Ruben Tommasi, PhDChief Scientific Officer

Entasis Therapeutics, Inc.

Improving our understanding of porin permeability in Gram-negative bacteria

Pew-NIAID Workshop7 February, 2017 – Washington, DC

The search for new antibacterials is stymied

“Drugs for bad bugs”•After 67 HTS campaigns searching for new antibacterial leads at GSK:

•16 efforts resulted in hits and only 6 resulted in viable leadsNote:

•Hit defined as chemically tractable, low-uM, >10-fold selective vs mammalian•Lead defined as having antibacterial activity, with MoA

GSK Experience: D. Payne et al., NRDD, 6, 2007, 29-41

AZ Experience: NRDD, 14, 2015, 529

65 HTS Campaigns57 w Hits

19 validated Leads

5 LO Programs

CD’s??

Of 65 HTS campaigns, 57 targets with confirmed hits, 19 with Leads, but only 5 progressed into Lead Optimization

Most targets failed due to our inability to identify compounds

with wt-MIC MIC ≠ IC50

Ultimately, these efforts have not resulted in Candidate Drugs.

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Multiple factors affect antibacterial permeation in Gram-negative bacteria

Passive Barrier: Outer Membrane (OM)• Diffusion through porins (aqueous)

• Diffusion across LPS-phospholipid (non-aqueous)

Energy-driven Barrier: Active efflux• Families of multidrug resistance transporters

• Both intrinsic and acquired antibiotic resistance

Tommasi et al (2015) NRDD 14:529

Porins differ largely across Gram-negative pathogens

E. coli, OmpF“general porin”

P. aeruginosa, OprD/OccD1“substrate-specific porin”Ampicillin

Active against E. coliInactive against P. aeruginosa

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TOMAS: Titrable Outer Membrane protein Assay SystemAn optimized cell-based porin over-expression assay

Porin-deficientE. coli

[L-arabinose]

[L-arabinose] a [porin] a permeability

OprD

Porin-deficientE. coli

OprD

OprD

Porin-deficientE. coli

• E. coli K-12 genetically modified for more sensitive and uniform response to inducer (L-arabinose)

• Deleted native porins (OmpF, OmpC and OmpA)

• Heterologous porin expression

• Increasing porin concentration in the OM would increase OM permeability• Tuning OM permeability with selective inducer in a controlled fashion • Read out using fold-change in MIC, independent of target activity level

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L-arabinose (µM)

Less Permeation rel. to Mero Similar4-8x shifts

Expanded SAR of carbapenem uptake by OprD:Proof of Concept for the approach

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More Permeant than Meropenem – 16-32x shifts

molLogPPSA

1.2561

-0.78102

-1.27119

-0.73110

-0.79119

-1.21110

-0.51102

-0.11102

+0.42 73

+0.78 73

-0.48 107

-0.10 102

-0.75 90

-1.76 119

-0.91 102

-2.04 147

-3.06 147

-1.56 147

-2.12 131

-1.41 102 -0.62

135

-1.60 149

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Meropenem translocating OprD

Extracellular

Periplasm

Recognitionpocket

Encountercomplex

Conclusions and future directions

• Identification of new antibiotics with good outer membrane permeation and optimized target inhibition is challenging

• Subtle changes in LogP, PSA and presentation of charges can make a large difference in relative permeation.• Therefore we shouldn’t only seek a set of Lipinski-like rules• Makes sense given the diversity among Gram-negative bacteria versus

gut endothelium or blood-brain barrier.

• Entasis is redefining antibacterial design by incorporating definition of the molecular drivers of compound uptake• Unique multidisciplinary approach using a combination of med chem,

in vitro/in vivo biology and in silico tools• POC established with SAR of carbapenem uptake by OprD• Now further developed with novel inhibitors

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Acknowledgements

Better understanding Gram-negative porin permeation: A cross-functional endeavor

CADD computational chemistry• Porin models• Docking• MD simulation

Chemistry (synthetic/medchem)• Carbapenem library• Exploratory compounds

Biology (enzymology, microbiology, genomics)• Transposon libraries• Target activity • Genetic tools

• KO and OE strains• Cell-based, titrable, porin over-expression assay

• Expression profiling

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