First-in-class inhibitors targeting the interaction between bacterial RNA polymerase and sigma initiation factor affect the viability and toxin release of streptococcus pneumoniae

Article abstract of DOI:10.3390/molecules24162902

Novel antimicrobial classes are in desperate need for clinical management of infections caused by increasingly prevalent multi-drug resistant pathogens. The protein-protein interaction between bacterial RNA polymerase (RNAP) and the housekeeping sigma ini

Full text of DOI:10.3390/molecules24162902

molecules
Article
First-In-Class Inhibitors Targeting the Interaction
between Bacterial RNA Polymerase and Sigma
Initiation Factor Affect the Viability and Toxin
Release of Streptococcus pneumoniae
Jiqing Ye 1
,†
, Adrian Jun Chu
2,†
, Lin Lin , Xiao Yang * and Cong Ma *
2
2,
1,
1
State Key Laboratory of Chemical Biology and Drug Discovery, Department of Applied Biology and
Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hong Kong
Department of Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital,
Shatin, Hong Kong
2
*
†
Correspondence: xiaoyang@cuhk.edu.hk (X.Y.); cong.ma@polyu.edu.hk (C.M.)
These authors contributed equally to this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 15 July 2019; Accepted: 8 August 2019; Published: 9 August 2019
Abstract: Novel antimicrobial classes are in desperate need for clinical management of infections
caused by increasingly prevalent multi-drug resistant pathogens. The protein-protein interaction
between bacterial RNA polymerase (RNAP) and the housekeeping sigma initiation factor is essential
to transcription and bacterial viability. It also presents a potential target for antimicrobial discovery,
for which a hit compound (C3) was previously identified from a pharmacophore model-based in
silico screen. In this study, the hit compound was experimentally assessed with some rationally
designed derivatives for the antimicrobial activities, in particular against Streptococcus pneumoniae
and other pathogens. One compound, C3-005, shows dramatically improved activity against
pneumococci compared to C3. C3-005 also attenuates S. pneumoniae toxin production more strongly
than existing classes of antibiotics tested. Here we demonstrate a newly validated antimicrobial
agent to address an overlooked target in the hit-to-lead process, which may pave the way for further
antimicrobial development.
Keywords: RNA polymerase; sigma factor; transcription; inhibitor; antimicrobial discovery
1
. Introduction
Infections by multi-drug resistant (MDR) bacteria (superbugs) have become an increasingly
significant health burden worldwide [
pneumonia, septicemia, and meningitis, but its susceptibility to existing classes of antimicrobials is also
on a decline [ ]. S. pneumoniae mediates disease through a wide range of well-characterized virulence
factors such as pneumolysin to facilitate colonization, nutrient scavenging, and immunoevasion [ ].
Commonly-used bacteriolytic antimicrobials such as -lactams have often been criticized for their
1]. S. pneumoniae causes serious febrile illnesses such as
2
3
β
role in the undesired elevation of S. pneumoniae toxin levels into host environments and affecting the
treatment outcome of S. pneumoniae infections [4].
The identification of unprecedented targets is crucial to the discovery of novel antimicrobial
agents for treatment against infections caused by superbugs. Amongst the antimicrobial classes,
agents specifically targeting bacterial transcription are under-represented with only rifamycins and
fidaxomicin in current clinical use [
RNAP core enzyme is responsible for binding to DNA template and RNA synthesis. This process
is facilitated by the formation of a crucial RNAP holoenzyme by the core enzyme with a factor
5]. For bacterial transcription, the well-characterized bacterial
σ
Molecules 2019, 24, 2902; doi:10.3390/molecules24162902
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2902
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(
Figure 1A), which is responsible for the initiation of promoter-dependent transcription [
6
]. The
70
A
essential house-keeping
σ
factor is referred to as
σ
(in Escherichia coli) or σ (in Bacillus subtilis)
and one of the major binding sites occurs between the highly conserved clamp-helix (CH) region of
RNAP and the N-terminal domain of (Figure 1B) [ ]. These sigma factors have been exploited for
upregulation or inhibition of bacterial transcription through engineered or synthetic modulators [8].
σ
7
Figure 1. Inhibitors of RNAP-
σ
interaction. (
A) The crystal structure of E. coli RNAP holoenzyme (PDB:
7
0
4
LJZ; 27) with the RNA polymerase core enzyme colored in gray, the CH region in yellow and
σ
in
) The interaction between RNAP CH region (surface in mesh) and the N-terminal domain of
C3 docked in the pharmacophore model.
Green spheres, H-bond acceptors; pink spheres, H-bond donor; cyan spheres, hydrophobic groups. (
The docking model C3 (left) and C3-005 (right) with the CH region in yellow helix and mesh surface.
blue. (
B
7
0
σ
(blue helix) with key amino acid residues labeled. (C)
D)
The interaction between RNAP and
discovery [ 13] as opposed to other inhibitors which target RNAP enzyme activities (such as
rifampicin binding near the active site, lipiarmycin leading to allosteric inhibition of template DNA
binding, myxopyronin and squaramides blocking the switch region of the RNAP clamp open-close) [ ].
