New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans

Article abstract of DOI:10.1016/j.ijantimicag.2015.03.015

Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC₅₀) of 454.0 ± 10.2 nM for the biofilm and 8.7 ± 1.9 nM for DHFR of S. mutans. In contrast, the IC₅₀ of this compound for human DHFR was ca. 1000 nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries.

Full text of DOI:10.1016/j.ijantimicag.2015.03.015

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Contents lists available at ScienceDirect
International Journal of Antimicrobial Agents
journal homepage: http://www.elsevier.com/locate/ijantimicag
New small-molecule inhibitors of dihydrofolate reductase inhibit
Streptococcus mutans
Qiong Zhanga,b,1, Thao Nguyenc,1, Megan McMichaelc, Sadanandan E. Veluc, Jing Zoua,
Xuedong Zhoua, Hui Wub,∗
a State Key Laboratory of Oral Diseases, Department of Pediatric Dentistry, West China Hospital of Stomatology, Sichuan University,
Chengdu, Sichuan 610041, PR China
b Department of Pediatric Dentistry, University of Alabama at Birmingham School of Dentistry, Birmingham, AL 35294, USA
c Department of Chemistry, University of Alabama at Birmingham, Birmingham, AL 35294, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 January 2015
Accepted 31 March 2015
Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence
factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate
reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus
potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of
analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and
three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had
a 50% inhibitory concentration (IC50) of 454.0 10.2 nM for the biofilm and 8.7 1.9 nM for DHFR of S.
mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000 nM, a >100-fold decrease
in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least
potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further
indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking
of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed
selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are
thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries.
© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
Keywords:
Streptococcus mutans
Biofilm
Dihydrofolate reductase
Trimetrexate analogues
1. Introduction
immunomodulatory monoclonal antibodies [6]. However, in vivo
application of these promising approaches is uncertain.
Dental caries is initiated by cariogenic bacteria under the
carbohydrate-rich environment of the oral cavity. Streptococcus
mutans is the principal causative agent for the development of den-
tal caries. The ability of S. mutans to adhere to the tooth surface and
to incorporate into a polymicrobial consortium is paramount for
the development of the disease; thus, reducing the number of cari-
ogenic bacteria in dental biofilms is the key to devising therapeutic
and preventive strategies [1].
Various strategies aimed at preventing dental caries have been
attempted on the basis of various criteria: increasing antimicro-
bial activity [2]; replacement of sucrose with other sweeteners
[3]; inhibition of the key matrix-producing enzyme glucosyltrans-
ferases either by vaccine approaches [4] or enzymatic inhibitors [5];
and targeting another important surface protein antigen I/II using
Folate is essential for all organisms. It contributes to the produc-
tion of cofactors required for the synthesis of DNA, RNA and amino
acids. Dihydrofolate reductase (DHFR) is the key enzyme involved
in the production of the cofactors, thereby playing a critical role in
regulating folate metabolism [7].
The importance of DHFR in the folate cycle has been explored
in order to develop drugs for tumour therapy as well as therapy
against opportunistic infections of diverse origin [7–9]. Inhibition
of DHFR leads to partial depletion of reduced intracellular folate,
thereby limiting cell growth. Functionally, DHFR is highly con-
served in all domains of life, however they are divergent in amino
acid sequence, offering an opportunity to impart a high degree of
selectivity for certain antifolate drugs against one organism versus
another [10].
Antifolate drugs have been successful in the treatment of bacte-
rial and parasitic infections and for cancer chemotherapy. A number
of antifolate drugs have been developed to date [9,11–13]. Trime-
trexate (TMQ) has been used clinically for treatment of the parasites
Pneumocystis carinii and Toxoplasma gondii infections in acquired
∗
Corresponding author. Tel.: +1 205 996 2392; fax: +1 205 975 4430.
E-mail address: hwu@uab.edu (H. Wu).
These two authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ijantimicag.2015.03.015
0924-8579/© 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
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immunodeficiency syndrome (AIDS) patients [13]. To date, no DHFR
inhibitor has been evaluated for its selectivity against the cario-
genic bacterium S. mutans. Therefore, it is imperative to design
new inhibitors that have high selectivity against S. mutans DHFR
(SmDHFR). Considering that the primary sequence of SmDHFR
shares only 28% identity with the human DHFR (hDHFR) sequence,
it is conceivable to rationally design selective and potent inhibitors
of S. mutans based on the structure of TMQ.
The goal of this study was to identify small-molecule inhibitors
of DHFR that are capable of inhibiting S. mutans. A library of ca.
100 small-molecule compounds, which were designed based on
TMQ, a US Food and Drug Administration (FDA)-approved drug
[13], was screened and compounds that potently inhibit S. mutans
were identified. More importantly, these compounds were selec-
tive against SmDHFR. Computer modelling and docking analysis
supported the finding that the identified small molecule possesses
much higher affinity for SmDHFR than for hDHFR. The study to
design more potent and selective compounds inhibiting S. mutans
is ongoing, which should facilitate the development of potent ther-
apeutic drugs against S. mutans.
and GCCGCTCGAGTCATTCCTTTTTCTCAAGTAC). The resulting
PCR product (513 bp) was digested with BamHI and XhoI
and was ligated into the pET28-SUMO vector to construct
pET28-SUMO-SmDHFR. The constructed plasmids were ver-
ified by DNA sequence analysis and were then transformed
into Escherichia coli BL21. The pET28-SUMO-hDHFR was
constructed using a similar experimental procedure with hDHFR-
specific primers (CGCGGATCCATGGTTGGTTCGCTAAACTGC and
CCTCTCGAGTTAATCATTCTT CTCATATACTTCAAATTTGTAC) and
human cDNA.
