Prioritization and characterization of validated biofilm blockers targeting glucosyltransferase C of Streptococcus mutans

Abstract

To date, several Glucosyltransferase C (GtfC) inhibitors have been identified and experimentally validated. All these inhibitors have been validated at different experimental conditions like degree of purity, animal models, kinetic conditions, experimental environment etc.; and most of these inhibitors (ligands) proved to be quite effective in their respective validation environment. However, due to varied experimental validation conditions, and absence of molecular interaction data, there is no way to prioritize these validated ligands for their inhibition potential against GtfC. The present study is a novel attempt of comparative evaluation of the interaction of the validated ligands on a single platform and under similar conditions with a dual objective, i.e. ligand prioritization for their respective inhibitory potential and elucidation of the involved unknown molecular interactions. Carbohydrate derivatives (6-Deoxy sucrose and Trichloro-galactosucrose) were identified as the most promising GtfC inhibitors. In addition, Asp588, Trp517, and Asn481 amino acid residues of the domain A1 proved vital for the inhibitory effect. The study highlights the importance of the comparative analysis of the validated ligands in order to identify the most promising leads for drug discovery against dental caries.

Keywords:

• Glycosyltransferase C
• biofilm
• prioritization
• Streptococcus mutans
• D-acarbose
• quantum mechanical

Introduction

Biofilm exopolysaccharides (EPS also termed as glucan) are responsible for the growth and colonization of the cariogenic bacteria. Several Streptococci strains including S. mutans are capable of synthesizing EPS. Streptococci EPS are either homopolysaccharides or heteropolysaccharides. Streptococcus mutans secretes an EPS, which is a homopolysaccharide [1]. Glucosyltransferases (also known as Glycosyltransferases Gtfs; i.e. GtfB, GtfC and GtfD) are known to play central role in biofilm EPS (glucan) generation [2–5]. Streptococcus mutans synthesizes at the minimum 3 genetically different Gtfs, and each of them forms a structurally discrete glucan by using sucrose. The special features of the structural and functional organization of Gtfs have been reported earlier [6,7]. GtfB (generally known as GtfI) produces firstly an insoluble glucan with plenty of 1,3-linkages. Whereas, GtfD (GtfS) produces primarily a soluble glucan [8–10]. Unlike above two Gtfs, GtfC (GtfSI) synthesizes an amalgam of insoluble and soluble (having more α-1,6-linkages) forms of glucans. The reaction showing glucan generation catalyzed by GtfC has been presented in Figure 1. Earlier, Ooshima et al. [12] reported that the presence of GtfC intensifies the glucan formation in vitro model, which was later endorsed by the report of Tamesada et al. [13]. GtfC is adsorbed into pellicle, where – when exposed to sucrose, and under in situ condition it synthesizes glucan.

Figure 1. GtfC reaction scheme (Brenda enzyme database) [11].

The data generated from in situ and in vitro experimental studies suggest that Glucosyltransferase identified in pellicle is predominantly a GtfC from S. mutans (Venkitaraman et al., 1995) [14]. In addition to the above mentioned significant role of GtfC in EPS (glucan) generation, Tsumori and Kuramitsu [15] identified the essential role of GtfC in sucrose‐dependent colonization of tooth surfaces. Knocking out of the GtfC gene (while keeping GtfB and GtfD) radically reduced the adherence to smooth surfaces in quantitative sucrose dependent attachment system. GtfC alone was capable of catalyzing (enough sucrose to EPS) required for adherence to smooth surface and subsequent colonization [15]. These studies identify the unique and central role of GtfC in biofilm (EPS) generation. Sine GtfC is vital for biofilm generation determination and characterization of potential leads against GtfC is a significant step towards potent drug discovery against biofilm generation and associated diseases. Present study attempts to characterize the potent leads against the biofilm drug target, GtfC.

