Bio-prospecting for anti-mrsa leads. Dr. Venu Mukku University of Minnesota, Crookston

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1 Bio-prospecting for anti-mrsa leads Dr. Venu Mukku University of Minnesota, Crookston

2 utline of the talk Synopsis Natural products notable examples Why work on natural products? Aren t there enough antibiotics already? Past research experience Bioassay-guided fractionation Bioassays Summary

3 Natural Products are. rganic compounds produced by plants, microorganisms and marine invertebrates. Produced for the purposes of defense, offense, signaling etc. Used since ancient times to treat various disorders. The best sources for developing new drugs.

4 Notable Natural Products - Artemisinin Artemesia annua (sweet annie) was described (168 BC) in the Chinese medical treatise, 52 Remedies Artemisinin, isolated (1972) from A. annua, is a potent and effective antimalarial. Artemisinin when used in combination therapy produces high cure rates in 3 days. load/0/000/015/364/rbminfosh eet_9.htm H H H

5 Azadirachtin Insect antifeedant If you have just one tree to plant, what would it be? Azadirachta indica (neem). Its products are sold worldwide as natural and easily biodegradable pesticides. Azadirachtin, the principal component, inhibits the growth and development of many insect larvae at 1-10 ppm. Azadirachtin is used to protect about 50 different crops in CA. H H H H H H

6 Taxol Isolated by Drs. Wall and Wani of the Research Triangle Institute in 1971 from the bark of Taxus brevifolia (Pacific Yew tree). Taxol is now being used for the treatment of ovarian cancer, metastatic breast and lung cancers, and Kaposi s sarcoma. Taxol and its analogs may bring revenues up to a quarter of a billion dollars annually. (photos) eb.html (story) NH H H H H Ac

7 Natural Products based Drug Development A perpetual success story Drs. Newmann and Cragg of the National Cancer Institute (NCI) mention that out of the 109 antibacterial drugs approved by the FDA in the last 25 years, 76% owe their origin to a natural product. The approved drugs were divided into: Biologicals (B) Natural products (N) Drugs derived from natural products (ND) Totally synthetic drugs (S) Synthetic drugs but the pharmacophore is/was a natural product (S*) Natural product mimics (NM) and Vaccines (V) Newman, D. J; Cragg, G. M. J. Nat. Prod., 2007, 70,

8 Classification of approved drugs Indication Total B N ND S S/NM S*/NM V % Antibacterial Anticancer As can be seen from the table, Natural Products have played a crucial role in the development of a majority of antibacterial and anticancer drugs. Anticancer drug data is for the period 1950 to June 06

9 Antibiotic Resistance The FDA defines multidrug-resistant organisms (MDR) as micro-organisms, predominantly bacteria, that are resistant to one or more classes of antibiotics. Examples: MRSA (methicillin resistant Staphylococcus aureus) MRSE (methicillin resistant S. epidermidis) VRE (vancomycin resistant Enterococci) VISA (vancomycin intermediate S. aureus) VRSA (vancomycin resistant S. aureus)

10 Severity of MRSA Causes serious problems in premature infants and post-operative patients. Nosocomial infections prolong hospital stay. About 1 in 4 discharges have MRSA. CA MRSA strains are readily transmitted in crowded settings.

11 MRSA in North Dakota Data taken from ND Department of Health. (accessed Dec 4 th, 2008)

12 Research experience Chemically examined Soft corals, gorgonians Sponges Starfishes, sea cucumbers Bacteria Plants Isolated and characterized a number of compounds.

13 Ampelocissus sp. Collected from Malaysia. Ethanol extract of the roots was obtained from the NCI and partitioned according to the modified Kupchan procedure (shown on next slide). Series of fractionations on Sephadex LH-20 followed by RP HPLC yielded 22-epicalamistrin B, uvaribonin and a chalcone. Structures of the three compounds were determined by a study of their NMR and MS spectral data. The striking feature of the first two compounds is their selectivity to certain cancer cell lines.

