Discovery of novel N-aryl pyrrothine derivatives as bacterial RNA polymerase inhibitors

Article abstract of DOI:10.1111/cbdd.13736

Bacterial RNA polymerase (RNAP) is a validated drug target for broad-spectrum antibiotics, and its “switch region” is considered as the promising binding site for novel antibiotics. Based on the core scaffold of dithiolopyrrolone, a series of N-aryl pyrrothine derivatives was designed, synthesized, and evaluated for their antibacterial activity. Compounds generally displayed more active against Gram-positive bacteria, but less against Gram-negative bacteria. Among them, compound 6e exhibited moderate antibacterial activity against clinical isolates of rifampin-resistant Staphylococcus aureus with minimum inhibition concentration value of 1–2?μg/ml and inhibited Escherichia coli RNAP with IC₅₀ value of 12.0?±?0.9?μM. In addition, compound 6e showed certain degree of cytotoxicity against HepG2 and LO2 cells. Furthermore, molecular docking studies suggested that compound 6e might interact with the switch region of bacterial RNAP in a similar conformation to myxopyronin A. Together, the N-aryl pyrrothine scaffold is a promising lead for discovery of antibacterial drugs acting against bacterial RNAP.

Full text of DOI:10.1111/cbdd.13736

PROFESSOR YIPING CHEN (Orcid ID : 0000-0002-5198-3348)
DR XIANGDUAN TAN (Orcid ID : 0000-0001-8627-5483)
Article type
: Research Article
Discovery of novel N-aryl pyrrothine derivatives as bacterial
RNA polymerase inhibitors
Mo-Han Huang 1, 2, Bo Kong 3, Jie-Yun Meng 1, Yu-Bin Lv 1, Yan-Fen Peng 1, Yi-Ping Chen4,
5,*, Xiang-Duan Tan 1,*
1
College of Pharmacy, Guilin Medical University, Guilin 541004, China
2
Department of Pharmacy, Liuzhou People’s Hospital, Liuzhou 545006, China
3
School of Life Sciences, Guangzhou University, Guangzhou, 510006, China
4
School of Pharmaceutical Sciences, Guangxi University of Chinese Medicine, Nanning 530200, China
5
Guangxi Key Laboratory of Translational Medicine for Treating High-Incidence Infectious Diseases with Integrative
Medicine, Nanning 530200, China

Correspondence: E-mail: tandy@glmc.edu.cn (X.-D.T.); yipchen2009@foxmail.com (Y.-P.C).
Abstract:
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through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/CBDD.13736
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Bacterial RNA polymerase (RNAP) is a validated drug target for broad-spectrum antibiotics, and its
“switch region” is considered as the promising binding site for novel antibiotics. Based on the core scaffold
of dithiolopyrrolone, a series of N-aryl pyrrothine derivatives was designed, synthesized and evaluated for
their antibacterial activity. Compounds generally displayed more active against Gram-positive bacteria, but
less against Gram-negative bacteria. Among them, compound 6e exhibited moderate antibacterial activity
against clinical isolates of rifampin-resistant Staphylococcus aureus with MIC value of 1-2 μg/mL, and
inhibited Escherichia coli RNAP with IC50 value of 12.0 ± 0.9 μM. In addition, compound 6e showed
certain degree of cytotoxicity against HepG2 and LO2 cells. Furthermore, molecular docking studies
suggested that compound 6e might interact with the switch region of bacterial RNAP in a similar
conformation to myxopyronin A. Together, the N-aryl pyrrothine scaffold is a promising lead for discovery
of antibacterial drugs acting against bacterial RNAP.
Keywords: dithiolopyrrolone, N-aryl pyrrothine, bacterial RNA polymerase, inhibitors, antibacterial activity
1 INTRODUCTION
Multiple drug resistant (MDR) bacteria are one of the most important threats to public health worldwide (Oliva
et al., 2018), and lead to an increase in the number of infections and the costs associated with these infections
(Pendleton et al., 2013; Rossolini et al., 2014). Bacterial RNA polymerase (RNAP) acts as a key enzyme in
transcription regulation and gene expression (Mosaei & Harbottle, 2019), and is highly conserved among bacteria, but
less highly conserved with eukaryotic RNAP sequences, which makes RNAP an excellent target for broad-spectrum
antibacterial agent discovery (Ma, 2016). Rifampicin is the well-known clinical antibiotic targeting bacterial RNAP
and has been the first line antibiotics used for tuberculosis treatment since 1970s (Chopra 2007; Villain et al., 2007).
