Inhibition of DNA relaxation by ferulic acid
The inhibitory potential of ferulic acid against MttopoI was determined by DNA relaxation assay. The activity of the enzyme was inhibited by ferulic acid at a concentration of 1, 2, 3, 4 and 5 µM. Interestingly, ferulic acid at a concentration of 5 µM was able to inhibit completely the activity of MttopoI (Fig. 1). Surprisingly, it was shown that DNA relaxation activity was not inhibited by ferulic acid against EctopoI (Data not shown). Overall, these results indicate that ferulic acid may specifically inhibit the Mycobacterial enzyme.
Effect of ferulic acid on Antibacterial activity
To determine the inhibitory potential as well as inhibition specificity by ferulic acid on cell growth. M. tuberculosis and M. Smegmatis cells were treated with ferulic acid in a concentration dependent manner. The inhibition of M. Smegmatis cells were delayed and complete growth inhibition by ferulic acid at a concentration of 500 µM (Fig. 2 A). Resazurin assay was performed to determine the MIC value of ferulic acid for M. Smegmatis and M. tuberculosis which was 125 µM and 250 µM respectively (Fig. 2 B, C, D).
Ferulic acid triggers the activity of DNA cleavage of topo1
Next, we further investigated the mechanism of ferulic acid by cleavage assay on each step of enzyme in DNA relexation cycle. The Cleavage assays were conceded with different concentrations of ferulic acid, double-stranded 32-mer containing strong topoisomerase site (STS). The cleavage of DNA through topoisomerase I was 2 fold higher in ferulic acid treated samples (Fig. 3 A, B).
Cells overexpressing enzymes affected by ferulic acid
DNA cleavage asaay suggested that ferulic acid may be a cytotoxic agent. In order to find out the cytotoxic potential of ferulic acid, we have overexpressed the topo1 in mycobacterial cells. Interestingly, ferulic acid (125 µM) was able to inhibit the growth of M. smegmatis cells with normal level of topoI (WT). Whileas, the growth inhibition of the topo1 overexpressed cells (OE) was higher at lower concentration of ferulic acid (Fig. 4 A, B). These results suggested that MIC value of ferulic acid was lowed in topo1 overexpressing cells may be because of topoisomerase mediated DNA cleavage.
Effect of ferulic acid on metal binding mutant activity
Type 1A topoisimerases has a metal coordination motif DxDxE and TOPRIM domain in amino acid terminal region [22]. The Mg2+ coordination with these motifs is very important for the function of enzyme. The mutations in these acidic residues results in impairment of the catalytic activity and cytotoxicity which leads to the impairment of the enzyme function. The binding of ferulic acid with the topoisomerase may be abrogated in the metal binding site of the enzyme embracing mutations. Ferulic acid at a concentration 1 µM and 5 µM inhibited the activity of mutant D108A as compared with WT enzyme inhibition (Fig. 5). Intrestingly, it was shown that ferulic acid remains unaffected the relaxation activity of E112A (Fig. 5). These results suggested that the interaction of ferulic acids and enzymes proximal to metal coordination motif may be in TOPRIM region.
Ferulic acid enhances efficacy of rifampicin in LS-180 cells
Ferulic acid were tested for P-gp inhibition activity in LS-180 cells overexpressing P-gp by using rhodamine-123 at a concentration dependent manner (10 µM, 20 µM and 30 µM) in the presence and absence of rifampicin. It was observed ferulic acid retains rifampicin in a concentration dependent manner which was indicated by the increasing fluorescence of rhodamine-123 (Fig. 6A). Moreover, ferulic acid was non-toxic at 30 µM in LS180 (Fig. 6B).