Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria

Abstract

Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by Rel_Msm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.

Keywords: (p)ppGpp; mycobacteria; stringent response.

FIG 1

Structure of pppGpp and Relacin.

FIG 2

Reagents and conditions: scheme 1. (i) Acetic anhydride, 4-dimethylaminopyridine, pyridine, 0°C to room temperature, 12 h, 56%. (ii) Benzoyl chloride, 4-dimethylaminopyridine, pyridine, 0°C to room temperature, 12 h, 62%. (iii) a. Sodium hydroxide (0.1 M), methanol, room temperature, 12 h; b. acetic anhydride, 4-dimethylaminopyridine, pyridine, 0°C to room temperature, 12 h, 41% (after two steps). In the text, compound 1 is referred to as the acetylated compound (AC compound), and compound 3 is referred to as the acetylated benzoylated compound (AB compound).

FIG 3

MALDI-TOF analysis of AC and AB compounds. (Top) MALDI-TOF analysis of acetylated compound (AC compound). The 451.9 m/z value corresponds to the acetylated derivative of guanosine (AC compound). (Bottom) MALDI-TOF analysis of acetylated benzoylated guanosine (AB compound). The 513.8 m/z value corresponds to the mass of AB compound, and the 536.0 m/z value is its sodiated adduct. Intens., intensity; a.u., arbitrary units.

FIG 4

(a) Inhibitory effects of AC and AB compounds on in vitro pppGpp synthesis at a 100 μM concentration. Each experiment was performed in three biological replicates. Densitometric analysis was performed, and the values obtained were normalized with respect to the wild type (WT or 4). A Student t test was carried out to confirm the significance. The P value was <0.05 in the case of 6 and 7. (b) Dose-dependent inhibition of in vitro pppGpp synthesis ranging from 1 to 250 μM.

FIG 5

In vivo estimation of (p)ppGpp levels in M. smegmatis. The experiment was conducted in three biological replicates. A Student t test was performed to confirm the significance. The P value was found to be <0.05 for both the AC compound and the AB compound.

FIG 6

Binding of the acetylated benzoylated compound to the Rel enzyme from M. smegmatis. An ITC curve corresponding to the binding of the synthetic compound to the Rel enzyme at 25°C is presented here. The upper panel shows the raw data for the titration of Rel enzyme with the AB compound, and the lower panel indicates the integrated heat of binding obtained from the raw data. The model for one-site binding was implicated to fit the curve. The “N” represents the number of binding site that is 1, and the “K” represents the association constant (K_a). The dissociation constant, K_d, can be calculated by reciprocating the K_a and was determined to be nearly 10 μM.

FIG 7

Enzyme kinetics of Rel_Msm. The rate of formation of ppGpp as a function of substrate GTP is shown here. (a) Formation of product with various substrate concentrations; (b) phosphorimage data of the same. The table below quantifies the K_0.5 and V_max of the reaction in the absence or presence of the synthesized inhibitors. Lanes: -ve, negative control (assay buffer); WT, control (where inhibitor was not added); other lanes, assay mixture with increasing concentrations of GTP. The concentration of the protein (Rel_Msm) was kept at 200 μg/ml. A Student t test was performed to analyze the significant change in V_max and K_0.5 values. The graph was fitted using the Hill equation. The Hill coefficient value was determined to be 1.89 ± 0.17.

FIG 8

Long-term survival in the presence or absence of synthetic compounds (100 μM) with Rel KO and Rel complement controls in triplicates. This experiment was performed in three biological replicates. Inhibition was found to be significant for both AB and AC compounds.

FIG 9

Biofilm formation in the presence of AC and AB compounds (100 μg/ml) compared to a control experiment in which compound was not added to M. tuberculosis and M. smegmatis.

FIG 10

Biofilm quantification assay in the presence of compounds (100 μg/ml) in Sauton's media in three biological replicates. Biofilm formation was found to be decreased in the treated bacterial cells. Compounds were added at the zero time point.

FIG 11

MTT assay results in the presence of a range of concentrations of synthetic compounds using the H460 cell line. The x axis indicates the concentration of the compound; the y axis indicates the absorbance at 570 nm. (a) Acetylated benzoylated (AB) compound; (b) acetylated (AC) compound.

FIG 12

The crystal structure of Relseq385, Rel/Spo from S. equisimilis, was utilized for in silico docking. The GDP molecule was removed from the active site of the protein structure. The acetylated benzoylated compound structure (in a low energy state) was positioned at the active site in place of GDP, and all of the rotatable bonds were kept flexible. (A) Two-dimensional representation of the interactions at the active site. The red line indicates the stacking interaction, whereas the purple dotted line shows hydrogen bonding. (B) Three-dimensional depiction of the active site with bound acetylated benzoylated compound.