There are at least 2 subsites, i.e. glucosyl and fructosyl available on GtfC that might be the target for inhibitors [16–18]. The site includes 3 aspartic acid residues that has been discovered as the location for glucose conjugation. Whereas, Zn²⁺ has been shown to bind to the fructose site [19]. Also, the enzyme has numerous separate/unrelated glucan-binding sites, including one present on a 60kD domain of the C-terminus [20]. Distinctly, there are some additional sites present on Gtfs to which potential inhibitors could attach. Various chemical compounds have been used to inhibit GtfC. Initially, quaternary ammonium compounds, aliphatic amines and antibodies were found efficacious in inhibiting Gtfs to a certain extent in the solution.

Later, oleanic and oleic acids were found potential inhibitors of Gtfs activity in a solution of perborate, metaperiodate, sodium, metal ions and naturally available substances [21–23]. The activity of GtfB in a solution is drastically affected (reduced) by many commercially available agents, for e.g. alexidine dihydrochloride, cetylpyridinium chloride or triclosan. 4′,5,7-trihydroxyflavone (commonly known as Apigenin) is extremely efficient inhibitor (non-competitive inhibition) of Gtfs even at the concentrations of micromoles. Interestingly, only few agents had any effect on GtfC. GtfC has several hydrophobic domains especially at the C-terminus [24] and these hydrophobic domains possibly block the access through hydrophilic inhibitors to the active site that may describe its comparative resistance towards inhibitory agents.

In this study, an advanced Quantum Mechanical Molecular Mechanical (QM/MM) technique has been employed for the optimization of the conformations of the potential leads as many studies published in recent times have shown that there are significant advantages of QM/MM methods compared to other empirical molecular mechanics techniques [25].

Structural details of GtfC

In the crystal, GtfC is organized as tetramer, the outcomes of size-exclusion chromatography suggested that GtfC is probably a monomer in a solution (Figure 2). Also, the structures of the individual tetramer members were found nearly identical. The present model refined at 2.1Å resolution comprises amino acid residues 244–1087, as the entire protein could not be crystallized. The structure of GtfC encompasses four different domains, i.e. A, B, C and IV, and except domain C, each domain was made up of two separate regions of the polypeptide chain. Domain IV consisted of IV1 (residues 244–372) and IV2 (residues 1057–1087); domain A consisted of A1 (residues 443–692) and A2 (residues 829–961); and domain B consisted of B1 (residues 373–442) and B2 (residues 962–1056). Domain C was formed of a single stretch of amino acid (693–828) residues. Domain A composed the core of the GTF-SI catalytic domains [19].

Figure 2. GtfC monomer (A-chain) with active site.

Materials and methods

Structural details of validated ligands/inhibitors of GtfC

The ligands/inhibitors subjected to prioritization during this study are presented in Table 1. All the ligands were derived from references provided in Table 1. The structural details of these inhibitors are provided in the Supplementary Information (Figure SI L1–L15). The structural .sdf files of these inhibitors were obtained from PubChem database. Some structures (Flavonoid new, G16, G43 and Flavonoid (Z)-3-(2,4-Dihydroxyphenyl)-1-(3-hydroxyphenyl)-2-propen-1-one) were designed in Discovery Studio Visualizer. IUPAC names were generated using BIOVIA Draw, 2019. All the ligands were prepared following the ligand protocol of BIOVIA Discovery Studio (Accelrys^@). The ligands were typed with CHARMM36 force field [32].

Table 1. Details of the validated ligands: the compound name refers to the compound used in the respective study.