Previously, by rational design and pharmacophore model-based in silico screening, we have identified
three chemical compounds (Figure 2) that inhibit bacterial RNAP- interaction by binding to the CH
σ factors has been considered a target for novel antimicrobial
9–
5
σ
region of RNAP [14]. One of the three compounds (C5), composed of a steroidal ABC tricyclic ring and
an indolone moiety which commonly appear in natural products, was chosen for characterization. C5
was shown to inhibit RNAP-σ interaction in an ELISA-based assay as well as an in vitro transcription
assay [14]. C5 demonstrated mild bacterial growth inhibition against both Gram-positive Staphylococcus
aureus and Gram-negative E. coli. While the mechanism of C5 was established both at the molecular

Molecules 2019, 24, 2902
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and cellular level [14], the other two compounds, C3 and C4, require further studies for their
antimicrobial potential.
Figure 2. Three hit compounds C3, C4, and C5 previously identified by in silico screening.
2
. Results and Discussion
2
.1. Docking Study of C3 and Its Antimicrobial Activity
We are particularly interested in C3 as it is a small molecule with drug-like properties predicted
by Discovery Studio 2016 (Biovia, San Diego, California, United States). The substituted benzene rings
can be easily modified and are suitable for studying the structure-activity relationship and validating
our previously established pharmacophore models (Figure 1C). Nevertheless, modifications may be
made to improve the inhibitory and antimicrobial activity of C3. As shown in the docking model
(
Figure 1D, left), C3 fits into the pharmacophore model using the right benzoic acid to form an ionic
bond as the key anchor to R278 or R281 of E. coli RNAP CH, while the left substituted benzene ring may
form interactions with I291 of RNAP CH by van der Waals forces, which is appropriate for an initial
modification to probe the interaction with RNAP CH and identify a lead compound for further studies.
The antimicrobial activities of C3 were first tested to determine the minimum inhibitory
concentration (MIC) in accordance with the guidelines published by the CLSI using six bacterial
species from the most recent “WHO priority pathogens list for guiding R&D of new antibiotics”
consisting of three Gram-positive and three Gram-negative bacteria: Enterococcus faecalis, S. aureus,
S. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. [15]. C3 shows
very mild antimicrobial activity (MIC 256 µg/mL) against S. pneumoniae ATCC 49619 (Table 1).
Table 1. Antimicrobial activity of C3 and derivatives.
SAURb
Compound
EFAE
SAURa
SPNE
KPNE
ABAU
PAER
ECLO
ECOL
C3
>256
256
>256
256
32
>256
256
>256
128
16
>256
256
>256
256
16
0.5
256
64
128
64
>256
>256
>256
>256
>256
>64
>256
>256
>256
>256
>256
>64
>256
>256
>256
>256
>256
>64
>256
>256
>256
>256
>256
>64
>256
>256
>256
>256
>256
>64
C3-002
C3-003
C3-004
C3-005
VAN
8
1
4
1
0.25
0.063
RIF
0.063
0.063
32
4
32
≥64
64
a
b
EFAE: Enterococcus faecalis ATCC 19433, SAUR : S. aureus ATCC 25923, SAUR : S. aureus ATCC 29213, SPNE:
Streptococcus pneumoniae ATCC 49619, ABAU: Acinetobacter baumannii ATCC 19606, PAER: Pseudomonas aeruginosa
ATCC 27853, ECLO: Enterobacter cloacae ATCC 13047, ECOL: E. coli ATCC 25922, VAN: vancomycin, RIF: rifampicin.
2
.2. Molecular Mechanism of C3 by Inhibiting the Protein-Protein Interaction between RNAP CH-σ
We then confirmed the mechanism of C3 at the molecular level by assessing the inhibition against
the in vitro protein-protein interaction (PPI) at the major binding site between RNAP CH region
and
σ
. Previously established split-luciferase assay was employed [16], in which the B. subtilis CH
A
region of RNAP (amino acid 220-315) and full-length
σ
were each tagged with one of the luciferase
facilitates the
reformation of the luciferase indicated by the luminescence released. Reduction of the luminescence
complementation fragments. In the absence of inhibitors, the interaction between CH-
σ

Molecules 2019, 24, 2902
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signal due to inhibitor treatment reflects the percentage of inhibition of the PPI between CH-
compared to the control without inhibitor. As a result, the IC50 of C3 against the PPI between CH-
.05 M was measured as 6.40 0.71
inhibit CH- interaction as designed. The full data set of the assay was illustrated in Table S1. We also
measured the percentage of inhibition of C3 at 10 M against the PPI between CH- , which can be
σ
as
σ
at
0
µ
±
µM (Figure S1). This suggested the C3 compound was able to
σ
µ
σ
used to facilitate the activity comparison with C3 derivatives (Table 2) (17).
Table 2. Antimicrobial and inhibitory activities of C3 derivatives.
MIC a
µg/mL)
% Inhibition b
Compound
R
ClogP
(
C3-001
C3-002
C3-003
C3-004
C3-005
2-NH2
3-NH2
4-NH2
256
64
128
64
62.2 ± 2.6
81.3 ± 4.9
69.1 ± 10.7
60.5 ± 16.5
81.7 ± 0.9
N.D.
3.39
3.39
3.39
4.39
5.94
N.D.
N.D.
2-NH , 4-Cl
2
3-Cl, 4-Cl
N.A.
N.A.
8
Vancomycin
Rifampicin
0.25
0.0625
N.D.
a
The MIC values against S. pneumoniae ATCC 49619; b the percentages of inhibition of C3 and derivatives at 10
µM
against the PPI between RNAP CH-σ at 0.05 µM.
