A Coumarin-Based Analogue of Thiacetazone as Dual Covalent Inhibitor and Potential Fluorescent Label of HadA in Mycobacterium tuberculosis

Abstract

A novel coumarin-based molecule, designed as a fluorescent surrogate of a thiacetazone-derived antitubercular agent, was quickly and easily synthesized from readily available starting materials. This small molecule, coined Coum-TAC, exhibited a combination of appropriate physicochemical and biological properties, including resistance toward hydrolysis and excellent antitubercular efficiency similar to that of well-known thiacetazone derivatives, as well as efficient covalent labeling of HadA, a relevant therapeutic target to combat Mycobacterium tuberculosis. More remarkably, Coum-TAC was successfully implemented as an imaging probe that is capable of labeling Mycobacterium tuberculosis in a selective manner, with an enrichment at the level of the poles, thus giving for the first time relevant insights about the polar localization of HadA in the mycobacteria.

Keywords: HadA; Mycobacterium tuberculosis; coumarin; fluorescence; thiacetazone.

Figure 1.

The FAS-II pathway in M.tb with enzymes in bold.

Figure 2.

Coum-TAC probe as a covalent conjugate of an anti-TB agent (in blue, see ref for details) with a fluorescent moiety (in red).

Figure 3.

Steady-state kinetics of EthA as a function of Coum-TAC as a substrate. Data are mean ± SD of at least three independent determinations. In the inset the comparison between the steady-state kinetics of TAC (red curve) vs Coum-TAC (black curve) is reported.

Figure 4.

TLC analysis of (A) lipids and (B) methyl esters of fatty (FAME) and mycolic (MAME) acids isolated from ¹⁴C labeled M.tb H37Ra cells treated with Coum-TAC or TAC. Lipids were separated in chloroform/methanol/water (20:4:0.5) and detected by autoradiography. Different forms of methyl esters were separated in n-hexane/ethyl acetate (95:5; 3 runs) and detected by autoradiography (with TDM: trehalose dimycolates; TMM: trehalose monomycolates; PE: phosphatidylethanolamine; CL: cardiolipin; alpha-, methoxy- and keto- refer to the forms of MAMEs).

Figure 5.

Formation of HadA dimer under cell-free conditions. SDS-PAGE analysis of proteins from reaction mixtures containing: (A) isolated proteins HadA and EthA, as well as TAC or Coum-TAC. The samples were analyzed in non-reducing environment. Up: gel stained with Coomassie Brilliant Blue; Down: immunodetection using anti-His antibodies; (B) only isolated protein HadA and TAC or Coum-TAC. The samples were analyzed in non-reducing environment. Up: gel stained with Coomassie Brilliant Blue; Down: immunodetection using anti-His antibodies; (C) isolated proteins HadA and EthA, as well as TAC or Coum-TAC. The samples were analyzed in the presence of 2-mercaptoethanol. Left: gel stained with Coomassie Brilliant Blue / Right: immunodetection using anti-His antibodies.

Figure 6.

Covalent binding of EthA-activated Coum-TAC to HadA. (A) UV-vis spectrum of HadA protein, re-purified after the reaction with EthA and Coum-TAC (black line), compared with the blank reaction (red line). Green line represents the spectrum of Coum-TAC, while yellow line is the spectrum of HadA before incubation. (B) UV-vis spectrum of HadA/Coum-TAC adduct after heat denaturation (black line), compared with the blank reaction (red line). (C) Emission spectra (excitation at 470 nm) of HadA/Coum-TAC adduct at 0.5 mg/mL (green line), 1 mg/mL (red line) and 2 mg/mL (black line). (D) Emission spectra (excitation at 470 nm) of HadA/Coum-TAC adduct after heat denaturation (black line), compared with the blank reaction (red line).

Figure 7.

Imaging of M.tb H37Rv strain incubated with Coum-TAC. (A) Monitoring of fluorescence in M.tb H37Rv mycobacteria expressing m-Cherry (red) with time-dependent fluorescence increase of Coum-TAC (in green) at the poles and septa. (B) Time-dependent Coum-TAC distribution along the medial axis of the mycobacteria. (C) Lattice-SIM microscopy of coumarin 466, C2 and Coum-TAC (in green) in M.tb H37Rv mycobacteria expressing m-Cherry (red). (D) M.tb was segmented using mCherry fluorescence and fluorescence intensity profiles of each compound was measured along the medial axis of each bacilli using the MicrobeJ plugin of ImageJ. Results show variations of intensity along the medial axis from the first pole (P₀) to the opposite pole at regular intervals (P_x). Results show Mean±SEM of >100 individual bacteria per condition. (E) Chemical structures of coumarin 466 and C2.

Scheme 1.

Thiacetazone (TAC): structure and suggested mechanism of HadA inactivation.

Scheme 2.

Synthesis and photophysical properties of Coum-TAC. ^a Maximum absorption wavelength in nm. ^b Molar extinction coefficient in M⁻¹ cm⁻¹. ^c Maximum emission wavelength in nm. ^d Fluorescence quantum yield at 461 nm.