Compound name*	Figure SI	PubChem ID/IUPAC*	Molecular formula	Molecular weight	Reference
D-acarbose	L1	41774	C25H43NO18	645.6	[19]
6-Deoxysucrose	L2	146,374	C12H22O10	326.3	[26]
Trichloro-galactosucrose	L3	71,485	C12H19Cl3O8	397.6	[23]
Flavonoid myrecitin	L4	5281672	C15H10O8	318.23	[27,28]
Flavonoid new	L5	(6Z)-3-hydroxy-6-[(Z)-3-hydroxy-3-(3-hydroxyphenyl)prop-2- enylidene]cyclohexa-2,4-dien-1-one	C15H12O4	256.253	[27,28]
2-N,3-N-Bis[2-(4-methoxyphenyl)ethyl]quinoxaline-2,3-diamine	L6	1,023,772	C26H28N4O2	428.5	[28]
Flavonoid apigenin	L7	5,280,443	C15H10O5	270.24	[27,28]
D-mannosamine	L8	440,049	C6H13NO5	179.17	[29]
Stilbene piceatannol	L9	667,639	C14H12O4	244.24	[30]
1-Deoxynojirimycin	L10	29,435	C6H13NO4	163.17	[28]
Flavonoid (Z)-3-(2,4-Dihydroxyphenyl)-1-(3-hydroxyphenyl)- 2-propen-1-one	L11	(Z)-3-(2,4-Dihydroxyphenyl)-1-(3-hydroxyphenyl)-2-propen-1-one	C15 H11O4	255.253	[27,28]
D-galactosamine	L12	24,154	C6H13NO5	179.17	[29]
G16	L13	(6E)-3-(dihydroxymethyl)-6-[5-[4-[(4-fluorophenyl)-hydroxy-methylene]cyclohexa-2,5-dien- 1-ylidene]-4-phenyl-imidazol-2-ylidene]cyclohexa-2,4-dien-1-one	C29H21FN2O4	480.499	[31]
G43	L14	(2Z)-N-(2-carbamoylphenyl)-5-nitro-benzothiophene-2-carboximidic acid	C16H11N3O4S	341.351	[31]
Epiclusianone	L15	5,471,610	C33H42O4	502.7	[28]

Target structure and active site

The structural coordinates of GtfC was retrieved from RCSB-PDB (3AIC). This .pdb file represents the crystal structure of Glucansucrase from Streptococcus mutans in complex with D-Acarbose [19]. This structure was deciphered by x-ray diffraction @3.11 Å resolution. The native state of GtfC is known to be a monomer [19]. A GtfC monomer complexed with alpha D-Acarbose, 2-(n-morpholino)-ethanesulfonic acid and calcium was selected from the crystallised octamer .pdb file. The target structure was free of ligands (D-Acarbose and 2-(n-morpholino)-ethanesulfonic acid) but include the water molecules found to be linked with the crystal structure. The target GtfC monomer was prepared according to the protein preparation protocol of BIOVIA Discovery Studio. The input parameters have been given in the Supplementary Information (SI) file. The target protein monomer was typed with CHARMM36 force field [32]. The active site was defined based on pdb site record (Figure 2). Includes Glu515, Asp477, Asp588, Arg475, His587, Tyr916, Tyr430, Leu433, Asn481, and Trp517.

Customized flexible docking protocol

An exhaustive flexible docking protocol was implemented (Supplementary Information: Figure SI 1) on BIOVIA Discovery Studio platform. Briefly, this protocol permits for receptor flexibility during the docking of flexible ligands up to some extent [33]. The side-chains of specified amino acids (in this case Glu515, Asp477, Asp588, and Asn481) were allowed to move during the docking process. This movement allows the receptor to adapt to different ligands in an induced-fit model (Supplementary Information: Figure SI 2). The protocol employs a combination of components from other protocols to perform the docking, and is based on methods within CHARMm [34] to sample side-chain and ligand conformations. The detailed input parameters are given in Supplementary Information (SI) file. Flexible docking protocol performs the following steps: (i) Calculation of receptor side-chain conformations: Initially, the protocol creates protein side-chain conformations using the ChiFlex algorithm [35], (ii) Create ligand conformations: Low energy ligand conformations are created for the docking process using Generate Conformations protocol of BIOVIA Discovery Studio platform, (iii) Perform initial placement of the ligand conformations: The ligand conformations are docked into the active site of each receptor side-chain conformation using LibDock [36], (iv) Clustering to remove similar ligand poses: Poses are clustered irrespective of the protein conformation as the protein conformations are rebuilt during the next step, (v) Refine side-chains: In the presence of the ligand, the specified receptor side-chain residues are refined using the ChiRotor algorithm [35], and (vi) Refine docking: A final simulated annealing and energy minimization of each ligand pose was done by using CDOCKER [37]

Scoring ligand poses

BIOVIA Discovery Studio implementation of the empirical scoring functions LigScore1, LigScore2 [38] was used to score the ligand poses including the reference ligand (D-Acarbose). Table SI1 represents detailed scores of the ligands on the two scales (Supplementary Information)

ADMET properties calculation

ADMET properties were calculated on Discovery studio using ADMET Descriptor predictor tool. Table SI3 and SI3E (supplementary Information) represents the summarized ADMET properties of the Ligands.