Expression, production and purification of recombinant
SmDHFR and hDHFR proteins were carried out using the experi-
mental procedures described previously [15]. In brief, cell lysates
prepared from induced recombinant strains were loaded into
a Ni-NTA resin column (Novagen, USA) for initial purification
and the 6×His-SUMO tag was removed from the fusion proteins
with SUMO proteinase. The fractions were then subjected to
size exclusion chromatography on a Superdex75 column (GE Life
Science, USA). Concentrations of purified SmDHFR and hDHFR
were determined by the Bradford method (Bio-Rad Protein Assay).
2.5. Assay of enzymatic activity of SmDHFR and hDHFR
2. Materials and methods
The activity of purified SmDHFR and hDHFR was determined
by monitoring the decrease in absorbance at 340 nm due to
the oxidation of NADPH to NADP+ accompanying the reduc-
tion of dihydrofolate (DHF) to tetrahydrofolate (THF) (coupled
ε = 12.260 M−1 cm−1) using a UV–visible spectrophotometer. Reac-
2.1. Synthesis of small-molecule compounds and assembly of a
screening library
Protocols used to synthesise small-molecule compounds are
described in the Supplemental material. The small-molecule library
was assembled by the Velu Research Group.
tion velocities were measured at 25 ◦C for 1 min.
A DMSO
concentration of ≤0.125% was used in the assays as the effect
of the solvent at this concentration is negligible. Under opti-
mised conditions for each enzyme, SmDHFR activity was measured
in 50 mM Tris–HCl buffer (pH 7.0), whilst hDHFR activity was
measured in 50 mM KH2PO4 (pH 7.3) containing 250 mM KCl
and 5 mM ␤-mercaptoethanol [9]. The inhibitory activities of the
compounds were determined by measuring reaction velocities at
several fixed concentrations of DHF (15, 20, 25, 30 and 40 ␮M),
NADPH (150 ␮M) and various concentrations of compounds (dis-
solved in 0.1% DMSO). The 200 ␮L reaction was initiated with 0.2 ␮g
of purified SmDHFR or 0.3 ␮g of hDHFR. Each measurement was
performed in triplicate.
The synthetic processes and schemes are described in the main
text and the Supplementary material (Fig. 1A; Supplementary Fig.
S1). The chemical structures of TMQ and its analogues are depicted
in the results section (Fig. 1B).
2.2. Bacterial strains and culture conditions
Streptococcus strains, including S. mutans UA159, Streptococcus
sanguinis SK36 and Streptococcus gordonii DL1, were grown as pre-
viously described [14].
2.3. Biofilm formation and inhibition assays
The double-reciprocal plots were used to calculate the reac-
tion constant (Km) and inhibitory constant (Ki) for inhibition of
SmDHFR and hDHFR by each compound. The concentration of each
Exponentially grown S. mutans and S. sanguinis bacteria were
inoculated at 1:100 dilution with chemically-defined biofilm
medium (CDBM) containing 1% sucrose for biofilm assays, whilst S.
gordonii was inoculated at 1:50 dilution. Compounds at indicated
concentrations were added to the inoculated bacterial cultures. The
incubation time was 16 h for S. mutans and S. sanguinis and 12 h
for S. gordonii to obtain reproducible and comparable biofilms. For
control cultures, the corresponding volume of dimethyl sulphoxide
(DMSO) was added. Crystal violet staining measured at an optical
density of 562 nm was used to monitor biofilm formation described
previously [14]. Minimum inhibitory concentrations (MICs) of the
compounds were examined using a previously described method
[14]. The concentration of potent compounds that inhibited S.
mutans biofilm formation by 50% (IC50) was determined by serial
dilution. Each assay was carried out with duplicate samples and
was repeated three times.
inhibitor required for inhibition of enzyme activity by 50% (IC50
)
was determined using the following equation: IC50 = Ci/(Vo/Vi − 1),
where Vo and Vi are the initial velocities in the absence and presence
of inhibitor, respectively, and Ci is the concentration of inhibitor.
The selectivity index (SI) was calculated using the equation:
SI = Ki(hDHFR)/Ki(SmDHFR) or IC50(hDHFR)/IC50(SmDHFR). The SI
of each analogue was compared with that of TMQ to assess the
selectivity of each compound.
2.6. Sequence alignment, homology modelling and validation
The SmDHFR sequence was submitted to Phyre2 (Protein
Homology and Analogy Recognition Engine v.2.0) (http://www.
sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) and Swiss-Model
(http://www.swissmodel.expasy.org) for structural prediction and
modelling. Streptococcus pneumoniae DHFR (SpDHFR) (PDB code,
2.4. Cloning, expression and purification of recombinant DHFR
from Streptococcus mutans and human cells
˚
3ix9; resolution, 1.95 A) was selected for structural modelling,
and the whole chain B of SpDHFR served as the template for
the homology modelling of SmDHFR using Phyre2 normal mode.
The model was superposed with the SpDHFR template by VMD
1.9.1 to establish the agreed and disagreed regions [11]. The
overall calculated root-mean-square deviation (RMSD) was taken
The coding sequence for the S. mutans UA159 DHFR
protein was amplified by PCR (GoTaq® DNA Polymerase;
Promega) using genomic DNA as template and SmDHFR-
specific
primers
(CGCGGATCCATGAACAATAAGCGAGAAAAG
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Fig. 1. (A) Synthetic scheme of trimetrexate (TMQ) analogues and (B) molecular structures of TMQ, trimethoprim (TMP) and their analogues #66, #151, #153 and #154.