However, the emerging rifampin-resistant mycobacteria has threatened the usefulness of this drug in treating
mycobacterial diseases (Campbell et al., 2001; Darst, 2004).
Dithiolopyrrolones are a class of antibiotics that possess the unique pyrrolinonodithiole (4H- [1,2] dithiolo [4,3-b]
pyrrol-5-one) skeleton (Qin et al., 2013). Currently, there are approximately 30 naturally occurring dithiolopyrrolone
derivatives identified, including thiolutin, holomycin, aureothricin, xenorhabdin I and thiomarinols (Figure 1) (Li et
al., 2014; Gao et al., 2014; Maffioli et al., 2017). These derivatives belong to N-methyl, N-acyl pyrrothine (thiolutin
type), N-acyl pyrrothine (holomycin type) and thiomarinols. Natural dithiolopyrrolones exhibit relatively
broad-spectrum antibiotic activity against many Gram-positive and Gram-negative bacteria (Qin et al., 2013). In
recent years, N-aryl pyrrothine derivatives have been reported to have anticancer (Li et al., 2007) and anti-leukopenia
activity (Tan et al., 2013; Li et al., 2014).
Recently, the bacterial RNAP “switch region” was considered as a new binding site for drug discovery (Sahner
et al., 2013). The “switch region” located at the base of the clamp serves as a hinge that facilitates the clamp swinging
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between the open and closed state (Irschik et al., 1983). The switch region is distinct from the rifamycins binding site
on RNAP and is highly conserved in Gram-positive and Gram-negative bacteria (Li et al., 2014). Myxopyronins,
corallopyronins and ripostatins target the switch region of the bacterial RNAP and prevent extension of RNA during
transcription initiation by restraining the entry of the promoter DNA, which interacts with RNAP (Figure 1)
(Mukhopadhyay et al., 2008; Ho et al., 2009). Currently, natural dithiolopyrrolone compounds, such as thiolutin and
holomycin, show antibacterial activity that is attributed to the inhibition of RNA synthesis (Qin et al., 2013). The
hybrid-type antimicrobial agent 29 also shows antibacterial activity, and might binds to be the switch region according
to molecular docking studies (Yakushiji et al., 2013). Dithiolopyrrolones derivatives are popular structures for
developing the next generation of bacterial RNAP inhibitors (Qin et al., 2013).
Previous reports have not described the antibacterial activity of N-aryl pyrrothine derivatives. In this study, the
core scaffold of natural pyrrothine was used as a template to do further structural optimization. A series of N-aryl
pyrrothine derivatives was designed and synthesized (Figure 2), which bear the aryl group at the N-4 position, and a
variety of alkyl or aromatic groups in the amino group at the C-6 position of the pyrrothine core scaffold, followed by
structure-activity relationship study and biological evaluation.
OH R3
O
S
O
H
S
HO
O
R2
N
NH
O
O
O
OH
O
n
N
R1
O
O
HN
O
S
R1
O
N
S
S
H
R2
HN
O
S
Thiomarinol A: R1= H, R2= OH, R3= OH, n= 7
Thiomarinol C: R1= H, R2= OH, R3= H, n= 7
Thiomarinol D: R1= Me, R2= OH, R3= H, n= 7
Thiomarinol E: R1= H, R2= OH, R3= OH, n= 9
Thiolutin: R1= Me, R2= Me
Holomycin: R1= H, R2= Me
Aureothricin: R1= Me, R2= Et
Xenorhabdin I: R1= H, R2= n-Pentyl
Hybrid-type 29
O
O
OH
O
O
O
OH
O
OH
NHCOOMe
R
NHCOOMe
R
Corallopyronin A: R= H
Corallopyronin B: R= Me
Myxopyronin A: R= n-Pr
Myxopyronin B: R= n-Bu
Figure 1. Chemical structures of dithiolopyrrolones, myxopyronins and corallopyronins
O
O
H
H
R1
X
N
N
6
R1
N
4
R2
N
Ar
O
O
S
S
1
3
R2= Me or H
S
2
S
Natural Dithiolopyrrolones
N-aryl pyrrothine Derivatives
Figure 2. General design strategy for N-aryl pyrrothine derivatives.
