Antibiotic resistance: a real and present danger in our hospitals

Transcription

1 Antibiotic resistance: a real and present danger in our hospitals 2

2 The search for new antibacterials HTS based on genetically validated targets GSK Experience: D. Payne et al., NRDD, 6, 2007, 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 leads Note: Hit defined as chemically tractable, low-um, >10-fold selective vs mammalian Lead defined as having antibacterial activity, with MoA AZ Experience: NRDD, 14, 2015, 529 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 IC 50 Ultimately, these efforts have not resulted in Candidate Drugs. 65 HTS Campaigns 57 w Hits 19 validated Leads 5 LO Programs CD s?? 3

3 Multiple factors affect antibacterial permeation in Gram-negative bacteria Tommasi et al (2015) NRDD 14:529 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 4

4 Porins differ largely across Gram-negative pathogens E. coli, OmpF general porin Ampicillin Active against E. coli Inactive against P. aeruginosa P. aeruginosa, OprD/OccD1 substrate-specific porin 5

5 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 6

6 Visualizing imipenem translocating OprD MD simulations Recognition Pocket Encounter complex Extracellular Periplasm 7

7 Meropenem translocating OprD Recognition pocket Encounter complex Extracellular Periplasm 8

8 Similar recognition elements: Arginine and imipenem OprD substrate arginine imipenem Tyr 282 Ser 296 Arg 410 Arg 131 Arg 410 Arg 30 Asp 307 Arg 30 Asp 307 Simulation interactions diagram 9

9 TOMAS: Titrable Outer Membrane protein Assay System An optimized cell-based porin over-expression assay 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 Porin-deficient E. coli Porin-deficient E. coli OprD Porin-deficient E. coli Readout: OprD OprD [L-arabinose] MIC rel = MIC ara MIC 3ko [L-arabinose] a [porin] a permeability E. coli K-12 genetically modified for more sensitive and uniform response to inducer (L-arabinose) Deleted native porins (OmpF, OmpC and OmpA) Deleting TolC to test porins with/without efflux (in progress) Heterologous porin expression Iyer et al ACS Infect Dis Feb 17 L-arabinose (µm) 11

10 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 OprD, meropenem and control antibiotics Antibacterial activity of control antibiotics is NOT affected by increasing OprD levels Meropenem uses both OprD and OpdP 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 O p d P S u l b a c t a m M e r o p e n e m O p r D [ A r a b i n o s e ], M [ L - A r a b i n o s e ], M Compound 7 does not permeate through OprD as well as meropenem M e ro p e n e m C o m p o u n d 7 Compound [A ra b in o s e ], u M 12

11 Cell permeation & activity: Meropenem matched pair P. aeruginosa OprD with meropenem in recognition pocket Meropenem PBP3 k on (M -1 s -1 ) = 49,000 P.a. wt MIC (µg/ml) = 0.25 P.a. oprd (µg/ml) MIC = 4 Compound 7 PBP3 k on (M -1 s -1 ) = 24,000 P.a. wt MIC (µg/ml) = 4 P.a. oprd (µg/ml) MIC = 8 Addition of acid moiety creates mismatch in OprD recognition pocket Result is diminished permeation despite good biochemical potency MIC (µg/ml) strain Meropenem Cmpd 7 Imipenem Cefixime E. coli PB22 (ompa,c,f - ) PB22 + pvi-oprd WT 0.25 (32X) 0.5 (8X) 0.5 (x16) 1.0 PB22 + pvi-oprd Y282R, D307H 4.0 (2x) NT 4.0 (2x) Isabella V., et al., Chemistry & Biology, 2015, 22, 535

12 Expanded SAR of carbapenem uptake by OprD: Proof of Concept for the approach mollogp PSA Less Permeation rel. to Mero Similar 4-8x shifts More Permeant than Meropenem 16-32x shifts

13 Expanded SAR of carbapenem uptake by OprD: POC for the approach Our goal is not to design in uptake for a single porin but across multiple porins to improve WCA and decrease potential for resistance emergence ~30 different porin-encoding genes in P. aeruginosa Several crystal structures available How to prioritize? Determine MOU of very potent WCA cmpds (TNseq) Determine relative abundance (RNAseq) 15

14 R e l a t i v e e x p r e s s i o n Relative porin expression in P. aeruginosa: in vitro - in vivo correlation R i c h m e d i a M o u s e l u n g o p r F o p r D o p r Q o p r G o p d P o p r E o p r M o p d Q In vitro: PAO1 grown in rich media; harvested during exponential phase In vivo: PAO1 murine lung infection, harvested 8 hours post-infection 16

