WalK inhibitory activity and antibacterial activity of synthesized naphthoquinones
The inhibitory activities of naphthoquinones 2 and 3 on the HK (WalK) were evaluated and expressed as IC50 values. Upon analyzing the relationship between structure and HK inhibitory activity, several histidine kinase inhibitors were identified. These include 2-aminonaphthoquinones with a cyclic amino group (2a, 2b, 2d), an aromatic amino group (2g, 2h, 2j, 2l, 2m, 2o), and naphtho[2,3-d]isoxazole-4,9-diones (3a, 3b) (Table 1). Their IC50 values against WalK ranged from 12.6 to 305 µM, and their MICs against B. subtilis strain 168 ranged from 0.5 to 128 µg ml−1. Compounds 2c, 2e, 2f, 2i, 2k, 2n, and 3c did not show any inhibitory activity against WalK. However, all naphthoquinone derivatives, except for 2k, exhibited antibacterial activity.
The structural features of naphthoquinone derivatives targeting WalK (HK) were found to be as follows: (1) The presence of hydroxyl groups on the 5,8-position of 2 or 3 was a crucial factor for inhibitory activity against WalK. Derivatives lacking hydroxyl groups (2c and 2i) were inactive. (2) The effect of the amino group (2o) was confirmed, but the nitro group substituent (2d and 2n) had no or only a weak effect. (3) Aliphatic amino derivatives (2e and 2f) were inactive even if hydroxyl groups were introduced into the structure. (4) A noninhibitory compound such as 2k has hydroxyl groups at both 5 and 8 positions, suggesting that the functional substituents on the aromatic amino group also affect the affinity and the inhibition ability. (5) The structure-activity relationship suggested that prototype structures as WalK (HK) inhibitors for 2-(arylamino)naphthoquinones 2 can be proposed (Fig. 3).
Prototype of H-box inhibitors. Z (OH or NH2, C5, C8) and R (polar groups) are essential for interacting with amino acid residues conserved in the H-box region
As shown in Table 1, correlation between WalK inhibitory activity and antimicrobial activity against B. subtilis was observed for some compounds. Compounds with WalK inhibitory activity comparable to waldiomycin (2h and 2l) showed similar level of antimicrobial activity to waldiomycin. 2a, 2m, and 2o showed weaker WalK inhibitory activity and antimicrobial activity. 2k and 2n did not inhibit WalK and showed no or a very weak antimicrobial activity. However, the other compounds with weaker or no WalK inhibitory activity exhibited antimicrobial activity equal to waldiomycin or even stronger (2b, 2c, 2d, 2e, 2g, 2f, 2i, 2j, 3a, 3b, and 3c). These compounds may exert antibacterial activity by interacting with proteins other than WalK, including other HKs and proteins involved in cell proliferation. Other factors may be the difference in permeability across the cell membrane and the accessibility to WalK. To further understand the nature of naphthoquinone derivatives that interact with WalK, a RT-qPCR (reverse transcription quantitative PCR) experiment was conducted.
Effect against the expression of WalK/WalR regulated genes
Among the naphthoquinone derivatives that demonstrated in vitro WalK inhibitory activity and antimicrobial effect against B. subtilis (Table 1), six compounds (2a, 2b, 2h, 2l, 2o, and 3b) were chosen to verify if they also inhibited WalK in vivo, i.e., in B. subtilis cells. The WalK/WalR system in B. subtilis is active during the exponential growth phase of the cells [19]. Therefore, exponentially growing B. subtilis cells were treated with subMIC or MIC levels of the selected compounds. The changes in the expression of a WalK/WalR negatively regulated gene, iseA (formerly yoeB) [20], were analyzed using RT-qPCR. The iseA gene codes a negative regulator of D,L-endopeptidases and is associated with cell separation [21]. The expression of rpoB (a housekeeping gene) and iseA in each sample was normalized by the expression of 16S rRNA and was shown as relative expression against their DMSO treated control (Fig. 4). The expression of iseA significantly increased with the treatment of 2a, 2h, 2l, and 3b at subMIC or MIC levels, whereas the expression of iseA did not change with the treatment of 2b and 2o (data not shown). To confirm the inhibition of the WalK/WalR system, the changes in the expression of another WalK/WalR negatively regulated gene, pdaC (formerly yjeA), were also measured. This gene codes a polysaccharide deacetylase and is associated with lysozyme sensitivity [22]. As shown in Fig. 4, the expression of pdaC also tended to increase with the treatment of the four compounds, supporting the in vivo inhibition of WalK.
