Discovery of lead compounds targeting the bacterial sliding clamp using a fragment-based approach

Article abstract of DOI:10.1021/jm500122r

The bacterial sliding clamp (SC), also known as the DNA polymerase III β subunit, is an emerging antibacterial target that plays a central role in DNA replication, serving as a protein-protein interaction hub with a common binding pocket to recognize linear motifs in the partner proteins. Here, fragment-based screening using X-ray crystallography produced four hits bound in the linear-motif-binding pocket of the Escherichia coli SC. Compounds structurally related to the hits were identified that inhibited the E. coli SC and SC-mediated DNA replication in vitro. A tetrahydrocarbazole derivative emerged as a promising lead whose methyl and ethyl ester prodrug forms showed minimum inhibitory concentrations in the range of 21-43 μg/mL against representative Gram-negative and Gram-positive bacteria species. The work demonstrates the utility of a fragment-based approach for identifying bacterial sliding clamp inhibitors as lead compounds with broad-spectrum antibacterial activity.

Full text of DOI:10.1021/jm500122r

Article
pubs.acs.org/jmc
Discovery of Lead Compounds Targeting the Bacterial Sliding Clamp
Using a Fragment-Based Approach
Zhou Yin,† Louise R. Whittell,† Yao Wang,† Slobodan Jergic,† Michael Liu,‡ Elizabeth J. Harry,‡
Nicholas E. Dixon,† Jennifer L. Beck,† Michael J. Kelso,† and Aaron J. Oakley*,†
†School of Chemistry and Centre for Medical and Molecular Bioscience, University of Wollongong, Wollongong, New South Wales
2522, Australia
‡ithree Institute, University of Technology, Sydney, New South Wales 2007, Australia
S
* Supporting Information
ABSTRACT: The bacterial sliding clamp (SC), also known as the
DNA polymerase III β subunit, is an emerging antibacterial target
that plays a central role in DNA replication, serving as a protein−
protein interaction hub with a common binding pocket to recognize
linear motifs in the partner proteins. Here, fragment-based screening
using X-ray crystallography produced four hits bound in the linear-
motif-binding pocket of the Escherichia coli SC. Compounds
structurally related to the hits were identified that inhibited the E.
coli SC and SC-mediated DNA replication in vitro. A tetrahy-
drocarbazole derivative emerged as a promising lead whose methyl
and ethyl ester prodrug forms showed minimum inhibitory
concentrations in the range of 21−43 μg/mL against representative
Gram-negative and Gram-positive bacteria species. The work demonstrates the utility of a fragment-based approach for
identifying bacterial sliding clamp inhibitors as lead compounds with broad-spectrum antibacterial activity.
considered challenging due in part to the shape and large
surface areas of such sites.24 Nevertheless, an increasing number
of inhibitors of PPIs are being reported, with recent examples
including inhibitors of clathrin,25 the polo-box domain of polo-
like kinase 1,26 and 14-3-3 protein.27
INTRODUCTION
■
The emergence of antibiotic-resistant bacteria has presented a
great challenge to humanity,1 with new classes of antibiotics
acting on novel targets being urgently needed.2−4 The bacterial
sliding clamp (SC), also known as the DNA polymerase III
(Pol III) β subunit, is a torus-shaped homodimeric protein5
that is conserved across bacterial species.6−8 The SC serves as a
protein−protein interaction (PPI) hub during bacterial DNA
replication and repair,9,10 surrounding double-stranded DNA
and subsequently recruiting a diverse range of protein binding
partners, including the δ and α subunits of DNA Pol III, DNA
Pols I, II, IV, and V, and MutS.5,9 The pivotal role it plays in
bacterial DNA replication and repair, its conserved structure
across bacterial species,6−8 and its structural divergence from
the equivalent human protein (proliferating cell nuclear
antigen, PCNA)9,11 make the SC a highly attractive target for
producing new antibiotics acting via a novel mechanism.
