alan lesniewicz memorial lecture at uic - july 2015
TRANSCRIPT
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: [email protected]
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