Potent 1,2,4-Triazino[5,6 b]indole-3-thioether Inhibitors of the Kanamycin Resistance Enzyme Eis from Mycobacterium tuberculosis

Abstract

A common cause of resistance to kanamycin (KAN) in tuberculosis is overexpression of the enhanced intracellular survival (Eis) protein. Eis is an acetyltransferase that multiacetylates KAN and other aminoglycosides, rendering them unable to bind the bacterial ribosome. By high-throughput screening, a series of substituted 1,2,4-triazino[5,6 b]indole-3-thioether molecules were identified as effective Eis inhibitors. Herein, we purchased 17 and synthesized 22 new compounds, evaluated their potency, and characterized their steady-state kinetics. Four inhibitors were found not only to inhibit Eis in vitro, but also to act as adjuvants of KAN and partially restore KAN sensitivity in a Mycobacterium tuberculosis KAN-resistant strain in which Eis is upregulated. A crystal structure of Eis in complex with a potent inhibitor and CoA shows that the inhibitors bind in the aminoglycoside binding site snugly inserted into a hydrophobic cavity. These inhibitors will undergo preclinical development as novel KAN adjuvant therapies to treat KAN-resistant tuberculosis.

Keywords: aminoglycoside resistance; antitubercular agent; combination therapy; high-throughput screen; structure-activity relationship (SAR).

Figure 1.

Stepwise cone diagram showing the winnowing of ~23 000 compounds to the four Eis inhibitors that restored KAN activity in Mtb K204.

Figure 2.

Structures of all molecules used in this study along with their origins. Note: At the bottom of the figure, the compound numbers listed in orange indicate that the compounds were tested in the HTS only. Those listed in pink were tested in the HTS and repurchased for validation. Those listed in gray were compounds that were not present in the HTS but were purchased to expand the preliminary SAR study. Those in green were new derivatives synthesized to further expand our SAR study. Compound 37b is in purple as it was present in the HTS, repurchased, and resynthesized to confirm the values obtained for purchased material. Compound 39b is in blue as, not only was it present in the HTS, repurchased, and resynthesized, but was additionally crystallized with Eis. All compounds (except those listed with orange numbers) had an IC₅₀ or/and MIC_KAN value determined as presented in Table 1.

Figure 3.

(A) Crystal structure of one of the six monomers of the EisC204A-CoA-inhibitor 39b (shown as yellow sticks in a box) complex. Note: For the sake of simplicity, the CoA is omitted in this figure, but is shown in panel C. (B) Zoom-in view of the binding pocket of compound 39b. The strong omit F₀-F_c electron density map contoured at 3σ generated without compound 39b is shown by a magenta mesh. The amino acid residues interacting with compound 39b are depicted in dark turquoise. The conserved residues that interact with 39b and previously published Eis inhibitors are depicted as orange sticks. The Asp26-Ser32 loop is shown as dark turquoise sticks. The C-terminus is labeled as C-ter. A water molecule is shown as a green sphere. The distances between atoms of the inhibitor 39b and those of amino acid residues of the Eis protein are shown by a dark blue dashed line and are in Å. For compound 39b, the carbon, oxygen, nitrogen, fluorine, and sulfur atoms are colored pale green, red, blue, dark green, and orange, respectively. (C) Compound 39b is overlapped with tobramycin (TOB) and previously published Eis inhibitors. The previously published structure of bound TOB (PDB ID 4JD6¹²) is in blue. Bound CoA is depicted as red sticks. (D) Inhibitor “a”, a pyrrolo[1,5-a]pyrazine-based Eis inhibitor (labeled 2k* in ref 20, PDB ID 5TVJ), is depicted as green sticks. (E) Inhibitor “b”, a sulfonamide-based inhibitor (labeled 39 in ref 18 PDB ID 5IV0), is depicted as gray sticks. (F) Inhibitor “c”, an isothiazole S,S-dioxide heterocyclic core (labeled 11c in ref 17 PDB ID 5EBV) is depicted as purple sticks. Note: In panels D−F, amino acid residues that interact specifically with the inhibitors presented are depicted in lilac. (G) Chemical structures of TOB as well as inhibitors 39b and “a−c”.

Figure 4.

Mammalian cytotoxicity of selected compounds (34b, 37b, 39b, and 43d) alone (depicted as dark color columns) or in the presence of 50 μg/mL (86 μM) KAN (depicted as light color column directly to the right of the dark color column for the compound alone) against (A) A549, (B) HEK-293, and (C) J774A.1 cells. Note: No cytotoxicity was observed from 0 to 12.5 μM.

Scheme 1.

Synthetic Schemes Used for the Synthesis of 24 Compounds Generated in This Study