2 RESULTS AND DISCUSSION
2.1 Chemistry Synthesis
The synthesis procedures for N-aryl pyrrothine derivatives is shown in Scheme 1, following the synthesis routes
on previous reports with minor modifications (Tan et al., 2013; Li et al., 2014). The starting material of compound 1
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was obtained according to previous procedure (Tan et al., 2013). Compounds 2a–j were synthesized from compound 1
and chlorocarbonate derivatives (R1OCOCl) by amidation reaction at room temperature for 3–10 h. Treatment of
compound 1 with triphosgene in the presence of THF afforded intermediate 3, Glycol (4) was then reacted with
intermediate 3 to obtain 2k. Reaction of compound 1 with phenyl chloroformate at room temperature for 3–8 h
afforded intermediate 5 (Thavonekham, 1997). Compounds 6a–i were synthesized by nucleophilic substitution of
compound 5 with the corresponding amines (R2R3NH) (Li et al., 2014). The synthesized compounds were confirmed
by 1H NMR,13C NMR and mass spectrometry (see Supporting information).
O
H
HO
Cl
N
OH
4
N
Ar
O
2k
c only for
S
S
3
2a
2b
2c
2d
2e
: R1= ethyl, Ar= 2,4-dimethoxyphenyl
: R1= propyl, Ar= 2,4-dimethoxyphenyl
: R1= ethyl, Ar= 4-chlorophenyl
: R1= phenyl, Ar= 4-chlorophenyl
: R1= ethyl, Ar= 4-flurorophenyl
: R1= phenyl, Ar= 4-flurorophenyl
b
O
O
H
O
N
H2N
R1
2f
N
Ar
N
Ar
2g
2h
2i
2j
: R1= ethyl, Ar= 2-methylphenyl
: R1= i-propyl, Ar= 2-methylphenyl
: R1= phenyl, Ar= 2-methylphenyl
: R1= phenyl, Ar= benzyl
O
2a-j
a for
S
S
S
S
2a-k
2k
: R1= 2-hydroxyethyl, Ar= 2,4-dimethoxyphenyl
1
6a
6b
6c
: R2= methyl, R3= methyl, Ar= 2,4-dimethoxyphenyl
: R2= 2-hydroxylethyl, R3= H, Ar= 2,4-dimethoxyphenyl
: R2= (1-methylpyrrolidin-2yl)ethyl, R3= H,
Ar= 2,4-dimethoxyphenyl
d
R3
N
O
H
O
H
O
N
6d
6e
6f
: R2R3N= 1-pyrrolidino, Ar= 2,4-dimethoxyphenyl
: R2R3N= 1-piperidino, Ar= 2,4-dimethoxyphenyl
: R2R3N= 1-piperidino, Ar= 4-methoxyphenyl
N
R2
e
N
Ar
N
Ar
O
O
S
6g
6h
6i
: R2= ethyl, R3= H, Ar= benzyl
S
S
: R2= 2-hydroxylethy, R3= H, Ar= benzyl
: R2=R3= hydroxylethyl, Ar= benzyl
S
6a-i
5
Scheme 1. Synthesis of N-aryl pyrrothine derivatives 2a–k and 6a–i. Reagents and conditions: (a) R1OCOCl, Et3N, THF, rt, 3–10
h, 45–63%; (b) triphosgene, THF, 0–5 °C, 1 h; (c) Et3N, THF, 0 °C–rt, 2 h, 49% in two steps; (d) phenyl chloroformate, Et3N,
THF, rt, 3–8 h; (e) amines (R2R3NH), THF, rt, 8–12 h, 40–56% in two steps.
2.2 Biological Evaluation
2.2.1 Disk Diffusion Test
Disk diffusion test was used to assess antibacterial activity for all the synthesized compounds with rifampin and
ciprofloxacin as positive controls. Paper wafer containing 30 μg of test compound was placed on Muller–Hinton agar
plate where bacteria of Staphylococcus aureus (S. aureus) or Escherichia coli (E. coli) had been inoculated. The
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diameter of the clear zones around the paper wafer was measured and shown in Table 1. Interestingly, all the
compounds showed stronger inhibition against Gram-positive bacteria of S. aureus than the Gram-negative bacteria of
E. coli. Among them, four compounds (2k, 6a,6e and 6f) exhibited strong activity against S. aureus (inhibition zone >
20 mm) than the other ten compounds (2a, 2b, 2f, 2g, 2h, 6b, 6c, 6d, 6h and 6i) (16 mm < inhibition zone < 20 mm).
