Anti-infective Drugs from Nature: The next generation of antimicrobial therapy? Cassandra L. Quave, Ph.D. Assistant Professor

Emory University

Department of Dermatology (SOM)

Center for the Study of Human Health (ECAS)

E-mail: cquave@emory.edu

Website: http://etnobotanica.us/

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Clinical Relevance • On the precipice of the post-

antibiotic era? ▫ 2M serious infections, 23k fatalities

linked to MDR infection in US

• Antibiotic pipeline nearly empty ▫ Last line therapies for Shigella and

Neisseria gonorrhoeae

▫ CDC Urgent threat level: CRE, Clostridium dificile, N.

gonorrhoeae

▫ CDC Serious threat level includes drug resistant strains of:

Acinetobacter, Camplyobacter, VRE, MRSA, Pseudomonas aeruginosa, Salmonella, Shigella, Streptococcus pneumonia, TB

• We already face a high economic burden for infectious disease (HAI’s in US = $28.4-45 B)

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The Problem: Drug Resistance

Intrinsic vs. acquired

resistance: 17M new

biofilm infections per

year in US = 550k

fatalities

Staphylococcus aureus as a Pathogen • Opportunistic pathogen

• Colonizes nasal passages of 30% healthy adults in US

• Leading cause of: ▫ Bacteremia

▫ Sepsis

▫ Brain abscesses

▫ Medical device infections

▫ Skin and soft tissue infections (SSTI)

• Commonly implicated in: ▫ Bone and joint infections

▫ Surgical site infections

▫ Pneumonia

▫ Endocarditis

• HA-MRSA vs. CA-MRSA

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The Current Approach • More of the “same” ▫ bacteriostatic and

bactericidal agents ▫ Are we asking the right

questions??? • Sources of new drugs ▫ Chemical library screens ▫ Environmental samples ▫ Genome sequencing

and combinatorial chemistry

▫ Animal proteins ▫ Plants and fungi

Natural products (or small molecules

derived from them) account for 75%

of antibacterial drugs!

Why look to plants?

• Natural botanical

products: ▫ Have rich structural diversity,

chirality, and extensive

functional group chemistry

In other words, they are very

cool chemicals!

▫ Are likely produced by the

plant to fill a specific need

(i.e. defense against

pathogens)… there is

potential for efficacy against

human pathogens

Secondary Metabolites

• Organic compounds not

directly involved in basic survival

of the organism (growth,

development, or reproduction)

• In plants, these are used for:

▫ Defense against predation and

herbivory

▫ Competitive “warfare” with

other organisms in the

community

▫ Pollinator attractors

▫ Dispersal

▫ Responsible for plant colors,

flavors, and odors

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Tree creature: Lord of the Rings

Since plants are sessile, secondary metabolites are critical in either attracting or deterring other organisms as needed and can increase fitness of the plant.

Deter other

plant species

from growing

nearby

Fight off

microbial

invasion/infection

Attract pollinators Defense against

herbivory

Secondary Metabolites

History of plants as a source of medicine

• Ancient tradition, dating to prehistory ▫ Pollen samples found in cave

rich in medicinal species Yarrow, cornflower, hollycock,

bachelor’s button, ragwort, grape hyacinth, and woody horsetail

Shanidar Neanderthal cave

site, Iraq (60k-80k years b.p.)

Ephedra altissima Desf.

History of plants as a source of medicine

Hippocrates Engraving by Peter Paul Reubens, 1638

Hippocrates mentions the use

of 300-400 medicinal plants in

his writings (ca. 460-370 B.C.)

Dioscorides De Materia Medica (reproduction in Arabic - Spain, 12th-13th century)

Pedanius Dioscorides seminal work “De

Materia Medica” recorded medicinal

practices of ancient Greece and the Roman

Empire during the time of Nero (ca. 40-90

A.D.). Multiple uses of more than 600

medicinal plants are described.

