.; Benson, J.; Watanabe, C. M. H. "Synthesis and Cellular Effects of Cycloterpinals: Cyclohexadienal-Based Activators of Neurite Outgrowth," Bioorg. Med. Chem. 2008, 16, 7573-7581.

Bench, B. J.; Suarez, V. H.; Watanabe, C. M. H. "An Efficient One-Pot Synthesis of Tethered Cyclohexadiene Enaminonitriles from Methyl-Ketones: An Effective Route to Quinozolines," Bioorg. Med. Chem. Lett. 2008, (Special Issue: Symposium in Print, Wilfred van der Donk), 18, 3126-3130 (Epub 2007, Oct. 18).

Kim, E. J.; Angell, S.; Janes, J.; Watanabe, C. M. H. "Estimating P-Coverage of Biosynthetic Pathways in DNA Libraries and Screening by Genetic Selection: Biotin Biosynthesis in the Marine Microorganism Chromohalobacter," 2008 (Special Issue: Emerging Investigators, invited submission), 4, 606-613 (Epub 2007, Nov 22).

Kelly, G. T.; Sharma, V.; Watanabe, C. M. H. "An Improved Method for Culturing Streptomyces sahachiroi: Biosynthetic Origin of the Enol Fragment of Azinomycin B Firmly Established," Bioorg. Chem. 2008 (Special Issue: Ian Scott Dedication) 36, 4-15 (Epub 2007, Sept 27).

Liu, C.; Kelly, G. T.; Watanabe, C. M. H. "In vitro Biosynthesis of the Antitumor Agent Azinomycin B," Org. Lett. 2006, 8, 1065-1068.

Kelly, G. T.; Liu, C., Smith III, R.; Coleman, R. S.; Watanabe, C. M. H. "Cellular Effects Induced by the Anti-tumor Agent Azinomycin B," Chem. & Biol. 2006, 13, 485-492.

Angell, S.; Bench, B. J.; Watanabe, C. M. H. "Pyocyanin Isolated from a Marine Microbial Population: Synergistic Production Between Two Distinct Bacterial Species and Mode of Action," 2006, Chem. Biol. 2006, 13, 1349-1359.

• Previewed in Bode, H. G. No Need to be Pure: Mix the Cultures Chem. Biol. 2006, 13, 1245-1246.
• Research Highlight in Nature Chem. Biol. 2007, February issue.
• Featured in C&EN News 2007, January 15^th issue.

Bench, B. J.; Liu, C.; Evett, C. R.; Watanabe, C. M. H. "Proline Promoted Synthesis of Ring-Fused Homodimers: Self-Condensation of α, β-Unsaturated Aldehydes," J. Org. Chem. 2006, 71, 9458-9463 (article)

Williams, B.; Watanabe; C. M. H.; Schultz, P. G.; Krucker, T. "Age-dependent effects of Ginkgo biloba extract (EGb 761) on synaptic transmission and plasticity in mouse CA1 hippocampus," Neurobiol. Aging 2004, 25, 955-962.

Watanabe, C. M. H.; Supekova, L.; Schultz, P. G. "Transcriptional Effects to the Potent Enediyne Anti-Cancer Agent Calicheamicin Gamma γ1^I," Chem. Biol. 2002, 9, 245-251.

Watanabe, C. M. H.; Townsend, C. A. "Initial Characterization of a Type I Fatty Acid Synthase and Polyketide Synthase Multienzyme Complex NorS in the Biosynthesis of Aflatoxin B1," Chem. Biol. 2002, 9, 245-251.

Watanabe, C. M. H.; Wolffram, S.; Ader, P.; Rimbach, G.; Packer, L.; Maguire, J. J.; Schultz, P. G.; Gohil, K. "The In vivo Neuromodulatory Effects of the Herbal Medicine Ginkgo Biloba," Proc. Nat. Acad. Sci. USA 2001, 98, 6577-6580.