Results

The active site of GtfC (Figure 2) was derived from the pseudo-tetrasaccharide, αD-Acarbose, binding site of the crystallized GtfC-α D-Acarbose complex (Active site BC8: A chain, 3AIC.pdb). αD-Acarbose was removed from the complex and subjected to same docking protocol (as discussed above, Figure 3) while keeping the crystallized (3AIC.pdb) protein conformation as constant. Figure 3 compares the interaction profile of αD-Acarbose in the crystallized complex (3AIC) with highest scoring docked pose of αD-Acarbose at the active site. The root mean square deviation (RMSD) of the redocked highest scoring acarbose pose was determined by aligning it to the bound D-acarbose in the experimental PDB structure (3AIC). A low RMSD value of 0.6536 for the redocked D-acarbose underscores the efficiency of the applied docking protocol.

Figure 3. Interaction profile of GtfC-D-Acarbose. (a) From 3AIC.pdb structure. (b) Docked complex.

The binding efficiency, of the docked poses in the order of their inhibitory potential, determined as per the scoring algorithm (Scoring ligand poses as discussed above) is listed in Table 2. The binding score and interaction profile of αD-Acarbose in complex with GtfC serve as reference (Table 2, Figure 3(b)). Table 3 represents interacting chemical fragments of the top 3 Ligands w.r.t Acarbose. Table SI2 represents interacting chemical fragments of the rest of the ligands w.r.t Acarbose (Supplementary Information). The ADMET properties of all the ligands discussed in this study are reported in Tables SI3 and SI3E (Supplementary Information).

Table 2. Summary of ligand interactions.

Ligand (inhibitor)	H	O	U	ALIS	%Efficiency @D-Acarbose	Target-ligand 2-D Interaction Diagram
D-Acarbose	10	3	0	7.66	100.00	Figure 3(b)
6-Deoxysucrose	4	6	0	5.84	76.24	Figure 5(a)
Trichloro-galactosucrose	6	9	0	5.81	75.88	Figure 5(b)
Flavonoid Myrecitin	2	10	0	5.79	75.59	Figure 5(c)
2-N,3-N-Bis[2-(4-methoxyphenyl)ethyl]quinoxaline-2,3-diamine	2	9	0	5.75	75.10	Figure 5(d)
Flavonoid new	3	4	0	5.58	72.85	Figure SI C1
G16	3	6	0	5.55	72.42	Figure SI C2
Flavonoid Apigenin	4	9	1	5.46	71.28	Figure SI C3
D-Mannosamine	6	6	0	5.20	67.82	Figure SI C4
G43	1	11	1	5.09	66.45	Figure SI C5
Stilbene Piceatannol	5	5	1	5.07	66.22	Figure SI C6
1-Deoxynojirimycin	3	7	0	4.62	60.28	Figure SI C7
Flavonoid (Z)-3-(2,4-Dihydroxyphenyl)-1-(3-hydroxyphenyl)-2-propen-1-one	3	2	2	4.32	56.36	Figure SI C8
D-Galactosamine	4	3	0	4.20	54.77	Figure SI C9
Epiclusianone	0	8	0	3.80	49.61	Figure SI C10

H: Number of possible HYDROGEN BONDS with GtfC Target; O: other possible non-covalent interactions, i.e. Van der Waal, Pi-Sigma, Carbon–Hydrogen, Pi-cation, Pi-anion, alkyl interaction and Pi-alkyl with GtfC Target; U: unfavourable Interaction. ALIS=(LigScore1_drieding+Ligscore2_drieding+LigScore1_CFF+ LigScore2_CFF)/4. (refer to Table 1-SI for ligand Pose Score) $\mathrm{\%}\mathrm{ }\mathit{efficiency}@\mathrm{Acarbose}=\left(\frac{{\mathit{ALIS}}_{\mathit{Ligand}}}{{\mathit{ALIS}}_{\text{Acarbose }}}\right)\times 100.$

Table 3. Summary of Interacting chemical fragments.

