Structure-based virtual screening to identify small-molecule compounds that target Gtfs and inhibit biofilm formation

Taking advantage of the available crystal structure of the GtfC catalytic domain complexed with acarbose, we conducted a structure-based in silico screening of 500,000 drug-like compounds using the FlexX/LeadIT software. The top ranked small molecules, as calculated using the binding energy scores in the FlexX software, were considered based on their binding pose, potential interactions with key residues, and ease of synthesis. Due to the abundance of polar residues in the GtfC active site, several of the top scored docking scaffolds contain aromatic rings, nitro groups, and polar functional groups such as amides and heteroatoms such as sulfur, etc. A total of 90 compounds with diverse scaffolds which vary in their functional groups, hydrophobicity, and H-bond accepting/donating capacity were then purchased and subjected to in vitro biofilm assays using cariogenic S. mutans. Seven potent low micromolar inhibitors were identified (Fig. 1A). Two of these compounds (#G16 and #G43) were the most potent, as they inhibited more than 85% of S. mutans biofilms at 12.5 μM (Fig. 1B). Compounds #G16 and #G43 share several functional groups including a nitro group, heterocyclic rings, and polar carbonyl functional property.

Figure 1 (A) Structures of seven most potent Gtf inhibitors of S. mutans biofilms. (B) Biofilm inhibitory activities of the potent inhibitors at 12.5 µM as determined by the crystal violet assay. Full size image

Inhibition of Gtfs by lead compounds

Zymographic enzymatic assay was used to determine whether the lead compounds inhibited the activity of Gtfs that are responsible for the production of glucans and biofilm formation. Supernatants containing Gtf proteins prepared from S. mutans bacterial cultures were subjected to SDS-PAGE analysis and zymographic assay. Treatment of the SDS-PAGE gels with lead compounds #G16 and #G43 in a zymographic assay revealed that both #G16 and #G43 drastically reduced the glucan production by the Gtfs, #G43 was more potent (Fig. 2A, bottom panels). The same amount of the protein sample was used as controls and visualized by protein staining (Fig. 2A, top panels). The lead compounds were also tested against individual Gtfs using supernatant proteins harvested from cultures of various double mutants. Compound #G43 consistently inhibited the activity of both GtfB and GtfC (Fig. 2B and C), ImageJ analysis of the intensities suggest 80% inhibition of both enzymes, while compound #G16 had a smaller effect on the activity of GtfB (60% inhibition) (Fig. 2B) and GtfC (70% inhibition) (Fig. 2C). Overall #G43 is more potent than #G16 in inhibiting Gtfs.

Figure 2 Gtf patterns of S. mutans UA159 and its mutant variants. Culture supernatants were prepared from S. mutans UA159 wild type and gtf double mutants, and then subjected to SDS-PAGE analysis with equivalent amount of proteins in each lane. The upper panel was stained with Coomassie blue to monitor the total protein amounts while the lower panel shows enzymatic activities of Gtfs with the treatment of the lead by the zymographic assay. The intensity of the bands were quantified using ImageJ in comparison to DMSO. (A) Effects of lead compounds #G16 and #G43 at 25 µM on the activity of Gtfs from wild type S. mutans. (B) Effects of lead compounds #G16 and #G43 at 25 µM on the activity of GtfB from S. mutans GtfCD mutants. (C) Effects of lead compounds #G16 and #G43 at 25 µM on the activity of GtfC from S. mutans GtfBD mutants. Full size image

Binding kinetics of #G43 lead compound determined by OctetRed Analysis

Zymograhic assays suggest that the lead compound #43 inhibited the activity of both GtfB and GtfC. To determine if the inhibition is attributed by the binding of the lead compound to the enzymes, the OctetRed96 system was used to characterize protein-small molecule binding kinetics. The his-tagged catalytic domains of GtfB and GtC were immobilized separately onto an anti-penta-HIS (HIS1K) biosensor which consists of high affinity, high specificity penta-his antibody pre-immobilized on a fiber optic biosensor. This sensor was then exposed to varying concentrations of #G43. Assay data fit to a 1:1 binding model with a fixed maximum response, which produced a K D value of 3.7 µM for GtfB. The K D value for GtfC was more potent at 46.9 nM (Fig. 3A and B). These data suggest that the lead compound is selective toward GtfC, the protein used in the in-silico analysis. It should be noted that the catalytic domain of GtfC is less soluble compared to that of GtfB’s, which may be responsible for the inherent higher off rate of the his-tag from the sensor, leading to a weaker association curve when compared to GtfB. Nevertheless, consistent nanomolar K D values were obtained from three independent experiments.

