Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity

Abstract

A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC₅₀ ∼ 1 μM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc² 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.

Keywords: Crystal structure; Drug resistance; Enhanced intracellular survival; Enzyme kinetics; Mycobacterium tuberculosis; Structure-activity relationship; Tuberculosis.

Fig. 1.

Scaffolds of previously studied Eis inhibitors. Structure-activity relationship studies were conducted by changing R groups. The eight scaffolds are A. isothiazole S,S-dioxides, B. sulfonamides, C. methyl 4H-furo[3,2-b]pyrrole-5-carboxylate (left) and 3-(1,3-dioxolano)-2-indolinone (right), D. pyrrolo[1,5-a]pyrazines, E. 1,2,4-triazino[5,6b]indole-3-thioethers, F. thieno[2,3-d]pyrimidines, and G. haloperidol derivatives.

Fig. 2.

Synthetic scheme for the preparation of compounds 19-56, used in this study. Intermediates 1-18 were used to generate compounds 19-56. Ph denotes phenyl, Py denotes pyridyl. The reaction yields are indicated in parentheses.

Fig. 3.

Acetylation rates as a function of concentration of KAN at different concentrations of inhibitors 26, 27 and 28. The steady-state reaction rates (left) and their double-reciprocal Lineweaver-Burk plots (right). The concentrations of the inhibitors are shown inside the panels.

Fig. 4.

Crystal structures of Eis in complexes with compounds 32, 33, and 38. The overall structures of an Eis protomer in complex with 38 and 32 are shown as surface views in panels A, and B, respectively. The Eis inhibitors (represented by green sticks) overlap with the binding site of aminoglycoside substrate (boxed). Tobramycin (TOB) and CoA (PDB: 4JD6), represented as light and dark grey sticks, respectively, were superimposed onto the Eis-inhibitor structures. The Eis interfaces are shown in zoomed in views for complexes with compounds 38, 32 and 33 in panels C, D, and E, respectively. The residues surrounding the inhibitor are represented as purple sticks, while the inhibitors are shown as green sticks. A subtle difference between compounds 32 and 33 is the position of the nitrogen in the pyridinyl ring at R₂, this is noted by the red arrow. The 2D ligand-protein diagrams were automatically generated by the LigPlot+ for compounds 38 (panel F) and 33 (panel G). As clearly seen in panels D and E, the environment surrounding compounds 32 and 33 were comparable, hence, we only showed LigPlot+ diagram for compound 33. A red arrow denotes the location of the change of position of nitrogen (from N2 in compound 33) in compound 32. The nomenclature used by LigPlot+ for the atoms is based on the PDB format.

Fig. 5.

Evaluation of cytotoxicity for compounds 22 (yellow), 25 (orange), 26 (red), 29 (blue), 31 (purple), 46 (turquoise), and 48 (gray) against A. A549, B. HEK-293, and C. J774A.1. The corresponding graphs with non-normalized data are presented in Fig. S107.