History of plants as a source of medicine

• Many of these medical traditions persist today worldwide in both indigenous groups and popular culture: ▫ Ayurveda ▫ Unani ▫ Jamu ▫ Kampo ▫ Ancient Egyptian medicine ▫ Traditional Chinese Medicine ▫ Shamanism ▫ Native American traditions ▫ African traditional medicine ▫ Folk medicine

Unani Medicine

Painting by Mahaveer Swami

http://www.hinduonnet.com/

TM can be found in every region of

the globe where humans live.

Ethnobotanical Approach to Drug Discovery

• Ethnobotany (from

ethnology, study of

culture, and botany,

study of plants) is the

scientific study of the

relationships that exist

between peoples and

plants.

• Ethnobotany is the

science of survival.

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Ethnobotanical-directed study of plants used for infectious disease is often more

effective than a random approach.

The Ethnobotanical Approach

Study Sites in the Mediterranean

Mount Vulture – Basilicata, Italy

Southern Italy

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Truffle Hunting in Southern Italy

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Plant Collecting

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Plant Collecting

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Mountain Landscapes – NE Albania

Kukes District, Albania

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Local economy

Local Economy

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Strategy for New Drug Identification Field-work & Lit. Review of Medicinal Plants for skin infections

Plant collection (bulk & voucher)/ Taxonomic identification

Process materials & prepare crude extracts

MIC/ MBC Cytotoxicity In vitro

bioassays

Bioassay-guided fractionation of active extracts

In vivo models Clinical trials

Isolation of active principle(s)

QSI Biofilm

• Layers of Consent:

▫ IRB

▫ Prior informed consent

▫ Country & community

agreements

▫ Plant permits

• Data Collection:

▫ Semi-structured

interviews

▫ Focus groups

▫ Participant-observation

• Biological Sampling:

▫ Voucher collection

▫ Bulk specimen collection

Methods: Ethnobotanical Research

Lulë balsami

Hypericum perforatum L., Hypericaceae

St. John’s Wort

Plantago spp., Plantaginaceae

Plantago lanceolata L.

Plantago major L.

Topical therapies

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Juglans regia L.

Evil-eye amulets of Albania

Zootherapy & Evil-eye Amulets

Methods: Plant Extraction

Dried 48-72 hrs

Vacuum-sealed

with silica packets Pulverized with a

grinder

Methods: Plant Extraction

1:10 extraction in

95% EtOH for 72 hrs.

or boiled in water

for 30 minutes

Plant materials

separated from

extract with

vacuum

filtration

Solvent removed

under reduced

pressure with a

rotary evaporator

Methods: Plant Extraction

After freezing at -80°C,

extracts were

lyophilized Dried extracts

were scraped

out and

weighed

DMSO added,

creating a stock

concentration of

10 mg/ml &

sterile filtered

(0.2 μm)

On Tipping the Balance…

We can not expect

antimicrobials to sterilize the

body, but to tip the balance

back in the body’s favor.

What threatens this: Drugs (including natural products) that confer

extreme selective pressure, quickly yielding resistance.

Example: Resistance to the broad spectrum antimicrobial Tea Tree Oil

(Melaleuca alternifolia Cheel, Myrtaceae) is easily acquired after repeat

exposure.

McMahon et al. 2007. Letters in Applied Microbiology 5:958-965

Example: Garlic

• Allium sativum L., Amaryllidaceae

• Inhibits communication and toxin production in P. aeruginosa

• Decreases hyphae formation (budding in C. albicans)

• Improves response of C. albicans to Amphotericin B in a synergistic fashion

Allicin

IJAA, 33:258-63; FEMS Immun. & Med. Micro., 58: 161-8;

J. Applied Micro., 105: 2169-2177

Example: Cranberry

• Vaccinium macrocarpon Ait.,

Ericaceae

• >1 million women in US get UTI’s each

year

• Proanthocyanidins inhibit adhesion of

P-fimbriated E. coli to uroepithelial

cells

Howell. 2007. Mol. Nutr. Food Res. 51, 732-737

Why Target Pathogenesis & Virulence?

• Attacking microbial defense and offense

mechanisms makes them more vulnerable to

attack by both the host immune response and

existing antimicrobial therapies.