Figure 3 Binding curves of compound #G43 at varying concentrations with (A) GtfB, and (B) GtfC catalytic domain. Full size image

Expression of gtfs was not significantly affected by compound #G43

We have also examined the effect of this potent small molecule on the gene expression of gtfs. The relative expression level of gtfs was evaluated by real time RT-PCR. Compared to the DMSO control group, expression of gtfs were marginally down-regulated after the treatment with compound #G43 at different concentrations. However, no significant difference was observed between the treated and control groups, suggesting that compound #G43 inhibited Gtfs via binding to the targets rather than altered expression of its targeting genes, gtfs (Fig. 4).

Figure 4 Effect of the compound #G43 on expression of gtfs in S. mutans. S. mutans UA159 cells treated with different concentrations of #G43 were harvested and used to prepare RNA. The expression of gtfs was examined by real time RT-PCR. The mRNA expression levels were calibrated by 16S rRNA. Values represent the means ± standard deviations from three independent experiments. NS indicates no significant difference between DMSO control and compound-treated groups. The P value > 0.05 is considered to be not significant. Full size image

The most potent compound is not bactericidal and did not inhibit the growth of commensal streptococcal species, and other oral bacteria

To determine the selectivity of the lead compound toward S. mutans biofilm formation versus bacterial growth, we evaluated effects of the compound on bacterial growth and viability. No significant difference in S. mutans cell viability was observed between the control group and #G43 treated groups up to 200 µM (Fig. 5A), suggesting that the compound is not bactericidal towards S. mutans. This compound was also evaluated for its ability to inhibit two oral commensal streptococci: S. sanguinis and S. gordonii as the goal was to develop non-bactericidal and species-selective agents. The compound did not have any effect on bacterial growth (Fig. 5B) of both streptococcal species. In addition, we evaluated effects of the compound on other oral bacteria including Aggregatibacter actinomycetemcomitans VT1169, a Gram-negative, facultative anaerobe, and Actinomyces naeslundii T14VJ1, a gram-positive, facultative anaerobe (Fig. 5C). At 200 µM, the compound had no significant inhibition of Aggregatibacter actinomycetemcomitans. Slight inhibition (>20%) was observed of Actinomyces naeslundii growth at 200 µM, suggesting the selectivity towards S. mutans.

Figure 5 Effects of the lead compound #G43 on cell viability. (A) Effects on S. mutans. S. mutans was treated with DMSO and a serial dilution of #G43. The cell viability was determined by the numbers of CFU in a logarithmic scale. (B) Effects on commensal species. S. gordonii, S. sanguinis, and S. mutans were treated with 200 µM of the compound or DMSO, and bacterial growth was measured at OD 470 , and normalized to the DMSO control (100%). (C) Effects on Aggregatibacter actinomycetemcomitans and Actinomyces naeslundii. A. actinomycetemcomitans and A. naeslundii were treated with the lead compound at 200 µM or 25 µM or DMSO control, bacterial growth was measured at OD 470 , and normalized to the DMSO control (100%). Values represent the means ± standard deviations from three independent experiments. NS indicates that the cell viability between DMSO control and compound-treated groups was not significantly different. The P value > 0.05 is considered to be not significant. Full size image

#G43 did not inhibit the biofilm formation by commensal streptococci but inhibit S. mutans in the dual species biofilms

To determine the selectivity of the lead compound toward S. mutans biofilm formation over the biofilms of other species, we evaluated effects of the compound on the biofilm formation by two oral commensal bacteria: S. sanguinis and S. gordonii. No significant difference in S. sanguinis biofilm formation was observed between the control group and #G43 treated groups up to 200 µM (Fig. 6A). A slight increase in the biofilm formation by S. gordonii was observed when treated with the lead compound. Further, experiments using a dual species model was conducted using S. mutans with either S. sanguinis (Fig. 6B), or S. gordonii (Fig. 6C). We observed a reduction in the overall biofilm formation with the treatment of the compound. Moreover, the lead compound shifted the bacterial composition ratio of commensal streptococcus to S. mutans from untreated 1:4 to either 4:1 (Fig. 6D) for S. sanguinis, or 3:2 for S. gordonii (Fig. 6E). The increase in commensal bacteria by the treatment again suggests that the lead selectively affects S. mutans biofilm.

