Bio-prospecting for anti-MRSA...
Transcript of Bio-prospecting for anti-MRSA...
Bio-prospecting for anti-MRSA leads
Dr. Venu Mukku
University of Minnesota, Crookston
Outline 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
Natural Products are ….
• Organic compounds produced by plants, micro-organisms and marine invertebrates.
• Produced for the purposes of defense, offense,signaling etc.
• Used since ancient times to treat variousdisorders.
• The best sources for developing new drugs.
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.
• http://www.rbm.who.int/cmc_upload/0/000/015/364/RBMInfosheet_9.htm
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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. O
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• http://www.pesticideinfo.org/Detail_ChemUse.jsp?Rec_Id=PC35467
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, meta-static breast and lung cancers, and Kaposi’s sarcoma.
• Taxol and its analogs may bring revenues up to a quarter of a billion dollars annually.
• http://www.rti.org
• http://www.usda.gov (photos)
• http://www.phcog.org/Taxus/Taxus_Web.html (story)
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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 FDAin 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, 461-477
Classification of approved drugs
Indication Total B N ND S S/NM S*/NM V %
Antibacterial 109 10 64 23 1 11 76
Anticancer 100 17 9 25 18 12 17 2 77
Anticancer drug data is for the period 1950 to June 06
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.
Antibiotic Resistance
• The FDA defines multidrug-resistant organisms (MDRO) 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)
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.
MRSA in North Dakota
• Data taken from ND
Department of Health.
http://www.ndhealth.gov/disease/Info/mrsa.aspx#Historical_Data
(accessed Dec 4th, 2008)
Research experience
• Chemically examined ……
• Soft corals, gorgonians
• Sponges
• Starfishes, sea cucumbers
• Bacteria
• Plants
• Isolated and characterized a number of compounds.
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.
Isolation scheme
Crude ethanol
extract (9.87 g)
Hexane soluble
fraction
DU-145 0.07 μg/mL
Methylene chloride
soluble fraction (3 g)
DU-145 0.04 μg/mL
Butanol soluble
Fraction
DU-145 > 1 μg/mL
Remaining
fraction
DU-145 > 1 μg/mL
Active fraction A
DU-145 0.003 μg/mL
Active fraction B
DU-145 0.66 μg/mL
Kupchan partition
Sephadex LH-20 column
22-Epicalamistrin B
• 22-epicalamistrin B, colorless oil; IR (CHCl3) 3450, 1755, 1740 cm-1; HRMS (APCI positive) m/z 651.5163 [M + H]+
(calcd for C39H70O7, 651.5200)
• 1H NMR (CDCl3, 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);
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Mass spectral fragmentation
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OOHOAcOH
169 (C12H25)
481 (C27H45O7) 421 (C25H41O5) 403 (C25H39O4)
355 (C21H39O4)295 (C19H35O2)
395 (C18H31O3)
Human cancer cell line inhibitory values
(GI50 in μg/mL)
Compound BXPC3 MCF7 SF268 NCIH460 KM20L2 DU145
22-epicalamistrin B 7.3 0.12 11.9 1.2 0.034 0.006
Uvaribonin 1.3 0.002 1.6 1.4 0.005 0.009
Chalcone 0.3 0.3 0.55 0.52 0.36 0.33
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 MDROs
• About 75% of the antibacterial and anticancer drugs are
either natural products or are derived from natural
products
Proposed research
• Antibacterial ‘Lead’ compound discovery
• Anticancer ‘Lead’ compound discovery
• Plant collection
• Extraction and Partition
• Library of plant extracts
Plant collection, Extraction and
Partition
• Plant collection
– Random collection
– Targeted collection – ethnobotanical approach
• Extraction methods
– Aqueous extraction
– Extraction with organic solvents
• Partitioning using the modified Kupchan method
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.
http://web.indstate.edu/thcme/micro/basic.html
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.
http://www.cbs.umn.edu/CGC/what.html
A pivotal study
• Researchers at Harvard medical School , Massachusetts General Hospital and Northeastern University have used this host-pathogen 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, 10414-10419. http://www.pnas.org/cgi/reprint/103/27/10414
C.elegans and Staph strains
• Normal life span of C. elegans is 2 weeks.
• Normal food E. coli OP 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.
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.
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 52955 (Dr. Schmitz, OU, Norman)
• 4. NCI Grant CA 90441 (Dr. Pettit, ASU, Tempe)
• Thanks are also due to
• Dr. Hoffmann for the opportunity to visit UND
• To all of you for coming
Indian Ocean Fauna
http://www.tnenvis.nic.in/bio_faunalgallery.htm
http://people.hws.edu/mitchell/cards01/LEI-015.jpg