A major resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. Originally recognized for their ability to hydrolyze penicillins, emergent β-lactamases can now confer resistance to other β-lactam drugs, including both cephalosporins and carbapenems. The emergence and global spread of β-lactamase-producing multi-drug-resistant “superbugs” has caused increased alarm within the medical community due to the high mortality rate associated with these difficult-to-treat bacterial infections. To address this unmet medical need, we initiated an iterative program combining medicinal chemistry, structural biology, biochemical testing, and microbiological profiling to identify broad-spectrum inhibitors of both serine- and metallo-β-lactamase enzymes. Lead optimization, beginning with narrower-spectrum, weakly active compounds, provided 20 (VNRX-5133, taniborbactam), a boronic-acid-containing pan-spectrum β-lactamase inhibitor. In vitro and in vivo studies demonstrated that 20 restored the activity of β-lactam antibiotics against carbapenem-resistant Pseudomonas aeruginosa and carbapenem-resistant Enterobacteriaceae. Taniborbactam is the first pan-spectrum β-lactamase inhibitor to enter clinical development.

As shifts in the epidemiology of β-lactamase-mediated resistance continue, carbapenem-resistant Enterobacterales (CRE) and carbapenem-resistant Pseudomonas aeruginosa (CRPA) are the most urgent threats. Although approved β-lactam (BL)–β-lactamase inhibitor (BLI) combinations address widespread serine β-lactamases (SBLs), such as CTX-M-15, none provide broad coverage against either clinically important serine-β-lactamases (KPC, OXA-48) or clinically important metallo-β-lactamases (MBLs; e.g., NDM-1). VNRX-5133 (taniborbactam) is a new cyclic boronate BLI that is in clinical development combined with cefepime for the treatment of infections caused by β-lactamase-producing CRE and CRPA. Taniborbactam is the first BLI with direct inhibitory activity against Ambler class A, B, C, and D enzymes. From biochemical and structural analyses, taniborbactam exploits substrate mimicry while employing distinct mechanisms to inhibit both SBLs and MBLs. It is a reversible covalent inhibitor of SBLs with slow dissociation and a prolonged active-site residence time (half-life, 30 to 105 min), while in MBLs, it behaves as a competitive inhibitor, with inhibitor constant (Ki) values ranging from 0.019 to 0.081 μM. Inhibition is achieved by mimicking the transition state structure and exploiting interactions with highly conserved active-site residues. In microbiological testing, taniborbactam restored cefepime activity in 33/34 engineered Escherichia coli strains overproducing individual enzymes covering Ambler classes A, B, C, and D, providing up to a 1,024-fold shift in the MIC. Addition of taniborbactam restored the antibacterial activity of cefepime against all 102 Enterobacterales clinical isolates tested and 38/41 P. aeruginosa clinical isolates tested with MIC90s of 1 and 4 μg/ml, respectively, representing ≥256- and ≥32-fold improvements, respectively, in antibacterial activity over that of cefepime alone. The data demonstrate the potent, broad-spectrum rescue of cefepime activity by taniborbactam against clinical isolates of CRE and CRPA.

There is an urgent need for oral agents to combat resistant gram-negative pathogens. Here we describe the characterization of VNRX-5236, a broad-spectrum boronic acid β-lactamase inhibitor (BLI) and its orally bioavailable etzadroxil prodrug, VNRX-7145. VNRX-7145 is being developed in combination with ceftibuten, an oral cephalosporin, to combat strains of Enterobacterales expressing extended spectrum β-lactamases (ESBLs) and serine carbapenemases. VNRX-5236 is a reversible covalent inhibitor of serine β-lactamases, with inactivation efficiencies on the order of 104 M−1. sec−1, and prolonged active site residence times (t1/2, 5 to 46 min). The spectrum of inhibition includes Ambler class A ESBLs, class C cephalosporinases, and class A and D carbapenemases (KPC and OXA-48, respectively). Rescue of ceftibuten by VNRX-5236 (fixed at 4 μg/mL) in isogenic strains of E. coli expressing class A, C or D β-lactamases demonstrated an expanded spectrum of activity relative to oral comparators including investigational penems, sulopenem and tebipenem. VNRX-5236 rescued ceftibuten activity in clinical isolates of Enterobacterales expressing ESBLs (MIC90 = 0.25 μg/mL), KPCs (MIC90 = 1 μg/mL), class C cephalosporinases (MIC90 = 1 μg/mL) and OXA-48-type carbapenemases (MIC90 = 1 μg/mL). Frequency of resistance studies demonstrated a low propensity for recovery of resistant variants at 4× the MIC of the ceftibuten/VNRX-5236 combination. In vivo, whereas ceftibuten alone was ineffective (ED50, >128 mg/kg), ceftibuten/VNRX-7145 administered orally protected mice from lethal septicemia caused by K. pneumoniae producing KPC carbapenemase (ED50, 12.9 mg/kg). The data demonstrate potent, broad-spectrum rescue of ceftibuten activity by VNRX-5236 in clinical isolates of cephalosporin-resistant and carbapenem-resistant Enterobacterales.

