Hydroxychalcone inhibitors of Streptococcus mutans glucosyl transferases and biofilms as potential anticaries agents

Article abstract of DOI:10.1016/j.bmcl.2016.06.033

Streptococcus mutans has been implicated as the major etiological agent in the initiation and the development of dental caries due to its robust capacity to form tenacious biofilms. Ideal therapeutics for this disease will aim to selectively inhibit the biofilm formation process while preserving the natural bacterial flora of the mouth. Several studies have demonstrated the efficacies of flavonols on S. mutans biofilms and have suggested the mechanism of action through their effect on S. mutans glucosyltransferases (Gtfs). These enzymes metabolize sucrose into water insoluble and soluble glucans, which are an integral measure of the dental caries pathogenesis. Numerous studies have shown that flavonols and polyphenols can inhibit Gtf and biofilm formation at millimolar concentrations. We have screened a group of 14 hydroxychalcones, synthetic precursors of flavonols, in an S. mutans biofilm assay. Several of these compounds emerged to be biofilm inhibitors at low micro-molar concentrations. Chalcones that contained a 3-OH group on ring A exhibited selectivity for biofilm inhibition. Moreover, we synthesized 6 additional analogs of the lead compound and evaluated their potential activity and selectivity against S. mutans biofilms. The most active compound identified from these studies had an IC₅₀value of 44?μM against biofilm and MIC₅₀value of 468?μM against growth displaying >10-fold selectivity inhibition towards biofilm. The lead compound displayed a dose dependent inhibition of S. mutans Gtfs. The lead compound also did not affect the growth of two commensal species (Streptococcus sanguinis and Streptococcus gordonii) at least up to 200?μM, indicating that it can selectively inhibit cariogenic biofilms, while leaving commensal and/or beneficial microbes intact. Thus non-toxic compounds have the potential utility in public oral health regimes.

Full text of DOI:10.1016/j.bmcl.2016.06.033

Accepted Manuscript
Hydroxychalcone inhibitors of Streptococcus mutans glucosyl transferases and
biofilms as potential anticaries agents
Bhavitavya Nijampatnam, Luke Casals, Ruowen Zheng, Hui Wu, Sadanandan
E. Velu
PII:
S0960-894X(16)30644-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.033
BMCL 23986
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 April 2016
12 June 2016
13 June 2016
Please cite this article as: Nijampatnam, B., Casals, L., Zheng, R., Wu, H., Velu, S.E., Hydroxychalcone inhibitors
of Streptococcus mutans glucosyl transferases and biofilms as potential anticaries agents, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.033
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Hydroxychalcone inhibitors of Streptococcus mutans glucosyl
transferases and biofilms as potential anticaries agents
Bhavitavya Nijampatnama, Luke Casalsa, Ruowen Zhenga, Hui Wub*, Sadanandan E. Velua*
aDepartment of Chemistry, University of Alabama at Birmingham, Birmingham, Alabama 35294,
b
USA; Department of Pediatric Dentistry, UAB School of Dentistry, Birmingham, Alabama
35294, USA
* Corresponding authors:
Sadanandan Velu, Mailing address: Department of Chemistry, University of Alabama at
Birmingham, 901, 14th Street S. Birmingham, AL 35294, USA, Phone: (205) 975-2475, Fax:
(205) 934-2543, Email: svelu@uab.edu.
Hui Wu, Mailing address: Department of Pediatric Dentistry, University of Alabama at
Birmingham, School of Dentistry, Birmingham, Alabama 35294, Phone: (205) 996-2392, Fax:
(205)975-4430, E-mail: hwu@uab.edu.
Keywords: Hydroxychalcone, inhibitors, glucosyltransferase (Gtfs), Streptococcus mutans,
biofilms, and dental caries.
1