Proteins that interact with the SC recognize a surface binding
pocket that consists of two subsites (I and II),6,12,13 with one
pocket located on each of the SC monomers (Figure 1). These
protein binding partners interact with the SC using linear
motifs (LMs) located on their surfaces.5,13,14 LMs are short (4−
10-residue), intrinsically disordered sequences most often
found at the termini of proteins but sometimes in loop
regions.15−17 Binding of LMs to rigid protein domains
represents a distinct category of PPIs that generally display
weak affinities (Kd ≈ 1−100 μM).18−23 PPIs are attractive drug
targets, but the development of small-molecule inhibitors is
A consensus LM sequence, QLx1Lx2F/L (S/D preferred at
x1; x2 may be absent), has been identified that interacts with the
SC LM-binding pocket, and isolated peptides based on this
sequence have been shown to bind the SC (Figure 1A) with
affinities similar to those of their parent proteins,28−30 and
conversely, when LMs are removed from the parent proteins,
they lose all SC affinity.13,31 Our recent work proposed a
mechanism of sequential binding of LMs to the Escherichia coli
SC highlighting the role of subsite I as the “anchor site” for LM
recognition.30 Two classes of small molecules have previously
been reported that bind to subsite I (Ki > 10 μM), but no
antibacterial activities were provided for these compounds.12,28
In this work, X-ray crystallography was used as a primary
screen to identify fragment hits against the E. coli SC. Binding
poses adopted by these hits were used in combination with
chemoinformatics to guide subsequent inhibitor design, with
several promising compounds being identified that bind to the
SC, disrupt LM−SC interactions, and inhibit SC-dependent
DNA replication in vitro. Antibacterial activity was demon-
Received: January 23, 2014
Published: March 4, 2014
© 2014 American Chemical Society
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Several other fragments were identified that bind in subsite I,
but they showed only weak electron density and were not
investigated further. An additional six fragments were identified
that bound only in the binding pocket of chain B, where they
were stabilized by a symmetry-related protein molecule. These
were considered to be crystallographic artifacts and were not
pursued.
The four fragment hits bound in the deep region of subsite I,
forming van der Waals interactions with the hydrophobic side
chains of V360, V247, L177, and M362 (Figure 2). The shallow
region of subsite I, comprising residues T172, P242, and L155,
remained vacant (Figures 1B and 2). Each fragment positioned
an aromatic ring in the narrow groove of subsite I, with the
fluoro and chloro substituents of 1 and 2 being buried in the
deep hydrophobic region (Figure 2A,B). Binding of both 1 and
2 caused rotation of the side chain of S346 relative to the apo-
SC structure. Fragments 3 and 4 bound similarly to 1 and 2,
placing their NO2 groups in the positions occupied by the
halogen atoms of 1 and 2 (Figure 2C,D). The NO2 groups
appeared to accept H-bonds from the side chain of S346,
allowing this residue to retain the same conformation it adopts
in the apo-SC structure.30
A fluorescence polarization (FP)-based competition assay
was used to quantify inhibition of an LM−SC interaction by the
four fragment hits.30 In this assay, a fluorescently labeled SC-
binding tracer peptide based on an LM derived from the
consensus-binding motif (5-carboxyfluorescein-QLDLF, Kd =
70 nM)12,32,33 was used as the competitive probe ligand.
Inhibition of tracer binding to the E. coli SC was plotted against
inhibitor concentrations, and IC50 values were calculated and
transformed into inhibition constants (Ki) using the Kenakin
correction for ligand depletion.34 None of the four hits 1−4
showed any significant inhibition of probe binding below 1 mM
under the assay conditions (data not shown).
Identification of Fragments That Fully Occupy
Subsite I. Following identification of fragment hits 1−4, we
sought to improve binding affinity by identifying fragments that
could more completely occupy subsite I, including the shallow
region. It was noted that the fluoroaryl ring found in 1 was also
present in the previously reported biphenyl inhibitor,28
triggering further investigations with compound 5, a readily
available analogue containing the fluorobiphenyl moiety (Table
1). A Ki of 280 μM was measured for 5 using the FP assay30
(Table 1), and its X-ray cocrystal structure was solved in
complex with the SC (denoted SC5). Compound 5 was found
to fully occupy subsite I (Figure 3A; crystallographic data in
Table S2, Supporting Information), with its fluoroaryl ring
adopting a binding pose similar to that of 1. The side chain of
M362 was observed to rotate toward subsite II, possibly due to
steric crowding. The second phenyl ring of 5 occupied the
shallow region of subsite I, with its carboxylate group involved
in a salt bridge and/or H-bonds with the side chains of R152
and Y154. Both of these side chains move slightly toward the
carboxylate of 5 relative to their positions in the apo structure.