Figure 6 Effects of compound #G43 on commensal single and dual species biofilms. (A) S. mutans, S. gordonii, and S. sanguinis were treated with DMSO or 25 µM of compound #G43, and the biomasses of each treated biofilm were quantitated by crystal violet staining and measured at OD 562 . (B) The cell viability of dual species biofilms was determined by the numbers of CFU in a logarithmic scale using S. mutans, and S. sanguinis. (C) The cell viability of dual species biofilms was determined by the numbers of CFU in a logarithmic scale using S. mutans, and S. gordonii. (D) Species distribution in dual species biofilms with S. mutans and S. sanguinius. Bars represent the mean and standard deviations of three independent experiments. (E) Species distribution in dual species biofilms with S. mutans and S. gordonii. Bars represent the mean and standard deviations of three independent experiments. *P < 0.05. Full size image

Docking analysis, the facile synthesis of #G43 and its inactive analog to establish that the ortho primary benzamide moiety is crucial for its potency

To explore the underlying mechanism of #G43’s bioactivity, the compound was docked into the active site of GtfC to elucidate plausible interactions. The top docking pose of #G43 within the GtfC active site revealed several key interactions. The nitro group on the benzothiophene ring interacts with Arg540, the amide linker is within close proximity of Gln592, and pi-pi stacking interactions are observed between Trp517 and the benzene ring. Of particular importance is the interaction of the primary ortho amide group on the benzene ring with Glu515, Asp477, and Asp588. While the mechanism of the glucan formation is not fully understood, Glu515, Asp477, and Asp588 are assumed to function as a nucleophile, a general acid/base catalyst, and a stabilizer of the glucosyl intermediate, respectively15. Thus, we hypothesized that this functional ortho amide group is crucial for the binding of the compound to the protein.

In order to test this, we designed an analog (#G43-D) with a 3D structure (Fig. 7A) that does not contain the primary amide group and subjected it to docking analysis, as a theoretical design. This scaffold failed to produce a good docking score in FlexX (greater than −25 kJ/mol) and yielded a weak binding pose (Fig. 7B). Due to the absence of the primary amide group, the scaffold takes on a different orientation and possesses poor interactions with the active site.

Figure 7 Effects of the lead compound #G43 and its inactive analog #G43-D. (A) Chemical structures of lead and its inactive analog. Docking poses of (B) Compound #G43 in blue skeleton and (C) compound #G43-D in pink skeleton. Three key residue interactions are depicted by displaying residue chains. (D) Effects of active and inactive compound on the activity of Gtfs by zymographic assays. Glucan zymographic assays (bottom panel) were performed using SDS-PAGE analysis of Gtfs from culture supernatants of S. mutans UA159 incubated with vehicle control DMSO, the synthesized active #G43, and its derivative at 50 µM. SDS-PAGE analysis of Gtfs (top panel) was used as a loading control. (E) Fluorescent microscopy images of S. mutans UA159 biofilms treated with DMSO control, the synthesized #G43, and its derivative #G43-D at 100 µM. Viable bacterial cells were stained with 2.5 µM Syto9 (green). Full size image

The lead compound was re-synthesized in one step using commercially available reagents, anthranlinamide and 5-nitro-1-benzothiophene -2-carboxylic acid, in an excellent yield and fully characterized (see supplemental data). We also synthesized the “inactive” analog (#G43-D) in one step by replacing the anthranilinamide with aniline in the EDAC coupling synthesis. Zymographic analysis consistently showed that the lead compound #G43 drastically reduced the glucan production, especially of those produced by GtfC. However the designed “inactive” compound #G43-D significantly reduced the ability to inhibit the glucan production (Fig. 7D). Additionally, in vitro biofilm assay and fluorescence microscopy revealed that the inactive analog #G43-D did not inhibit S. mutans biofilms at concentrations up to 200 µM (Fig. 7E). Binding studies of this analog against GtfB yielded a K D value of 68 µM, compared to a K D value of 3.7 µM by the active analog (see supplemental data). Our data demonstrates that not only is the inhibition of biofilms by selectively targeting Gtfs plausible, but the inclusion of the primary ortho amide group is crucial to maintain potent anti-biofilm activity. Further structure and activity relationship studies are ongoing to improve the potency of #G43.

#G43 reduced S. mutans virulence in vivo

To evaluate in vivo efficacy of the lead compound #G43, we tested the compound using a rat model of dental caries27 (Table 1). All rats from the two experimental groups were colonized with S. mutans. The bacterial colonization appears to be reduced in #G43 treated rats, however the reduction did not reach the statistically significant difference when compared with the control group. The buccal, sulcal, and proximal surface caries scores of the treated animals were significantly reduced. These data suggest that the lead small molecule selectively targets virulence factors, Gtfs and Gtf- mediated biofilm formation, rather than a simple inhibition of bacterial growth. Furthermore, the #G43 treated rats did not lose weight over the course of the study in comparison with the control group, suggesting that the compound is not toxic.