Drug discovery from natural products can be prohibitively expensive and inefficient due to the labor and materials required for preparative-scale isolation of an active component, often before its novelty and potency can be determined. We propose to apply 10-fold more sensitive methods of mass spectrometry (MS) and nuclear magnetic resonance (NMR) analysis early in the isolation of a bioactive natural product. The novel MS interface provides a high sensitivity and resolution previously attainable only with nanobore LC-MS, with the use of larger LC columns, which allow enough material to be collected for NMR analysis. The micro-NMR method can acquire NMR spectra of all collected fractions under full automation, with a limit of quantitation below 1 mu/g, and recover the samples for bioassay. Together, these methods enable identification and quantitation of an active species concurrent with its initial isolation, eliminating the lengthy and expensive large-scale purification needed by traditional methods. The proposed methodology will be demonstrated and developed in the isolation of 3 recently-discovered protease inhibitor activities in extracts of cyanobacteria, a slow-growing, low-yield organism that has not been practical to study by traditional methods. The higher sensitivity and non-destructive nature of the new analytical methods enables enough material for analysis and bioassay to be obtained from 100-fold smaller growths, or for 100 times more strains to be grown with the same resources. The ability to perform extraction and purification on an analytical scale rather than preparative scale reduces the time required from weeks to days. These dramatic improvements have the potential to reinvigorate the field of natural products discovery.
The specific aims are: 1) To implement more-sensitive, higher-recovery LC-MS methods using the recently-developed nano-splitter device. 2) To implement high-throughput NMR of fractions collected from LC-MS analysis, combining use of microcoil probes with a method for lossless loading of small volumes (2 uL) from 96-well plates. 3) To provide bioguided fractionation of cyanobacterial extracts, which are optimized for high-throughput micro-NMR and LC-MS analysis. The objective of this proposal is to improve on natural product discovery. This has the potential to directly impact on public health, by overcoming current limitations of traditional methods and reinvigorate the important field of drug discovery from natural sources.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-R (50))
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Schwab, John M
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University of Illinois at Chicago
Schools of Pharmacy
United States
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Kruni?, Aleksej; Orjala, Jimmy (2015) Application of high-field NMR spectroscopy for characterization and quantitation of submilligram quantities of isolated natural products. Magn Reson Chem 53:1043-50
Kautz, Roger; Wang, Poguang; Giese, Roger W (2013) Nuclear magnetic resonance at the picomole level of a DNA adduct. Chem Res Toxicol 26:1424-9
Chlipala, George E; Mo, Shunyan; Orjala, Jimmy (2011) Chemodiversity in freshwater and terrestrial cyanobacteria - a source for drug discovery. Curr Drug Targets 12:1654-73
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Sturdy, Megan; Krunic, Aleksej; Cho, Sanghyun et al. (2010) Eucapsitrione, an anti-Mycobacterium tuberculosis anthraquinone derivative from the cultured freshwater cyanobacterium Eucapsis sp. J Nat Prod 73:1441-3
Krunic, Aleksej; Vallat, Armelle; Mo, Shunyan et al. (2010) Scytonemides A and B, cyclic peptides with 20S proteasome inhibitory activity from the cultured cyanobacterium Scytonema hofmanii. J Nat Prod 73:1927-32
Mo, Shunyan; Krunic, Aleksej; Santarsiero, Bernard D et al. (2010) Hapalindole-related alkaloids from the cultured cyanobacterium Fischerella ambigua. Phytochemistry 71:2116-23
Chlipala, George; Mo, Shunyan; Carcache de Blanco, Esperanza J et al. (2009) Investigation of antimicrobial and protease-inhibitory activity from cultured cyanobacteria. Pharm Biol 47:53-60
Mo, Shunyan; Krunic, Aleksej; Pegan, Scott D et al. (2009) An antimicrobial guanidine-bearing sesterterpene from the cultured cyanobacterium Scytonema sp. J Nat Prod 72:2043-5

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