Targeting Siderophore-Mediated Iron Uptake in M. abscessus: A New Strategy to Limit the Virulence of Non-Tuberculous Mycobacteria

Abstract

Targeting pathogenic mechanisms, rather than essential processes, represents a very attractive approach for the development of new antimycobacterial drugs. In this context, iron acquisition routes have recently emerged as potentially druggable pathways. However, the importance of siderophore biosynthesis in the virulence and pathogenicity of M. abscessus (Mab) is still poorly understood. In this study, we investigated the Salicylate Synthase (SaS) of Mab as an innovative molecular target for the development of inhibitors of siderophore production. Notably, Mab-SaS does not have any counterpart in human cells, making it an interesting candidate for drug discovery. Starting from the analysis of the binding of a series of furan-based derivatives, previously identified by our group as inhibitors of MbtI from M. tuberculosis (Mtb), we successfully selected the lead compound 1, exhibiting a strong activity against Mab-SaS (IC₅₀ ≈ 5 µM). Computational studies characterized the key interactions between 1 and the enzyme, highlighting the important roles of Y387, G421, and K207, the latter being one of the residues involved in the first step of the catalytic reaction. These results support the hypothesis that 5-phenylfuran-2-carboxylic acids are also a promising class of Mab-SaS inhibitors, paving the way for the optimization and rational design of more potent derivatives.

Keywords: antimicrobial resistance; cystic fibrosis; drug design; grating-coupled interferometry (GCI); homology model; siderophores.

Figure 1

Scheme highlighting the reactions catalyzed by SaS in mycobacteria. In the first step, chorismate is converted to isochorismate, which is then transformed to salicylate, with the release of pyruvate.

Figure 2

Inhibitor 1 does not behave as a PAIN compound. The IC₅₀ value determined in the absence of any additive (black) was consistent with those measured in the presence of 1 mg/mL BSA (red) or 0.01% Triton X-100 (green), confirming that the compound does not induce aggregation, and in the presence of 100 μM DTT (blue), excluding an interaction of 1 with the cysteine residues of Mab-SaS.

Figure 3

Quantitative binding kinetics of Mab-SaS vs. 1 (A), 2 (B), and 3 (C), measured by GCI. All curves were blank subtracted. Experiments were performed in triplicate. All the quality assessments (i.e., χ² shown in Table 1, parameter errors, and residual plots were acceptable; the sensorgrams had sufficient curvatures and the kinetic constant k_off were within the measurable range) were fulfilled. Descriptive Statistical Analysis was employed to calculate the mean of the obtained values of k_on, k_off, and K_D as a measure of central tendency; standard deviations of the former values were computed as a measure of dispersion.

Figure 4

Compound 1 inhibits the production of siderophores and mycobactins by M. bovis BCG in iron-limiting conditions. (A) Siderophore units in culture media of cells grown in the presence of different concentrations of 1, determined by the Universal CAS assay. (B) Determination of mycobactins extracted and measured in the above-mentioned cells. Data are mean ± SD of three replicates.

Figure 5

Top-scored docking poses of 1 (A), 2 (B), 3 (C), and chorismate (D) within the binding site of the developed Mab-SaS homology model. Ligands and important residues are rendered as sticks, while the protein as cartoon. H-bond and salt-bridge interactions are depicted by a dotted black line and a blue line, respectively. For the sake of clarity, only polar hydrogen atoms are shown. Notice that the fluorine atoms belonging to the trifluoromethyl substituents of 1 and 3 are depicted as cyan sticks.