Slides from CDD's March 22 Webinar - Penetrating Gram Negative Bacteria. Hosted by Brad Sherborne (Merck) featuring Derek Tan (Memorial Sloan Kettering Cancer Center) and Helen Zgurskaya (University of Oklahoma).

1. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Antibiotic Discovery Webinar:
Penetrating Gram Negative Bacteria
March 22, 2018

2. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Today’s Guest
Moderator
Brad Sherborne
Director of Computer Aided Drug
Design at Merck and Co. Inc.
Today’s Panelists
Helen Zgurskaya
Professor of Chemistry &
Biochemistry at University of
Oklahoma
Derek Tan
Chairman, Member, and
Tri-Institutional Professor, and
Chairman of the Chemical Biology
Program at Memorial Sloan
Kettering Cancer Center

3. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Adapted from Lynn L. Silver, “Challenges of Antibacterial Discovery,” Clinical Microbiology Reviews 24, no. 1. (2011): 71-109.
Please ask a question of our panelists by
opening the GoToWebinar Questions
Panel and typing in your question at
anytime !
We’ll be sure to save time for them later!

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Gram-negative pathogens are protected from antibiotics by two-membrane
cell envelopes studded by active multidrug efflux pumps
Outer membrane
Inner membrane
Peptidoglycan
Efflux pump

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Examples of Gram-Negative Antibiotics
5
ceftriaxone gentamicin
imipenem trimethoprim–sulfamethoxazole
ciprofloxacin

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Hiroshi Nikaido, UC Berkeley

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The most interesting latest results
• Hyperpotination of the outer membrane enables
analyses of contributions of the outer membrane barrier
and active efflux in accumulation and activities of
antibiotics (Krishnamoorthy et al., 2017, mBio, 8).
Δ3-
Pore
PAO1-
Pore
P. aeruginosa
IPTG - +
+
Δ3-Pore
• New mathematical model that accurately describes permeation of
antibiotics across two membranes with active efflux (Westfall et al.,
PLOS One, 2017, 12(9):e0184671)
Valentin Rybenkov, OU

8. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
8
Activity-Independent Analysis of Compound Accumulation
• Davis, T. D.; Gerry, C. J.; Tan, D. S. “General platform for systematic quantitative evaluation of small-
molecule permeability in bacteria.” ACS Chem. Biol. 2014, 9, 2535–2544; doi: 10.1021/cb5003015
• LC-MS/MS quantitation: Cai, H.; Rose, K.; Liang, L. H.; Dunham, S.; Stover, C. Anal. Biochem. 2009, 385, 321−325.
Incubate bacteria with compound
(± efflux pump inhibitors)
Wash (4x PBS) & centrifuge
 Washes
Lyse (sonication) & centrifuge
 Lysate  Intracellular
LC-MS/MS analysis vs.
internal standard
linear range of detection:
0.0025–100 mM (PBS)
CFU
determination
Centrifuge
 Supernatant
100 mM extracellular
30 min, tryptic soy broth
0.01–1000 mM extracellular
20 min, PBS
100 mM extracellular
0–60 min, PBS
100 mM extracellular
15 min pre-load, PBS + CCCP
• Salicyl-AMS accum/efflux • Concentration effects
• Accumulation kinetics • Efflux kinetics
passive
activeefflux
linear
range
25 mM

9. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery

10. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Equilibrium and kinetic drivers of drug accumulation
𝑂
𝑘1
𝑘6+𝑣
𝑀
𝑘5
𝑘2+𝑣
𝑃
𝑘3
𝑘4
𝐼
Westfall et al., 2017
• Drug uptake is governed
by permeation rates and
drug efflux
• Multiple transporters with
diverse mechanisms
contribute to efflux (and
sometimes uptake)
• Efficiencies and
specificities of active
transporters define to
what extent the internal
concentration of the drug
is reduced/increased as
compared to that at the
equilibrium.

11. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Testing Predictions Based on Cheminformatic Analysis
11
salicyl-(2-phenyl-AMS)
salicyl-(2-phenylamino-AMS)
• Davis, T. D.; Gerry, C. J.; Tan, D. S. “General platform for systematic quantitative evaluation of small-molecule
permeability in bacteria.” ACS Chem. Biol. 2014, 9, 2535–2544; doi: 10.1021/cb5003015
60 237 358mM:
salicyl-AMS
E. coli accumulation
correlates with size,
hydrophobicity,
aromatic ring content
Physicochemical
Properties
Accumulation in E. coli

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Physicochemical Properties Affecting Accumulation in E. coli
12
• Richer, M. F.; Drown, B. S.; Riley, A. P.; Garcia, A.; Shirai, T.; Svec, R. L.; Hergenrother, P. J. “Predictive compound accumulation rules
yield a broad-spectrum antibiotic.” Nature 2017, 545, 299–340; doi: 10.1038.nature22308
Primary amines

13. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
component
loadings
13
Modeling the Unique Physicochemical Properties of Antibiotics
non-
antiinfectives
• Davis, T. D.; Gerry, C. J.; Tan, D. S. “General platform for systematic
quantitative evaluation of small-molecule permeability in bacteria.”
ACS Chem. Biol. 2014, 9, 2535–2544; doi: 10.1021/cb5003015
• Krishnamoorthy, G., Leus, I.V., Weeks, J., Wolloscheck, D., Rybenkov, V.V.,
Zgurskaya, H.I. Synergy between active efflux and outer membrane diffusion defines
rules of antibiotic permeation into Gram-negative bacteria. mBio. 2017, 8, e01172-17.
doi: 10.1128/mBio.01172-17.

14. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Adapted from Lynn L. Silver, “Challenges of Antibacterial Discovery,” Clinical Microbiology Reviews 24, no. 1. (2011): 71-109.
Reminder to:
Pose your questions
to the panelists
here!

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…If you had a Magic Wand?

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… and a source of money?

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…what could you fund?

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The Pew Charitable Trust’s SPARK Initiative

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AIM of SPARK from the Pew Roadmap
• Overcome the cellular defenses of drug-resistant Gram-
negative bacteria
• Generation of new chemical matter designed for antibiotic
discovery.
• Tools and methodologies to evaluate promising alternatives to
traditional antibiotic use.
Those interested in SPARK can contact Frank Cole
Frank@collaborativedrug.com

20. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Remember that Poll?
What is the most promising approach to understanding penetration of small
molecules in Gram-negative bacteria?
1) Developing kinetic models integrating penetration and efflux
2) Testing a wider range of compounds for penetration and efflux
3) Understanding molecular mechanisms of penetration and efflux
4) Studying known Gram-negative antibiotics

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22. Copyright © 2018 All Rights Reserved Collaborative Drug Discovery
Today’s Guest
Moderator
Brad Sherborne
Director of Computer Aided Drug
Design at Merck and Co. Inc.
Today’s Panelists
Helen Zgurskaya
Professor of Chemistry &
Biochemistry at University of
Oklahoma
Derek Tan
Chairman, Member, and
Tri-Institutional Professor, and
Chairman of the Chemical Biology
Program at Memorial Sloan
Kettering Cancer Center
brad.sherborne@merck.com elenaz@ou.edu tand@mskcc.org

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