The Gram-negative bacterium *Acinetobacter baumannii* is a significant nosocomial pathogen causing infections like sepsis, wound infections, and pneumonia. The World Health Organization identifies carbapenem-resistant *A. baumannii* as a critical threat due to its high mortality rate and resistance to many antibiotics, including carbapenems and colistin. The limited pipeline of new antibiotics necessitates the urgent development of novel antibacterial agents. Benzisoxazole scaffolds are known for their presence in various FDA-approved drugs with diverse biological activities, including antimicrobial properties. This study focuses on identifying and characterizing a novel benzisoxazole antibiotic produced by a marine bacterium, *Bradyrhizo-bium denitrificans*, against multi-drug-resistant *A. baumannii*. The research investigates the compound's mechanism of action and explores structure-activity relationships to potentially optimize its potency.

Previous research highlights the serious threat posed by multi-drug resistant *A. baumannii*, particularly carbapenem-resistant strains. The lack of new antibiotics entering the pipeline underscores the need for novel therapeutic strategies. Benzisoxazoles have shown promise in various pharmacological applications, including antimicrobial activity. However, benzisoxazole antibiotics from natural sources are uncommon. This study builds upon existing knowledge of benzisoxazole structures and their potential, aiming to identify a novel antibiotic with activity against this clinically important pathogen.

The study involved bioassay-guided fractionation of a crude organic extract from *B. denitrificans* (Isolate B158). The extract was screened using the *p*-iodonitrotetrazolium chloride (INT) assay to assess multi-drug resistance reversal potential. Phylogenetic analysis identified the isolate. Compound 1, 3,6-dihydroxy-1,2-benzisoxazole, was isolated, and its structure was confirmed by HRESIMS and NMR. Due to limited quantity (1 mg from 14.1 g crude extract), the compound was also synthesized. Antibacterial activity was tested against a panel of clinical *A. baumannii* strains, using minimum inhibitory concentration (MIC) assays in both Muller-Hinton broth (MHB) and a defined minimal medium (DM01). A series of synthetic analogs (2-10) were produced and tested to investigate structure-activity relationships. To explore the mechanism of action, the effects of 4-hydroxybenzoic acid (4-HB) were examined. Molecular docking studies using a homology model of *A. baumannii* chorismate pyruvate-lyase (CPL) were conducted to assess potential target interactions.

Compound 1, 3,6-dihydroxy-1,2-benzisoxazole, demonstrated potent antibacterial activity against multiple *A. baumannii* strains, with MICs ranging from 6.25 to 50 µg ml⁻¹. The compound was significantly more potent in minimal media (DM01) compared to MHB, suggesting that components in MHB might antagonize its activity. Structure-activity relationship (SAR) studies revealed that a hydrogen bond donor at the C6 position is crucial for activity, with a hydroxyl group being optimal. Modifications to the isoxazole ring abolished activity. 4-HB antagonized the antibacterial effect of compound 1, suggesting involvement of 4-HB metabolic pathways. Molecular docking studies indicated that compound 1 interacts favorably with the active site of *A. baumannii* CPL, potentially acting as a competitive inhibitor. The interaction involves hydrogen bonding with conserved residues, similar to 4-HB binding in *E. coli* CPL. A secondary hypothesis suggests that compound 1 could also competitively inhibit 4-hydroxybenzoate octaprenyltransferase.

The findings demonstrate that 3,6-dihydroxy-1,2-benzisoxazole is a potent antibiotic against multi-drug resistant *A. baumannii*. The SAR analysis indicates that the natural compound may already be near optimal for activity. The reversal of the antibacterial effect by 4-HB suggests that compound 1 targets enzymes in the 4-HB metabolic pathway. Molecular docking supports the hypothesis that compound 1 inhibits chorismate pyruvate-lyase (CPL), a key enzyme in 4-HB biosynthesis. The conserved binding interactions between compound 1 and CPL in *A. baumannii* and 4-HB in *E. coli* CPL suggest a competitive product inhibition mechanism. The absence of CPL in mammals may account for the low toxicity observed in previous studies. An alternative hypothesis suggests that compound 1 may also inhibit 4-hydroxybenzoate octaprenyltransferase.

This study identified and characterized a novel benzisoxazole antibiotic, 3,6-dihydroxy-1,2-benzisoxazole, with potent activity against multi-drug resistant *A. baumannii*. The compound's mechanism of action likely involves inhibition of chorismate pyruvate-lyase, and potentially 4-hydroxybenzoate octaprenyltransferase. Further optimization of the structure and investigation of the exact mechanism are warranted. This compound represents a promising lead for the development of new antibiotics to combat this critical pathogen.

The study primarily focused on in vitro assays. Further in vivo studies are needed to assess the efficacy and toxicity of the compound. The molecular docking studies were based on a homology model of *A. baumannii* CPL, and experimental validation is required. The relatively small number of analogs tested limits the scope of the SAR analysis. More research is needed to fully explore the mechanism of action, especially concerning the potential inhibition of 4-hydroxybenzoate octaprenyltransferase.