Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis

Abstract

A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.

Figure 1

(a) Schematic representation of the winnowing of ~123 000 small organic molecules to 18 showing inhibition of both Eis enzymatic activity and growth of Mtb K204 in the presence of KAN. (b) Structures of the 41 Eis inhibitors (with an isothiazole S,S-dioxide heterocyclic scaffold) for which IC₅₀ against pure Eis enzyme and MIC values against strains H37Rv and K204 of Mtb have been determined. Note: Compounds highlighted in turquoise are those for which MIC values of KAN against Mtb K204 were found to be <2.5–5 µg/mL. Compounds highlighted in gray are those for which MIC values of KAN against Mtb K204 were found to be >5–10 µg/mL. Compounds highlighted in fuchsia are those for which the X-ray structure in complex with EisC204A and CoA has been determined.

Figure 2

Crystal structures of EisC204A from Mtb in complex with 13g (a and b) and 11c (d and e). The overall views of the Eis monomer with the inhibitors bound (a and c) and zoom-in views of the active site (b and e) show that the inhibitors (green sticks with atoms labeled as follow: C = green, N = blue, O = red, S = orange, and Cl = dark green) occupy a site overlapping with the aminoglycoside substrate-binding site. The F_o−F_c omit map contoured at 3σ is shown by the purple mesh. Inhibitor interacting residues are shown as orange sticks (with N and O in blue and red, respectively). Water molecules mediating inhibitor binding are shown as navy blue spheres. The side chain of Asp26 and the backbone of this residue and its neighbors adopt different conformations in the two complexes. The CoA molecule is not shown in panels a, b, d, and e to simplify the view. Superposition of these respective inhibitor structures (in green) with the previously reported crystal structure of EisC204A (pale orange) in complex with CoA and tobramycin is shown in panels c and f. The tobramycin is shown in red, and the ordered part of CoA (from the structures of Eis-inhibitor-CoA complexes) is in blue. The CoA molecule from the complex of Eis with tobramycin is bound similarly and is not shown.