Generally, the synthesis involved a reductive amination of 2-fluoro-5-formylbenzonitrile 4 with appropriate amines (RNH2) in the presence of NaCNBH3 and ZnCl2 in methanol
to afford the aminated product 5 (59–100% yield). The aminated compound 5 was refluxed with guanidine carbonate in N,N-dimethyl acetamide to give the final products
#66, #151, #153 and #154 (13–76% yield).
from overlapping the backbone of all aligned residues, whilst the
active-site RMSD was obtained from overlapping the homology
model with the SpDHFR backbone containing residues 8–119. The
SmDHFR model generated by Phyre2 was validated by PROCHECK
and VERIFY3D provided by Structure Analysis and Verification
Server (SAVES v.4.0) and was also assessed by Swiss-Model Assess-
ment for QMEAN and QMEAN Z-Score.
within 1 standard deviation from the scores of high-resolution
X-ray structures of similar size (Supplementary Table S1) [21]. Eval-
uation validated the model [20] since the scores for SmDHFR were
similar or better than those of the SpDHFR X-ray crystal structure
˚
with the resolution of 1.95 A.
2.8. Statistical analysis
Statistical tests were performed using SPSS v.16.0 software
(SPSS Inc., Chicago, IL) and the results are presented as the
mean standard deviation. Two-group comparisons were per-
formed using Student’s t-test. A P-value of <0.05 was considered
statistically significant.
2.7. Molecular docking and energy analysis
The computer system Ubuntu 10.04 LTS on Quad Core OptiPlex
780 was used to dock the lead compound #66 into the active sites
of hDHFR (PDB code, 1pd8) and the modelled SmDHFR. The three-
dimensional structure of compound #66 was obtained from VEGA
ZZ program using TRIPOS and Gasteiger for force field and charge
calculations, respectively [16].
3. Results
3.1. Synthesis of SmDHFR inhibitor library
Preliminary docking was carried out using FlexX. The top ten
structures with the best scores from FlexX were redocked and their
binding energy distribution was visualised by HYDE. LeadIT 2.0.1
package that contains FlexX and HYDE is commercially available
via BioSolveIT. HYDE is an innovative redocking and lead optimi-
sation program that determines protein–ligand binding energies
based on two factors: hydrogen bonding and dehydration [17]. Here
we exploited HYDE to understand binding of the lead compound
to the homology model SmDHFR, since the HYDE scoring function
is known to be suitable for substantially improving docking and
reducing false positives [17]. The docking results were visualised
and illustrated by Chimera 1.8.1 [18]. In PROCHECK, the Ramachan-
dran plot contained 87% residues in favourable regions, whilst most
of the remaining residues were in allowed regions and none were
in disallowed regions. In addition, the overall G-factor measur-
ing the stereochemical property was 0.17, significantly above the
threshold of −0.5 [19]. Furthermore, VERIFY3D showed 98% of the
residues with an averaged 3D-1D score >0.2 [20]. Likewise, the
QMEAN of the model was 0.79 and the QMEAN Z-Score was 0.26.
Thus the QMEAN score of the homology model SmDHFR differed
To identify new derivatives that inhibit S. mutans biofilms, a new
library of TMQ analogues was assembled and used in the screening.
The general scheme employed for the synthesis of the new deriva-
tives is represented by the synthesis of four TMQ analogues (#66,
#151, #153 and #154) identified in this study (Fig. 1A), and the
structure of each derivative is outlined (Fig. 1B).
3.2. Identification of small-molecule compounds that selectively
inhibit biofilm formation of Streptococcus mutans
To identify desirable inhibitors for S. mutans biofilms, the library
of 100 TMQ derivatives was screened by a biofilm assay described
previously [14]. Three of the most potent small-molecule ana-
logues (#66, #151 and #153) (Fig. 1B) were identified and were
further evaluated in biofilm assays and were found to inhibit S.
mutans biofilm formation as well as cell growth. The MICs of the
three analogues were 0.781, 1.56 and 1.56 ␮M, respectively, whilst
MICs for the control compound TMQ and the least active analogue
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(A)
100
#
growth
biofilm
80
60
40
20
0
#
#
#
#
*
*
#
*
*
*
#
*
#
*
*
#
#
TMQ
#66
Compounds
#151
#153
(B)
100
80
60
40
20
0
*
*
#
*
#
*
*
#
*
#
*
*
#
#
growth
biofilm
TMQ
#66
#151
#153
Compounds
(C)
100
80
60
40
20
0
#
growth
biofilm
#
*
*
*
#
#
*
#
#
#
#
*
*
*
*
TMQ
#66
Compounds
#151
#153
Fig. 2. Effects of trimetrexate (TMQ) and its analogues (#66, #151 and #153) on cell growth and biofilm formation of commensal streptococci. (A) Streptococcus mutans, (B)
Streptococcus sanguinis and (C) Streptococcus gordonii were treated with TMQ and its analogues at different concentrations and cell growth and biofilm formation of each
strain were determined. The percentage cell growth or biofilm formation was normalised to the dimethyl sulphoxide (DMSO) control groups (100%). Values represent the
mean standard deviation from three independent experiments. *Statistically significant difference observed in cell growth; #statistically significant difference observed in
biofilm formation.
#154 were 48.8 nM and 50 ␮M, respectively. The follow-up dose-
dependent studies determined the precise IC50 for each compound
(Table 1). Analogue #66 inhibited S. mutans growth and biofilm at a
low submicromolar range, whereas a higher dose of #151 and #153
was required to inhibit biofilm formation. TMQ inhibited bacterial
growth and biofilm formation more effectively than its analogues.
