alan lesniewicz memorial lecture at uic - july 2015

Medical Ethnobotany & the

Discovery of New Drugs for

Antibiotic Resistant Infections

Cassandra L. Quave, Ph.D. Assistant Professor of Dermatology & Human Health

Curator, Emory University Herbarium

E-mail: cquave@emory.edu

Lab Website: http://etnobotanica.us/

Twitter: @QuaveEthnobot

Plants as a source of medicine

Willow Aspirin

Foxglove Digoxin/Digitoxin

Mayapple Podophyllin/ Etoposide

Poppy Codeine/ Morphine

Overview

• Ethnobotanical approach to drug discovery

• Antibiotic resistant bacteria as a target

• New strategies for evaluating old remedies

▫ Quorum quenchers

Chestnut

▫ Biofilm inhibitors

Elmleaf Blackberry

• Next generation of anti-infectives

The Science of Ethnobotany

• 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.

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

effective than a random approach.

Ethnobotany is Multidisciplinary

Ethnobotany

Botany

Chemistry

Microbiology

Anthropology

Linguistics

Pharmacology

• Medicine • Conservation • Food security

Deter other plant species from growing nearby

Fight off microbial invasion & infection

Attract pollinators Defense against herbivory

Why do plants make medicinal compounds?

How to find new medicines?

Approach to New Lead Identification

Fie

ld-w

ork

& L

it.

Rev

iew

of

Med

icin

al

Pla

nts

fo

r S

ST

I

Plant collection & Taxonomic

identification

Process materials & prepare extracts

Isolation/elucidation of active leads or

marker compounds Animal Studies

DM/PK and Formulation

Studies

Proof of Concept

Clinical Trials

Bioassay-guided fractionation

MIC/MBC

Quorum Sensing

Cytotoxicity

Biofilms

Study Sites in the Mediterranean

Mount Vulture – Basilicata, Italy

Plant Collecting

Plant Collecting

Plant Collecting

Origanum heracleoticum L., Lamiaceae

Plant Collecting

DNA barcoding

Herbarium Vouchers

Plant Extraction

Dried 48-72 hrs

Vacuum-sealed with silica packets

Pulverized with a grinder

1:10 extraction in 95% EtOH or MeOH 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

Plant Extraction

After freezing at -80°C, extracts are lyophilized Dried extracts

scraped out and weighed

DMSO added, creating a drug solution for antibacterial testing

Plant Extraction

Laboratory studies • Antibacterial testing

• Antibiotic resistance

• Bacterial communication pathways

• Research targets: ▫ MRSA

▫ Eczema

▫ Acne

▫ Pneumonia

▫ Ear infections

▫ Skin infections

▫ Medical device infections

Clinical Relevance • On the precipice of the post-

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

linked to MDR infection in US

• Intrinsic vs. acquired resistance

▫ 17M new biofilm infections/year in US = 550k fatalities

• Antibiotic pipeline nearly empty

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

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

S. aureus exotoxins cause serious disease

Toxic Shock Syndrome Toxin

(TSST-1) Pyrogenic Toxin Superantigens

Scalded Skin Syndrome

Exfoliative Toxins

Abscesses, Necrosis, Sepsis

Hemolytic Toxins, Proteases, Lipases

S. aureus immune evasion mechanisms

Rigby and De Leo. 2012 Semin. Immunopathology 34:237-29

S. aureus immune evasion mechanisms

Rigby and De Leo. 2012 Semin. Immunopathology 34:237-29

Quorum Quenching Approach

• Quorum quenching

▫ “Disarming” bacteria

▫ Protect the host

▫ Adjuvant to existing lines of antibiotics

• Accessory gene regulator (agr) system

▫ controls virulence

Be Toxic!

Be Toxic!

Be Toxic!

Be Toxic!

Quorum Quenching Approach

• Quorum quenching

▫ “Disarming” bacteria

▫ Protect the host

▫ Adjuvant to existing lines of antibiotics

• Accessory gene regulator (agr) system

▫ controls virulence

X

X

X

X X

Not Bactericidal

Be Toxic!

