Abstract

Background: There is an urgent need to develop improved therapies for the treatment of hospital infections caused by carbapenem-resistant Gram-negative pathogens. Of particular concern is the spread of class D OXA-type carbapenemases among carbapenem-resistant Acinetobacter baumannii (CRAB) and Enterobacteriaceae (CRE). We here describe the rational design and optimization of a novel diazabicyclooctanone (DBO) β-lactamase inhibitor, ANT3310, with broad-spectrum activity against class A, C and D serine β-lactamases (SBLs), including KPC- and OXA-type carbapenemases. Materials/methods: Compound synthesis was carried out by GVKBio (India). MIC determinations on clinical isolates were performed using the CLSI broth micro-dilution method with fixed concentrations of SBL inhibitors. Enzyme inhibition assays were performed using purified SBL enzymes; the hydrolysis of nitrocefin was followed using a Perkin Elmer Envision plate reader and IC50 values determined using Dotmatics analytical software. Molecular modelling was performed with Flare, part of the Cresset™ suite of packages. Results: A novel series of DBO analogues bearing halogen-containing substituents was prepared. Lead optimisation was guided by enzymatic inhibition of representative SBLs, potentiation of meropenem against carbapenemase-producing clinical strains (including A. baumannii, Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae) expressing a variety of SBLs (including AmpC, ESBL, KPC and OXA variants) and modelling of the covalent products formed by reaction of the active serine of OXA-48 and the inhibitor compound. Exploration of the SAR led to insights into both enzyme inhibition and efficient entry into the periplasm. Smaller, electron-withdrawing substituents, restricted to one carbon combined with various halogens, led to improved meropenem potentiation. Taking the small size of the substituent to the limit, a simple fluorine substituent led to ANT3310, which showed broad-spectrum inhibition of relevant SBLs (IC50 values between 1 – 175 nM), excellent potentiation of meropenem against clinical isolates (MICs between 0.06 – 1 μg/mL) and promising drug-like properties. Conclusions: Rational-design and optimization led to the identification of a novel DBO compound, ANT3310. This compound shows broad-spectrum SBL inhibition and restores the activity of meropenem against CRAB and CRE pathogens and is a candidate for preclinical development.