# Stringent Response in Mycobacteria: From Biology to Therapeutic Potential
## 7 Chemical Inhibition of Stringent Response as a Therapeutic Tool  
Since the stringent response is important for several processes, such as persistence, virulence, antibiotic resistance, and biofilm formation, its chemical inhibition might be an attractive way to address the problem of drug resistance. In this direction, relacin, a synthetic (p)ppGpp analog, has been shown to inhibit stringent response in _B. subtilis_ and _B. anthracis_. Relacin binds to Rel protein near the active site, which inhibits (p)ppGpp synthesis [122] and inhibits stringent response both in vivo and in vitro. Moreover, it also blocks the sporulation process in both _B. subtilis_ and _B. anthracis_, when added to sporulating cultures, irrespective of the stage of sporulation. Since relacin blocks (p)ppGpp synthesis, it also inhibits biofilm formation in _Bacillus_ species. In an alternative approach, an anti-biofilm peptide, 1018, was found to interact with (p)ppGpp [123]. The peptide was identified in a screen and was previously labeled as an innate defense regulator due to its immunomodulatory activities; 1018 was able to degrade (p)ppGpp, the mediator of stringent response in several clinically important species. These include pathogens such as _P. aeruginosa, E. coli, Acinetobacter baumannii, Klebsiella pneumoniae_, methicillin-resistant _Staphylococcus aureus_, Salmonella typhimurium_, and _Burkholderia cencopacia_, which when treated with 1018, failed to form biofilms. A low dosage of 1018 triggered biofilm dispersal, while the high dosage caused the death of bacterial cells in the biofilms. Additionally, overproduction of (p)ppGpp in _P. aeruginosa_ and _S. aureus_ imparted resistance to 1018 [123]. These studies demonstrated that chemical inhibition of stringent response can be used to inhibit pathogenic bacteria.  
Recently, such approaches have also been applied to inhibit mycobacterial stringent response [124; 125]. In one such study, acetylated and benzohydated (p)pGpp--N2,2',3',5'-O-Tetraacetylguanosine and N2,2'-O,3'-O,5'-O-Tetrabenzoylguanosine--were synthesized to assess if these compounds can inhibit stringent response in mycobacteria. Both the compounds significantly inhibited the activity of RelMsm protein in vitro and in vivo. These compounds also inhibited biofilm formation in both _M. singematis_ and _M. tuberculosis_. Moreover, these compounds were not toxic in cell culture assays, thus demonstrating a potential to be used in in vivo studies in mice. Furthermore, vitamin C was also shown to be used as a chemical inhibitor of stringent response in _M. singematis_[126]. Vitamin C-treated _M. singematis_ cultures show lower levels of (p)ppGpp compared to untreated control. Vitamin C also inhibited the activity of RelMsm, possibly leading to decreased synthesis of (p)ppGpp. Interestingly, treatment with vitamin C also inhibited biofilm formation by _M. singematis_. However, whether the inhibition of stringent response by vitamin C is responsible for the disruption of biofilm formation remains to be elucidated. In another study, a chemically synthesized compound called DMNP [4-(4,7-DiMethyl-1,2,3,4-tetrahydrooNphthalene-1-y)] Pentanoic acid--an analog of natural marine diterpene ergorgiae--could bind to RelMsm protein and inhibit its (p)ppGpp synthase activity. Moreover, when _M. singematis_ cultures were treated with DMNP, they failed to form biofilms and their persistence was reduced [124]. Hence, these studies demonstrate that inhibition of stringent response is an attractive target to design novel antimyobacterial compounds.