A molecular model to study FosA enzyme inhibition

Highlights

• FosA reaction leads fosfomycin to lose its pharmacological effect and promotes fosfomycin resistance.

• We have elucidated the dynamic interactions between the enzyme FosA and the inhibitor ANY1.

• The molecular model developed here describes the protein-ligand affinity with R² of 77.51%.

• Additional residues should be considered in the efforts to design novel attractive FosA inhibitors.

Abstract

Fosfomycin resistance protein (FosA) is a metalloenzyme known for catalyzing a nucleophilic addition reaction of glutathione to the epoxide ring of Fosfomycin, a broad-spectrum antibiotic used to combat Gram-positive pathogens. The reaction leads fosfomycin to lose its pharmacological effect, thus promotes antibiotic resistance. A small-molecule FosA inhibitor has been discovered. ANY1 (3-bromo-6-[3-(3-bromo-2-oxo-1H-pyrazolo[1,5-a]pyrimidin-6-yl)-4-nitro-1H-pyrazol-5-yl]-1H-pyrazolo[1,5-a]pyrimidin-2-one) is competitive with the antibiotic for binding the active site of the enzyme. Through Molecular Mechanics methods, using the AMBER force field, we carry out molecular dynamics simulations and binding free energy calculations to investigate the most important interactions between the enzyme and inhibitor. Our results were able to reproduce the trend of experimental data with R² of 77.51%. Furthermore, we have shown that electrostatic and van der Waals interactions, as well as cavitation energies, are favorable for maintaining the enzyme-inhibitor complex, while reactive field energies and non-polar interactions act in an unfavorable way for interactions between FosA and ANY1.

Introduction

Fosfomycin (FOF) is a broad-spectrum antibiotic with a safety and effectiveness history administration in humans [1]. Its bactericidal activity is exerted by covalent binding to UDP-N-acetylglucosamine-enolpyruvyl transferase (MurA), an enzyme that catalyzes the first step in cell wall biosynthesis. It is important to note that Fosfomycin is highly active against Escherichia coli, including beta-lactamase producers [2]. In gram-negative bacteria, the clinically relevant resistance mechanism involves the presence of the FosA metalloenzyme, which is capable of catalyzing the degradation of the fosfomycin epoxide ring by adding nucleophilic glutathione (GSH), metabolizing the antibiotic structure and thus breaking its pharmacological effect [3,4].

The FosA enzyme is a homodimer composed of two active sites coordinating Mn²⁺ ions that bind to fosfomycin and act as Lewis acids during the nucleophilic attack by glutathione. A K⁺ ion also binds near the active site, balancing negative charges and increasing the rate of reaction [3]. The fosA gene is often found on chromosomes of many Gram-negative bacteria, such as Klebsiella pneumoniae, conferring intrinsic resistance to fosfomycin. Escherichia coli has already been reported presenting the fosA3 gene in its plasmid. This gene is responsible for the synthesis of FosA3 enzymes. FosA^KP and FosA^PA are enzymes derived from the chromosomes of the bacterias Klebsiella pneumoniae and Pseudomonas aeruginosa. Experimental results report that FosA3 and FosA^KP exhibit higher resistances to fosfomycin than FosA^PA, since the previous enzymes have larger loops at the interface of their dimers, compared with FosA^PA, and that the interface of these loops has an influence on FosA activity [3].

A competitive small-molecule inhibitor was found to increase fosfomycin activity against Gram-negative bacteria [2]. Among several molecules with potential inhibition of FosA, ANY1 (3-bromo-6-[3-(3-bromine-2-oxo-1H-pyrazolo [1,5-a]pyrimidin-6-yl)-4-nitro-1H-pyrazole-5-yl]-1H-pyrazolo [1,5-a]pyrimidin-2-one) (Fig. 1) was the most potent of them with IC₅₀ of 5.1 ± 2.2 μM. This inhibithor can bind to the active site of the metaloenzyme, ceasing the metabolization of FOF in the reaction with GSH. Furthermore, the citotoxicity of ANY1 in association with Fosfomycin was experimentally evaluated and it was found that administration of this compound with the antibiotic is safe and ANY1 had a minor impact in cell viability both in human kidney cells and human peripheral mononuclear cells [2]. Thus, this small-molecule is an advantageous booster of Fosfomycin antibacterial activity (see Fig. 1).

Therefore, we conjecture that the discovery and development of FosA inhibitors can significantly expand the use of fosfomycin against gram-negative pathogens that have the gene responsible for the production of this metalloenzyme. In this work, the interactions between two FosA enzymes and the inhibitor ANY1 were evaluated. Their affinities were estimated by the calculation of binding free energies based on molecular mechanics (MM) methods and molecular dynamics (MD) trajectory. Our results show at a molecular level the dynamics of the interactions between ANY1 and the active site residue that contribute to the inhibitor's affinity with the protein. Additionally, we found a great correlation between the predicted results in silico and the results obtained experimentally.