This revised grant application is focused on experimental studies to elucidate the biosynthesis of three biogenetically related families of natural products: (1) the Paraherquamides, Asperparalines, Malbranchemaides, Marcfortines and Chrysogenamide (the monooxopiperazines); (2) the Stephacidins, Notoamides, Waikialoids and Brevianamides (the dioxopiperazines); and (3) the Citrinadins, Citrinalins, Cyclopiamines and PF1270 alkaloids (the decarbonylated alkaloids). The goals are to exploit the powerful synergies of total synthesis, whole genome sequencing, bioinformatics analysis, genome mining, functional expression and X-ray crystallography of biosynthetic enzymes to fully elucidate the corresponding biosynthetic pathways to all three families of alkaloids. I. Paraherquamides, Asperparalines, Malbranchemaides, Marcfortines and Chrysogenamide: the Monooxopiperazines. Provocative evidence has been elucidated in our laboratory indicating that this class of alkaloids, are constructed by a rare biosynthetic intramolecular [4+2] cycloaddition reaction resulting from the reductive release of a Trp-Pro (or Trp-Pip) dipeptide amino-aldehyde from a NRPS module that upon reverse prenylation, suffers a cascade of cyclization, dehydration, tautomerization and intramolecular Diels-Alder cycloaddition to construct the monooxopiperazine bicyclo[2.2.2]diazaoctane ring system common to this family. Through the use of total chemical synthesis of isotopically labeled intermediate metabolites, genome mining, biosynthetic gene cluster identification and functional expression of biosynthetic enzymes, key features of the biosynthetic pathways to these complex secondary metabolites will be experimentally elucidated. In a multi-PI relationship and sub-award with Prof. David Sherman's laboratory (University of Michigan), we are actively engaged in the high-resolution elucidation of the entire biosynthetic pathway to these biomedically significant alkaloids. II. Stephacidins, Notoamides, Waikialoids and Brevianamides: The Dioxopiperazines. The dioxopiperazine family of bicyclo[2.2.2]diazaoctane alkaloids are constructed by a net oxidative transformation of a fully prenylated dioxopiperazine substrate. We have discovered that two orthologous species of Aspergillus produce the opposite enantiomers of Stephacidn A and Notoamide B. This fascinating enantiodivergent biogenesis will be further evaluated using bioinformatics analysis by a new collaborator, Prof. Martin Kreitman, a renowned evolutionary geneticist, to determine the evolutionary mechanisms that resulted in this rare production of opposite enantiomers of these complex alkaloids. III. Citrinadins, Citrinalins, Cyclopiamines and PF1270 Alkaloids: The Decarbonylated Alakloids. As a natural out-growth of our work on the Paraherquamide family of prenylated indole alkaloids, we propose to initiate a new project to study the total synthesis and biosynthesis of these structurally related alkaloids that appear to have arisen biogenetically from the reductive decarbonylation of bicyclo[2.2.2]diazaoctane progenitors. Here also, we shall deploy the powerful synergies of total synthesis, whole genome sequencing, bioinformatics analysis, genome mining and functional expression of biosynthetic enzymes to fully elucidate the corresponding biosynthetic pathways to all three families of alkaloids. In all three sub-projects, total synthesis of the natural products and isotopically-labeled biosynthetic intermediates and probe molecules will be utilized to confirm pathway transformations. New chemical entities generated in this program, either from the biological sources or through chemical synthesis, will be extensively screened and evaluated for biological activities at the Univ. of Michigan Center for Chemical Genomics, the National Human Genome Research Institute, and to Prof. Sachiko Tsukamoto (Japan) for analysis of biological activity using a series of biochemical and cell-based assays relevant to cancer and parasitic disease targets. Additional collaborators include: Prof. Sachiko Tsukamoto, Kumamoto University, Japan; Prof. Jens Frisvad, Technical University, Denmark; Prof. Martin Kreitman, University of Chicago; and Prof. Janet Smith, University of Michigan.

Public Health Relevance

The purpose of this application is to utilize the tools of chemical synthesis, whole genome sequencing, genome mining, biosynthetic gene cluster identification and functional expression of biosynthetic enzymes to study the molecular details of how Nature biosynthesizes biomedically significant natural substances that includes anti-cancer drugs. In addition, the chemical technologies being developed will be applied to the design and synthesis of new biologically active agents, with a focus on anti-cancer drugs, and anti-infective agents.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA070375-20
Application #
9110016
Study Section
Synthetic and Biological Chemistry B Study Section (SBCB)
Program Officer
Fu, Yali
Project Start
1997-01-15
Project End
2019-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
20
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Colorado State University-Fort Collins
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
785979618
City
Fort Collins
State
CO
Country
United States
Zip Code
80523
Newmister, Sean A; Romminger, Stelamar; Schmidt, Jennifer J et al. (2018) Unveiling sequential late-stage methyltransferase reactions in the meleagrin/oxaline biosynthetic pathway. Org Biomol Chem 16:6450-6459
Newmister, Sean A; Li, Shasha; Garcia-Borràs, Marc et al. (2018) Structural basis of the Cope rearrangement and cyclization in hapalindole biogenesis. Nat Chem Biol 14:345-351
Klas, Kimberly R; Kato, Hikaru; Frisvad, Jens C et al. (2018) Structural and stereochemical diversity in prenylated indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring system from marine and terrestrial fungi. Nat Prod Rep 35:532-558
Fraley, Amy E; Garcia-Borràs, Marc; Tripathi, Ashootosh et al. (2017) Function and Structure of MalA/MalA', Iterative Halogenases for Late-Stage C-H Functionalization of Indole Alkaloids. J Am Chem Soc 139:12060-12068
Sugimoto, Kayo; Sadahiro, Yusaku; Kagiyama, Ippei et al. (2017) Isolation of amoenamide A and five antipodal prenylated alkaloids from Aspergillus amoenus NRRL 35600. Tetrahedron Lett 58:2797-2800
Kato, Hikaru; Kai, Aika; Kawabata, Tetsuro et al. (2017) Enantioselective inhibitory abilities of enantiomers of notoamides against RANKL-induced formation of multinuclear osteoclasts. Bioorg Med Chem Lett 27:4975-4978
Li, Shasha; Lowell, Andrew N; Newmister, Sean A et al. (2017) Decoding cyclase-dependent assembly of hapalindole and fischerindole alkaloids. Nat Chem Biol 13:467-469
Kagiyama, Ippei; Kato, Hikaru; Nehira, Tatsuo et al. (2016) Taichunamides: Prenylated Indole Alkaloids from Aspergillus taichungensis (IBT 19404). Angew Chem Int Ed Engl 55:1128-32
Newmister, Sean A; Gober, Claire M; Romminger, Stelamar et al. (2016) OxaD: A Versatile Indolic Nitrone Synthase from the Marine-Derived Fungus Penicillium oxalicum F30. J Am Chem Soc 138:11176-84
Le, V H; Inai, M; Williams, R M et al. (2015) Ecteinascidins. A review of the chemistry, biology and clinical utility of potent tetrahydroisoquinoline antitumor antibiotics. Nat Prod Rep 32:328-47

Showing the most recent 10 out of 57 publications