The ZINC library35 was subsequently searched for
compounds displaying structural similarity to fragments 1−5.
Selected molecules were subjected to a docking-based screen
using UCSF DOCK6.5,36 and candidates were purchased for
screening using the FP assay. Initial efforts failed to yield hits
with Ki < 1 mM, but it was noted during the work that 5-
chlorocarbazoles appeared promising. Further searches of the
ZINC library35 identified several 5-chlorocarbazoles, including
6, which showed a Ki of 199 μM (Table 1 and Table S3,
Figure 1. (A) X-ray crystal structures of consensus pentapeptide
AcQLALF bound to the E. coli SC (PDB entry 4K3P30). The complex
is shown with the SC colored orange and the peptide carbon atoms
light green. All other atoms are presented in CPK colors. Dashed lines
in red represent H-bonds. (B) Stereo view of the SC binding pocket
(PDB entry 4K3S,30 chain A). SC carbon atoms are shown shaded
white. All other atoms are presented in CPK colors. Electrostatic
potential surfaces of the binding site are shown with blue = positive
and red = negative. A crystallographic water molecule is shown as a red
sphere; arrows indicate the deep (red) and shallow (green) regions of
subsite I in (A) and (B).
strated for a lead inhibitor against two Gram-negative and two
Gram-positive bacterial species.
RESULTS
■
Fragment-Based Screening Using X-ray Crystallog-
raphy. A total of 352 fragment compounds from the First Pass
Screen (Zenobia Fragment Libraries) were soaked into E. coli
SC crystals as 4-in-1 cocktails and screened using X-ray
crystallography. Four fragment hits showing good electron
density, 3,4-difluorobenzamide (1), 5-chloroisatin (2), 6-
nitrobenzopyrazole (3), and 5-nitroindole (4), were found to
bind in subsite I of the binding pocket on chain A of the SC
dimer (complexes denoted as SC1, SC2, SC3, and SC4,
respectively, in Figure 2; crystallographic data are in Table
S1, Supporting Information). No significant changes in the SC
main-chain structure were observed upon fragment binding.
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Figure 2. X-ray cocrystal structures showing binding of fragment hits 1−4 to subsite I of the E. coli SC LM-binding pocket. The complexes denoted
(A) SC1, (B) SC2, (C) SC3, and (D) SC4 are shown with the SC carbon atoms colored orange and the bound fragment carbon atoms light green. All
other atoms are presented in CPK colors. Apo-SC structures (PDB entry 4K3S,30 chain A) are superimposed on the SC structures in the complexes
for comparison (shown in white). Hydrogen bonds from S346 to fragment nitro groups are represented as dashed red lines. Electron density maps
(2mFo − DFc) contoured at 1σ are shown in blue wire-basket form.
a
Table 1. Follow-Up Fragment Inhibitors of the E. coli SC Identified from Initial Hits 1−4
a
n/a = not available.
Supporting Information). The X-ray cocrystal structure of 6
was obtained, but the compound, although clearly binding in
subsite I, showed only weak electron density (data not shown).
The slight curvature at the base of subsite I (due to P242)
appeared likely to impair binding of rigid/planar compounds
such as 6, and it was postulated that ligands with increased
flexibility might better complement the curvature. Searches of
the ZINC library35 (Table S3, Supporting Information)
identified tetrahydrocarbazoles 7 and 8, which were found to
have Ki values of 216 and 64 μM, respectively (Table 1).
Compounds 9 and 10, the methyl and ethyl esters of 8,
displayed significantly weaker binding, highlighting the
importance of the acid group. Eighteen additional tetrahy-
drocarbazoles were synthesized and evaluated using the FP
assay, but all showed poor SC binding affinities (11−29, Table
S3 and Scheme S1, Supporting Information).
Compounds 7 and (R)-8 were both found to completely
occupy subsite I with their tetrahydrocarbazole rings nestled
against P242. Binding of 7 preserved the position of all residues
in subsite I and had no effect on the overall protein structure.