All the synthesized compounds displayed weak activity against E. coli with an inhibition zone < 12 mm.
Table 1. Disk diffusion test using S. aureus and E. coli.
Inhibition zone (mm)a
Inhibition zone (mm)a
Compd.
Compd.
S. aureus
E. coli
ATCC 25922
8.23
S. aureus
E. coli
ATCC 25922
9.34
ATCC 29213
16.16
16.23
12.23
10.56
16.63
9.33
ATCC 29213
20.23
2a
2b
2c
2d
2e
2f
6a
6b
6c
8.45
19.62
9.02
9.45
18.23
9.14
9.78
6d
6e
18.46
8.70
8.41
23.34
8.39
8.34
6f
20.83
8.51
2g
2h
2i
19.87
19.10
10.49
14.32
25.48
8.65
6g
11.82
8.54
8.16
6h
6i
19.16
8.25
8.41
18.84
8.36
2j
7.25
Rifb
Cipb
36.16
17.20
38.84
2k
11.36
33.42
a The diameter of inhibition zones for compounds 2a–k and 6a–i in the disk diffusion test. All compounds were dissolved in MeOH
and tested at a dose of 30 μg/wafer.
b Used as a positive control. Rifampin (Rif), ciprofloxacin (Cip).
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2.2.2 Determination of Minimum Inhibition Concentration (MIC)
An MIC assay against five bacteria was further performed for the compounds showing antibacterial activities in
disk diffusion test (inhibition zone against S. aureus> 16 mm), and presented in Table 2. Compounds 2g, 2k, 6d, 6e
and 6f showed the lowest MIC value (1 µg/mL) against S. aureus, and four compounds (2g, 2k, 6e and 6f) showed the
lowest MIC value (2 µg/mL) against Bacillus subtilis (B. subtilis). In addition, compounds 2g and 6e displayed the
strong inhibitory activity against Enterococcus faecalis (E. faecalis) with MIC values of 1 µg/mL, and compounds 2g,
2h, 6e and 6f displayed the strong inhibitory activity against against M. smegmatis with MIC value of 4 µg/mL.
Consistent with previous disk diffusion test, all the compounds showed weak activity against the Gram-negative
bacteria of E. coli with MIC values more than 64 µg/mL. All these results indicate that these synthesized compounds
only inhibit Gram-positive bacteria.
Among all the test compounds, compound 6e showed the lowest MIC values against most of the bacteria. When
the 2,4-dimethoxylphenyl group at the N-4 position of compound 6e was replaced with 4-methoxylphenyl group,
compound 6f showed slightly decreased activity against E. faecalis. Compound 6b with the 2-hydroxylethyl group at
the C-6 side chain and the 2,4-dimethoxylphenyl group at the N-4 position showed moderate antibacterial activity
against all the five tested bacteria. However, the corresponding compound 6h with the benzyl group at the N-4
position was less potent than the compound 6b. A similar phenomenon was also found between compounds 2a and 2e.
These results suggest that the introduction of the 2,4-dimethoxylphenyl group at the N-4 position plays an important
role in antibacterial activity, while introduction of the 4-benzyl, 4-chlorophenyl or fluorophenyl groups reduce
antibacterial activity.
Side chain substituents at the C-6 position also affected antibacterial activity. For the carbamate derivatives
2a–k, compound 2i with a phenyl group was inactive, whereas the corresponding compounds 2g and 2h with ethyl
and i-propyl groups, respectively, showed antibacterial activity against the tested bacteria. These results suggest that
the introduction of the phenyl group at the side chain is not crucial for antibacterial activity. For the urea derivatives
6a–i bearing the polar heterocycle ring at the side chain, compounds 6d, 6e and 6f but not compound 6c, displayed
antibacterial activity to certain degree.
Table 2. Antimicrobial activity (MIC, μg/mL) of compounds 2a–b, 2e, 2g–2k, 6a–f and 6h–i.
