Addressing the Challenge of Gram-negative Permeation in Antibacterial Discovery

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1 Addressing the Challenge of Gram-negative Permeation in Antibacterial Discovery Thomas Durand-Réville ASM Microbe Atlanta June 8, 2018

2 Disclosures Thomas Durand-Réville: Full-time Employee; Self; Entasis Therapeutics. 2

3 The Failed Search for New Antibacterials GSK Experience: Drugs for bad bugs (D. Payne, et al., NRDD 2007, 6, 29) After 67 HTS campaigns searching for new antibacterial leads, 16 efforts resulted in hits and only 6 resulted in viable Leads. Notes: Hit defined as chemically tractable, low µm, >10-fold selective vs mammalian. Lead defined as having antibacterial activity, with MoA. AZ Experience: ESKAPEing the labyrinth of antibacterial discovery (R. Tommasi, et al., NRDD 2015, 14, 529) Of 65 HTS campaigns, 57 targets with confirmed Hits, but only 5 progressed into Lead Optimization. Notes: Most targets failed due to inability to identify compounds with wildtype MIC. Ultimately, these efforts did not result in Drug Candidates. 65 HTS Campaigns 57 with Hits 19 Validated Hits 5 LO Programs 0 CDs The industry abandoned this approach as a lost decade. Are we working on the right mechanisms, do we need better compounds, other approaches? 3

4 Many Factors Affect Antibacterial Permeation in Gram-negative Bacteria Gram-negative cell envelop represents a formidable barrier to small molecule entry and provides intrinsic resistance To maximize target engagement, compounds must reach sufficient concentrations in the target s compartment (periplasm or cytoplasm) Cytoplasm Manchester, J.I., et al., J. Med. Chem., 2012, 55, 2532 Silver, L.L., Bioorg. Med. Chem., 2016, 24,

5 Better Understanding Gram-negative Porin Permeation: A Cross-functional Endeavor Biology (enzymology, microbiology, genomics) Target activity Genetic tools KO and OE strains Cell-based, titrable, porin OE assay Transposon libraries Expression profiling and sequencing Chemistry (synthetic/medchem) Carbapenem library Exploratory compounds CADD computational chemistry Porin models Docking MD simulation 5

6 TOMAS: An Optimized Cell-based Porin Over-expression Assay (Titrable Outer Membrane protein Assay System) Increasing porin concentration in the OM would increase OM permeability Tuning OM permeability with selective inducer in a controlled fashion [L-arabinose] [L-arabinose] a [porin] a permeability L-arabinose (µm) E. coli K-12 genetically modified for more sensitive and uniform response to inducer Deleted native porins (ompf, ompc and ompa) Optionally deleted tolc to test porins with/without efflux Heterologous porin expression (10 P. aeruginosa, 1 A. baumannii and 2 K. pneumoniae porins) OmpA Ab Iyer R. et al., ACS Infect. Dis. (2017) 3, 310 6

7 R e l a t i v e C h a n g e i n M I C R e l a t i v e C h a n g e i n M I C R e l a t i v e C h a n g e i n M I C R e l a t i v e C h a n g e i n M I C R e l a t i v e C h a n g e i n M I C Validation Using P. aeruginosa OprD: Meropenem, Control Antibiotics and Analogs Meropenem Compound E m p t y C h l o r a m p h e n i c o l R i f a m p i n S u l b a c t a Om p d P M e r o p e n e m O p r D E m p t y O p d P O p r D M e ro p e n e m C o m p o u n d 71 E m E O p O O p O [ L [- A r a b iin o s e ]],, M Opposite to meropenem, antibacterial activity of control antibiotics is NOT affected by increasing OprD levels [ A r a b i n o s e ], M Meropenem uses both OprD and OpdP [A ra b in o s e ], u M [ A r a b i n o s e ], M [ A r a b i n o s e ], M Compound 1 does not permeate through OprD as well as meropenem 7

8 Expanded SAR of Carbapenem Uptake by OprD: Proof of Concept for the Approach 4x less permeant than meropenem analogs synthesized with various thioether sidechains Broad chemical diversity: 271<MW<496 83<PSA<199-6<logD<1 5<HBA<12 2<HBD<7 4<rotatable bonds<11 various pkas, number of charges LogD PSA x more permeant than meropenem 8

9 Several OprD Crystal Structures Available in PDB Monomeric water-filled 18-stranded β-barrel Recognition pocket (L7 loop) Central channel constricted by two mobile loops (L3 and L7) Extracellular Second most abundant porin in P. aeruginosa, specific for basic amino acids Bottom view Periplasm Constriction zone Side view Use Steered Molecular Dynamics (fully atomistic) to simulate permeation. Eren, E., et al., PLOS Biology, 2012, 10, e Samanta, S., et al., Phys. Chem. Chem. Phys., 2015, 17,

10 Visualizing Meropenem Translocation Through OprD Recognition pocket Encounter complex Extracellular Periplasm 10

11 Electronic Signature of OprD Substrates: Understanding the Recognition Elements Apo OprD Asp 307 Asp 295 Recognition pocket + _ Arginine Histidine L7 loop Basic ladder Imipenem Meropenem 11

12 Free energy change ΔG (kcal/mol) H-bond frequency (% of MD frames) Attempts at Predicting OprD Permeation and Identifying Potential Mechanistic Insights Steered MD vs. TOMAS > 8-fold MIC shift (++), no MIC shift (-) from 0 to 120µM [arabinose] Steered MD simulation predicts correct binning of compounds in TOMAS _ H-bond frequency per residue OprD residue number H-bond frequency varies across compounds for key OprD residues New theoretical approaches needed to significantly improve sampling and computational efficiency. 12