Ligand (inhibitor)	Interacting chemical fragment/s	Interacting critical amino acid residues
D-Acarbose	Hydroxyl group of the tetrahydropyran ring	Arg54O
	Hydroxyl and hydroxymethyl groups of the Cyclohex-2ene fragment	Gln960, Asp477, Asp909, His587, Arg475, Glu515, Asp588
	Amino Nitrogen interaction of cyclohex-2-en-1-yl] amino tetrahydropyran	Glu515, Leu433
	Carbonyl oxygen of the pyran ring	Asn481
6-Deoxysucrose	Hydroxyl group of the tetrahydropyran ring	Asp588
	Hydroxyl groups and hydrogen atoms of the tetrahydrofuran ring	Asp588, Asp909, Gln960, Asp477
Trichloro-galactosucrose	Hydroxyl groups of the chloro- tetrahydropyran ring	Arg475, His587, Asp588, Asp477
	Chlorine atoms of the chloro- tetrahydropyran ring	Tyr916, His587
	Hydroxyl groups of the di chloro- tetrahydrofuran ring	Asp588, Glu515
	Chlorine atoms of the di-chloro- tetrahydrofuran ring	Ala478, Tyr610, Trp517
Flavonoid Myrecitin	Hydroxyl groups of the chromen-4-one ring	His587, Tyr916, Arg475, Ala478
	Carbonyl oxygen of the chromen-4-one ring	Arg475
	pi Interactions of chromen-4-one ring	Tyr916, Asp588, Ala478
	pi Interactions of trihydroxyphenyl ring	Leu433, Asn481

A uniform scoring algorithm, Score ligand poses, was applied to both the reference (αD-Acarbose) and the selected ligands. Tables 2 and 3 summarizes the interaction profile of the top scoring ligand pose representing a ligand group (ligand group represents all the docked poses of a single ligand) along with Average ligand Interaction Score (ALIS). Figure 4 summarizes the percentage efficiency of the validated ligands @αD-Acarbose. None of the validated ligands could match the inhibitory potential of αD-Acarbose.

Figure 4. Percentage efficiency of the validated ligands @αD-Acarbose.

The detailed interaction profile of the top four validated ligands having binding efficiency within 75%@αD-Acarbose is summarized in Figure 5(a–d). The interaction profile of the rest of the ligands is shown in the Supplementary Information (Figure SI C1–C10). The amino acid residues interacting with the ligands were classified on the basis of the type and frequency of the interaction. All the ligand poses were analyzed to determine the amino acid residues frequently involved in hydrogen bonding (Figure 6(a)). The amino acid residues responsible for maximum favourable interactions are summarized in Figure 6(b) and also as favourable heat map in the Supplementary Information (Figure SI H1) file. The amino acids playing central role in hydrophobic and ionic interactions are summarized in the histogram (Figure SI AI, and Figure SI AH) in Supplementary Information.

Figure 5. GtfC-interaction profile. (a) 6-Deoxysucrose, (b) trichloro-galactosucrose, (c) flavonoid myrecitin, (d) 2-N,3-N-Bis(2-(4-methoxyphenyl) ethyl) quinoxaline-2,3-diamine.

Figure 6. Frequently interacting residues. (a) Hydrogen bonding; (b) favourable count.