MIC (μg/mL)a
S. aureus
B. subtilis
E. faecalis
M. smegmatis
ATCC 700084
128
E. coli
ATCC 25922
128
Compd.
ATCC 29213
ATCC 6633
ATCC 29212
64
64
32
64
64
64
2a
2b
128
128
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2e
2g
32
64
64
1
128
4
128
64
64
64
64
64
64
64
64
64
64
64
8
1
2
2h
2
4
4
4
2k
1
2
2
32
8
6a
4
4
8
6b
4
8
2
8
6c
16
16
16
8
64
8
6d
1
4
2
6e
1
1
4
6f
1
2
4
4
6h
4
16
8
64
64
64
0.25
16
6i
4
≤0.0625
0.5
32
8
Rifb
Cipb
Vanb
≤0.0625
0.0625
0.5
0.5
1
≤0.0078
64
1
4
a Minimum inhibitory concentration (MIC) was represented as the mean values of three independent experiments.
b Used as a positive control. Rifampin (Rif), ciprofloxacin (Cip) and vancomycin (Van).
Based on the in vitro antibacterial activity above, five potent compounds were selected for further evaluation
against four clinical isolates of rifampin-resistant S. aureus. As illustrated in Table 3, all the test compounds displayed
antibacterial activity against rifampin-resistant S. aureus with MIC values in the range of 1-4 μg/mL, which was much
lower than rifampin. In addition, compounds 6e and 6f exhibited similar activity as vancomycin (MIC= 1-2 μg/mL).
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These results suggest that these five compounds may have different mechanism from that of rifampin to inhibit
bacteria, and they might have the potential to overcome the rifampin-resistance in clinics.
Table 3. In vitro antibacterial activity against rifampin-resistant S. aureus
MIC (μg/mL)
Compd.
Structure
Rifampin-resistant S. aureus (4)a
O
H
N
O
2g
2h
2k
6e
6f
2-4
N
O
S
S
O
H
N
O
2-4
2-4
1-2
1-2
N
O
S
S
O
O
H
N
O
HO
N
O
O
S
S
O
O
H
N
N
N
O
O
S
S
O
H
N
N
N
O
O
S
S
Rifampin
>64
1-2
Vancomycin
a Number of bacterial strains tested are given in parentheses.
2.2.3 In vitro Inhibitory Activity against E. coli RNAP and Cytotoxicity
The five most potent compounds (2g, 2h, 2k, 6e and 6f) and compound 6i with weak antibacterial activity as
negative control were evaluated for their inhibitory activities against E. coli RNAP in vitro. A commercially available
E. coli RNAP assay kit was used since bacteria RNAP subunit sequences are highly conserved between Gram-positive
and -negative bacteria (Irschik et al., 1983). As shown in Table 4, compound 6e showed the most potent inhibitory
activity against E. coli RNAP, with an IC50 value of 12.0 ± 0.9 μM (Figure 3). Compounds 2h, 2k and 6f inhibited E.
coli RNAP with IC50 values of 94.4 ± 2.1, 65.6 ± 1.2 and 87.6 ± 1.4 μM, respectively. However, compound 2g
exhibited weaker inhibitory activity against E. coli RNAP (IC50 = 158.5 ± 3.0 μM), and compound 6i showed no
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activity against E. coli RNAP (IC50 > 1 mM) as expected. Moreover, the activities here were much lower than those
based on the in vivo antibacterial assay (Table 3), indicating that these selected compounds might inhibit the bacterial
growth through some other mechanisms in addition to the RNAP inhibition. In summary, the weaker antibacterial
activity against Gram-negative bacteria than Gram-positive bacterial for these compounds might be due to the
difference in cellular-uptake barriers and efflux (Kadakia et al., 2017), and the selected synthesized compounds might
bind to different sites of E. coli RNAP from that of rifampin. Further studies are needed to reveal the exact mechanism
of action.
The cytotoxicity of the selected compounds on HepG2 and LO2 cells was determined by methyl thiazoly
tetrazolium (MTT) assay (Table 4). Rifampin showed no cytotoxicity against HepG2 and LO2 cells with IC50 value of
145.3 and 823.2 µM, respectively. While all the selected compounds showed certain degree of cytotoxicity on both
cells. In particular, compound 6e displayed cytotoxic activity against HepG2 and LO2 cells with IC50 values of 6.3
and 9.8 µM, respectively. These results suggest that we should consider the toxicity in the future for the
N-2,4-dimethoxyphenyl dithiolopyrrolones derivatives.