The carboxylate group of 7 made two H-bonds to the backbone
carbonyl oxygen atom of V247 and the hydroxyl group of S346.
Interestingly, while the commercially obtained carboxy-5-
chlorotetrahydrocarbazole (8) was racemic, only the (R)-
enantiomer appeared in the electron density map (Figure 3C).
The binding of (R)-8 caused conformation changes in the
M362 and S346 side chains relative to the apo structure and
buried the 5-chloro group in the hydrophobic deep region of
subsite I. The carboxyl group of 8 appeared to interact with the
guanidinium group of R152, which may explain the preference
for binding of the (R)-enantiomer in the crystal.
Ligand efficiency (LE) and ligand lipophilicity efficiency
(LLEAT)37 were calculated (Supporting Information) to assess
the suitability of these fragments for further medicinal
chemistry optimization (Table 1), where it is considered that
The X-ray cocrystal structures of 7 and 8 in complex with the
E. coli SC were solved (denoted as SC7 and SC8 in Figure 3B,C;
crystallographic data in Table S2, Supporting Information).
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Figure 3. X-ray cocrystal structures of 5, 7, and 8 bound to subsite I of the E. coli SC LM-binding pocket. Complexes are denoted (A) SC5, (B) SC7,
and (C) SC8 and are shown with the SC carbon atoms colored orange and the ligand carbon atoms light green. All other atoms are presented in
CPK colors. Apo-SC structures (PDB entries 4K3S,30 chains A in (B), and 1MMI,38 chain A in (A) and (C)) are superimposed (shown in white) on
the SC structures of the complexes for comparison. H-bonds are represented as dashed red lines. Electron density maps (2mFo − DFc) contoured at
1σ are shown in blue wire-basket form.
compounds with LE and LLEAT values greater than 0.3 are
desirable.37 Compounds 7 and 8 showed LLEAT values >0.4,
suggesting they are attractive scaffolds for further optimization.
Two E. coli SC inhibitors, a thioxothiazolidinyl derivative
(RU7)12 and a biphenyloxime ether derivative,28 that bind to
subsite I have previously been identified. Their binding poses
are compared with that of (R)-8 in Figure S1 (Supporting
Information). It is noteworthy that when superimposed, the
aryl ring systems of all three compounds are approximately
coplanar and project their halogens into the deep region of
subsite I (Figure 1B). While all three compounds show low-
micromolar affinity, RU7 and the biphenyloxime ether
derivative project large moieties toward bulk solvent that
appear to contribute few (if any) binding interactions. In the
case of RU7, the thiocarbonyl and carboxymethyl moieties are
oriented toward solvent, while the biphenyloxime ether
derivative projects its substituted cyclohexyl moiety toward
the solvent. Compound (R)-8, on the other hand, projects no
groups out into the solvent and better complements the
geometry of subsite I, thus suggesting it is a more suitable lead
for optimization studies.
incomplete dsDNA products. A complete dsDNA product is
formed when the whole of the ssDNA circle is replicated (see
Figure S4, Supporting Information, for the control experiment).
Compounds 5, 6, and 7 were found to inhibit replication at 500
or 1000 μM (Figure 4A−C), while compound 8 produced
complete inhibition at 250 μM (Figure 4D), consistent with its
higher SC affinity (Table 1).
The antibacterial effects of compounds 5 and 7−10 were
examined against two representative Gram-negative (E. coli and
Acinetobacter baylyi) and two Gram-positive (Bacillus subtilis
and Staphylococcus aureus) species. The background-corrected
E. coli SC Inhibitors Disrupt in Vitro DNA Replication
and Show Antibacterial Effects. An in vitro DNA
replication assay was used to assess inhibition of E. coli SC
function by compounds 5−8. The reaction system contained
the SC, Pol III α subunit, and reconstituted clamp loader
complex (γ3δδ′).39 During E. coli replication, the SC is loaded
onto DNA by the clamp loader complex (composed minimally
of the Pol III δ, γ/τ, and δ′ subunits)40−42 and confers high
processivity upon the Pol III α subunit (the replicase) by acting
as a mobile tether.43,44 Circular single-stranded M13 DNA
(ssDNA) with an annealed RNA primer was used as the
template for DNA synthesis and was coated with single-
stranded DNA-binding protein (SSB). The SC is loaded onto
the primed DNA by the clamp loader complex, and the α
subunit (bound to the SC) catalyzes extension of the primer
through incorporation of dNTPs using ssDNA as a template. A
schematic illustration of this assay is shown in the Supporting
Information (Figure S3).