Abstract:
Streptococcus mutans has been implicated as the major etiological agent in the initiation
and the development of dental caries due to its robust capacity to form tenacious biofilms. Ideal
therapeutics for this disease will aim to selectively inhibit the biofilm formation process while
preserving the natural bacterial flora of the mouth. Several studies have demonstrated the
efficacies of flavonols on S. mutans biofilms and have suggested the mechanism of action
through their effect on S. mutans glucosyltransferases (Gtfs). These enzymes metabolize sucrose
into water insoluble and soluble glucans, which are an integral measure of the dental caries
pathogenesis. Numerous studies have shown that flavonols and polyphenols can inhibit Gtf and
biofilm formation at millimolar concentrations. We have screened a group of 14
hydroxychalcones, synthetic precursors of flavonols, in an S. mutans biofilm assay. Several of
these compounds emerged to be biofilm inhibitors at low micro-molar concentrations. Chalcones
that contained a 3-OH group on ring A exhibited selectivity for biofilm inhibition. Moreover, we
synthesized 6 additional analogs of the lead compound and evaluated their potential activity and
selectivity against S. mutans biofilms. The most active compound identified from these studies
had an IC50 value of 44μM against biofilm and MIC50 value of 468μM against growth displaying
>10 fold selectivity inhibition towards biofilm. The lead compound displayed a dose dependent
inhibition of S. mutans Gtfs. The lead compound also did not affect the growth of two
commensal species (Streptococcus sanguinis and Streptococcus gordonii) at least up to 200 μM,
indicating that it can selectively inhibit cariogenic biofilms, while leaving commensal and/or
beneficial microbes intact. Thus non-toxic compounds have the potential utility in public oral
health regimes.
2

Dental caries is a multifactorial disease evident by localized destruction of susceptible
dental surfaces. S. mutans is the primary etiological agent. 1 Through the function of its Gtfs, S.
mutans metabolizes sucrose into water insoluble and soluble glucans. 2 Three types of Gtfs are
reported: GtfB, GtfC, and GtfD. Both GtfB and GtfC predominantly synthesize water-insoluble
glucans whereas GtfD synthesizes water-soluble glucans. 3 These exopolymeric glucans are an
integral measure of the cariogenic biofilms that provide an extracellular matrix, shielding the
bacteria from the host immune responses, mechanical stresses, and antimicrobial agents. 4 As S.
mutans accumulate, the bacterium and other cariogenic species within the biofilm undergo active
sugar metabolisms and produce copious amounts of their end-product, lactic acid, leading to the
dissolution of the hydroxyapatite crystal lattice of the enamel. 5
Current preventive approaches for dental caries include tooth-brushing and the use
antimicrobial mouthwashes. 6 Unfortunately, the removal of bacterial biofilms through brushing
demands frequent repetition because the tooth surfaces are rapidly re-colonized by bacteria.
Furthermore, the drawback of existing antimicrobial mouthwashes lies in their lack of selectivity,
affecting pathogenic species as well as commensal beneficial species, which give rise to some
undesired side effects.7 Therefore, emerging innovative approaches of dental caries prevention
need to be selective and should strive to preserve the natural bacterial flora in the mouth.
Several studies have established the importance of Gtfs in the S. mutans biofilm
formation. 8-11 Mutants defective in genes gtfB and gtfC, which express GtfB and GtfC
respectively, exhibited markedly reduced levels of smooth surface caries lesion compared to the
parental S. mutans. 8 Thus, S. mutans Gtfs are valid targets for the discovery of inhibitors of
cariogenic biofilms, which will have potential applications in the treatment of dental caries.
3

Efforts have been placed in search for S. mutans biofilm inhibitors by several research
groups. 12-15 Since diet is one of the key factors that define oral health, research has mainly been
focused on widely consumed food products. Numerous studies have been conducted comparing
the efficacies of flavonols and low-molecular-weight polyphenols found in cranberries.16
Flavones and flavonols were inhibitors of Gtfs; of these, a compound called apigenin (4’,5,7-
trihydroxyflavone, Fig.1) was the most effective inhibitor of Gtfs.17 Flavonols and their
glycosides such as myricetin and myricetin 3-rhamnoside exhibited significant but moderate
effects (Fig. 1). 18, 19 In addition, scaffolds found in tea (Camellia sinensis) that inhibit S. mutans
biofilms attract attention due to their unique polyphenol compositions and their prevalence in the
human diet. 20 Early studies were carried out on extracts containing multiple constituents
however recent reports focus on effects of single defined component in vitro and in vivo. 21
Furthermore, these natural product compounds inhibited S. mutans biofilms at high micromolar
and even millimolar concentrations.
Figure 1: Known inhibitors of S. mutans biofilm and S. mutans Gtfs and general structures of
flavone and hydroxychalcone
Undoubtedly, flavonols are a promising class of scaffolds for the development of
anticaries therapeutics as they may selectively inhibit the formation of cariogenic biofilms. By
targeting S. mutans’ virulence instead of its viability, the agents developed will be non-
4