Among the new analogues, #66 exhibited the most potent activ-
ity with an inhibitory IC50 for biofilm at 454.0 10.2 nM, whilst
the IC50 for planktonic cells was much higher at 604.1 4.5 nM
(P < 0.05). Consistently, #151 and #153 also possessed similar mod-
est selectivity toward inhibiting biofilm formation, whilst #154 and
the control TMQ did not have any selectivity.
To determine whether the identified inhibitors had any selec-
tivity against S. mutans, the effect of these TMQ analogues on cell
growth and biofilm formation of commensal streptococci (S. san-
guinis and S. gordonii) at the concentration that inhibited biofilm
formation of S. mutans was evaluated. TMQ did not inhibit biofilm
formation of S. sanguinis and S. gordonii (P > 0.05) at 1.56 ␮M. Sim-
ilarly #66, #151 and #153 had no inhibitory effect on biofilm
formation of S. sanguinis and S. gordonii (P > 0.05) at 6.25 ␮M. #153
failed to inhibit biofilm formation of S. sanguinis even at a higher
concentration of 12.5 ␮M (P > 0.05) (Fig. 2). These data suggest that
the identified compounds did possess selectivity towards inhibiting
S. mutans.
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Table 1
the inhibition acted via inhibiting DHFR activity. The inhibitory
activities of four analogues (#66, #151, #153 and #154) and TMQ,
used as a positive control, were determined by kinetic assays estab-
lished above.
Inhibitory activity against Streptococcus mutans growth, biofilm and S. mutans dihy-
drofolate reductase (SmDHFR).
Compound
IC50 (nM)
Growth
Four inhibitors that were potent in vivo were also potent in vitro
in the enzymatic kinetic assay (Tables 1 and 2). The biofilm IC50 val-
ues of potent analogues #66, #151 and #153 ranged from 0.45 ␮M
to 1.1 ␮M, whilst the SmDHFR IC50 was ca. 10 nM. The IC50 values of
the least effective analogue #154 for the biofilm and SmDHFR were
>100-fold higher than those of the analogue #66. Compared with
the active analogues #66, #151 and #153, the analogue #154 had
much higher IC50 for both SmDHFR and biofilm, suggesting that the
biofilm IC50 correlated with the SmDHFR IC50. These data suggest
that SmDHFR is the target for TMQ analogues in the inhibition of
bacterial growth and biofilm formation.
The SIs of these inhibitors were determined by the IC50 ratio
of hDHFR over SmDHFR. Although the IC50 value of SmDHFR for
analogue #66 (8.7 1.9 nM) was ca. 4-fold higher than that of
TMQ (2.2 0.7 nM), the selectivity of the analogue #66 (117.8) was
much greater than that of TMQ (3.1) towards inhibiting S. mutans
(Table 2). In addition, the DHFR selectivity by the analogue #66
was also greater than that of analogues #151 and #153 (117.8 vs.
7.5 and 6.5) (Table 2). Furthermore, the inhibitor constant Ki as
an indicator for the potency of each analogue was also evaluated
(Fig. 4; Supplementary Fig. S2; Table 2). The Ki of SmDHFR for ana-
logue #66 was only ca. 2-fold higher than that of TMQ, but analogue
#66 had the best SI(Ki) (26.7 vs. 1.4). The SI(Ki) of compound #66
(26.7) was much greater than that of the analogues #151 (1.8) and
#153 (1.4), further indicating that analogue #151 and #153 had
lower selectivity against hDHFR. These data further demonstrated
that compound #66 was the most potent and selective analogue
towards inhibiting SmDHFR.
Biofilm
SmDHFR
8.7 1.9
7.1 0.9
8.2 0.9
1074.8 150.4
2.2 0.7
#66
604.1 4.5
454.0 10.2*
#151
#153
#154
TMQ
1505.5 28.5
1352.7 57.5
51 518.5 260.2
42.48 3.22
1127.9 13.1*
942.1 3.5*
71 026.3 360.4
49.08 4.1
IC50, 50% inhibitory concentration; TMQ, trimetrexate. Streptococcus mutans was
treated with dimethyl sulphoxide (DMSO) (a negative control), TMQ (a positive con-
trol) or the library analogues. Cell growth and biofilm formation were determined.
Lead compounds (TMQ and its analogues #66, #151, #153 and #154) were selected
to examine their effects on SmDHFR enzymatic activity. The IC50 value of cell growth,
biofilm formation and SmDHFR in the analogue-treated groups was calculated with
a series of diluted analogue compounds. Values represent the mean standard devi-
ation from three independent experiments.
*
Statistically significant difference observed from the various comparisons.
3.3. Characterisation of recombinant Streptococcus mutans and
human DHFR
To determine whether the identified analogues inhibited biofilm
formation of S. mutans via inhibiting the activity of DHFR as
designed, and to evaluate the selectivity of the analogues against
hDHFR, recombinant SmDHFR and hDHFR were first expressed and
purified and the recombinant enzymes were then characterised.
Both DHFRs exist as a monomer as observed from the gel filtration
profile with an estimated molecular weight of 17 kDa and 18 kDa,
respectively (data not shown). Enzymatic parameters were charac-
terised as described previously [9].
First, the optimal concentration of SmDHFR to reach linear reac-
tion velocity at the first 2 min was determined (0.001 ␮g/␮L). This
concentration was then used to characterise the enzyme. SmDHFR
has its maximal activity at pH 7.0. The activity decreased by ca.