BackgroundVNRX-5133 is a cyclic boronate β-lactamase inhibitor (BLI) currently in clinical development with cefepime to treat multidrug-resistant (MDR) infections caused by ESBL- and carbapenemase-producing Enterobacteriaceae (ENT) and P. aeruginosa (PSA). VNRX-5133 has direct inhibitory activity against serine-active site β-lactamases (Ser-BL) and emerging VIM/NDM metallo-β-lactamases (MBL). We show herein that cefepime/VNRX-5133 is highly active against MDR-K. pneumoniae and P. aeruginosa clinical isolates producing BL-variants evolved during therapy that compromise activity of ceftazidime/avibactam and ceftolozane/tazobactam.MethodsSusceptibility testing was performed according to CLSI methods with cefepime, ceftolozane, and ceftazidime alone or in combination with VNRX-5133, avibactam, or tazobactam, respectively, fixed at 4 mg/L. Five clinical isolates of K. pneumoniae producing KPC variants impacting ceftazidime/avibactam and five clinical isolates of P. aeruginosa producing Pseudomonas-derived cephalosporinase variants impacting ceftolozane/tazobactam activity were collected in 2016 and 2017, respectively, from United States and Spanish hospitals. All other clinical isolates of Enterobacteriaceae and P. aeruginosa (n = 40) were collected in 2016.ResultsCefepime/VNRX-5133 was highly active against five ceftazidime/avibactam-resistant K. pneumoniae clinical isolates producing KPC variants with MIC ranging from 0.5 to 4 mg/L relative to ceftazidime/avibactam MIC range of 16 to >128 mg/L. Cefepime/VNRX-5133 was also active against all five clinical isolates of ceftolozane/tazobactam-resistant P. aeruginosa, where 4/5 isolates had MIC of 4–8 mg/L relative to ceftolozane/tazobactam MIC range of 32–128 mg/L. The elevated cefepime/VNRX-5133 MIC (16 mg/L) in the remaining P. aeruginosa isolate was not due to the PDC-221 variant, as an engineered strain of P. aeruginosa producing this enzyme had a cefepime/VNRX-5133 MIC of 1 mg/L.ConclusionVNRX-5133 is a potent BLI possessing a unique broad spectrum of activity, including Class A, C, and D Ser-BLs, clinically evolving variants of Ser-BLs (e.g., KPC, PDC) and emerging VIM/NDM-type MBLs. Cefepime/VNRX-5133 is highly active against emerging multidrug-resistant Enterobacteriaceae and P. aeruginosa.Disclosures D. Daigle, VenatoRx Pharmaceuticals Inc.: Employee and Shareholder, Salary. J. Hamrick, VenatoRx Pharmaceuticals Inc.: Employee, Salary. C. Chatwin, VenatoRx Pharmaceuticals Inc.: Employee, Salary. N. Kurepina, VenatoRx Pharmaceuticals Inc.: Research Contractor, Research support. B. N. Kreiswirth, VenatoRx Pharmaceuticals Inc.: Research Contractor, Research support. R. K. Shields, VenatoRx Pharmaceuticals Inc.: Research Contractor, Research support. A. Oliver, VenatoRx Pharmaceuticals Inc.: Research Contractor, Research support. C. J. Clancy, VenatoRx Pharmaceuticals Inc.: Research Contractor, Research support. M. H. Nguyen, VenatoRx Pharmaceuticals Inc.: Research Contractor, Research support. D. Pevear, VenatoRx Pharmaceuticals Inc.: Employee, Salary. L. X...

A major antimicrobial resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. The increasing emergence of β-lactamase-producing multi-drug-resistant “superbugs” has resulted in increases in costly hospital Emergency Department (ED) visits and hospitalizations due to the requirement for parenteral antibiotic therapy for infections caused by these difficult-to-treat bacteria. To address the lack of outpatient treatment, we initiated an iterative program combining medicinal chemistry, biochemical testing, microbiological profiling, and evaluation of oral pharmacokinetics. Lead optimization focusing on multiple smaller, more lipophilic active compounds, followed by an exploration of oral bioavailability of a variety of their respective prodrugs, provided 36 (VNRX-7145/VNRX-5236 etzadroxil), the prodrug of the boronic acid-containing β-lactamase inhibitor 5 (VNRX-5236). In vitro and in vivo studies demonstrated that 5 restored the activity of the oral cephalosporin antibiotic ceftibuten against Enterobacterales expressing Ambler class A extended-spectrum β-lactamases, class A carbapenemases, class C cephalosporinases, and class D oxacillinases.

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