bactericidal, preserving the natural bacterial flora of the mouth, and will also be less likely to
develop resistance to therapy. Encouraged by reported findings on flavonols, we were interested
to examine the effect of hydroxychalcones (Fig. 1), precursors of flavonols and isoflavonols, for
their effect on S. mutans biofilm and Gtfs. Chalcones were originally isolated from natural
sources and are abundant in edible plants. Structurally, they can be defined as open-chain
flavonoids in which two aromatic rings are joined by a three carbon α,β-unsaturated carbonyl
system. 22 Being a minor subgroup of the flavonoid family, like other members, chalcones have
been reported responsible for a variety of biological activities, including antiviral, anticancer,
antimicrobial, anti-inflammatory, antioxidative, antimalarial, anti-leishmania, antinociceptive,
and antiproliferative activities. 23, 24 Hence, chalcones are considered to be a class of compounds
with important therapeutic potential. To our knowledge, there are no studies that report the
antibiofilm activity of chalcones against S. mutans. We therefore investigated this class of
compounds to identify novel selective anti-biofilm agents that can have potential applications in
dental caries prevention.
Although the inhibition of both GtfB and GtfC are required for the maximum reduction in
biofilm formation, only the structural information of GtfC’s active site is currently known.
However, genes encoding these two Gtfs have a very similar amino acid composition (76%
homology), and are subject to the same regulatory processes. 25 Thus, we used GtfC as a target
for the drug discovery efforts. We initially performed docking analysis using FlexX/LeadIT
package against GtfC active site (resolution: 3.11Å, PDB code: 3AIC) on a polyphenol database
which included various scaffolds such as flavonols, chalcones, stilbenes, and other complex
structures. Of these compounds, we focused on chalcones due to their properties discussed
above. A total of 14 chalcones with various hydroxyl substitution patterns were obtained from
5

National Cancer Institute (NCI) compound library and screened for their effect on S. mutans
growth and its biofilm. Table 1 summarizes the results of the preliminary screen conducted at
200 µM.
Table 1: Biofilm and growth inhibition profiles of chalcone derivatives obtained from the NCI
library
Biofilm
inhibition 200
µM (%)a
Growth
inhibition200
µM (%)a
S. mutans
Biofilm IC50
(µM)a
S. mutans
Growth IC50
(µM)a
Compd
No
NSC code
R1
R2
1
2
3
4
5
6
7
73255
73257
46672
94615
401492
636790
636810
2-OH
2-OH
2,5-di-OH
2-OH
2,4-di-OH
2,4-di-OH
3- OH
4'-OH
3'OH
4'-OMe
27
84
87
86
NI
NI
NI
5
3
8
2
48
8
NA
NA
32 0.3
NA
86 0.5
76 0.9
85 0.6
NI
NI
NI
64
NA
61
NA
NA
NA
4
2
4'-OH, 3'-OMe
H
44
NA
3
2'-OH
2'-OH
NA
NA
2'-OH, 3’,5' di-
Cl
2,4'-di-OH
3',4',5'-tri-OMe
2'-OH
8
640543
3-OH
81
93
NI
85
NI
27
NI
2
1
82
NI
NI
71
NI
2
5
NA
NA
9
10
11
17051
90932
640536
3-OH
4-OH
4-OH, 5-OMe
4-OH, 3-NMe2, 5-
NMe2
32
NA
34
5
2
NI
NA
3
4
40
5
12
677786
4'-OMe
NA
NA
13
14
196539
196540
4- OMe, 5- OMe,
4,5- OCH2O
4'-OMe, 3'-OH
4'-OMe, 3'-OH
30 0.7
NI
NA
NA
NA
NA
aAverage of at least 5 measurements; NINo inhibition; NANot available.
The initial screen highlighted chalcone activities ranging from almost complete inhibition
of both S. mutans growth and biofilm (Compounds 2-4, 8, 11), selective inhibition of S. mutans
biofilm (Compound 9), to no effect on either S. mutans growth or biofilm (Compounds 5-7, 10,
12, 14). Active compounds inhibited biofilm in the range of 27-95% at 200μM concentration.
However, several of these compounds also proportionally affected the growth of S. mutans,
indicating that these small molecules may not be selective biofilm inhibitors. Nonetheless, four
compounds that showed promise by exhibiting greater than 80% biofilm inhibition were pursued
further into serial dilutions in order to determine their bactericidal and anti-biofilm IC50 values,
6