With this assay system, binding of the DNA Pol III δ and α
subunits to the SC is essential for replication, meaning impaired
SC binding due to the presence of inhibitors will hinder DNA
synthesis, as shown by the presence of ssDNA templates and
Figure 4. Inhibition of SC-dependent in vitro DNA synthesis by 5
(A), 6 (B), 7 (C), and 8 (D). Ladders on the left show molecular size
markers. Circles with a dash above represent primed ssDNA templates,
and concentric circles represent fully replicated dsDNA product.
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Table 2. Antibacterial Activities of Compounds 5−10
MIC
E. coli
A. baylyi
B. subtilis
S. aureus
compd
μM
μg/mL
μM
μg/mL
μM
μg/mL
μM
μg/mL
5
7
2500
2500
313
156
78
540
623
78
5000
5000
313
78
1080
1246
78
2500
2500
156
78
540
623
39
5000
2500
156
156
78
1080
623
39
8
9
41
21
21
41
10
22
156
43
78
22
22
optical density was measured after 24 h of bacterial growth
(Table S4, Supporting Information). The minimum inhibitory
concentrations (MICs), defined as background-corrected
optical densities of less than 0.1, are shown in Table 2.
Compounds 5 and 7 showed only slight antibacterial activity
(MICs of 540−1246 μg/mL), consistent with their modest E.
coli SC-binding affinities (Table 1). Their carboxylic acid
groups may have reduced membrane penetration. The MIC of
8 (39−78 μg/mL) was lower than those of 5 and 7 against all
four species, consistent with its higher SC affinity (Table 1).
Compounds 9 and 10 were considered as possible prodrug
forms of 8 (i.e., methyl and ethyl esters, respectively) that
might show improved membrane penetration properties. Esters
9 and 10 displayed increased antibacterial activity relative to 8
(MICs of 21−43 μg/mL) despite having lower affinities for the
SC (Table 1), consistent with their acting as membrane-
permeable prodrugs.
using the SC from E. coli, yet similar antibacterial potencies
were observed with compounds 9 and 10 against E. coli, A.
baylyi, B. subtilis, and S. aureus. This finding is consistent with
the high conservation of SC structure across bacterial species6
and suggests that the E. coli SC serves as an excellent model
system for identifying SC inhibitors that could potentially be
developed into broad-spectrum antibiotics.
Current work in our laboratories aims to improve the SC
affinities of the tetrahydrocarbazoles by targeting both subsites I
and II. Our previous work suggested that residues of peptide
ligands occupying subsite II contribute significantly to the
binding affinity and that binding at subsite II is subsequent to
binding at subsite I (the anchor site).30 A conformational
change in the side chain of M362 was proposed to act as a gate
in the channel region connecting subsites I and II.30 A similar
conformational change in M362 was observed with the binding
of (R)-8 in subsite I (Figure 3C). Therefore, the synthesis of
tetrahydrocarbazole derivatives substituted at the nitrogen atom
may expand the inhibitor-binding area beyond subsite I, into
the channel region and subsite II.
DISCUSSION
■
PPIs are often considered difficult targets to inhibit with small
molecules.45 Nevertheless, several PPI inhibitors have been
successfully developed.46 Studies indicate that PPIs rely on so-
called “hot spots” for most of the interaction energy, and
accordingly, these are usually the most productive sites for
inhibitor design.24 We previously identified subsite I as the
“anchor site” of consensus peptide binding to the E. coli SC,
indicating that this site acts as the SC hot spot.30 We also
showed that the affinity of peptide LMs for the E. coli SC arises
from residues occupying both subsites I and II,30 suggesting
that the potential of these individual subsites as drug targets is
probably limited. The close proximity of the two subsites,
however, suggests that fragment-based approaches are highly
suited to identifying inhibitors of this target.