15 R e l a t i v e e x p r e s s i o n Acinetobacter baumannii Causes infections in critically ill patients Mortality rates up to ~ % > 60% of A. baumannii isolates are MDR Outer membrane is ~100-fold less permeable than E. coli Crystal structures of CarO and OprD (Occ) orthologs recently been solved R e l a t i v e A. b a u m a n n i i p o r i n e x p r e s s i o n i n v i t r o Am. J. Respir. Crit. Care Med Int. J. Antimicrob. Agents Poirel, L AAC. 54: Lancet J. Glob. Infect. Dis A 1 S _ ( o m p A ) A 1 S _ ( o p r B - l i k e ) A 1 S _ ( c a r O ) A 1 S _ A 1 S _ A 1 S _ A 1 S _ A 1 S _ A 1 S _

16 OmpA: a multifunctional, highly expressed OM protein Required for outer membrane stability, biofilm formation, conjugation Virulence factor (in vivo fitness and persistence) Unique two domain structure N-terminal domain: 8 β-barrel narrow pore C-terminal domain: peptidoglycan interaction & OM stabilization Predicted to form a single large pore domain with rearrangement of domains predicted small % of the population similar to P. aeruginosa OprF N-terminal and C-terminal domains have been crystallized separately (no full-length structure available) Reusch, R.N. FEBS J. 2012; 27(6) : 894 Krishnan, S. & Prasadarao, N.V. FEBS J. 2012; 27(9): 919 Pautsch, A. & Schulz, G.E. Nat. Struct. Biol. 1998; 5: 1013 Park et al (2012) FASEB J. 26:

17 A. baumannii OmpA: Role as a porin Role in transport of small molecule substrates or antibiotics not well-defined Reconstitution assays have shown some limited pore-forming function Similar to E. coli OmpA and P. aeruginosa OprF about 70-fold lower than that of E. coli OmpF ATCC COL NOVO ATM CMP CAZ MEM IPM SUL wildtype > ompa > Can we demonstrate A. baumannii OmpA porin function in TOMAS? Sugawara, E. & Nikaido, H., J. Bacteriol. 2012; 194(15):

18 Discovery of ETX2514, a novel broad-spectrum serine BLI A combination of innovative chemistry, structure-based design, and quantum mechanics calculations culminated in the discovery of ETX2514. Compound (MIC 90, mg/l) Imipenem Meropenem Aztreonam Ceftazidime Sulbactam E. coli n = 202 K. p. n = 198 P. a. n = 202 A. b. n = 195 alone >64 + ETX alone >64 + ETX alone >64 + ETX >64 alone 16 >64 >64 >64 + ETX alone 64* >64 > ETX * 0.12 >64 4 ETX2514 alone 1 8 >64 >64 *n = 21 strains n = 20 strains MIC 90 across recent clinical isolates (+/- ETX2514 at 4 mg/l) 20

19 N o rm a liz e d A n tib a c te ria l A c tiv ity Antibiotic uptake by A. baumannii OmpA in TOMAS OmpA Porin-deficient E. coli Porin-deficient E. coli Porin-deficient E. coli OmpA OmpA [L-arabinose] N o v o b io c in * Im ip e n e m S u lb a c ta m E T X [A ra b in o s e ], u M *similar results for RIF, TET, MER, VAN, ETX

20 N o r m a l i z e d A n t i b a c t e r i a l A c t i v i t y ETX2514 uptake is mediated by OmpA in A. baumannii Sulbactam antibacterial activity vs. A. baumannii strains in the presence of increasing amounts of ETX2514 A T C C ( M D R, B L + ) M I C = 1 g / m L M I C = 8 g / m L w i l d t y p e o m p A [ E T X ] m g / L [ E T X ] m g / L 22

21 N o rm a liz e d A n tib a c te ria l A c tiv ity SAR of A. baumannii OmpA-mediated uptake of novel small molecules in TOMAS a n a lo g 1 a n a lo g 2 a n a lo g 3 a n a lo g 4 a n a lo g [A ra b in o s e ], M Informs rational design of small molecules optimized for OmpA-mediated uptake 23

22 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 SAR of carbapenem uptake by OprD Now further developed with novel inhibitors First demonstration of A. baumannii OmpA porin function in whole cells (and structure-permeation-relationship across this porin) 24

23 Acknowledgements 25