2
.3. Antimicrobial and Inhibitory Activities of C3 Derivatives
We proceeded to construct chemical derivatives of C3 (named C3-001 in the library) to probe the
inhibitory and antimicrobial activities by modification of the position of -NH on the left benzene ring
2
(
Scheme 1). As shown in Table 2, when the amine moved from 2-position of the left benzene ring to
-position (C3-002), both the inhibitory and antimicrobial activity against S. pneumoniae improved,
which can be explained by the additional interaction with I291 of RNAP CH. As shown in Figure 1D, the
-position of the left benzene ring of C3 is closer to I291 than the 2-position. While C3-003 with 4-NH2
3
3
gave similar results to C3-001, we decided to add one chloride group at 4-position of C3-001 to form
C3-004, intending to probe the van der Waals interaction with N294 of RNAP CH. The result showed
that C3-004 maintained the inhibitory activity but improved the antimicrobial activity. The increased
antimicrobial activity may be the effect on logP by replacing amine with chloride to improve cell
permeability. As the substitution at the 3-position (C3-002) was preferred compared to the 2-position
(C3-001) for improving both the inhibitory and antimicrobial activity, based on the data, we synthesized
a 3,4-dichloro compound C3-005, which demonstrated a dramatically improved antimicrobial activity
(MIC 8
µg/mL; Tables 1 and 2) and similar inhibitory activity against RNAP CH and σ to C3-002
(Table 2).
Scheme 1. Synthesis of C3-001–005.
2
.4. Antimicrobial Activity of C3-005 against Representative Gram-Positive Bacterial Pathogens
We expanded the antimicrobial activity testing of the C3 derivatives to a selected panel of clinically
relevant pathogens, as shown above. In our screening, the Gram-positive bacteria were generally more

Molecules 2019, 24, 2902
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responsive to the anti-
σ
compound than the Gram-negative pathogens (Table 1)–a result normally
attributable to altered permeability and efficient efflux mechanisms. Nonetheless, this warrants further
investigation and synthesis in the near future.
Since S. pneumoniae showed particular susceptibility to the compound series, we extended
our antimicrobial activity testing to groups A and B Streptococci: Streptococcus pyogenes (Group A
Streptococcus, GAS) causing strep throat, localized skin infection and necrotizing fasciitis [17], and;
Streptococcus agalactiae (Group B Streptococcus, GBS) causing neonatal infections [18]; as well as clinically
significant Gram-positive pathogens Staphylococcus epidermidis and Staphylococcus saprophyticus. The
MIC of C3-005 against GAS and GBS strains (MIC 16
at a similar level to that of S. pneumoniae (Table 3).
µg/mL) and other Gram-positive pathogens was
Table 3. Antimicrobial activity (MIC
µg/mL) of C3-005 against representative Gram-positive
bacterial pathogens.
MIC (µg/mL)
Compound
SPYO
SAGA
SEPI
SSAP
C3-005
VAN
RIF
16
0.5
0.031
16
1
0.031
16
2
≤0.063
32
1
≤0.063
SPYO: Streptococcus pyogenes ATCC 19615, SAGA: Streptococcus agalactiae ATCC 12386, SEPI: Staphylococcus epidermidis
ATCC 12228, SSAP: Staphylococcus saprophyticus ATCC 15305, VAN: vancomycin, RIF: rifampicin.
2
.5. Time-Kill Kinetics
The time-kill kinetics study reflects the effect of antimicrobial agents to the growth of bacteria
at diverse concentrations over time. We constructed time-kill curves by administering C3-005 to
S. pneumoniae at various concentrations and to assess its in vitro antimicrobial activity. The S. pneumoniae
cells were grown in liquid culture with agitation and 5% CO pursuant to the CLSI guidelines [15].
2
C3-005 was bacteriostatic at 1
×
MIC, while at 4
×
MIC a decrease in CFU counts could be observed
(
Figure 3A). At 16 MIC eradication of colonies below the level of detection (200 CFU/mL) was
×
achieved from 2 h onwards (Figure 3A). This suggested that C3-005 acts primarily in a bacteriostatic
manner at lower concentrations but was capable of rapid bactericidal effects (>3-fold log10 decrease) at
higher concentrations.
Figure 3. The effect of C3-005 on (A) the time kill kinetic and (B) ATP production of S. pneumoniae cells
when challenged at 1 ×, 1×, 4× and 16× MIC.
4
2
.6. ATP Production
The inhibition of vital components central to bacterial metabolism, such as respiratory ATP
synthesis, is one of the hallmarks of an effective antibiotic [19]. We therefore monitored the ATP
production over time in the presence of C3-005 at various concentrations in S. pneumoniae cells. The

Molecules 2019, 24, 2902
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same culturing conditions as that of the time-kill assay were followed. The rate of ATP production saw
a marked decrease at 1
MIC compared to the untreated control, whereas higher concentrations of
×
4
C3-005 further arrested cellular respiration (Figure 3B). This trend mirrors the previously reported
impact of rifampicin, the leading transcription inhibitor drug, on pathogen respiration, as well as its
established correlation with antimicrobial efficacy [20,21].
