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

Abstract

Hyperphosphorylated guanosine nucleotide (p)ppGpp, synthesized by Rel proteins, regulates the stringent response pathway responsible for biofilm growth and persister cell formation in the stationary phase of mycobacteria. The discovery of vitamin C as a potent inhibitor of Rel protein activities raises the prospect of such a tetrone lactone to prevent biofilm growth and persister cell formation. The closely related isotetrone lactone derivatives are identified in the present study as potent inhibitors of the above processes in a mycobacterium. Isotetrone lactone derivatives are synthesized from appropriate α-ketocarboxylic acids, derived from the a-amino acids. Aldol condensation with formaldehyde, followed by the lactone formation, completes synthesis of isotetrone derivatives, possessing varied substituents atC-4 carbon, in good yields. A series of biochemical evaluations of biofilm growth and persister cell formation inM. smegmatisis conducted. Among the derivatives, isotetrone possessing phenyl substituent atC-4 carbon completely inhibit the biofilm formation at 400 μg mL-1concentration, 84 h of post-exposure, followed by a moderate inhibition by the isotetrone possessingp-hydroxyphenyl substituent. Whereas, the latter isotetrone inhibits the growth of cells at 400 μg mL-1f.c. when monitored for 2 weeks, under PBS starvation condition. Isotetrones also potentiate the inhibition of antibiotic tolerant regrowth of cells by ciprofloxacin antibiotic (0.75 μg mL-1) and thus act as bio-enhancers. The combination is shown to significantly arrest the emergence of ciprofloxacin-resistant genetic mutants. The observations suggest that isotetrones in combination with ciprofloxacin are therapeutically superior when administered together. Systematic molecular dynamics studies show that isotetrone derivative binds to Rel protein more efficiently than vitamin C and the binding is aided by hydrogen bonding, van der Waals and electrostatic interactions at a binding site possessing serine, threonine, lysine and arginine residues. The present study establishes that the identified isotetrone derivatives (i) act as inhibitors ofM. smegmatisbiofilm growth and (ii) arrest the re-emergence of recalcitrant persister cells when administered together with ciprofloxacin antibiotic. Results of this study establish that isotetrones as new chemical entities that interfere with stringent response pathways in a mycobacterium under stress and permit overcoming the multidrug-resistant persister cell emergence in the bacterium.

A novel vitamin C analog acts as a potent bio-enhancer to augment the activities of anti-tuberculosis drugs againstMycobacterium tuberculosis

“…We screened the selected C-4 modified isotetrones (16) (Figure S1) for their potential activity against M. tuberculosis H37Rv by minimum inhibitory concentration (MIC) assay The compounds were screened alone and also in combination with first-line TB drugs. Simultaneously, the selected C-4 modified isotetrones were analyzed for the planktonic growth inhibitory activity against M. tuberculosis H37Rv (Figure S2) .…”

“…In the present study, we identified the specific bio-enhancer compound that could improve the bactericidal activity of existing TB drugs. As the compound C11 was synthesized by attaching L-phenylalanine to the vitamin C core structure, those could also be regarded as vitamin C analogs (16). The identified bio-enhancer C11 has been shown to have a potent effect in combination with first-line and second-line injectable drugs.…”