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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OHOAcOH

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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