2
.7. S. pneumoniae Toxin Secretion
Release of the toxin pneumolysin into the extracellular milieu is a signature virulence factor
of S. pneumoniae and non-lytic antimicrobials such as macrolides and rifampicin have been shown
to repress pneumolysin release at sub-MIC concentrations [ 22 23]. In this study we explored the
impact of C3-005 on S. pneumoniae pneumolysin secretion. Adapting from the CLSI guidelines, S.
pneumoniae cells were cultured overnight in Brain-Heart Infusion (BHI) media at 12 and 1
of the
4, ,
×
×
4
corresponding pre-determined MICs of C3-005, the non-lytic antimicrobial rifampicin, clindamycin,
and the bacteriolytic agent ceftriaxone, along with a drug-free control [15]. The cultures were then
centrifuged, and the supernatant harvested for Western blot analysis. There were significant differences
between Control and 1 x and 1 x MIC of the treatment groups, Rifampicin (p = 0.0018), Clindamycin
2
4
(p < 0.0001), Ceftriaxone (p = 0.0026) and C3-005 (p < 0.0001) analyzed by the one-way ANOVA
method. The three control drugs performed as previously reported, where rifampicin and clindamycin
decreased toxin release while the bacteriolytic antimicrobial agent ceftriaxone drastically promoted the
level of pneumolysin [24]. Significant reduction of post-culture toxin levels by C3-005 was observed
over the untreated control, as well as more toxin reduction than rifampicin (Figure 4). The trends were
also highly comparable with that of the bacteriostatic lincosamide clindamycin, indicative of growth
repression mechanism without the induction of pneumolysin (Figure 4).
Figure 4. The effect of C3-005 on the secretion of S. pneumoniae pneumolysin. (A) Western blot showing
pneumolysin levels extracted from culture supernatant following overnight incubation of S. pneumoniae
1
(checkered bars) and 1
with
×
×
(light grey bars) MICs of C3-005, rifampicin (Rif), clindamycin (Clin),
) relative intensity of blotted bands normalized against the drug-free control
solid black bar). One-way ANOVA was used to compare data across the treatment groups. Data was
2
4
ceftriaxone (Cef), and; (
B
(
presented in GraphPad Prism style: p ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***), ≤ 0.0001 (****).
2
.8. Cytotoxicity of C3-005
With the best antimicrobial activity among the C3 derivatives, C3-005 was subjected to cytotoxicity
testing against HepG2 human liver cancer cell line (HB-8065
™, the American Type Culture Collection,
Manassas, Virginia, United States) and A549 human lung carcinoma cell line (CCL-185
™, the American
Type Culture Collection, Manassas, Virginia, United States). As shown in Table 4, C3-005 did not show
significant cytotoxicity against the mammalian cell lines compared to the anti-cancer drug cisplatin
control, indicating a promising clinical prospect base on the lead optimization of C3-005.

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Table 4. Cytotoxicity of C3-005 against Human HepG2 and A549 cell lines.
CC50 (µM)
Compound
HepG2
A549
C3-005
cisplatin
63.82 ± 9.32
6.81 ± 0.61
76.65 ± 11.37
7.70 ± 0.58
3
. Materials and Methods
3
.1. Chemistry
Starting materials and regents, unless otherwise stated, were of commercial grade and used
without further purification. All reactions were monitored by thin-layer chromatography (TLC) on
glass sheets (Silica gel F254) which can be visualized under UV light. Flash chromatography was
1
13
carried out using silica gel (200–300 mesh). H-NMR (400 MHz) and C-NMR (100 MHz) spectra
were measured on BRUKER AVANCE-III spectrometer with TMS as an internal standard. Chemical
shifts are expressed in
δ (ppm) and coupling constants (J) in Hz. High resolution MS spectra were
measured using a QTOF-2 micromass Spectrometer by electron spray ionization. HPLC analysis was
performed on an Agilent 1260 HPLC apparatus.
3
.1.1. Synthesis of Methyl 2-(4-chloro-3-nitrobenzoyl)benzoate (2)
A solution of 2-(4-chloro-3-nitrobenzoyl) benzoic acid
1 (3.057 g, 10 mmol) in MeOH was cooled
◦
to 0 C followed by a dropwise addition of thionyl choloride (0.5 mL). The mixture was refluxed
for 24 h. After evaporation of the volatiles, the residue was treated with 5 mL MeOH and stirred at
room temperature for 10 min. The precipitate was collected by filtration and dried in vacuum to give
compound 2 as a white solid (2.877 g, 90%).
3
.1.2. General Procedure for the Synthesis of Compound C3-001a and its Derivatives
To a flask was added compound (64 mg, 0.2 mmol), benzenethiol (0.24 mmol), NaOAc (82 mg,
mmol.) and EtOH 5 mL. The mixture was heated to reflux for 4h. After cooling to room temperature,
2
1
the precipitate was collected via filtration and washed with appropriate EtOH and water successively,
then dried in vacuum to give the titled compounds. Otherwise, water was added and the aqueous
layer was extracted by EtOAc. The combined organic layers were dried over Na SO and purification
2
4
by column chromatography to provide the titled compounds.
Methyl 2-(4-((2-aminophenyl)thio)-3-nitrobenzoyl)benzoate (C3-001a): Yellow solid, 60 mg, 73% yield.
1
H-NMR (400 MHz, CDCl3)
H), 7.68 (td, J = 7.4, 1.0 Hz, 1H), 7.61 (td, J = 7.5, 0.9 Hz, 1H), 7.43 (dd, J = 7.6, 1.0 Hz, 1H), 7.38–7.33
m, 2H), 6.94 (d, J = 8.5 Hz, 1H), 6.85 (m, 2H), 4.29 (s, 2H), 3.74 (s, 3H).
