The WHO lists Acinetobacter baumannii (Ab) as the most problematic pathogen infecting patients. In this renewal, we will build upon our successful accomplishments in our first two R01s in which we developed an entirely new class of boronic acid transition state inhibitors (BATSIs), explored the structure activity relationships (SAR) of the chromosomal cephalosporinase of Ab, ADC-7, as well as the class D carbapenemases OXA-23, - 24/40 and -66/51, and defined the efflux pumps governing antibiotic resistance. Herein, we will embark upon innovative ways to overcome β-lactam resistance by proposing two novel approaches. Firstly, we propose that structural and mechanistic similarities exist between class C and D β-lactamases present in Acinetobacter spp. that reveal a common intermediate. This notion will drive efforts to synthesize carbapenem and cephem based BATSIs that will inactivate both β-lactamase classes. Secondly, we advance that chromosomal blaADC and blaOXA overexpression in resistant isolates creates new cellular vulnerabilities that can be identified by genetic approaches such as Tn-Seq. These unique vulnerabilities will be exploited in high-throughput screens (HTS) to identify novel compounds that specifically target highly β-lactam resistant isolates. The novel targets and new inhibitors identified could lead to the “next generation” of therapeutics that overcome high-level β-lactam resistant Ab and avoid the indiscriminate killing of all bacteria. To meet these goals, we will build upon our insights into the structures of ADC and OXA β-lactamases and the chemistry of α-triazoles and amide bioisostere BATSIs to explore the SAR of these two series of compounds that will identify inhibitory activity toward both class C and D β-lactamases. As carbapenems are known to be both substrate (class D) and inhibitors (class C) of these β- lactamases, we will first synthesize novel BATSI compounds based upon a carbapenem backbone, as this scaffold provides a common core from which to build a broad-spectrum inhibitor of both C and D β-lactamases. These new inhibitors will be evaluated microbiologically, biochemically, and structurally against emerging clinical variants of ADC and OXA β-lactamases. We will then validate the selected inhibitors against a panel of clinical strains. Simultaneously, we will also use Tn-Seq to identify mutations that confer a synthetic lethal phenotype in an Ab strain overexpressing the chromosomal blaADC or blaOXA β-lactamase. A genetic screen will be performed to confirm that the genes identified confer a synthetic lethal phenotype. Lastly, we will utilize high throughput screening (HTS) to identify new therapeutic agents that impact the interplay of chromosomal blaADC or blaOXA expression and bacterial viability. The current prevalence of MDR and pan-resistant strains of Ab, combined with the lack of new antibiotics, underscores the critical need for the development of novel therapeutics to combat this bacterium. Our g...