In response to NIH NOT-OD-09-058 titled """"""""NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications,"""""""" we wish to extend studies spearheaded during the funding of NIH grant 2R01 CA78747 titled """"""""Enediyne Biosynthesis and Engineering."""""""" Cancer causes one of every four deaths in the US. The development of fundamentally new, clinically useful anticancer drugs therefore constitutes a national health and research imperative. The enediynes are the most potent, highly active anticancer agents in existence today, and their use as anticancer drugs has been demonstrated clinically. A great challenge is to develop ways to prepare enediynes and their structural analogs and to discover new enediyne natural products for mechanistic studies and clinical development. We propose in this Competitive Revision application to (1) structurally (by X-ray crystallographic means) characterize the enediyne polyketide synthases (PKSEs) and their associated enzymes for enediyne core biosynthesis as well as other enzymes from selected 9- and 10- membered enediyne biosynthetic pathways;(2) produce and further analyze engineered enediynes with distinct exploitable biophysical properties lending themselves to potential clinical applications;and (3) isolate and characterize new enediynes from microbial sources identified on the basis of genome mining. Our hypotheses are that: (1) characterization of selected novel enzymes involved in enediyne biosynthesis especially C-1027, neocarzinostatin (NCS), maduropeptin (MDP), calicheamicin (CAL), esperamicin (ESP), and dynemicin (DYN)) biosynthesis will make fundamental contributions to mechanistic enzymology and natural product chemistry;(2) enediynes produced by combinatorial biosynthetic methods can and do display biological activities superior to those displayed by the parent compound;such compounds warrant further study enabled only through increased production;and (3) new microorganisms identified on the basis of genome mining produce novel, and potentially medically important, enediynes.
The specific aims for this Competitive Revision application are: (1) In vivo and in vitro characterization of the selected enediyne PKSs and associated enzymes and their roles in both 9- (C-1027, NCS, and MDP) and 10-membered (CAL, ESP, and DYN) enediyne core biosynthesis;(2) Structural characterization of selected enzymes from enediyne (C- 1027, NCS, MDP, CAL, and DYN) biosynthetic machineries by X-ray crystallography;(3) Isolation of engineered C-1027 analogs to evaluate them as anticancer agents in vivo;and (4) Isolation and structural elucidation of novel 9- or 10-membered enediyne natural products from S. ghanaensis NRRL B-12104, A. orientalis ATCC43491, and S. citricolor IFO13005. The outcomes from these studies will greatly accelerate the tempo of our enediyne biosynthesis, engineering, and drug discovery program by (1) defining the minimal enzymes necessary to convert a nascent linear polyene intermediate from the enediyne PKSE to the characteristic enediyne core structure, (2) demonstrating, on a pilot scale, the feasibility of a """"""""structural genomics"""""""" approach to enediyne biosynthesis by solving the structures of key enzymes from selected pathways, (3) advancing C-1027 and its engineered analogs into in vivo testing to realistically develop them into clinically useful, new anticancer drugs, and (4) expanding the portfolio of enediyne anticancer drugs and drug leads by isolating new enediyne natural products.

Public Health Relevance

Cancer causes 1 of every 4 deaths in the US, and 565,650 Americans are expected to die of cancer in 2008. It is therefore a critical research goal to optimize available drugs and to develop fundamentally new, clinically useful anticancer drugs. The enediynes are the most potent, highly active anticancer agents in existence today. Although the natural enediynes have seen limited use as clinical drugs, polymer-based delivery systems and enediyne-antibody conjugates have shown great clinical success or promise in anticancer chemotherapy, demonstrating that the enediynes can be developed into powerful drugs when their extremely potent cytotoxicity is harnessed and delivered to specific cancer cells. A great challenge is to develop methods to make enediynes and their structural analogs and to discover new enediyne natural products for mechanistic studies and clinical developments. This research will study enediyne biosynthesis and engineered novel enediyne analogs. The outcomes include development of enediynes and their analogs into potential anticancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA078747-10S1
Application #
7811497
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (95))
Program Officer
Fu, Yali
Project Start
2009-09-30
Project End
2011-01-31
Budget Start
2009-09-30
Budget End
2011-01-31
Support Year
10
Fiscal Year
2009
Total Cost
$178,357
Indirect Cost
Name
University of Wisconsin Madison
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Chang, Chin-Yuan; Lohman, Jeremy R; Huang, Tingting et al. (2018) Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027. Biochemistry 57:3278-3288
Chang, Chin-Yuan; Yan, Xiaohui; Crnovcic, Ivana et al. (2018) Resistance to Enediyne Antitumor Antibiotics by Sequestration. Cell Chem Biol 25:1075-1085.e4
Rudolf, Jeffrey D; Chang, Chin-Yuan; Ma, Ming et al. (2017) Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function. Nat Prod Rep 34:1141-1172
Annaval, Thibault; Rudolf, Jeffrey D; Chang, Chin-Yuan et al. (2017) Crystal Structure of Thioesterase SgcE10 Supporting Common Polyene Intermediates in 9- and 10-Membered Enediyne Core Biosynthesis. ACS Omega 2:5159-5169
Yan, Xiaohui; Ge, Huiming; Huang, Tingting et al. (2016) Strain Prioritization and Genome Mining for Enediyne Natural Products. MBio 7:
Huang, Tingting; Chang, Chin-Yuan; Lohman, Jeremy R et al. (2016) Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027. J Antibiot (Tokyo) 69:731-740
Smanski, Michael J; Zhou, Hui; Claesen, Jan et al. (2016) Synthetic biology to access and expand nature's chemical diversity. Nat Rev Microbiol 14:135-49
Rudolf, Jeffrey D; Yan, Xiaohui; Shen, Ben (2016) Genome neighborhood network reveals insights into enediyne biosynthesis and facilitates prediction and prioritization for discovery. J Ind Microbiol Biotechnol 43:261-76
Li, Wenli; Li, Xiuling; Huang, Tingting et al. (2016) Engineered production of cancer targeting peptide (CTP)-containing C-1027 in Streptomyces globisporus and biological evaluation. Bioorg Med Chem 24:3887-3892
Chang, Chin-Yuan; Lohman, Jeremy R; Cao, Hongnan et al. (2016) Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier Protein-Tethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus. Biochemistry 55:5142-54

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