δ 8.60 (d, J = 1.6 Hz, 1H), 8.10 (d, J = 7.7 Hz, 1H), 7.76 (dd, J = 8.5, 1.7 Hz,
1
(
Methyl 2-(4-((3-aminophenyl)thio)-3-nitrobenzoyl)benzoate (C3-002a): Yellow solid, 62 mg, 76 % yield.
1
H-NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.69 (t, J =
7
7
.5 Hz, 1H), 7.62 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 7.4 Hz, 1H), 7.32–7.25 (m, 3H), 7.09 (t, J = 7.8 Hz, 1H),
.05 (d, J = 8.6 Hz, 1H), 6.97 (d, J = 7.6 Hz, 1H), 6.89 (d, J = 8.2 Hz, 2H), 6.82 (d, J = 9.9 Hz, 2H), 6.54 (d,
J = 7.7 Hz, 1H), 3.85 (s, 2H), 3.75 (s, 3H).
Methyl 2-(4-((4-aminophenyl)thio)-3-nitrobenzoyl)benzoate (C3-003a): Yellow solid, 80 mg, 98 % yield.
1
H-NMR (400 MHz, CDCl3)
δ 8.49 (d, J = 1.7 Hz, 1H), 8.10 (d, J = 7.8 Hz, 1H), 7.77 (dd, J = 8.6, 1.8 Hz,
1
1
H), 7.68 (t, J = 7.4, 0.9 Hz, 1H), 7.61 (td, J = 7.5, 0.9 Hz, 1H), 7.35 (t, J = 7.6 Hz, 3H), 6.99 (d, J = 8.6 Hz,
H), 6.78 (d, J = 8.4 Hz, 2H), 4.01 (s, 2H), 3.74 (s, 3H).
Methyl 2-(4-((2-amino-4-chlorophenyl)thio)-3-nitrobenzoyl)benzoate (C3-004a): Yellow solid, 70 mg,
1
7
9 % yield. H-NMR (400 MHz, CDCl )
δ
8.60 (s, 1H), 8.10 (d, J = 7.5 Hz, 1H), 7.78 (d, J = 8.5 Hz, 1H),
3

Molecules 2019, 24, 2902
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7
.67 (dd, J = 13.5, 6.2 Hz, 1H), 7.62 (t, J = 6.9 Hz, 1H), 7.35 (d, J = 7.0 Hz, 2H), 6.93 (dd, J = 8.3, 1.2 Hz,
1
H), 6.85 (s, 1H), 6.81 (d, J = 8.1 Hz, 1H), 4.39 (s, 2H), 3.74 (s, 3H).
Methyl 2-(4-((3,4-dichlorophenyl)thio)-3-nitrobenzoyl)benzoate (C3-005a): Yellow solid, 85 mg, 92 %
1
yield. H-NMR (400 MHz, CDCl )
δ
8.55 (d, J = 1.6 Hz, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.82 (dd, J = 8.6,
3
1
1
.7 Hz, 1H), 7.72 (d, J = 1.9 Hz, 1H), 7.69 (d, J = 6.7 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.61 (d, J = 8.3 Hz,
H), 7.45 (dd, J = 8.2, 1.9 Hz, 1H), 7.37 (d, J = 7.4 Hz, 1H), 6.95 (d, J = 8.5 Hz, 1H), 3.76 (s, 3H).
3
.1.3. General Procedure for the Synthesis of Compound C3-001 and Its Derivatives
The methyl esters of the title compounds (C3-001a and its derivatives) were hydrolyzed with 1 M
NaOH in THF (1:1) at room temperature overnight. The mixture was then diluted with a small amount
of water and washed twice with CH Cl . The aqueous solution was acidified by the addition of 2 M
2
2
HCl. The precipitate was collected by filtration and washed with water to afford the titled compounds.
If the compound was not pure at this stage of the procedure, it was purified by column chromatography.
2-(4-((2-Aminophenyl)thio)-3-nitrobenzoyl)benzoic Acid (C3-001): The title compound was prepared
from the hydrolysis of C3-001a (60 mg, 0.15 mmol) in 1N NaOH (0.8 mL) and THF (0.8 mL). Yellow
◦
1
solid, 50 mg, 85 % yield, mp 217–219 C. H-NMR (400 MHz, DMSO-d6)
δ
13.30 (s, 1H), 8.33 (d, J =
1
7
.4 Hz, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.82–7.72 (m, 2H), 7.69 (t, J = 7.2 Hz, 1H), 7.44 (d, J = 7.3 Hz, 1H),
.35 (d, J = 7.5 Hz, 1H), 7.28 (t, J = 7.3 Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 6.83 (d, J = 8.1 Hz, 1H), 6.65 (t, J
7.3 Hz, 1H), 5.62 (s, 2H). 13C-NMR (100 MHz, DMSO-d6)
167.4, 151.4, 145.1, 143.1, 141.2, 137.7,
=
δ
1
34.8, 133.3, 132.9, 132.9, 130.9, 130.5, 130.2, 127.8, 127.4, 126.1, 117.3, 115.7, 109.1. HRMS (ESI): calcd
−
for C H N O S, (M−H) 393.0551, found 393.0547. HPLC purity: 97.00%.