Expression of WalK/WalR down-regulated genes in B. subtilis cells after treatment with naphthoquinone derivatives. Mid-exponential phase cells were treated with subMIC or MIC doses of 2a (a), 2h (b), 2l (c) and 3b (d) for 5 min, their total RNA extracted, and analyzed by RT-qPCR. Expression of genes was normalized with that of 16S rRNA, and shown as relative expression against each DMSO control. Note the difference in the y-axis scale. Error bars represent the SD of at least three biological repeats. Statistical analyses were performed by Tukey’s multiple comparison test. *p < 0.05; **p < 0.01, ***p < 0.001
The results showed that 2a, 2h, 2l, and 3b significantly induced iseA gene expression, while 2b and 2o did not. These findings suggest that 2b and 2o may have other targets besides WalK in B. subtilis cells, which prevent them from binding to WalK.
Affinity selection/mass spectrometry
Inhibitors binding to a similar region show competitive binding. If the binding of naphthoquinone derivatives to WalK is competitive with that of waldiomycin, it will be a strong indication of those compounds binding to the H-box region. An affinity selection/mass spectrometry (AS/MS) binding analysis system was developed to evaluate the binding of waldiomycin to WalK (Fig. 5). Waldiomycin was quantitatively detected on a chromatogram by isolating the WalK-bound waldiomycin through size-exclusion chromatography from the mixture with WalK and applying it to LC/MS. However, a sample containing only waldiomycin without WalK did not yield a detectable signal (Fig. 6, column 1) because it eluted much slower than the protein. The effects of selected naphthoquinone derivatives on the binding of waldiomycin to WalK were further analyzed. The addition of 2a, 2o, or 3b reduced the signal of waldiomycin in a dose-dependent manner (Fig. 6, columns 3, 4, 7, 8, 13, 14). Compared to the signal detected by mixing only waldiomycin with WalK (Fig. 6, column 2), only about 10% of the signal was observed in the presence of these compounds at 100 µM (Fig. 6, columns 4, 8, 14). These results suggest that the binding of 2a, 2o, or 3b to WalK is competitive with that of waldiomycin, indicating that the binding sites of these compounds overlap with that of waldiomycin. In contrast, the addition of 2b, 2h, or 2l increased the MS signal of the collected waldiomycin by about twofold compared to the signal in the presence of only waldiomycin (Fig. 6, columns 5, 6, 9, 10, 11, 12). These results suggest that the binding of 2b, 2h, or 2l to WalK is not competitive with that of waldiomycin, but rather enhances the binding of waldiomycin.
Principle of affinity selection/mass spectrometry
Affinity selection/mass spectrometry analysis. WalK (1 µM) was incubated with waldiomycin (5 µM, column 2) or with both waldiomycin (5 µM) and each compound (30 µM, columns 3, 5, 7, 9, 11, 13; 100 µM, columns 4, 6, 8, 10, 12, 14). In the absence of WalK, only waldiomycin (5 µM, column 1) was incubated. After mixing, AS/MS analysis was performed as described in “Materials and methods”. The value of peak area was expressed as relative values, with the value of waldiomycin and WalK protein as 100% (column 2). Data were the average of the quadruplicate results ± standard deviation
A previous study showed that a single molecule of waldiomycin binds to an EnvZ dimer, even though the dimer potentially contains two H-box regions [8]. It is possible that the binding of waldiomycin to one chain of the EnvZ DHp region results in a conformational change in the other chain of the EnvZ dimer, inhibiting the binding of another waldiomycin molecule. Assuming that the WalK dimer similarly binds to a single molecule of waldiomycin, it is plausible that 2b, 2h, and 2l may bind to the vicinity of the waldiomycin-binding site, thereby canceling the conformational change caused by waldiomycin. It is predicted that 100% level of the detected signal (Fig. 6, column 2) corresponds to one molecule of waldiomycin against the WalK dimer. Although this idea lacks sufficient experimental evidence, it well explains why 2b, 2h, and 2l contribute to the binding of twice the amount of waldiomycin to WalK (Fig. 6, columns 5, 6, 9, 10, 11, 12). An ongoing structural biology study is expected to provide unambiguous evidence for this prediction.
Naphthoquinone derivatives inhibiting HK activity of class I HKs
As previously reported [8], waldiomycin inhibited 13 class I HKs, including WalK, PhoQ, EnvZ, and CpxA. This broad-spectrum inhibition is attributed to waldiomycin binding to the H-box, which contains conserved amino acid residues found in the DHp domain of class I HKs. If the naphthoquinone derivatives exhibiting WalK inhibitory activity also target the conserved H-box region, they may inhibit the activity of other HKs. Therefore, we selected naphthoquinone derivatives to assess their inhibitory activity against other class I HKs, namely EvgS, EnvZ, PhoQ, and CpxA. These HKs play essential roles in the virulence and drug resistance of pathogens such as Shigella, Salmonella, Klebsiella, and Pseudomonas [2, 23, 24]. As indicated in Table 2, the chosen compounds, 2h, 2l, 2o, and 3b, inhibited all five HKs with IC50 values ranging from 0.1 to 186 µM. This implies that these compounds target a conserved region among HKs, similar to waldiomycin.