Biofilms and Intrinsic Resistance

• 5-step Process:

1. Initial attachment

2. Irreversible attachment

3. Maturation I

4. Maturation II

5. Dispersion

• Uni- or Poly-microbial

• Heightened gene exchange

• Slow growth/metabolism

• Matrix presents a physical barrier to host immune response and antibiotic

therapy

James et al. (2008) Biofilms in chronic wounds. Wound Repair Regen. 16(1):37-44

Scanning electron micrograph demonstrating the presence of

mixed species biofilm in a chronic wound. Both cocci and bacilli

are seen embedded in an amorphous matrix characteristic of

biofilm formation.

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Models for Biofilm Formation & Dispersal

microtiter plate

catheters in vivo

(Imaging with IVIS) flow cells

catheters in vitro

Elmleaf Blackberry • Traditional uses in S. Italy:

▫ Leaves: furuncles, abscesses,

and other skin inflammations

▫ Roots: hair loss

▫ Fruits: eaten fresh and in

marmalades

• One of 116 remedies related to SSTIs and other topical

dermatological treatments

identified

• 168 extracts screened

• Anti-biofilm activity first identified & published in 2008 and # 220

marked as possible lead

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Quave et al. Journal of Ethnobiology & Ethnomedicine. 2008. 4(5)

Quave et al. Journal of Ethnopharmacology. 2008. 118:418-428

Rubus ulmifolius Schott. (Rosaceae):

The source of the bioactive

composition “220D-F2”.

220D-F2 is effective against all clonal lineages of S.

aureus, regardless of antibiotic resistance profile and is

nontoxic to mammalian cell lines.

Quave et al., PLoS One. 2012: 7(1)

Biofilm Prevention

Biofilm Inhibitor: 220D-F2

220D-F2 improves response to functionally distinct classes of antibiotics,

including daptomycin, clindamycin, vancomycin, and oxacillin.

Quave et al., PLoS One. 2012: 7(1)

Biofilm Treatment

Summary

• There is great promise in nature!

• Pathogenesis is an important drug target

• Our biofilm inhibitor improves antibiotic

activity

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Conclusions

Single drug/single

target,

Bacteriostatic

Bactericidal

New Direction for Drug

Discovery

Immune adjuvants

Conjugation

Biofilms

Quorum sensing

Efflux pumps

Synergy

Clinically relevant isolates

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Acknowledgments

• Collaborators: Dr. Andrea Pieroni (UNSIG, Italy), Dr. Brad Bennett

(FIU), Dr. Lisa Plano (Univ. of Miami), Dr. Michael Otto

(NIH/NIAID), Dr. Mark Smeltzer (UAMS), Dr. Cesar Compadre

(UAMS), Dr. Alex Horswill (Univ. of Iowa)

• All of the study participants in the Mediterranean

• Emory Mentors and Collaborators: Dr. Michelle Lampl, Dr. Dennis

Liotta, Dr. Jorge Vidal, Dr. Sharmilla Talekar, Dr. Bill Shafer, Dr.

Joanna Goldberg, Dr. John Varga, Dr. Effrosyni Seitaridou, Dr.

Maira Goytia, Dr. David Weiss, Dr. Phil Rather, Dr. Emily Weinert,

Dr. Effrosyni Seitaridou, Dr. Brian Pollack and Dr. Jack Arbiser

• Current Lab Team: Kate Nelson, Dr. James Lyles, Parth Jariwala,

Janessa Aneke, Sam Anderson, Boru Wang, Samir Hussaini,

Sandy Jiang, Michelle Paine, Matt Dorian, Sarah Meadows,

Paula Tyler, Ian Buller and Amelia Muhs

• National Institutes of Health, National Center for Complementary

and Alternative Medicine (PI: Quave)

▫ R01 AT007052

▫ F32 AT005040

▫ F31 AT004288

• Georgia Research Alliance

▫ GRA.VL13.C7 (Phase IA & IB)

• Center for the Study of Human Health

People

Funding Sources

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To support our work through philanthropic donations, see

http://etnobotanica.us/ for details!