20
13
2
5
2-(4-((3-Aminophenyl)thio)-3-nitrobenzoyl)benzoic Acid (C3-002): The title compound was prepared
from the hydrolysis of C3-002a (60 mg, 0.15 mmol) in 1N NaOH (0.7 mL) and THF (0.7 mL). Yellow
◦
1
solid, 40 mg, 69 % yield, mp 123-125 C. H-NMR (400 MHz, DMSO-d6)
δ
13.31 (s, 1H), 8.31 (d, J =
.1 Hz, 1H), 8.02 (d, J = 7.4 Hz, 1H), 7.85–7.73 (m, 2H), 7.69 (t, J = 7.4 Hz, 1H), 7.46 (d, J = 7.2 Hz,
H), 7.20 (t, J = 7.7 Hz, 1H), 7.08 (d, J = 8.5 Hz, 1H), 6.79 (s, 1H), 6.74 (t, J = 8.7 Hz, 2H), 5.53 (s, 2H).
1
1
13
C-NMR (100 MHz, DMSO-d6)
δ
194.5, 167.2, 151.1, 144.8, 144.4, 140.7, 134.5, 133.6, 133.2, 131.5, 130.7,
30.4, 130.2, 129.3, 128.9, 127.9, 126.0, 122.4, 120.1, 116.5. HRMS (ESI): calcd for C20 H N O S, (M
93.0551, found 393.0544. HPLC purity: 95.24%.
−
1
3
−
H)
13
2
5
2-(4-((4-Aminophenyl)thio)-3-nitrobenzoyl)benzoic Acid (C3-003): The title compound was prepared
from the hydrolysis of C3-003a (80 mg, 0.2 mmol) in 1N NaOH (1.0 mL) and THF (1.0 mL). Yellow
◦
1
solid, 60 mg, 76 % yield, mp 129–131 C. H-NMR (400 MHz, DMSO-d6)
δ 13.31 (s, 1H), 8.29 (d, J
=
1.5 Hz, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.80–7.71 (m, 2H), 7.67 (t, J = 7.3 Hz, 1H), 7.44 (d, J = 7.3 Hz,
13
1H), 7.23 (d, J = 8.4 Hz, 2H), 6.98 (d, J = 8.6 Hz, 1H), 6.69 (d, J = 8.5 Hz, 2H), 5.79 (s, 2H). C-NMR
(
1
3
100 MHz, DMSO-d6)
30.2, 128.4, 127.6, 125.9, 115.7, 111.8. HRMS (ESI): calcd for C20 H N O S, (M
93.0548. HPLC purity: 99.73%.
δ
194.3, 167.3, 151.8, 146.9, 143.9, 141.1, 137.6, 134.36, 133.39, 133.1, 130.6, 130.4,
−
−H) 393.0551, found
13
2
5
2
-(4-((2-Amino-4-chlorophenyl)thio)-3-nitrobenzoyl)benzoic Acid (C3-004): The title compound was
prepared from the hydrolysis of C3-004a (70 mg, 0.16 mmol) in 1N NaOH (0.8 mL) and THF (0.8 mL).
◦
1
Yellow solid, 41 mg, 60 % yield, mp 118–120 C. H-NMR (400 MHz, DMSO-d6)
J = 1.6 Hz, 1H), 8.02 (d, J = 7.5 Hz, 1H), 7.76 (dd, J = 15.0, 7.8 Hz, 2H), 7.68 (t, J = 7.2 Hz, 1H), 7.44 (d, J
7.3 Hz, 1H), 7.37 (d, J = 8.2 Hz, 1H), 6.92 (d, J = 8.5 Hz, 1H), 6.86 (d, J = 2.0 Hz, 1H), 6.66 (dd, J = 8.2,
.1 Hz, 1H), 5.93 (s, 2H). 13C-NMR (100 MHz, DMSO-d6)
194.6, 167.3, 152.5, 145.3, 142.6, 141.0, 139.3,
δ 13.32 (s, 1H), 8.33 (d,
=
2
δ
1
37.4, 134.7, 133.6, 133.2, 130.6, 130.3, 127.9, 127.6, 126.04, 116.8, 114.6, 108.1. HRMS (ESI): calcd for
−
C H ClN O S, (M−H) 427.0161, found 427.0152. HPLC purity: 100.00%.
20
12
2
5
2-(4-((3,4-Dichlorophenyl)thio)-3-nitrobenzoyl)benzoic Acid (C3-005): The title compound was
prepared from the hydrolysis of C3-005a (80 mg, 0.17 mmol) in 1N NaOH (0.9 mL) and THF

Molecules 2019, 24, 2902
9 of 13
◦
1
(
(
7
0.9 mL). Yellow solid, 45 mg, 62 % yield, mp 247
−
248 C. H-NMR (400 MHz, DMSO-d6)
δ
13.34
s, 1H), 8.37 (s, 1H), 8.02 (d, J = 13.0 Hz, 2H), 7.84 (d, J = 8.3 Hz, 1H), 7.76 (dd, J = 14.9, 7.7 Hz, 2H),
.72–7.67 (m, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 6.8 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H). 13C-NMR
(
1
100 MHz, DMSO-d6)
30.8, 130.7, 130.5, 130.1, 129.5, 127.8, 125.8. HRMS (ESI): calcd for C H Cl NO S, (M
δ
194.4, 167.2, 144.8, 142.7, 140.7, 137.4, 136.1, 135.0, 134.4, 134.1, 133.3, 132.9,
−
−
H) 445.9662,
20
10
2
5
found 445.9651. HPLC purity: 99.73%.