50% from pH 6.8 to pH 8.0 (Fig. 3). The hDHFR optimal reaction
conditions were also determined in the same manner. The hDHFR
enzyme reaction reached maximal velocity at pH 7.3 in 50 mM
KH2PO4 buffer with 0.0015 ␮g/␮L of purified hDHFR. The purified
hDHFR is stable and soluble at concentrations up to 25 mg/mL at
4 ◦C and is catalytically active at room temperature. These param-
eters allowed us to determine the effects of the TMQ analogues on
both DHFRs.
3.5. Analogue #66 bound tighter to SmDHFR than to hDHFR
To explain the biological activity and observed selectivity of
TMQ analogues towards inhibiting SmDHFR versus hDHFR, homol-
ogy modelling and docking analysis were employed since the
crystal structure of SmDHFR is not available. A homology model of
SmDHFR was generated based on an available X-ray crystal struc-
ture of SpDHFR (PDB code, 3ix9) [22]. SpDHFR shares 55% sequence
identity with SmDHFR (Fig. 5). The overall predicated structure of
SmDHFR is very similar to the X-ray crystal structure from SpDHFR.
Superposition of the two enzymes showed that the RMSD differ-
ence only concentrated in one loop region (SmDHFR, 135 FPLRDFSS
142, and SpDHFR 130 FPAEFDLSL 138) (Fig. 6). It is known that
loops have a tendency to change their conformation upon crys-
tal contact, thus the model prediction is likely to be correct. In
addition, the loop is far away from the active site, and the super-
position of the active site residues exhibited significantly small
RMSD.
3.4. Trimetrexate analogues selectively inhibited SmDHFR
Analogues identified for their ability to inhibit bacterial growth
and biofilm formation were further assessed to determine whether
25
#
20
The structure of the analogue compound #66 generated by
VEGA ZZ was docked into the active sites of hDHFR and SmDHFR
using FlexX and HYDE (Fig. 7). A summary of the results (Table 3)
shows the free energy contribution of each atom and fragment
to the binding of SmDHFR and hDHFR to analogue #66. The free
energy calculation indicated that analogue #66 bound to SmDHFR
at a nanomolar range and to hDHFR at a micromolar range. To
analyse the docking details, the contribution of each numbered
atom to the ꢀG values was assessed. The accumulated score for
2,4-diaminoquinazoline scaffold (atoms 1–12) was −19.6 kJ/mol
for SmDHFR versus −12.5 kJ/mol for hDHFR, and the methyl amine
linker (atoms 13–14) contributed −3.0 kJ/mol to SmDHFR versus
0.7 kJ/mol to hDHFR, whilst phenyl groups (atoms 15–36) exhibited
almost the same binding energy.
15
10
5
0
6.8
7.0
7.5
8.0
pH values
Fig. 3. Effect of pH on Streptococcus mutans dihydrofolate reductase (SmDHFR)
enzymatic activity. The assay was carried out in 30 ␮M dihydrofolate (DHF) and
0.001 ␮g/␮L SmDHFR with varying pH values. #SmDHFR activity in the reaction at
pH 7 was significantly different from the activities measured at other pH conditions.
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Table 2
Inhibitory activity against Streptococcus mutans dihydrofolate reductase (SmDHFR) and human DHFR (hDHFR).
Compound
SmDHFR
hDHFR
SIc
IC50 (nM)a
Ki (nM)b
IC50 (nM)a
Ki (nM)b
IC50
Ki
#66
8.7 1.9
7.1 0.9
8.2 1.4
2.2 0.7
3.4 0.7
3.0 0.1
3.6 0.1
1.3 0.4
1027.0 137.8
52.8 6.9
53.5 6.8
91.7 4.6
5.5 1.0
4.9 1.5
1.88
117.8
7.5
6.5
26.7
1.8
1.4
#151
#153
TMQ
6.87 0.52
3.1
1.4
IC50, 50% inhibitory concentration; Ki, inhibitory constant; SI, selectivity index.
a
IC50 = Ci/(Vo/Vi − 1), where Vo and Vi are the initial velocities in the absence and presence of inhibitor, respectively, and Ci is the concentration of inhibitor.
b
c
Ki calculation uses the Michaelis–Menten equation for competitive inhibition.
SI is defined as IC50(hDHFR)/IC50(SmDHFR) or Ki(hDHFR)/Ki (SmDHFR).
(A)
0.06
0
0.05
7.5nM
15nM
20nM
0.04
0.03
0.02
Vmax = 166.67
0.01
Km = 19.58
Ki = 3.43
0
-0.01
0
0.01
0.02
0.03
0.04
0.05
1/[DHF] (μM)
(B)
0.05
0.04
0.03
0.02
0.01
0
0
500nM
750nM
1000nM
Vmax = 68.97
Km = 2.83
Ki = 91.69
-0.02
0
0.02
0.04
0.06
1/[DHF] (μM)
Fig. 4. Inhibition of (A) Streptococcus mutans dihydrofolate reductase (SmDHFR) and (B) human DHFR (hDHFR) by analogue #66. The enzymatic activity of DHFR was measured
at a series of fixed concentrations of dihydrofolate (DHF) (20–40 ␮M) with varying concentrations of analogue #66. The reaction rate was determined by monitoring
the decrease in absorbance at 340 nm [oxidation of NADPH to NADP+ and reduction of DHF to tetrahydrofolate (THF)] (coupled ε = 12.260 M−1 cm−1) using a UV–visible
spectrophotometer. The double-reciprocal plots were used to calculate the reaction constant (Km) and the inhibitory constant (Ki) for the inhibition of SmDHFR and hDHFR
by the analogue.
