Rational design of a new antibiotic class for drug-resistant infections.

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 ß-lactamase enzymes, stringent response and outer membrane permeation. Diazabicyclooctane inhibitors retain activity in the presence of ß-lactamases, the primary resistance mechanism associated with ß-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.

N-Hydroxyformamide LpxC inhibitors, their in vivo efficacy in a mouse Escherichia coli infection model, and their safety in a rat hemodynamic assay.

UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase (LpxC), the zinc metalloenzyme catalyzing the first committed step of lipid A biosynthesis in Gram-negative bacteria, has been a target for antibacterial drug discovery for many years. All inhibitor chemotypes reaching an advanced preclinical stage and clinical phase 1 have contained terminal hydroxamic acid, and none have been successfully advanced due, in part, to safety concerns, including hemodynamic effects. We hypothesized that the safety of LpxC inhibitors could be improved by replacing the terminal hydroxamic acid with a different zinc-binding group. After choosing an N-hydroxyformamide zinc-binding group, we investigated the structure-activity relationship of each part of the inhibitor scaffold with respect to Pseudomonas aeruginosa and Escherichia coli LpxC binding affinity, in vitro antibacterial potency and pharmacological properties. We identified a novel, potency-enhancing hydrophobic binding interaction for an LpxC inhibitor. We demonstrated in vivo efficacy of one compound in a neutropenic mouse E. coli infection model. Another compound was tested in a rat hemodynamic assay and was found to have a hypotensive effect. This result demonstrated that replacing the terminal hydroxamic acid with a different zinc-binding group was insufficient to avoid this previously recognized safety issue with LpxC inhibitors.

Discovery of an Orally Available Diazabicyclooctane Inhibitor (ETX0282) of Class A, C, and D Serine ß-Lactamases.

Multidrug resistant Gram-negative bacterial infections are an increasing public health threat due to rapidly rising resistance toward ß-lactam antibiotics. The hydrolytic enzymes called ß-lactamases are responsible for a large proportion of the resistance phenotype. ß-Lactamase inhibitors (BLIs) can be administered in combination with ß-lactam antibiotics to negate the action of the ß-lactamases, thereby restoring activity of the ß-lactam. Newly developed BLIs offer some advantage over older BLIs in terms of enzymatic spectrum but are limited to the intravenous route of administration. Reported here is a novel, orally bioavailable diazabicyclooctane (DBO) ß-lactamase inhibitor. This new DBO, ETX1317, contains an endocyclic carbon-carbon double bond and a fluoroacetate activating group and exhibits broad spectrum activity against class A, C, and D serine ß-lactamases. The ester prodrug of ETX1317, ETX0282, is orally bioavailable and, in combination with cefpodoxime proxetil, is currently in development as an oral therapy for multidrug resistant and carbapenem-resistant Enterobacterales infections.

5-Carboxytetramethylrhodamine-Ampicillin Fluorescence Anisotropy-Based Assay of Escherichia coli Penicillin-Binding Protein 2 Transpeptidase Inhibition.

The high-molecular mass penicillin-binding proteins (PBPs) are the essential targets of the ß-lactam classes of antibacterial drugs. In the Gram-negative pathogen Escherichia coli, these include PBP1a, PBP1b, PBP2, and PBP3. Techniques that enable facile measurement of the potency of inhibition of these targets are valuable for understanding structure-activity relationships in programs aimed at discovering new antibiotics to combat drug-resistant infections. Continuous fluorescence anisotropy-based assays for inhibition of soluble constructs of PBP1a, PBP2, and PBP3 from the serious Gram-negative bacterial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii and PBP3 from E. coli using the fluorescent phenoxypenicillin analogue BOCILLIN FL have been described previously, but this technique was not useful for PBP2 from E. coli due to a lack of change in fluorescence anisotropy or intensity upon reaction. Here, we report that a fluorescent analogue of ampicillin, 5-carboxytetramethylrhodamine-ampicillin (5-TAMRA-ampicillin), was useful as the indicator in a continuous fluorescence anisotropy-based kinetic assay for inhibition of a soluble construct of PBP2 from E. coli. The assay enables measurement of the bimolecular rate constant for inhibition kinact /Ki. This measurement was made for representative drugs from four classes of ß-lactams and for the diazabicyclooctenone ETX2514. 5-TAMRA-ampicillin was also useful in a fluorescence anisotropy-based assay for P. aeruginosa PBP2 and in fluorescence intensity-based assays with PBP1a and PBP3 from P. aeruginosa and A. baumannii and PBP3 from E. coli.

ETX2514 is a broad-spectrum ß-lactamase inhibitor for the treatment of drug-resistant Gram-negative bacteria including Acinetobacter baumannii.