3
.2. Biology
3
.2.1. Bacterial Strains and Antibiotics
The following bacterial strains were used in this study for the microdilution assay: Enterococcus
faecalis ATCC 19433, Streptococcus pneumoniae ATCC 49619, Klebsiella pneumoniae ATCC 700603,
Acinetobacter baumannii ATCC 19606, Pseudomonas aeruginosa ATCC 27853, Enterobacter cloacae ATCC
1
1
3047, Escherichia coli ATCC 25922, Streptococcus pyogenes ATCC 19615, Streptococcus agalactiae ATCC
2386, Staphylococcus epidermidis ATCC 12228 and Staphylococcus saprophyticus ATCC 15305 (the
American Type Culture Collection, Manassas, Virginia, United States). The antibiotic controls were
purchased from Sigma-Aldrich (St. Louis, Missouri, United States).
3
.2.2. Determination of Minimum Inhibitory Concentration (MIC)
The antimicrobial activity of the compounds was determined by broth microdilution according to
the Clinical & Laboratory Standards Institute (CLSI) guidelines [15]. The test medium was brain heart
infusion (BHI) for S. pneumoniae, S. pyogenes and S. agalactiae, and Mueller-Hinton (MH) broth for the
rest of the organisms. Serial two-fold dilutions were performed for the tested chemicals starting from
6
256
µ
g/mL to 0.5
µ
g/mL, and the bacterial cell inoculum was adjusted to approximately 1.5
×
10 CFU
◦
per mL. Results were taken after 16–20 hrs of incubation at 37 C (with 5% CO supplementation for
2
the Streptococcus spp.). The MIC was defined as the lowest concentration of antibiotic with no visible
growth. Experiments were performed in duplicate.
3
.2.3. Protein-Protein Interaction Inhibition Assay
Previously established protocols were used for inhibitor testing with modifications [14]. Vectors
were made in which B. subtilis RpoC clamp-helix (CH)-domain (220–315 aa) was tagged with SmBiT
NanoLuc fragment at its N-terminal (pCU252) and full-length SigA tagged with LgBiT NanoLuc
fragment at its C-terminal (pCU251) [14]. Protein overproduction and purification were performed as
detailed previously [16]. 40
and then mixed with 20 L compound (50
L N-LgBiT-SigA (0.125
µ
L of purified C-SmBiT-CH (0.125
µ
M in PBS) was added to 96-well plates
◦
µ
µM in PBS). The mixture was incubated for 10 min at 37 C.
4
0
µ
µM in PBS) was then added to each well, followed by incubation for 10 min
◦
at 37 C. The final concentration of the compounds was at 10
µ
M. After the final incubation step, equal
®
volume of Nano-Glo Luciferase Assay Substrate (Promega, Madison, Wisconsin, United States) was
added to the reaction mixture. Luminescence emitted was measured using a Victor X3 Multilabel plate
reader (Waltham, Massachusetts, United States). Experiment was performed in triplicate. Technical
repeats were taken to ensure consistent results were obtained.
3
.2.4. Time-Kill Kinetics
6
S. pneumoniae cells were suspended to ~1.5
×
10 CFU/mL at log phase in BHI medium with
compounds at various concentrations. As an untreated control, bacteria were incubated in BHI medium
without compounds. The cultures were grown at 37 C with shaking at 200 rpm supplemented with
◦
5
% CO , where 20 µL samples were taken at defined time points (0, 2, 4 and 6 h) for each treatment
2
group, followed by 10-fold serial dilutions. 5
on blood agar plate. After overnight incubation at 37 C with 5% CO , the number of viable bacteria
µ
L sample were taken from each dilution and spotted
◦
2

Molecules 2019, 24, 2902
10 of 13
in each sample was counted and expressed as CFU/mL. The experiment was performed in triplicate.
Technical repeats were taken to ensure consistent results were obtained.
3
.2.5. Assessment of ATP Production
6
S. pneumoniae cells were suspended to ~1.5
×
10 CFU/mL at log phase in BHI medium with
compounds at various concentrations. As an untreated control, bacteria were incubated in BHI medium
without compounds. The cultures were grown at 37 C with shaking at 200 rpm and at 5% CO , where
◦
2
100
µL samples were taken at defined time points (0, 2, 4 and 6 h) for each treatment group. The
ATP production was measured using the BacTiter-Glo
™
Microbial Cell Viability Assay Kit (Promega,
Madison, Wisconsin, United States) according to the manufacturer’s instructions. Experiment was
performed in triplicate. Technical repeats were taken to ensure consistent results were obtained.
3
.2.6. S. pneumoniae Toxin Secretion
S. pneumoniae cells were grown overnight without agitation on round-bottomed 96-well plates in
the presence of serially diluted concentrations of compounds. The antibiotics were also added at serial
two-fold dilutions starting from 4
µ
g/mL to 0.003
µ
g/mL. After 16–20 h incubation, each corresponding
1
and 1
×
×
MIC values were determined. The cultures were then resuspended with their OD595
g for 3 min. The supernatants from the
MIC of the test compounds, antibiotics and drug-free
controls were transferred to a fresh plate and were subsequently used for Western blot assay.