SmDHFR
SpDHFR
6
1
EKKIVAIWAEDDRHLIGVKGSLPWHLPKELNHFKEMTMGQALLMGRVTFDGMNRRVLPGR 65
TKKIVAIWAQDEEGVIGKDNRLPWYLPAELQHFKETTLNHAIL--MGRVTFDGMGRRLLPKR 60
* * *
* * * * * *
*
** * * * *
*
*
*
*
*
*
SmDHFR 66 DTLILTHDPQFKAEGVSIVHSVPEALSWYRQQDKSLFIVGGASIYKAFESYYDKIIKTSV 125
SpDHFR 61 ETLILTRNPEEKIDGVATFHDVQSVLDWYSAQEKNLYIVGGKQIFQAFEPYLDEVIVTHI 120
*
*
SmDHFR 126 HGQFEGDTYFPL-RDFSSFQEISQAFFEKDENNSHDFTVTVLEKKE 170
SpDHFR 121 HARVEGDTYFPAEFDLSLFETVSSKFYTKDEKNPYDFTIQYRKRKE 166
Fig. 5. Sequence alignment of Streptococcus pneumoniae dihydrofolate reductase (SpDHFR) and Streptococcus mutans DHFR (SmDHFR). SpDHFR shares 55% overall sequence
identity and 79% active-site identity with SmDHFR. Stars highlight the identical active sites of SpDHFR and SmDHFR.
4. Discussion
diverse bacteria [14]. However, the inhibitory mechanisms of these
molecules are not well understood. In this study, we identified
new small molecules that inhibited a well-defined target, DHFR.
DHFR has been proven to be a viable target [8,10–13]. TMQ is a
A variety of natural products [23,24] and semisynthetic small
molecules have been shown to inhibit biofilm formation of
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Table 3
Analogue #66 with numbered atoms, and table with HYDE score for free energy
binding of the analogue molecule #66 to Streptococcus mutans dihydrofolate reduc-
tase (SmDHFR) and human DHFR (hDHFR).
36
33
35
21
15
11
O
31
16
34
26
NH2
17
23
32
25
7
13
1
14
N
H
6
2N
O
18
20
24
10
9
27
19
22
28
30
3
5
H2N
N
29
8
4
12
Atom #
HYDE score
SmDHFR
hDHFR
1
2
−2.2
1.6
−1.5
3.7
3
4
5
6
7
8
9
−0.9
−3.8
−0.2
−0.5
−2.9
−1.2
−2.7
−0.1
−2.5
−4.2
−3.9
0.9
−0.2
−2.2
−0.2
−0.7
−1.5
−0.4
−0.6
0.2
−1.1
−5.8
−0.3
−0.2
−0.2
−1.3
−4.0
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
5.6
−7.4
−0.5
1.2
−2.7
−1.4
−0.8
−1.9
−1.1
−1.0
0.6
−2.7
−2.2
−1.7
−3.4
−0.1
−1.2
−0.4
0.8
−0.4
−0.1
−1.3
0
Fig. 6. Superposition of the homology model of Streptococcus mutans dihydrofo-
late reductase (SmDHFR) and template Streptococcus pneumoniae DHFR (SpDHFR).
Homology model SmDHFR (green) and 3D structure of SpDHFR (orange) are super-
˚
posed. With an overall root-mean-square deviation (RMSD) value of 1.76 A, the only
difference between the model and the target is shown in the box. The location is far
away from the ligand MTX (purple), a potential enzymatic active site of SpDHFR.
The active site superposition of SpDHFR and SmDHFR has a root-mean-square devi-
ation (RMSD) of 4.97 × 10−4 (range: SmDHFR amino acid residues 12–123, SpDHFR
residues 8–119). The image and calculation were generated using VMD 1.9.1.
0
−6.6
−2.6
−0.3
−2.3
−2
[9]. Attempts to enhance the potency and selectivity of TMQ have
achieved limited success until recently [27,28]. In contrast to TMQ,
the SI of the analogue #66 against SmDHFR over hDHFR reached
117.8, indicating its potential as a leading compound to develop an
anti-S. mutans drug.
−0.3
−0.1
−0.1
−0.1
−0.1
0
−0.9
−0.7
−0.2
−0.7
Despite the low selectivity, TMQ is very potent against S. mutans.
This is partly due to the lipophilic nature of TMQ, which allows it to
readily permeate the cell envelop of S. mutans [29]. This feature can
be harnessed to increase the potency of our lead compounds. Ana-
logues #66, #151 and #153 did not inhibit cell growth or biofilm
formation of the commensal streptococci at the IC50 value, whereas
S. mutans was significantly inhibited, suggesting that the analogues
possessed good selectivity. Another unique feature of the analogue
#66 is its ability to selectively inhibit biofilm formation as well. It
exhibited a greater selectivity in inhibiting S. mutans biofilm forma-
tion than TMQ. These features can be explored in the further design
of more selective #66 derivatives.
