C-4-Modified Isotetrones Prevent Biofilm Growth and Persister Cell Resuscitation in Mycobacterium smegmatis

Abstract

Hyperphosphorylated nucleotide (p)ppGpp, synthesized by Rel protein, regulates the stringent response pathway responsible for biofilm and persister cell growth in mycobacteria. The discovery of vitamin C as an inhibitor of Rel protein activities raises the prospect of tetrone lactones to prevent such pathways. The closely related isotetrone lactone derivatives are identified herein as inhibitors of the above processes in a mycobacterium. Synthesis and biochemical evaluations show that an isotetrone possessing phenyl substituent at C-4 inhibit the biofilm formation at 400 μg mL^-1, 84 h post-exposure, followed by moderate inhibition by the isotetrone possessing the p-hydroxyphenyl substituent. The latter isotetrone inhibits the growth of persister cells at 400 μg mL^-1 f.c. when monitored for 2 weeks, under PBS starvation. Isotetrones also potentiate the inhibition of antibiotic-tolerant regrowth of cells by ciprofloxacin (0.75 μg mL^-1) and thus act as bioenhancers. Molecular dynamics studies show that isotetrone derivatives bind to the Rel_Msm protein more efficiently than vitamin C at a binding site possessing serine, threonine, lysine, and arginine.

Scheme 1. Synthesis of Isotetrones 10–12, from a-Amino Acids 1–3, through Oxazolones 4–6 and C-3 Substituted Pyruvic Acid 7–9 Intermediates

Figure 1

γ-Butyrolactones 13 and 14, and isotetronic acid 15 synthesized from valine, isoleucine, and tryptophan, respectively.

Figure 2

M. smegmatis growth curves in the presence of varying concentrations of (a) 11; (b) 12; and (c) 10.

Figure 3

Kill kinetics of M. smegmatis with ciprofloxacin (Cip) (2.5 μg mL^–1) and Cip in the presence of derivatives 10–12 (400 μg mL^–1).

Figure 4

Images of M. smegmatis biofilms after 84 h, in the presence of varying concentrations of compounds 10–12 with untreated control.

Figure 5

M. smegmatis delayed addition time-kill kinetics profile. (a) Reduction in the regrowth of ciprofloxacin (0.75 μg mL^–1) treated tolerant cells in the presence of compounds 11 and 12 (400 μg mL^–1); (b) reduction of resistant mutant generation during ciprofloxacin (0.75 μg mL^–1) treatment in the presence of 11 and 12 (400 μg mL^–1). Black arrows depict the time of compound addition.

Figure 6

Plots of inhibition under long-term starvation of M. smegmatis in PBS (untreated) and in the presence of compound 12.

Figure 7

2D interaction plots of various protein–ligand complexes, obtained from docking studies. (a) Rel_Msm-ppGpp; (b) Rel_Msm-derivative 12; (c) Rel_Msm-vitamin C; and (d) Rel_Msm-derivative 11 complexes at their lowest energy conformations, predicted by molecular docking.

Figure 8

(a) Rel_Msm-ppGpp, (b) Rel_Msm-12, (c) Rel_Msm-vitamin C, and (d) Rel_Msm-11 complexes after 10 ns simulation. The secondary structure of the protein has been represented with the color code: yellow: β sheet, pale blue: turns, and white: other residues. The ligands are presented using “licorice” representation scheme of VMD. Protein residues within a 5 Å distance from the ligands are presented using VdW spheres with a color code: White: nonpolar residues, blue: basic residues, red: acidic residues, and green: polar residues. Water molecules are removed for clarity.

Figure 9

(a) Average electrostatic and VdW components of binding energy. Contribution of different residues in the binding energy for (b) Rel_Msm-ppGpp, (c) Rel_Msm-compound 12, and (d) Rel_Msm-vitamin C complexes. Nomenclature: Tyr Lac: compound 12; Vit C: vitamin C.