and to observe if any selectivity for biofilm inhibition over growth inhibition arises at lower
doses. Both the growth and biofilm IC50 values remained in the low micromolar ranging from 32
- 64 µM. While compounds 2, 4, and 11 failed to demonstrate biofilm-selective inhibition,
compound 9 did elicit selectivity with biofilm IC50 value 32 µM, without affecting the growth of
S. mutans at 200μM concentration. Through this study, we observed several trends in the elicited
biofilm and bactericidal effects in relation to chalcone structures.
Chalcones that did not possess any bactericidal or anti-biofilm properties also did not
have any OH groups on the ring B of the chalcone skeleton (Compound 5). Despite containing
methyl protected hydroxyl groups on the ring B, compounds 10 and 12 also failed to produce any
biofilm inhibition. The only analogs that do not contain free hydroxyl groups on the ring A are
compounds 13 and 14, which contain methoxy and methylenedioxy groups in the 4th and 5th
positions. While compound 13 was marginally active, reducing biofilm and growth by 30% at
200 µM, compound 14 exhibited no anti-biofilm effects. These results suggest that the presence
of hydroxyl groups on either ring of the chalcone scaffold is crucial for the inhibition of S.
mutans biofilm.
Compounds 5 and 6 differ only in the 2’ position of OH group in ring A, where the latter
contains an OH group in place of hydrogen. Interestingly, both compounds are inactive,
suggesting the OH in 2’ position may not contribute to the studied bioactivities. While
compound 7 also follows this pattern of activity, this effect is not observed in the case of
compound 11.
Furthermore, analysis of compounds 1-4 which contain an OH group at the 2nd position
of ring A seemed to exhibit potent anti-biofilm and bactericidal activities. Contrary to these
compounds, chalcones that bare a hydroxyl group at the 3rd position of ring A (Compounds 7-9)
7

seemed to exhibit a range of potent and selective anti-biofilm activities. With the exception of
the inactive compound 7, other compounds exhibited greater than 81% inhibition of biofilm at
200 µM and presented some selectivity over growth inhibition. Strikingly, when tested at lower
concentrations, the best compound that emerged from this study (9) almost exclusively inhibited
S. mutans biofilm, suggesting the importance of OH group in the 3rd position in the ring A.
There was only one compound that contained a mono-hydroxyl group at the 4th position
of the ring A (compound 11). This compound was inactive against both S. mutans growth and
biofilm. Compounds 5-6, 10-14 contain OH group at the 4th position and majority of these
compounds also fared poorly in the preliminary screen asides from compound 11 which was
toxic to S. mutans at low micromolar concentrations. The importance of OH group at this
position remains unclear and warrants further investigation.
Since we are in search of compounds that selectively target S. mutans biofilm without
affecting the its growth, the best candidate to pursue further was compound 9, which has a
biofilm IC50 value 32 µM. No previous biological activities have been reported with regard to
this synthetic polyhydroxy analog. This compound did not affect the bacterial growth at least up
to a concentration of 200μM, showing that it is a selective biofilm inhibitor. While compound 11
exhibited comparable biofilm inhibition to compound 9 (IC50 value of 34µM, and 32µM
respectively,) compound 11 also inhibited S. mutans growth at this concentration.
8