To ascertain why the analogue compound #66 possessed high
selectivity, the homology modelled SmDHFR and docking free
energy were used to determine the binding affinity and selectiv-
ity of the analogue #66 by FlexX docking and HYDE redocking
(Fig. 7). The docking predicts that the analogue compound #66
binds to SmDHFR in a nanomolar range and to hDHFR in a micro-
molar range, with an estimated 100-fold selectivity. Based on the
free energy distribution, the discrimination largely stems from the
2,4-diaminoquinazoline scaffold and in part from the methyl amine
linker. Perhaps binding of the 2,4-diaminoquinazoline scaffold and
methyl amine linker are affected by the binding of bulky phenyl
−1.1
1–12
13–14
15–36
−19.6
−3
−12.5
0.7
−21.9
−22.8
Total ꢀG
−44.5
−34.6
promising lipophilic antifolate drug [25]. Despite being an FDA-
approved drug, TMQ has a very low selectivity against hDHFR and is
thus not a desirable anti-S. mutans drug. The three analogues (#66,
#151 and #153) identified retain the key structural components
of TMQ, namely a 2,4-diaminoquinazoline scaffold, N-linker and
a modified phenyl ring (Fig. 1B). As a result, all three compounds
exhibit excellent inhibition against SmDHFR despite the activity not
being as potent as that of TMQ. Importantly, analogue #66 is highly
selective against SmDHFR versus hDHFR, being 38-fold greater than
TMQ in its selectivity. In addition, it exhibits almost 26-fold higher
potency toward inhibiting SmDHFR. Although the cytotoxicity of
the analogue is unknown, it likely exhibits less adverse effects on
human cells than TMQ owning to its high selectivity.
TMQ blocks the DHFR activity both of Trypanosoma cruzi and P.
carinii [9,26]. However, the selectivity of this compound is very low
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Fig. 7. Docking orientation of the analogue compound #66 into the active sites of (A) human dihydrofolate reductase (hDHFR) and (B) Streptococcus mutans DHFR (SmDHFR).
Protein structural models are shown in cartoon diagrams. Ligand and active site residues are presented as stick models (colour code: orange/grey – C, red – O, blue – N). The
analogue ligand #66 was docked with FlexX and was then redocked by HYDE. Images were generated using Chimera 1.8.1.
rings, since the other analogues #151 and #153 did not have signif-
icant selectivity despite their structural resemblance. Free-energy
simulations and binding affinity determination based on homol-
ogy modelling have been successfully used in the study of DHFR of
Plasmodium falciparum [29]. The current model prediction is con-
sistent with the experimental finding, demonstrating the validity
of the approach. Recently structure-based design has led to the
development of new DHFR inhibitors that are potent and selective
against Staphylococcus aureus in vitro and in vivo [30], indicating
the potential of developing more potent and selective antifolate
drugs.
(NIDCR/NIH) [R01 DE022350] and an International Association for
Dental Research/GlaxoSmithKline (IADR/GSK) Innovation in Oral
Care Award.
Competing interests: None declared.
Ethical approval: Not required.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ijantimicag.2015.
03.015
Two strategies can be employed to improve the activity of
TMQ analogues against SmDHFR. One focuses on improving both
potency and selectivity of the inhibitors by a structure-based
design, whilst a complementary strategy will be to enhance sol-
ubility and permeability of the lead compounds. These efforts
would be greatly facilitated by obtaining X-ray crystal structures
of the DHFR–inhibitor complexes. Introduction of the bulky ben-
zyl groups dramatically increases the selectivity of SmDHFR over
hDHFR. Further structural modification may not only increase the
selectivity but also enhance their potency in inhibiting bacterial
biofilms.
In conclusion, the TMQ analogues identified in this study selec-
tively inhibited cell growth and biofilm formation of S. mutans
biofilms. The 100-fold increase in the selectivity of this antifo-
late analogue against SmDHFR versus hDHFR is greater than those
ever reported for DHFR inhibitors of a number of different bacteria
[8,9,11,13]. Therefore, this analogue is a promising lead to develop
effective antifolate therapeutic drugs for the prevention and treat-
ment of dental caries.
References
[1] Loesche WJ. Role of Streptococcus mutans in human dental decay. Microbiol Rev
1986;50:353–80.
[2] Hada LS, Kakiuchi N, Hattori M, Namba T. Identification of antibacterial
principles against Streptococcus mutans and inhibitory principles against glu-
cosyltransferase from the seed of Areca catechu L. Phytother Res 1989;3:140–4.
[3] Ooshima T, Izumitani A, Minami T, Yoshida T, Sobue S, Fujiwara T, et al. Non-
cariogenicity of maltitol in specific pathogen-free rats infected with mutans
streptococci. Caries Res 1992;26:33–7.
[4] Hamada S, Horikoshi T, Minami T, Kawabata S, Hiraoka J, Fujiwara T, et al. Oral
passive immunization against dental caries in rats by use of hen egg yolk anti-
bodies specific for cell-associated glucosyltransferase of Streptococcus mutans.
Infect Immun 1991;59:4161–7.
[5] Kakiuchi N, Hattori M, Nishizawa M, Yamagishi T, Okuda T, Namba T. Studies
on dental caries prevention by traditional medicines. VIII. Inhibitory effect of
various tannins on glucan synthesis by glucosyltransferase from Streptococcus
mutans. Chem Pharm Bull (Tokyo) 1986;34:720–5.
[6] Robinette RA, Heim KP, Oli MW, Crowley PJ, McArthur WP, Brady LJ. Alter-
ations in immunodominance of Streptococcus mutans AgI/II: lessons learned
from immunomodulatory antibodies. Vaccine 2014;32:375–82.
[7] Schnell JR, Dyson HJ, Wright PE. Structure, dynamics, and catalytic function of
dihydrofolate reductase. Annu Rev Biophys Biomol Struct 2004;33:119–40.
[8] Gonen N, Assaraf YG. Antifolates in cancer therapy: structure, activity and
mechanisms of drug resistance. Drug Resist Updat 2012;15:183–210.
[9] Senkovich O, Bhatia V, Garg N, Chattopadhyay D. Lipophilic antifolate trime-
trexate is a potent inhibitor of Trypanosoma cruzi: prospect for chemotherapy
of Chagas’ disease. Antimicrob Agents Chemother 2005;49:3234–8.