Discussion

GtfC is an established drug target; chemically diverse inhibitors have been tested/validated on GtfC from Streptococcus mutans in recent years. These inhibitors may be broadly classified as oligosaccharide derivatives (D-Acarbose, 6-Deoxysucrose and Trichloro-galactosucrose), Amino-monosaccharide derivatives (Galactosamine, Mannosamine), Flavonoids (Apigenin, Myrecitin, Flavonoid new: 6Z)-3-hydroxy-6-((Z)-3-hydroxy-3-(3-hydroxyphenyl)prop-2-enylidene)cyclohexa-2,4-dien-1-on, Flavonoid: (Z)-3-(2,4-Dihydroxyphenyl)-1-(3-hydroxyphenyl)-2-propen-1-one), Hydroxylated phenyls (Stilbene), Quinolaxine diamine deivatives (2-N,3-N-Bis(2-(4-methoxyphenyl)ethyl)quinoxaline-2,3-diamine), Prenyl benzophenone (Epiclusianone), Heterocyclic amine polyhydroxylated derivatives (Deoxynojirimycin), Carboximidic acid derivative (G43: (2Z)-N-(2-carbamoylphenyl)-5-nitro-benzothiophene-2-carboximidic acid), Pheny methyl fluoro cyclohexanes (G16:6E)-3-(dihydroxymethyl)-6-(5-(4-((4-fluorophenyl)-hydroxy-methylene)cyclohexa-2,5-dien-1-ylidene)-4-phenyl-imidazol-2-ylidene)cyclohexa-2,4-dien-1-one) etc.

These inhibitors have been tested or validated either as impure extracts or under diverse experimental conditions (Table 1). Owing to the dearth of uniform experimental conditions it is impossible to prioritize these validated ligands with respect to (w.r.t) each other. The knowledge of the efficiency of inhibitors w.r.t each other assist in the discovery of the specific pharmacophore properties, which will determine the foundation for screening exhaustive ligand libraries for superior leads. Also, the majority of the validation studies performed for these inhibitors failed to report any information regarding the type and degree of chemical interaction between the ligand and the receptor (i.e. target protein). Present study is a comprehensive analysis of validated ligands of GtfC. This study compares the efficiency of the active ligands under uniform experimental conditions and to characterize and explore their interaction. The binding efficiency and interaction details of the reference ligand D-Acarbose (from GtfC-D-Acarbose crystallized complex:3AIC) was found to be highest when compared with any of the validated ligands considered in this study (ALIS = 7.66, Tables 2 and 3]. No unfavourable interactions were absorbed alongside high degree of hydrogen bonding and other interactions (Figure 3(b)). Amino acids at GtfC subsite-1 (Arg475, Asp477, Glu515, His587, Asp588) establish hydrogen bonds with oxygen and nitrogen atoms of D-Acarbose, Leu434, Trp517 and Tyr916 are involved in weak van der Waals, carbon hydrogen and Pi-Donor hydrogen bond with Acarbose. The inhibition efficiency of D-Acarbose may be specifically attributed to its stable interaction with Glu515, Asp477, Asp588, Leu433, Trp517, and Asn481. For the natural substrate (sucrose), Asp477 undergoes a nucleophilic substitution reaction thereby establishing a covalent interaction with the positively charged glycosyl moiety resulting in a stable intermediate. Glu 515 placed above the substrate protonates the leaving group, fructose, at subsite + 1 [19]. Asp588 further stabilizes the covalently bonded Asp477-glycosyl intermediate. The glutamate anion (Glu515) attracts out the proton from the second sucrose molecule at Subsite +1, generating a hydroxyl anion attacking the glucosyl intermediate producing glucan at Subsite −1. D-Acarbose is involved in stable hydrogen bonding and weak Pi-Donor hydrogen bonding with Leu433 and Trp517, respectively at Subsite +1. Since, Ca²⁺ binding site Asn481 side chain hydrogen bonding with the acceptor maltose/sucrose plays a key role in the stabilization of the acceptor [19], hydrogen bonding of D-Acarbose with the Asn481 side chain defines its inhibitory potential. 