Figu
UA159 wild type was co-incubated with compound 9 at various concentrations and its
subsequent biofilms were stained with Syto9. B) S. mutans growth and biofilm affected by
re
2.
A)
Mic
ros
co
pic
ev
alu
ati
on
of
S.
mut
ans bi
ofil
ms in
hib
ite
d b
y c
om
po
und 9.
S.
mu
tans
compound 9. S. mutans UA159 was co-incubated with compound 9 at various concentrations and
their growth was measured at OD470. Using the crystal violet protocol, their biofilm was
measured at OD562. C) Effect of compound 9 on commensal streptococci. S. mutans UA159, S.
sanguinis, S. gordonii were co-incubated with compound 9 at various concentrations and their
growth was measured at OD470.
Thus, compound 9 was selected as the lead scaffold for the further development of the
selective antibiofilm agents. In order to further establish its potency, the biofilms produced by S.
mutans treated with this compound at various concentrations were imaged as shown in Fig. 2A.
Fig. 2B depicts the dose-dependent inhibition of S. mutans growth and biofilm at various
concentrations. Considerable selectivity can be seen, especially at higher concentrations. Finally,
the effect of it on the survival rates of S. mutans, and two commensal species (S. sanguinis, S.
gordonii) were used to assess the toxicity of this scaffold. At higher concentrations (200 μM),
approximately 10-18% inhibition is seen with all strains when compared to DMSO (Fig. 2C). It
is important to note that at the biofilm IC50 value of 32 µM, no significant bactericidal activity is
9

observed. Thus, we concluded that the compound 9 is a non-toxic and a biofilm-selective
inhibitor.
As compound 9 was obtained from the NCI library in a very a small quantity, we were
not able to ascertain its structure with NMR spectroscopic analysis. In addition, the
stereochemistry of the double bond (E or Z) present in this molecule was not reported in the NCI
database. So, we synthesized both E and Z isomers of the compound 9 and characterized them.
The preparation of the E isomer of 9 (9a) was achieved by the Claisen-Schmidt aldol
condensation 28 of the desired benzaldehyde (15a) and acetophenone (16a) in EtOH using
aqueous NaOH as the base in 93% yield. Demethylation of the intermediate methoxy substituted
compound 17a was accomplished by the treatment with BBr3 in CH2Cl2 under N2 atmosphere led
to the formation of compound 9a (E isomer) in 87% yield (Scheme 1).
Scheme 1: Synthesis of the E chalcones
The stereospecific synthesis of the Z isomer of compound 9b was performed using a
reported procedure of acid mediated aldol condensation 28 as shown in Scheme 2. BF3.Et2O
mediated condensation of 2,4-dihydroxybenzaldehyde (19) with m-hydroxyl acetophenone (20a)
resulted in the Z-isomer 9b in contrast with the results obtained when using NaOH. This method
was tolerant of pre-existing hydroxyl groups and thus required no protecting groups.
10

Scheme 2: Synthesis of the Z chalcones
Taking compound 9 as our lead, we prepared a small collection of both E and Z isomers
of a few hydroxychalcone derivatives (18b-d and 21b) with different substitutions in rings A, B
using the synthetic procedures described above. Selected examples and their activities are shown
in Table 2.
Table 2: Biofilm and growth inhibition profiles of synthesized analogs of compound 9
Biofilm
IC50
Growth
IC50
No
E/Z
R1
R2
SIb
(µM)a
(µM)a
3-
OH
3-
OH
4-
OH
4-
OH
2-
9a
E
Z
2',4' di-OH
2',4' di-OH
2',4' di-OH
2',4' di-OH
68
44
3
5
639 25
468 16
9.39
10.6
6.6
9b
1063
118
18b
21b
18c
18d
E
162 21
77
224 23
76.
Z
8
782 96
323 36
10.1
1.5
2',4',5' tri-
OH
2',4',5' tri-
OH
E
OH
4-
OH
E/Z
6
376
6
4.9
aAverage of at least 5 measurements. bSelectivity index calculated by
dividing growth IC50 value by biofilm IC50 value.
We have included the E and Z isomers of the lead compound, E and Z isomers of a
synthetic derivative and two other compounds that further explore the hydroxyl regiochemistry
11