RNAII

AgrB AgrC

agrA agrC agrD agrB P3 P2 RNAIII

PSMα1-4, β1-2

agrD

Extracellular environment

Intracellular environment

AIP

AIP

AIP

AIP

AIP

AIP

agrA P agrA

Cell wall associated proteins fibronectin binding proteins, Protein A

Exoproteins: TSST-1, haemolysins, exfoliative toxins,

enterotoxins, proteases Accessory Gene Regulator (agr) system

Castanea sativa Mill. (Fagaceae)

224

224C

224C-F2

Fractionation

Scheme

agr P3-GFP reporters used to guide fractionation

Quorum quenching by multiple small molecules

0.5

0.7

0.9

1.1

0 12 24 36 48 60 72 84 96

OD

60

0

Retention Time (min)

Growth Inhibition

0

50

100

150

200

250

300

0 12 24 36 48 60 72 84 96

Flu

or

es

ce

nc

e

Retention Time (min)

Quorum Sensing

DMSO control

Test concentration = 25 μg/ml 1 standard deviation 2 standard deviations

224C-F2 inhibits agr in a nonbiocide manner

X X

Not Bactericidal

Quorum Quenching

Growth (OD)

agr (Fluorescence)

224C-F2 blocks d-toxin production

224C-F2-treated S. aureus does not

harm human keratinocytes

224C-F2-treated S. aureus does not produce alpha-hemolysin or lyse RBCs

224C-F2 is non-toxic to human cells & murine skin

224C-F2 reduces dermatopathology & morbidity

Passaging experiments do not yield

resistant mutants

RT: 0.00 - 109.62

0 10 20 30 40 50 60 70 80 90 100

Time (min)

0

10

20

30

40

50

60

70

80

90

100

Re

lative

Ab

un

da

nce

NL:2.27E7

Base Peak F: FTMS - p ESI Full ms [150.00-1500.00] MS 05041503

35 36

1

2

3

37, 38

39, 40

41

42

43

44

46

45

47

48 49

50

57

52 54

53

55 58

56

51

59

60

61

62

63 64

65 66

Time (min)

Rel

ati

ve

Ab

un

dan

ce

224C-F2 LC-MS Chromatogram

94

Core Structures ursene oleanene

plus

Mechanism of action?

Conclusions

Ethnobotanical Lead Effective and Safe

Common Core Structure & New Compounds

Further Isolation and NMR

Quave et al., Submitted

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

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”.

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

Models for Biofilm

Formation & Dispersal

microtiter plate

catheters in vivo

(Imaging with IVIS) flow cells

catheters in vitro

Bioassay-Guided Fractionation Scheme

Following bioassay-guided fractionation steps,

ellagitannins and glycosylated ellagic acid derivatives

were isolated from the extract.

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

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)

220D-F2 improves response to functionally distinct

classes of antibiotics, including daptomycin,

clindamycin, vancomycin, and oxacillin.

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

220D-F2 kills Streptococcus pneumoniae

C

Talekar et al., PLoS One. 2014: 9(5)

Talekar et al., PLoS One. 2014: 9(5)

220D-F2 disrupts Streptococcus

pneumoniae biofilms & kills cells

Conclusions

Ethnobotanical Lead Effective and Safe

Common Core Structure Further Isolation and NMR

Quave et al., PLoS ONE 2012,; Talekar et al., PLoS ONE 2014

Traditional Medicine for SSTI

Herbarium voucher

Plant DNA

Botanical Extracts

δ-hemolysin

RBC lysis Biofilm formation

Planktonic growth

Quorum sensing

In vivo abscess

Va

lid

ati

on

of

TM

Cytotoxicity

Antibiotic Combos

Acknowledgements Quave Lab: James T. Lyles, PhD Kate Nelson Eugenia Addie-Noye Matt Dorian Avni Hajdari Parth Jarivala Rina Lee Tracy Li Emily Mapelli Nami Mottoghi Amelia Muhs Alex Pijeaux Bledar Pulaj Paula Tyler Horswill Lab: Alexander Horswill Heidi Crosby Kristopher Heilmann Jeffery Kavanaugh Corey Parlet

Philanthropic Donors

Questions? Traditional Medicine for SSTI

Herbarium voucher

Plant DNA

Botanical Extracts

δ-hemolysin

RBC lysis Biofilm formation

Planktonic growth

Quorum sensing

In vivo abscess

Va

lid

ati

on

of

TM

Cytotoxicity

Antibiotic Combos