2
4
readings measured and the plates were centrifuged at 3000
×
cultures which were challenged with 1 and 1
×
×
2
4
3
.2.7. Western Blot
Samples were separated in 10% polyacrylamide gels at 150 V for 1 h, before being transferred to a
PVDF membrane at 110 V for 1 h. The membrane was then blocked with 5% non-fat milk in TBST
buffer for 1 h, incubated overnight with 1:1000 rabbit polyclonal anti-pneumolysin primary antibody
◦
(
ab71811, abcam, Cambridge, United Kingdom) at 4 C with agitation, followed by 1 hr incubation with
1
:5000 goat polyclonal anti-rabbit HRP-conjugated secondary antibody (ab97051, abcam, Cambridge,
United Kingdom) at room temperature with agitation, complete with TBST-washing cycles prior
TM
and after. Blots were incubated with Bio-Rad Clarity Western ECL Substrates and the resulting
TM
bands were visualized in a Bio-Rad ChemiDoc
Touch imaging system in Chemiluminescence mode
(
Bio-Rad, Hercules, California, United States). The experiment was performed in triplicate. Technical
repeats were taken to ensure consistent results were obtained.
3
.2.8. Cytotoxicity Assay
Human cell lines A549 lung carcinoma and HepG2 hepatoblastoma were used in this study. The
5
cells were seeded at 2.5
2
×
10 per well. After 24 h incubation, the tested compounds were added in a
◦
-fold serial dilution ranging from 1562
µg/mL to 50
µg/mL. The plates were then incubated at 37 C. At
4
8 h and 72 h after adding the compound, the MTT assay was performed as described previously [25].
Cisplatin was used as the positive control and DMSO as the negative control.
3
.3. Molecular Modelling
Modelling was performed using UCSF chimera [26]. E. coli RNAP holoenzyme crystal structure
(PDB: 4LJZ) was employed [27]. Images were made with UCSF chimera [26]. The pharmacophore
model was generated with Discovery Studio 2016 (Biovia, San Diego, California, United States).
3
.4. Data and Statistical Analysis
Technical repeats were taken for the biochemical assays to ensure reproducibility. One-way
ANOVA was used to measure the statistical significance. The data was presented in GraphPad Prism
style: p ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***), ≤ 0.0001 (****).

Molecules 2019, 24, 2902
11 of 13
4
. Conclusions
Herein, we report the discovery and evaluation of novel analogues of inhibitors against bacterial
RNAP and factor interaction. By the pharmacophore model-based rational design followed by
σ
synthesis of analogues, we were able to obtain a C3 derivative with greater inhibitory activity by the
additional interaction with RNAP CH N294 through hydrophobic interaction with a chloride group,
and significantly improved antibacterial activity against Gram-positive pathogens. The latter may be
attributed to the greater cell permeability which can be reflected by an elevated ClogP value calculated
by Discovery Studio 2016 (Biovia, San Diego, California, United States) as shown in Table 2. The
logP values represent the logarithm of the ratio of compound solubility in octanol and water. As a
result, the molecule with high logP values may display an unprecedented bacterial cell permeability.
The mechanism of the C3 derivatives against the PPI between RNAP CH and
σ was confirmed at
the molecular level by an in vitro luciferase complementation assay. The compound with the best
antimicrobial activity, C3-005, has been shown to be bactericidal at higher concentrations and able to
suppress ATP production in S. pneumoniae cells, like rifampicin as a bacterial transcription inhibitor.
It also showed more suppression of the S. pneumoniae virulence factor pneumolysin secretion than
rifampicin. Since bacterial transcription is a proven but under-utilized target for antibiotics, our
approach may lead the way to a valid platform for novel antimicrobial discovery. On top of their
therapeutic potential, the compounds described in this report could also complement the development
of chemical probes to study the regulation of transcription by σ factors.
Supplementary Materials: Figure S1: IC50 measurement of C3 against the protein-protein interaction between
RNAP CH-
σ
, Table S1: The luminescence data of C3 inhibiting the RNAP CH–
σ
protein-protein interaction,
1
H- & 13C-NMR: C3-001
,
C3-002
,
C3-003
,
C3-004
,
C3-005, and HPLC spectra: C3-001 C3-002 C3-003 C3-004,
,
,
,
C3-005 (PDF).
Author Contributions: Conceptualization, X.Y. and C.M.; Data curation, J.Y., A.J.C., L.L., X.Y. and C.M.; Formal
analysis, J.Y., A.J.C., L.L., X.Y. and C.M.; Funding acquisition, X.Y. and C.M.; Investigation, J.Y., A.J.C. and L.L.;
Methodology, L.L., X.Y. and C.M.; Project administration, X.Y. and C.M.; Supervision, X.Y. and C.M.; Validation,
J.Y., A.J.C. and L.L.; Visualization, J.Y., A.J.C., X.Y. and C.M.; Writing—original draft, J.Y., A.J.C., X.Y. and C.M.;
Writing—review & editing, J.Y., A.J.C., X.Y. and C.M.
Funding: The research was supported by Hong Kong RGC Early Career Scheme grant No. 25100017 (C.M.), the
State Key Laboratory of Chemical Biology and Drug Discovery, HKPU (C.M.); Hong Kong RGC General Research
Fund GRF No. 14165917 (X.Y.), and CU Faculty of Medicine Faculty Innovation Award FIA2018/A/03 (X.Y.).
Conflicts of Interest: The authors declare no conflict of interest.
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σ
2
Sample Availability: Samples of the compounds C3-001–005 are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(