13 OmpA is the Major Outer Membrane Porin in A. baumannii Unique two-domain structure among porins, no full length crystal structure solved to date Multifunctional pore: maintaining cell shape, OM stability, biofilm formation, adhesion and F-dependent conjugation Very high degree of conservation across A. baumannii isolates; Required for optimal in vivo survival and pathogenesis during disease progression No loss of function alleles described to date Role in transport of small molecule substrates or antibiotics not well established Unrooted maximum-likelihood tree of 103 recent clinical A. baumannii OmpA sequences based on alignment to the ATCC17978 OmpA reference Reusch R., FEBS (2012) 279, 894; Krishnan S., Prasadarao N., FEBS (2012) 279, 919 Sugawara E., Nikaido H., J. Bacteriol. (2012) 194, 4089; Sánchez-Encinales V. et al., J. Infect. Dis. (2017) 215,

14 R e la tiv e C h a n g e in M IC Small Molecule Permeation through A. baumannii OmpA Ab in TOMAS OmpA Ab uninduced MIC SUL > 512 mg/l MIC IMI 2 mg/l MIC RIF 8 mg/l 4 mg/l MIC ETX2514 E. coli K-12 ΔFCA/P BAD -ompa Ab [arabinose] µm S U L I M I R IF E T X Sulbactam (SUL) & imipenem (IMI) show OmpA dependence while rifampin (RIF) does not. Effect also demonstrated with meropenem, but not seen for aztreonam, tetracycline or chloramphenicol. Iyer R. et al., ACS Infect. Dis. (2018) 4,

15 R e la tiv e C h a n g e in M IC Generating Structure-Porin Permeation Relationships (SPPR) ETX2514 and close analogs tested in OmpA Ab TOMAS OmpA Ab uninduced MIC ETX mg/l MIC ETX878 8 mg/l MIC ETX386 4 mg/l MIC ETX105 8 mg/l 4 mg/l MIC ETX019 E. coli K-12 ΔFCA/P BAD -ompa Ab [arabinose] µm E T X E T X E T X E T X E T X Analogs of ETX2514 illustrate how subtle changes in structure affect uptake through OmpA Ab. Iyer R. et al., ACS Infect. Dis. (2018) 4,

16 Optimizing TOMAS from a Traditional MIC Readout TOMAS identifies specific porin(s) involved in uptake with rank ordering of substrates. but MIC is not a kinetic readout and MIC ratio cannot be used determine permeation and killing rates. Interpretation of relative susceptibilities in TOMAS may be overly simplistic. Meropenem MIC in E. coli K-12 WT vs ΔtolC is 0.03 mg/l in both cases. Is meropenem not effluxed? Look at the kinetics via time kill assays 16

17 Log CFU/ml Log CFU/ml Meropenem (MEM) Kill Kinetics Using Colony Forming Units (CFU) 8.00 E. coli K-12 WT MIC (MEM) = 0.03 mg/l 8.00 E. coli K-12 ΔtolC MIC (MEM) = 0.03 mg/l µg/ml 0.5 µg/ml µg/ml 0.12 µg/ml µg/ml µg/ml µg/ml growth ctrl Time (hr) Time (hr) Starting inoculum: 3-4x10 6 CFU/ml LLQ 500 CFU/ml ΔtolC strain appear to be killed more rapidly than WT. Despite MIC ratio suggesting there is no efflux, there is in fact an effect. But time kill assays are labor, time and resource intensive! 17

18 Developing a Quicker Read-out to Monitor Permeation Use of P. luminescens luciferase (luxcdabe) to monitor cell viability/kill. Plasmid-encoded P. luminescens lux cassette transformed into E. coli K-12. K-12 luc + (gent R ) K-12 ompf, ompc, ompa, luc + (gent R ) WT Efflux + : tolc + tolc Efflux - : tolc::kan Efflux + : tolc + Efflux - : tolc::kan Advantages: Cells make the luciferase substrate (no exogenous addition like firefly). P. luminescens luciferase produces 8x more light than V. harveyi luciferase. Potential to track cell viability in real-time. Atosuo, J., et al., Luminescence, 2013, 5, 771 Hilpert, K., Hancock, R.E.W., Nature Protocols, 2007, 2,

19 L u c ife ra s e (lu m in e s c e n c e a.u.) L u c ife ra s e (lu m in e s c e n c e a.u.) Meropenem (MEM) Kill Kinetics using Luciferase Assay E. coli K-12 WT lux + E. coli K-12 ΔtolC lux g /m L g /m L g /m L g /m L MEM g /m L g /m L 0. 5 g /m L 1 g /m L g /m L g /m L g /m L g /m L MEM g /m L g /m L 0. 5 g /m L 1 g /m L tim e, h o u rs tim e, h o u rs ΔtolC cells appear to be killed more rapidly by meropenem than WT, same conclusions as CFU experiments. Shallower killed curves observed because dead cells still emit light for some time. Assay provides higher throughput and better resolution, cost effective. 19

20 Conclusions and Future Directions Identification of new antibiotics with good outer membrane permeation and optimized target inhibition is challenging 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 MIC-shifts now expanded to more sensitive kinetic readouts Demonstration of A. baumannii OmpA porin function in whole cells Structure-Porin Permeation Relationships (SPPR) across this porin identified for the first time These assays are being used in combination with MD and other computational methods to elucidate SPPR across multiple Gram-negative pathogens Pay attention to permeation early during your drug discovery programs! 20

21 Acknowledgements AstraZeneca Infection Discovery Pharmaron, Syngene 21