The development of new antibiotics to treat infections caused by drug-resistant Gram-negative pathogens is of paramount importance as antibiotic resistance continues to increase worldwide1. Here we describe a strategy for the rational design of diazabicyclooctane inhibitors of penicillin-binding proteins from Gram-negative bacteria to overcome multiple mechanisms of resistance, including β-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of β-lactamases, the primary resistance mechanism associated with β-lactam therapy in Gram-negative bacteria2,3. Although the target spectrum of an initial lead was successfully re-engineered to gain in vivo efficacy, its ability to permeate across bacterial outer membranes was insufficient for further development. Notably, the features that enhanced target potency were found to preclude compound uptake. An improved optimization strategy leveraged porin permeation properties concomitant with biochemical potency in the lead-optimization stage. This resulted in ETX0462, which has potent in vitro and in vivo activity against Pseudomonas aeruginosa plus all other Gram-negative ESKAPE pathogens, Stenotrophomonas maltophilia and biothreat pathogens. These attributes, along with a favourable preclinical safety profile, hold promise for the successful clinical development of the first novel Gram-negative chemotype to treat life-threatening antibiotic-resistant infections in more than 25 years.
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We thank D. McKinney, K. Thakur, S. Narayan and Pharmaron for their chemistry support; IHMA and JMI laboratories for microbiological surveillance studies; Neosome Life Sciences for in vivo efficacy studies; MPI Research for preclinical toxicology studies and J. Abendroth for help with crystallography. This research programme was partially supported by the Cooperative Agreement No. 4500002444 from ASPR/BARDA and by awards from Wellcome Trust and Germany’s Federal Ministry of Education and Research (BMBF), as administrated by CARB-X. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Department of Health and Human Services Office of the Assistant Secretary for Preparedness and Response, other funders, or CARB-X. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (grant 085P1000817).
T.F.D.-R., A.A.M., J.P.O., X.W., M.A.S., S.G., R.I., A.B.S., N.M.C., C.V.-V., S.H.M., S.M.M., A.C., A.M.T., J.Z., J.C.-P., J.A.R. and R.A.T. are current or former employees of Entasis Therapeutics. H.H., A.D.F., P.S.H., S.J.M., H.S.H., G.L.D., J.E.C. and R.A.S. have no competing interests to declare.
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Extended data figures and tables
Extended Data Fig. 1 Compound-dependent bacterial morphologies and evidence of porin overexpression in TOMAS.
a, Morphology of P. aeruginosa PAO1 without drug treatment (left panel, 5 µm scale bar) or treated with 0.5X MIC of either NXL-105 (middle panel, 5 µm scale bar) or Compound 2 (right panel, 10 µm scale bar). Micrographs are representative of results obtained for three biologically independent studies. b, SDS-PAGE analysis of overexpressed porins in TOMAS19 after overnight induction with 120 µM L-arabinose. 2 µg outer membrane extracts were loaded onto a 13% polyacrylamide-SDS gel in 6 M urea. Expression levels are comparable to those observed for OprD in TOMAS19. Gel is representative of two biologically independent studies. Bands corresponding to the overexpressed porins are highlighted in boxes. Upper panel = P. aeruginosa porins; lower panel = K. pneumoniae porins.
a, Percent cumulative growth inhibition of 40 MDR P. aeruginosa clinical isolates from the CDC MDR P. aeruginosa panel at increasing concentrations of ETX0462 (blue), meropenem-vaborbactam (MEM-VAB, red), imipenem-relebactam (IMI-REL, orange, ceftolozane-tazobactam (TOL-TAZ, purple) and ceftazidime-avibactam (CAZ-AVI, green). MICs were measured in singleton. b, A composite Emax fitting of the %fT>MIC vs change in CFU burden for 7 strains (MIC range of 0.25 − 4 mg/L, R2 = 0.87) evaluated in a murine neutropenic thigh model in vivo suggests > 1-log kill when unbound systemic concentrations of ETX0462 exceed the MIC for 60% of the dosing interval. c, Kaplan-Meier survival plot of Y. pestis CO92 infected mice using an aerosolized dose (20 X LD50 of 6.8 x 104 CFU) followed by treatment with ETX0462 vs. vehicle control, ciprofloxacin (CIP) or ceftazidime (CTZ) shows equivalent in vivo efficacy (Log-rank test p < 0.0001 vs. control).
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Durand-Reville, T.F., Miller, A.A., O’Donnell, J.P. et al. Rational design of a new antibiotic class for drug-resistant infections. Nature 597, 698–702 (2021). https://doi.org/10.1038/s41586-021-03899-0