MATERIALS AND METHODS
■
Compounds. Fragment compounds (including 1−4) were
purchased from Zenobia Therapeutics (First-Pass Screen library).
Compounds 5−10 and 6a−c were purchased from Sigma-Aldrich,
Labotest, Enamine, or Vitas-M Laboratory. Purchased compounds
were ≥95% pure as confirmed by proton NMR and/or LC−MS.
Compounds 11−29 (Table S3, Supporting Information) were
synthesized in house (Scheme S1, Supporting Information).
Protein Overexpression and Purification. Expression and
purification of the E. coli SC, Pol III α subunit, SSB, and clamp
loader complex (γ3δδ′) were as described previously.29,38,39,48
Crystallization and X-ray Data Collection. Crystals of the E. coli
SC were grown at 285 K by the hanging-drop vapor diffusion method.
The drop was composed of 1 μL of sliding clamp (53 mg/mL) mixed
with the same volume of reservoir solution of 100 mM MES (pH 6.5),
100−150 mM CaCl2, and 25−30% (v/v) PEG400. The reservoir
volume was 1 mL. SC crystals were moved to a CaCl2-free reservoir,
and ligands were soaked into the crystal at 2−5 mM with <10%
DMSO. Fragment compounds were mixed as 4-in-1 cocktails as
suggested by the supplier and soaked into crystals with each fragment
at 5 mM. All crystals were mounted using MiTeGen loops on pins
with magnetic caps. For in-house data collection, crystals were flash-
frozen at 100 K using an Oxford Cryo-stream. Diffraction data were
collected using an MAR345 desktop beamline using Cu Kα X-rays
from a Rigaku 007HF rotating anode generator with Varimax optics.
For synchrotron data collection, the SSRL automated mounting
system (SAM) was used. Mounted crystals were flash-frozen in liquid
nitrogen, and diffraction data were collected at the Australian
Synchrotron, Beamline MX1 (X-ray wavelength 0.95 Å) using Blu-
Ice.49
Fragment screening in the current work using X-ray
crystallography initially identified four hits, but these proved
to be weak binders (Ki > 1 mM). The high sensitivity of X-ray
crystallography and its ability to identify weak binders in
fragment-based screening efforts have previously been noted.47
All four hits were found to bind in subsite I, i.e., the LM
anchor site.30 Follow-up investigations led to the identification
of several SC-binding ligands, with the substituted tetrahy-
drocarbazole 8 showing the highest SC affinity (Ki = 64 μM)
and excellent ligand lipophilicity efficiency (LLEAT = 0.42).
Tetrahydrocarbazoles 7 and 8 were shown by crystallography to
bind in subsite I and to inhibit in vitro DNA replication.
Although compound 8 showed antibacterial activity (MICs of
39−78 μg/mL) against representative bacteria, these effects
were improved upon masking the carboxylic acid as methyl and
ethyl ester prodrugs (compounds 9 and 10, MICs of 21−43
μg/mL). In this study the X-ray crystallography screening and
FP and in vitro DNA replication assays were all carried out
Data Processing, Structure Solution, and Refinement. Crystal
data sets were integrated, merged, and scaled with either HKL200050
or MOSFLM and SCALA.51 The structures were solved by molecular
replacement with CCP4 using the Protein Data Bank entry 1MMI or
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4K3S as the starting model. Iterative cycles of model building and
refinement were performed in COOT52 and REFMAC5.53
ACKNOWLEDGMENTS
■
Parts of this research were undertaken on the MX1 beamline at
the Australian Synchrotron, Victoria, Australia. This research
was supported by an Australian Research Council Discovery
Project (Grant DP110100660) and Future Fellowship (Grant
FT0990287) to A.J.O.
Fluorescence Polarization Assay. FP experiments were carried
out according to the published protocol.30
Molecular Docking and Chemoinformatics. Details are given
in the Supporting Information.