[10] Roth B. Design of dihydrofolate reductase inhibitors from X-ray crystal struc-
tures. Fed Proc 1986;45:2765–72.
Acknowledgement
The authors would like to thank Dr Christian Lemmen for advice
related to LeadIT.
Funding: This study was funded by the National Institute of
Dental and Craniofacial Research, National Institutes of Health
[11] Suling W, Reynolds R, Barrow E, Wilson L, Piper J, Barrow W. Susceptibilities
of Mycobacterium tuberculosis and Mycobacterium avium complex to lipophilic
Please cite this article in press as: Zhang Q, et al. New small-molecule inhibitors of dihydrofolate reductase inhibit Streptococcus mutans.
Int J Antimicrob Agents (2015), http://dx.doi.org/10.1016/j.ijantimicag.2015.03.015

G Model
ANTAGE-4578; No. of Pages9
ARTICLE IN PRESS
Q. Zhang et al. / International Journal of Antimicrobial Agents xxx (2015) xxx–xxx
9
deazapteridine derivatives, inhibitors of dihydrofolate reductase. J Antimicrob
Chemother 1998;42:811–5.
[12] Rosowsky A, Mota CE, Wright JE, Freisheim JH, Heusner JJ, McCormack JJ, et al.
2,4-Diaminothieno[2,3-d]pyrimidine analogues of trimetrexate and piritrexim
as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydro-
folate reductase. J Med Chem 1993;36:3103–12.
[22] Lee J, Yennawar NH, Gam J, Benkovic SJ. Kinetic and structural characteriza-
tion of dihydrofolate reductase from Streptococcus pneumoniae. Biochemistry
2010;49:195–206.
[23] Duarte S, Gregoire S, Singh AP, Vorsa N, Schaich K, Bowen WH, et al. Inhibitory
effects of cranberry polyphenols on formation and acidogenicity of Streptococ-
cus mutans biofilms. FEMS Microbiol Lett 2006;257:50–6.
[13] Fischi MA, Dickinson GM, La Vole L. Safety and efficacy of sulfamethoxazole and
trimethoprim chemoprophylaxis for Pneumocystis carinii pneumonia in AIDS.
JAMA 1988;259:1185–9.
[24] Percival RS, Devine DA, Duggal MS, Chartron S, Marsh PD. The effect of cocoa
polyphenols on the growth, metabolism, and biofilm formation by Streptococ-
cus mutans and Streptococcus sanguinis. Eur J Oral Sci 2006;114:343–8.
[14] Liu C, Worthington RJ, Melander C, Wu H. A new small molecule specifically
inhibits the cariogenic bacterium Streptococcus mutans in multispecies biofilms.
Antimicrob Agents Chemother 2011;55:2679–87.
[15] Zhou M, Zhu F, Li Y, Zhang H, Wu H. Gap1 functions as a molecular chaperone
to stabilize its interactive partner Gap3 during biogenesis of serine-rich repeat
bacterial adhesin. Mol Microbiol 2012;83:866–78.
[16] Pedretti A, Villa L, Vistoli G. VEGA: a versatile program to convert, handle
and visualize molecular structure on Windows-based PCs. J Mol Graph Model
2002;21:47–9.
[17] Schneider N, Hindle S, Lange G, Klein R, Albrecht J, Briem H, et al. Substantial
improvements in large-scale redocking and screening using the novel HYDE
scoring function. J Comput Aided Mol Des 2012;26:701–23.
[18] Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al.
UCSF Chimera—a visualization system for exploratory research and analysis. J
Comput Chem 2004;25:1605–12.
[19] Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM. AQUA
and PROCHECK-NMR: programs for checking the quality of protein structures
solved by NMR. J Biomol NMR 1996;8:477–86.
[20] Eisenberg D, Lüthy R, Bowie JU. VERIFY3D: assessment of protein models with
three-dimensional profiles. Methods Enzymol 1997;277:396–404.
[21] Benkert P, Biasini M, Schwede T. Toward the estimation of the absolute quality
of individual protein structure models. Bioinformatics 2011;27:343–50.
[25] Blanke CD. A double-blind placebo-controlled randomized phase III trial
of 5-fluorouracil and leucovorin, plus or minus trimetrexate, in previously
untreated patients with advanced colorectal cancer. Ann Oncol 2002;13:
87–91.
[26] Kovacs JA, Allegra CJ, Swan JC, Drake JC, Parrillo JE, Chabner BA, et al. Potent
antipneumocystis and antitoxoplasma activities of piritrexim, a lipid-soluble
antifolate. Antimicrob Agents Chemother 1988;32:430–3.
[27] Schormann N, Velu SE, Murugesan S, Senkovich O, Walker K, Chenna BC, et al.
Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihy-
drofolate reductase. Bioorg Med Chem 2010;18:4056–66.
[28] White EL, Ross LJ, Cunningham A, Escuyer V. Cloning, expression, and character-
ization of Mycobacterium tuberculosis dihydrofolate reductase. FEMS Microbiol
Lett 2004;232:101–5.
[29] Gangjee A, Vidwans AP, Vasudevan A, Queener SF, Kisliuk RL, Cody V, et al.
Structure-based design and synthesis of lipophilic 2,4-diamino-6-substituted
quinazolines and their evaluation as inhibitors of dihydrofolate reductases and
potential antitumor agents. J Med Chem 1998;41:3426–34.
[30] Lam T, Hilgers M, Cunningham ML, Kwan BP, Nelson KJ, Brown-Driver
V, et al. Structure-based design of new dihydrofolate reductase antibac-
terial agents: 7-(benzimidazol-1-yl)-2,4-diaminoquinazolines. J Med Chem
2014;57:651–68.
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