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

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 -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 at C-4 carbon, in good yields. A series of biochemical evaluations of biofilm growth and persister cell formation in M. smegmatis is conducted. Among the derivatives, isotetrone possessing phenyl substituent at C-4 carbon completely inhibit the biofilm formation at 400 g mL-1 concentration, 84 h of post-exposure, followed by a moderate inhibition by the isotetrone possessing p-hydroxyphenyl substituent. Whereas, the latter isotetrone inhibits the growth of cells at 400 g mL-1 f.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 g 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 of M. smegmatis biofilm 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.