PROJECT SUMMARY Carbapenem-resistance in Enterobacterales has steadily increased over the past decade, leading to multidrug and pan-drug resistance (MDR/PDR), further emphasizing the need for new innovative therapies. Carbapenem- resistant Enterobacterales (CRE) is a serious global health problem classified by the US Centers for Disease Control and Prevention as an “Urgent threat” and by the World Health Organization as a “Priority 1 critical threat”. The β-lactams have long been the front line therapeutic option for such infections, but efficacy of these agents, including last resort carbapenems, is threatened by recent expansion of β-lactamases, particularly subtypes (e.g., NDM) spreading rapidly among Enterobacterales that are unaffected by clinically-available β-lactam/β- lactamase inhibitor combinations. To address the medical need, Venatorx has identified a novel series of highly selective cyclic boronates that bind to and disrupt penicillin-binding protein (PBP) transpeptidase (TPase) function while avoiding the action of all current and future β-lactamases. This approach creates the first prospect and “rare” new class gram negative agent to treat infections caused by any β-lactamase-producing CRE pathogen. Significant strides in microbiological activity have already been achieved within the series by the lead compound VNRX-6736, with an MIC90 of 32 µg/mL relative to 128 µg/mL for meropenem-vaborbactam and ≥1,024 µg/mL for ceftazidime-avibactam in a recent challenge set of 100 CRE isolates. Not only does VNRX- 6736 outperform these clinical comparators from an MIC90 perspective, but does so with a narrow range of MIC owing to β-lactamase avoidance, a feature that will ultimately benefit setting of breakpoints. The series is rapidly bactericidal, exhibits a low spontaneous mutational frequency (frequency of resistance at 4x MIC of <2.7 x 10-11 in E. coli ATCC 25922) and has favorable ADME and PK properties. Proof of concept efficacy has been achieved by VNRX-6736 in the murine thigh model of carbapenem-resistant E. coli infection and pharmacokinetics modeling suggests that 30-45% time above MIC is required to achieve efficacy. Optimization efforts proposed herein are targeting an 8-fold improvement in antibacterial activity driven by rational structure-guided design to improve PBP binding interaction kinetics to enable an MIC90 ≤ 4 µg/mL. Such an optimized cyclic boronate PBPi could be a 1st new class antibiotic addressing resistance to β-lactams for the treatment of infections caused by CRE and a long term therapeutic solution to resistance development in Enterobacterales.

PROJECT NARRATIVE The penicillin binding protein-targeting β-lactams have been a mainstay therapeutic option for infections caused by enteric gram negative bacteria for more than 50 years. Efficacy of these agents is threatened by the continued expansion of β-lactamases (e.g., NDM) that contribute to carbapenem resistance in Enterobacterales including subtypes that are refractory to clinically available β-lactams or β-lactam/β-lactamase inhibitor combinations. This application focuses on the development of a cyclic boronate PBP inhibitor that circumvents class-specific resistance to β-lactams, resets the resistance clock and offers a new intravenous therapeutic option for the treatment of infections caused by carbapenem-resistant Enterobacterales (CRE).