Identification of Potent DNA Gyrase Inhibitors Active against Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis DNA gyrase manipulates the DNA topology using controlled breakage and religation of DNA driven by ATP hydrolysis. DNA gyrase has been validated as the enzyme target of fluoroquinolones (FQs), second-line antibiotics used for the treatment of multidrug-resistant tuberculosis. Mutations around the DNA gyrase DNA-binding site result in the emergence of FQ resistance in M. tuberculosis; inhibition of DNA gyrase ATPase activity is one strategy to overcome this. Here, virtual screening, subsequently validated by biological assays, was applied to select candidate inhibitors of the M. tuberculosis DNA gyrase ATPase activity from the Specs compound library (). Thirty compounds were identified and selected as hits for in vitro biological assays, of which two compounds, G24 and G26, inhibited the growth of M. tuberculosis H37Rv with a minimal inhibitory concentration of 12.5 μg/mL. The two compounds inhibited DNA gyrase ATPase activity with IC50 values of 2.69 and 2.46 μM, respectively, suggesting this to be the likely basis of their antitubercular activity. Models of complexes of compounds G24 and G26 bound to the M. tuberculosis DNA gyrase ATP-binding site, generated by molecular dynamics simulations followed by pharmacophore mapping analysis, showed hydrophobic interactions of inhibitor hydrophobic headgroups and electrostatic and hydrogen bond interactions of the polar tails, which are likely to be important for their inhibition. Decreasing compound lipophilicity by increasing the polarity of these tails then presents a likely route to improving the solubility and activity. Thus, compounds G24 and G26 provide attractive starting templates for the optimization of antitubercular agents that act by targeting DNA gyrase.

Bioisosteric Design Identifies Inhibitors of Mycobacterium tuberculosis DNA Gyrase ATPase Activity

“…Overexpression expression and purification of M. tuberculosis DNA GyrA and GyrB were described in our previous work. 42 Briefly, PCR products encoding the GyrA and GyrB subunits were cloned into the pET21a(+) and pET28a(+) vectors (Novagen) using the NdeI/XhoI restriction sites, respectively, resulting in plasmids encoding GyrA with a C-terminal hexa-histidine tag and GyrB with an N-terminal hexa-histidine tag. These expression vectors were separately transformed by the heat-shock method into Escherichia coli BL21(DE3)pLysS (Novagen).…”

“…Inhibition of ATPase activity was measured using an enzyme-coupled assay 65 in which the hydrolysis of ATP to ADP is coupled to the oxidation of NADH to NAD + as described in our previous work. 42 Briefly, the fluorescence intensity of NADH at excitation λ = 340 nm and emission λ = 420 nm was monitored for measurement of ATPase activity. The relative inhibition of ATPase activity for each inhibitor expressed as % relative inhibition is calculated using eq 1.…”

“…The minimum inhibitory concentration (MIC) of hit compounds against growth of M. tuberculosis H37Ra was determined by a microplate Alamar blue assay (MABA) as described in our previous work. 42 Briefly, inhibitor concentrations ranging from 0.1 to 100 μg/ mL were obtained from a serial twofold dilution with a constant concentration of 0.156% v/v DMSO. A mycobacterial suspension in 7H9GC media was added to assay well microplate and incubated at 37 °C for approximately 7 days.…”

Virtual Screening Identifies Novel and Potent Inhibitors of Mycobacterium tuberculosis PknB with Antibacterial Activity

“…The molecular mechanics/Poisson−Boltzmann surface area (MM/PBSA) and molecular mechanics/generalized Born surface area (MM/ GBSA) methods 77 were used to estimate the binding free energies (ΔG PBSA and ΔG GBSA , respectively) of the receptor− ligand complexes obtained from MD simulations as described in our previous work. 78 These methods are approximate and have well-known limitations, but they can give an indication of binding affinity (e.g., for similar molecules), that includes the effects of protein dynamics. Here, we use them to investigate, e.g., binding affinities of enantiomers.…”

Identification of Potential DNA Gyrase Inhibitors: Virtual Screening, Extra-Precision Docking and Molecular Dynamics Simulation Study

“…Recently, Pakamwong et al ., have identified 2,2’-((2-hydroxy-3-(5-methyl-2,3-diphenyl-1H-indol-1-yl)propyl)azanediyl)bis(ethan-1-ol) as potential DNA gyrase inhibitor having an antimycobacterial activity (Pakamwong et al, 2022). Govender et al ., have reported some potent spiropyrimidinetrione derivatives as DNA gyrase inhibitors with potent activity against mycobacterium tuberculosis (Govender et al, 2022).…”