Antibacterial Activity. Determination of MICs was carried out
with bacterial strains E. coli DH5α, A. baylyi ADP1, B. subtilis SU5, and
S. aureus NCTC8325. Experiments followed the CLSI broth
microdilution method.54 Briefly, strains were grown overnight and
diluted to 5 × 104 CFU/mL with cation-adjusted Mueller Hinton II
broth and used as inoculated medium for bacterial growth. Serial 2-
fold dilution of candidate compounds (predissolved in DMSO at 100
mM) or antibiotics was carried out in sterile 96-well plates. Controls
consisted of the growth medium only (blank controls) or inoculated
medium with 5% DMSO or DMSO diluted as for the test compounds
(negative controls) and inoculated medium with antibiotics (positive
controls). Optical density values of each inoculated culture were
measured at a wavelength of 595 nM as the background using a plate
reader (Tecan Infinite M200 Pro). The plates were incubated for 24 h
at 37 °C, and then the optical density values of the bacterial culture
were measured again. MICs were defined as the lowest compound
concentration with a background-subtracted optical density of <0.1.
DNA Replication Assay. Each assay contained 20 mM Tris−HCl
(pH 7.6), 10 mM MgCl2, 0.8 mM ATP, 8.4 mM dithiothreitol, a 0.6
mM concentration of each dNTP, 211 nM Pol III α subunit, 700 nM
SSB (as tetramers), 210 nM Pol III β subunit (as dimers), 42 nM γ3δδ′
clamp loader complex, 120 mM NaCl, and 3 nM RNA primed M13
ssDNA template in a volume of 6.8 μL. The compounds were
dissolved in DMSO and diluted in series 2-fold (in 50% (v/v) DMSO)
before being added (0.5 μL) to the assay mixture at 0 °C. The final
DMSO concentration was 3.4% (v/v) in all assays. The assay mixtures
were treated at 30 °C for 60 min before being quenched by the
addition of EDTA to 150 mM and SDS to 1% (w/v). The DNA
products were separated by 0.7% agarose gel electrophoresis in TAE
buffer (80 mM Tris, 40 mM acetic acid, and 4 mM EDTA) and then
stained with 10000-fold diluted SYBR Gold (Invitrogen) for 60 min.
The DNA products were visualized using a UV transilluminator.
ABBREVIATIONS USED
■
dNTP, deoxyribonucleoside triphosphate; dsDNA, double-
stranded DNA; FP, fluorescence polarization; IC50, half-
maximum inhibitory concentration; Ki, inhibition constant;
LC−MS, liquid chromatography−mass spectrometry; log D,
distribution coefficient; LE, ligand efficiency; LLEAT, ligand
lipophilicity efficiency; LM, linear motif; MIC, minimum
inhibitory concentration; NMR, nuclear magnetic resonance;
PCNA, proliferating cell nuclear antigen; PDB, Protein Data
Bank; Pol, polymerase; PPI, protein−protein interaction; SC,
sliding clamp; ssDNA, single-stranded DNA
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Crystallographic data, additional chemical and biochemical data
and figures, and detailed description of the materials and
methods used. This material is available free of charge via the
Internet at http://pubs.acs.org.
Accession Codes
The atomic coordinates and structure factors for SC1, SC2, SC3,
SC4, SC5, SC7, and SC8 have been deposited with the Protein
Data Bank under accession codes 4N94, 4N95, 4N96, 4N97,
4N98, 4N99, and 4N9A, respectively.
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AUTHOR INFORMATION
■
́
(11) Lopez de Saro, F. J.; Georgescu, R. E.; Goodman, M. F.;
Corresponding Author
*E-mail: aarono@uow.edu.au. Phone: +61-42214347.
O’Donnell, M. Competitive processivity-clamp usage by DNA
polymerases during DNA replication and repair. EMBO J. 2003, 22,
6408−6418.
Author Contributions
A.J.O., M.J.K., N.E.D., E.J.H., and J.L.B. supervised the project.
Z.Y. performed the fluorescence polarization-based assays,
molecular docking, and chemoinformatics studies. Z.Y. and
A.J.O. performed X-ray crystallography. Antibacterial testing
was performed by Z.Y. and M.L. Organic syntheses were
performed by L.R.W. Replication assays were performed by
Y.W. and S.J. The manuscript was initially drafted by Z.Y.,
L.R.W., and Y.W. with input from all coauthors.
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Notes
The authors declare no competing financial interest.
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