6-Deoxysucrose was found to be the most efficient ligand (Table 1, ALIS score: 5.84, %Efficiency@D-Acarbose: 76.24, Figure 5(a), Table 3). The inhibitory effect of this ligand may be attributed to its multiple hydrogen bonding with Asp588 and highly conserved Asp909 [19] and multiple weak van der Waals and carbon hydrogen interactions with Asp477 at Subsite −1 (Figure 5(a)). Trichloro-galactosucrose appear to be establish an almost equally stable interaction with GtfC (Table 2, ALIS score: 5.81, %Efficiency@D-Acarbose: 75.88, Figure 5b, Table 3). This halogenated disaccharide appears to be the closest analogue of the natural substrate (i.e. sucrose). Multiple hydrogen bonding with the Subsite −1 amino acids including Glu515, Asp477, Arg475, His587, and Asp588 along with weak Pi-alkyl interactions with Tyr916 and Trp517 (at Subsite +1) stabilizes the inhibition complex of GtfC-Trichloro-galactosucrose. The inhibitory potential of the Flavonoid Myrecitin appears to closely follow Tricloro-galactosucrose. The stability of Myrecitin-GtfC complex may be especially ascribed to its multiple salt bridge and Pi-Anion interactions with Subsite −1 amino acids: Asp588, Arg475, His587 and Tyr916 (Table 2, ALIS score: 5.79, %Efficiency@D-Acarbose: 75.79, Figure 5(c), Table 3). Weak van der Waals and Pi-alkyl interactions are also observed with Asn481 and Subsite +1 (Leu433 and Trp517). A quinolaxine diamine derivative also exhibits an inhibitory effect >¾ of D-Acarbose (Table 1, ALIS score: 5.75, %Efficiency@D-Acarbose: 75.10, Figure 5(d), Table SI2). The stability of diamine derivative-GtfC complex is based on multiple interaction with Asp588 and Trp517. The Aspartate anion is attracted towards the aromatic rings of the quinoxaline diamine derivatives due to Pi-Anion interactions and possible hydrogen bonding amino hydrogen atoms. Trp517 undergoes Pi-Pi T shaped interactions with the aromatic rings of the ligand. The ADMET properties of the top three Ligands (6-Deoxysucrose, Trichlorogalactosucrose and Flavonoid Myrecitin) have favourable aqueous solubility, low blood brain barrier penetration, almost null Cytochrome P450 Inhihibitory potential, almost zero hepatotoxicity, null plasma protein binding and moderate to poor Intestinal absorption. The inhibitory potential of the rest of the inhibitors is <¾ of D-Acarbose (Table 1, Supplementary Information: Figure SI-6a–14a, Table SI2). The abstraction of the collective interaction of all the validated ligands considered in this study reveals the identity of the critically placed amino acid residues responsible for the inhibitory effect of the ligands. The amino acids residues at GtfC Subsite −1 (Arg475, 7, Glu515, His587, Asp588, Tyr 916), Subsite +1 (Leu433, Trp517) and Ca²⁺ binding site Asn481 were observed to have most frequent favourable interaction (including hydrogen bonding) with the validated ligands. Subsite −1, Asp588, and subsite +1, Trp517, were observed to have the highest count of favourable interaction. Asp588 and Trp517 were also found to have highest counts of charged and hydrophobic interactions (Supplementary Information: Figure SI AI and Figure SI AH). Asp 588, Trp517 and Asn481 belong to the domain A1 of GtfC [19]. Since, these amino acids play a vital role in the catalysis of the natural substrate (sucrose), any favourable interaction (including hydrogen) successfully interrupts its native catalytic role (discussed above) thereby inhibiting the enzyme.

Conclusion

The present study successfully prioritizes and characterizes the binding efficiency of the validated ligands. This study also focuses on the chemical interactions specifically responsible for the inhibitory effect of the potent inhibitors. Structural subdomain of GtfC vital for the inhibitory effect of were revealed. The structural interaction details shared in this study may be employed to determine better suited leads for combating biofilm generation and associated diseases of the oral cavity.

Author contributions

Conceived and designed the study and experiments: HAH SK MK PS MAM RKM SH. Performed the experiments: HAH SK MK PS MAM RKM SH. Analysed the data: HAH SK MK PS. Contributed reagents/materials/analysis tools: MAM RKM SH. Wrote the paper: SK MK MAM SH. All authors reviewed the manuscript.

Acknowledgements

The authors are grateful to Hail University, Saudi Arabia for providing the access of the Saudi Digital Library and other software related help for this research study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All the data required is available in supplementary Information and on request.

Additional information

Funding

This work was supported by the King Abdulaziz city for Science and Technology (KACST) under [Grant ID: 201-36] issued to Dr. Hazza A Alhobeira.