Acetohydroxamic acid (AHA) is a reversible urease inhibitor already used clinically in the treatment of urinary tract infections caused by urease-positive bacteria. Urease is a nickel-dependent enzyme that hydrolyzes urea into carbon dioxide and ammonia. In addition to providing carbon and energy for bacteria, the produced ammonia raises the pH and allows survival inside macrophage phagolysosomes or in acidic environments such as the stomach [1]. Although well studied in Helicobacter pylori and Proteus mirabilis, its role in Acinetobacter spp. remains poorly explored. In addition to gastric and urinary tract infections, AHA could also be associated with antibiotic therapy to attenuate virulence in other sites, especially in multidrug-resistant A. baumannii strains, if it is capable of inhibiting their urease.

To evaluate the binding of AHA to the urease of clinical Acinetobacter isolates using docking simulations in immature (apoenzyme) and mature (holoenzyme) forms.

The apo form of urease was modeled with AlphaFold3, and nickel ions were positioned at the active site of the holo form using AlphaFill. Molecular docking was performed with AutoDock Vina. The interactions between AHA and the protein were analyzed in PyMOL, and conservation of active site residues was assessed through multiple sequence alignment with MAFFT.

AHA showed similar affinities for both forms (–4.3 kcal/mol for apo, –3.9 kcal/mol for holo). However, the holoenzyme presented a more canonical binding mode, with AHA coordinating both Ni²⁺ cofactors and interacting with the main catalytic residues His218, Asp359, and Ala362. These residues correspond to those observed in the crystallographic structure of Klebsiella aerogenes urease bound to AHA (PDB ID: 1FWE). In the apo form, AHA interacted with conserved residues Gly276 and Thr297, suggesting possible inhibition even before enzyme maturation. Multiple sequence alignment of urease from 233 clinical Acinetobacter spp. isolates revealed high conservation rates in all residues involved in AHA binding, supporting the functional relevance of the observed interactions.

Our findings support the use of AHA to inhibit urease activity in clinical Acinetobacter spp. isolates from different clinical sites, and further clinical studies are still needed.