of chalcones. Due to the possibility of photo-isomerization of chalcones, which has been
previously demonstrated to be correlated with the substituents in the aromatic rings and pH
dependent,26 all samples were tested immediately upon making them. The biofilm and growth
inhibition profiles were screened using the assay protocols as described earlier. All compounds
asides from compound 18c demonstrated considerable selectivity in biofilm inhibition.
Compound 18c was the most toxic to planktonic cells with a selectivity index of 1.5; a result that
is consistent to the observation reported earlier. Comparing the activities of the E and Z pairs (9a
and 9b and 18b and 21b), the Z isomers demonstrate slightly better inhibitions of both biofilm
and growth. The most active compound (9) that emerged from the NCI screen had a biofilm IC50
value of 32 µM, while the synthesized cis isomer of this compound (9b) exhibited a slight
decrease in activity with an IC50 value of 44µM. Corresponding E isomer (9a) also had a
slightly decreased activity with an IC50 value of 68µM. Both 9a and 9b demonstrated a
selectivity index of about 10 towards biofilm inhibition. The Z isomer (21b) fared slightly better
at being selective compared to its E isomer when comparing compounds with a hydroxyl in the
4th position of ring A also. Compound 18d (mixture of E/Z) produced biofilm activity with
moderate selectivity. Thus, the demonstrated selectivity of these compounds makes them
amenable for the development of non-toxic biofilm inhibitors.
In order to establish the relationship between the biofilm inhibition and the effect on Gtfs,
we performed a zymogram assay on six of our active compounds and qualitatively observed the
inhibition of Gtfs on an SDS-page gel through the intensity of the glucan bands produced. Data
from selected compounds is presented in Fig. 3A. Each of these compounds was added to
overnight cultures at 50 µM concentrations, and the Gtfs were collected from supernatants. One
of the duplicate SDS-PAGE gel is Coomassie blue stained while the other is subjected to the
12

zymo
gra
m
ass
ay, w
her
e th
e g
lucan
ba
nds pr
od
uce
d r
epr
ese
nt
Gtf
s enz
ym
e activ
ities.
Gtf
B and
GtfD remain unresolved above the 150 kD reference ladder while a clear separation can be seen
with the single band of GtfC.
Figure 3: A) Gtf level and activity determined by SDS-PAGE analysis and zymographic assay.S.
mutans UA159 wild type were co-incubated with compounds of interest at 50 µM. Gtfs were
concentrated from S. mutans UA159 wild type and then ran through duplicate SDS-PAGE. I) The
upper panel is Coomassie blue staining of proteins analyzed by SDS-PAGE . II) The bottom
panel shows glucan bands produced in the zymographic assay. The intensity of the bands reflects
Gtf enzyme activity. B) Effects of compound 9b at different concentrations. S. mutans UA159
wild type bacteria were co-incubated with compound 9b various concentrations. Protein profiling
(I) and activity (II) of Gtfs were evaluated as described in (A). C) Docking poses of compounds
9a in blue and 9b in green and the key residues interactions of GtfC active site. D) Fluorescent
microscopy images of S. mutans colonization in Drosophila. a) Treatment with DMSO, b) ΔgtfB
mutant strain, c) Treatment with 50 µM 9b and d) Treatment with 50 µM 9a
With regards to effects on protein level, compounds 9b, 18b, 18c and 21b showed an
inhibitory effect on the production of Gtfs. Compound 18c shows the largest inhibition and this
result is logical given the bactericidal effects of the compound. Mechanism by which these
compounds affect the Gtf expression is unknown and warrants further investigation. With regards
to Gtf enzyme inhibitory activity, several of the polyhydroxy chalcones exhibited a prominent
effect. Comparing the activities of the E and Z pairs (9a, 9b and 18b, 21b), the Z isomers (9b
13