Multidrug-resistant (MDR) bacterial infections are a serious threat to public health. Among the most alarming resistance trends is the rapid rise in the number and diversity of ß-lactamases, enzymes that inactivate ß-lactams, a class of antibiotics that has been a therapeutic mainstay for decades. Although several new ß-lactamase inhibitors have been approved or are in clinical trials, their spectra of activity do not address MDR pathogens such as Acinetobacter baumannii. This report describes the rational design and characterization of expanded-spectrum serine ß-lactamase inhibitors that potently inhibit clinically relevant class A, C and D ß-lactamases and penicillin-binding proteins, resulting in intrinsic antibacterial activity against Enterobacteriaceae and restoration of ß-lactam activity in a broad range of MDR Gram-negative pathogens. One of the most promising combinations is sulbactam-ETX2514, whose potent antibacterial activity, in vivo efficacy against MDR A. baumannii infections and promising preclinical safety demonstrate its potential to address this significant unmet medical need.

Optimization of physicochemical properties and safety profile of novel bacterial topoisomerase type II inhibitors (NBTIs) with activity against Pseudomonas aeruginosa.

Type II bacterial topoisomerases are well validated targets for antimicrobial chemotherapy. Novel bacterial type II topoisomerase inhibitors (NBTIs) of these targets are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. We now disclose the optimization of a class of NBTIs towards Gram-negative pathogens, especially against drug-resistant Pseudomonas aeruginosa. Physicochemical properties (pKa and logD) were optimized for activity against P. aeruginosa and for reduced inhibition of the hERG channel. The optimized analogs 9g and 9i displayed potent antibacterial activity against P. aeruginosa, and a significantly improved hERG profile over previously reported analogs. Compound 9g showed an improved QT profile in in vivo models and lower clearance in rat over earlier compounds. The compounds show promise for the development of new antimicrobial agents against drug-resistant Pseudomonas aeruginosa.

Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II with reduced pK(a): antibacterial agents with an improved safety profile.

Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. N-Linked amino piperidines, such as 7a, generally show potent antibacterial activity, including against quinolone-resistant isolates, but suffer from hERG inhibition (IC(50) = 44 µM for 7a) and QT prolongation in vivo. We now disclose the finding that new analogues of 7a with reduced pK(a) due to substitution with an electron-withdrawing substituent in the piperidine moiety, such as R,S-7c, retained the Gram-positive activity of 7a but showed significantly less hERG inhibition (IC(50) = 233 µM for R,S-7c). This compound exhibited moderate clearance in dog, promising efficacy against a MRSA strain in a mouse infection model, and an improved in vivo QT profile as measured in a guinea pig in vivo model. As a result of its promising activity, R,S-7c was advanced into phase I clinical studies.

Novel N-linked aminopiperidine inhibitors of bacterial topoisomerase type II: broad-spectrum antibacterial agents with reduced hERG activity.

Novel non-fluoroquinolone inhibitors of bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are of interest for the development of new antibacterial agents that are not impacted by target-mediated cross-resistance with fluoroquinolones. Aminopiperidines that have a bicyclic aromatic moiety linked through a carbon to an ethyl bridge, such as 1, generally show potent broad-spectrum antibacterial activity, including quinolone-resistant isolates, but suffer from potent hERG inhibition (IC(50)= 3 µM for 1). We now disclose the finding that new analogues of 1 with an N-linked cyclic amide moiety attached to the ethyl bridge, such as 24m, retain the broad-spectrum antibacterial activity of 1 but show significantly less hERG inhibition (IC(50)= 31 µM for 24m) and higher free fraction than 1. One optimized analogue, compound 24l, showed moderate clearance in the dog and promising efficacy against Staphylococcus aureus in a mouse thigh infection model.

Exploring Left-Hand-Side substitutions in the benzoxazinone series of 4-amino-piperidine bacterial type IIa topoisomerase inhibitors.

An SAR survey at the C-6 benzoxazinone position of a novel scaffold which inhibits bacterial type IIa topoisomerase demonstrates that a range of small electron donating groups (EDG) and electron withdrawing groups (EWG) are tolerated for antibacterial activity. Cyano was identified as a preferred substituent that affords good antibacterial potency while minimizing hERG cardiac channel activity.

Potent and selective inhibitors of Helicobacter pylori glutamate racemase (MurI): pyridodiazepine amines.

An SAR study of an HTS screening hit generated a series of pyridodiazepine amines as potent inhibitors of Helicobacter pylori glutamate racemase (MurI) showing highly selective anti-H. pylori activity, marked improved solubility, and reduced plasma protein binding. X-ray co-crystal E-I structures were obtained. These uncompetitive inhibitors bind at the MurI dimer interface.

Exploring 8-benzyl pteridine-6,7-diones as inhibitors of glutamate racemase (MurI) in gram-positive bacteria.

A successful scaffold-hopping approach gave a novel series of inhibitors of bacterial glutamate racemase (MurI). Early SAR studies of the 8-benzyl pteridine-6,7-diones led to compounds with micromolar enzyme potency and antibacterial activity.

Exploring 9-benzyl purines as inhibitors of glutamate racemase (MurI) in Gram-positive bacteria.

An early SAR study of a screening hit series has generated a series of 9-benzyl purines as inhibitors of bacterial glutamate racemase (MurI) with micromolar enzyme potency and improved physical properties. X-ray co-crystal EI structures were obtained.