14 Isolation scheme Crude ethanol extract (9.87 g) Kupchan partition Hexane soluble fraction Methylene chloride soluble fraction (3 g) Butanol soluble Fraction Remaining fraction DU μg/ml DU μg/ml DU-145 > 1 μg/ml DU-145 > 1 μg/ml Sephadex LH-20 column Active fraction A DU μg/ml Active fraction B DU μg/ml

15 22-Epicalamistrin B 22-epicalamistrin B, colorless oil; IR (CHCl 3 ) 3450, 1755, 1740 cm -1 ; HRMS (APCI positive) m/z [M + H] + (calcd for C 39 H 70 7, ) 1 H NMR (CDCl 3, 400 MHz); 3.38 (1H, m, H-15); 5.08 (1H, m, H-17); 3.78 (m, 2H, H-18 & H-21); 3.34 (1H, m, H-22); 6.95 (1H, d, H-35); 4.92 (1H, dq, H-36); 22 H 17 Ac 15 H

16 Mass spectral fragmentation 295 (C 19 H 35 2 ) 169 (C 12 H 25 ) 355 (C 21 H 39 4 ) H Ac H 395 (C 18 H 31 3 ) 481 (C 27 H 45 7 ) 421 (C 25 H 41 5 ) 403 (C 25 H 39 4 )

17 Human cancer cell line inhibitory values (GI 50 in μg/ml) Compound BXPC3 MCF7 SF268 NCIH460 KM20L2 DU epicalamistrin B Uvaribonin Chalcone

18 Recap & continue.. Natural products are used since pre-historic times Research on natural products has evolved with times Urgent need to develop new drugs to fight MDRs About 75% of the antibacterial and anticancer drugs are either natural products or are derived from natural products

19 Proposed research Antibacterial Lead compound discovery Anticancer Lead compound discovery Plant collection Extraction and Partition Library of plant extracts

20 Plant collection, Extraction and Plant collection Random collection Partition Targeted collection ethnobotanical approach Extraction methods Aqueous extraction Extraction with organic solvents Partitioning using the modified Kupchan method

21 Bio-assays - Disc diffusion assay An in vitro assay; suffers from some inherent problems like solubility and toxicity. Some compounds which are inactive in vitro may exhibit activity in vivo. Some compounds may exhibit activity in vitro but may lose activity in vivo.

22 Caenorhabditis elegans Caenorhabditis elegans (a nematode), was found to survive infection with MRSA and Enterococcus faecalis when treated with an antibiotic such as tetracycline. There is a broad overlap between the virulence factors required for the onset of pathogenesis in humans and C. elegans.

23 A pivotal study Researchers at Harvard medical School, Massachusetts General Hospital and Northeastern University have used this hostpathogen model to study the antibacterial activity of 6000 synthetic compounds, and 1136 natural product extracts. Some of the active compounds and extracts did not exhibit any in vitro activity. Advantages of this model. Since nematode survival is the criteria, all compounds that are either ineffective or toxic are weeded out. It was found that the minimum inhibitory concentrations are much smaller than those found in in vitro assays. Moy, T. I.; Ball, A. R.; Anklesaria, Z.; Casadei, G.; Leis, K; Ausubel, F. M. PNAS, 2006, 103,

24 C.elegans and Staph strains Normal life span of C. elegans is 2 weeks. Normal food E. coli P 50. S. aureus strain NCTC 8325 kills C. elegans if the latter were exposed to the former for greater than 18 hours. C. elegans is able to clear the pathogens from its gut if the exposure is less than 8 hours. The killing is currently attributed to the accumulation of live bacteria in the digestive tract.

25 Summary Research proposal based on the past success of natural products as good leads for drug development. Majority of antibacterial drugs owe their origin to natural products. Currently used antibiotics are fast becoming ineffective due to emerging resistance in pathogens. Some strains such as MRSA are already resistant to multiple antibiotics. C. elegans was found to be a model host organism in which pathogenesis can be induced and then treated with antibiotics. Using C. elegans as a model host, I am proposing to examine MN flora for antibacterial compounds. Bugs beware! You will be drugged.

26 Acknowledgements The research mentioned in today s talk was at various times funded by (in chronological order) 1. Council of Scientific and Industrial Research (CSIR), New Delhi 2. Alexander von Humboldt Foundation, Bonn, Germany 3. NCI Grant CA (Dr. Schmitz, U, Norman) 4. NCI Grant CA (Dr. Pettit, ASU, Tempe) Thanks are also due to Dr. Hoffmann for the opportunity to visit UND To all of you for coming

27 Indian cean Fauna