and 21b) exhibit the best inhibitory effects, consistent with the biofilm inhibition. A decrease is
observed in the bottom GtfC band, but a decrease in the upper band GtfBD is also observed,
suggesting that the compound inhibited Gtfs. This is expected as GtfC and GtfB share 76%
homology. Surprisingly, the E isomers 9a and 18b did not exhibit a drastic effect, suggesting that
perhaps these isomers use a different pathway to illicit biofilm inhibition. Compound 18d
demonstrates some weak inhibition of the GtfC band. A dose-dependent zymogram experiment
of compound 9b was performed to determine if any selectivity would emerge (Fig. 3B). All
Gtfs were inhibited by compound 9b dose-dependently. Thus, we conclude this compound
affects the production and activity of all Gtfs.
Docking analysis of the two different isomers 9a and 9b within the GtfC active site
revealed slightly different binding modes (Fig. 3C), which may explain the differences observed
between the inhibitions seen in the zymograms and biofilm activity. A previous report indicated
that binding of acarbose to Glu515 compromised the acid/base catalyst function, while
interactions with Asp477 and Asp588 blocked the acceptor glycosyl moiety, explaining the
inhibitory properties shown by acarbose when bound to GtfC 27. While the docking analysis
showed that these three residues were within proximity to the both isomers, the Z isomer, 9b
showed the hydroxy groups positioned appropriately and closer to the three key residues when
compared to the E isomer, 9a.
We used a sucrose-dependent Drosophila colonization model13 to examine the effect of
both compounds 9a and 9b on S. mutans colonization in vivo. Briefly, Gfp tagged S. mutans
bacteria were infected into flies along with the treatment appropriate compound at 100 µM in a
feeding-assay. DMSO was used as a negative control (Fig. 3D) and gtfB mutant, a known
biofilm defective strain, was used as a positive control (Fig. 3D.a). The fluorescence intensity
14

was observed in guts of the flies fed with the Gfp tagged bacteria after 7 days of infection. A
marked reduction in fluorescence was observed by both compounds 9a and 9b (Figs. 3D.d and
3D.c), with 9b eliciting a more potent effect, producing an effect similar to that observed in the
gtfB mutant (Fig. 3D.b). This data is consistent with the zymogram and the biofilm inhibition
observed previously.
Thus, we have demonstrated the S. mutans antibiofilm activities of
polyhydroxychalcones, and have observed some trends in activities with respect to the
stereochemistry of the double bond and the regiochemistry and numbers of hydroxyl groups
present in the molecules. Several low micromolar biofilm inhibitors emerged. Chalcones which
contained a hydroxyl group in the third position of ring A exhibited higher selectivity for biofilm
over growth. The lead compounds were further evaluated for their effect on S. mutans Gtfs and
their effect on commensal bacteria and found that these scaffolds have an effect of Gtf
expression and activity. The relationship between the biofilm activity and Gtf inhibition was
established using a zymogram assay. These results were further validated by conducting a
docking analysis of Z and E isomers of one of the lead compounds. We have also demonstrated
the effect of two lead compounds on S. mutans colonization in vivo. Further studies to optimize
the activity and selectivity of the lead compounds and establish polyhydroxychalcones as
selective antibiofilm agents are currently in progress.
Acknowledgments
This study was funded by the National Institute of Dental and Craniofacial Research,
National Institutes of Health (NIDCR/NIH) R01DE022350 to Wu, R03DE025058-0 to Velu and
F31 DE025783-01A1 to Nijampatnam. Initial chalcone compounds were obtained from the Drug
15

Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, National Cancer Institute.
Supplementary data:
Docking protocols, synthetic procedures, assay protocols, and compound characterization
data associated with this article can be found in the attached supplementary data.
Abbreviations
DMSO, dimethylsulfoxide; E, trans isomer; Gfp, green fluo-rescent protein; Gtfs,
glucosyltransferases; kD, kilodalton; SAR, structure activity relationship; SDS-PAGE, sodium
do-decyl sulfate polyacrylamide gel electrophoresis; µM, micromolar; Z, cis isomer.
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Hydr
ox
yc
hal
con
e i
nhi
bit
or
s of St
re
pto
coccu
s
mut
an
s gl
uc
osy
l t
ran
sfe
rases
an
d b
iofilms as
potential anticaries agents
Bhavitavya Nijampatnam1, Luke Casals1, Ruowen Zheng1, Hui Wu2*, Sadanandan E. Velu1*
19

Highlights
• Low micromolar inhibitors of S. mutans biofilm and Gtf are identified.
• The lead compounds are E and Z isomers of hydroxychalcones.
• The Z isomers of chalcones produced more potent anti-biofilm activities.
• Chalcones with a 3-OH group on ring A showed better selectivity for biofilm inhibition.
• These compounds do not affect bacterial growth.
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