Table 4. Inhibitory activity against E. coli RNAP and cytotoxicity of the compounds
Compounds
Cytotoxicity IC50 (μM)
E. coli RNAP
IC50 (μM)a, b
158.5 ± 3.0
94.4 ± 2.1
65.6 ± 1.2
12.0 ± 0.9
87.6 ± 1.4
>1 mM
HepG2
LO2
8.3
2g
2.0
2.3
2h
5.6
2k
1.5
2.9
6e
6f
6.3
9.8
3.4
8.6
6i
3.8
4.5
Rifampin
0.15 ± 0.06
145.3
823.2
a IC50 values (SD < 20%) are expressed as mean ± SD. Each experiment was performed in triplicate.
b The reported IC50 values of myxopyronin A and myxopyronin B were 20.0 μM and 5.0 μM, respectively (Hu et al., 1998).
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Figure 3. IC50 curve for compound 6e against E. coli RNAP
2.3 Biochemical Properties and Lipinski’s Rule of Five (RO5) Validation
The biochemical properties of selected compounds (2g, 2h, 2k, 6e and 6f) were validated by free web tool
(http://www.molsoft.com/ and http://www.swissadme.ch/) in order to reveal the correlations between these parameters
and antibacterial activity. According to the well‐known RO5 (Lipinski et al, 2001) and its extensions (Keller et al,
2006), a molecular mass ≤ 500 Da, logP value ≤ 5, hydrogen bond donors (HBD; sum of NH and OH) ≤ 5, hydrogen
bond acceptors (HBA; sum of N and O) ≤ 10 and polar surface area ≤ 140 A2 are indicative of an appropriate
orally‐active drug molecule for human use (Martel et al., 2016). Moreover, hydrogen bonding capacity is an important
parameter for drug permeability. Table 5 showed that all selected compounds have < 10 HBA and < 5 HBD values,
and the logP and molecular weight values are less than standard values, indicating the selected compounds follow the
RO5. Since lipophilic agents often demonstrate better antibacterial activity (Lipinski et al., 2001), the higher activity
of compound 6e may arise because this compound had the highest LogP.
Table 5. Biological properties of selected compounds.
Properties
Mol. weight (g/mol)
HBA
2g
2h
2k
6e
6f
334.04
348.06
397.03
419.10
389.09
5
1
5
1
8
2
6
1
4
2
HBD
Mol. LogP
SC
3.0
0
3.27
0
1.62
0
3.32
0
3.09
0
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Mol. Vol (A3)
Mol. PSA (A2)
GI
343.78
46.81
high
0
359.09
46.49
high
0
395.67
78.94
high
0
443.58
56.01
high
0
407.68
51.80
high
0
Lipinski
logS
-3.37
No
-3.71
No
-2.55
No
-3.52
No
-3.44
No
BBB permeant
HBA, hydrogen bond acceptor; HBD, hydrogen bond donor; LogP, log partition coefficient; SC, stereo center; PSA, polar surface
area; LogS, water solubility in mg/L; BBB Score, the blood-brain barrier (BBB) score (0-Low, 6-High).
2.4 Molecular Docking Studies
All the selected compounds, myxopyronin A and rifampin were docked into the switch region of bacterial
RNAP (PDB ID: 3DXJ), and the binding energy was calculated by AutoDock 4.2 (Scripps Research Institute, La
Jolla, CA, USA) (Morris et al., 2009). The negative and low binding energy values indicate strong interactions
between the compounds and RNAP (Uddin et al., 2019). As illustrated in Table 6, compound 6e in complex with
RNAP had the lowest binding energy value of –8.92 kcal/mol, whereas compounds 2g, 2h, 2k, 6f and 6i had binding
energies of –7.15, –7.69, –8.44, –8.22 and –6.20 kcal/mol, respectively. Figure 4A, C provides the details of the
specific interactions between compound 6e and the switch region of bacterial RNAP. The methoxy group in the
aromatic ring at the N-4 position of compound 6e formed a hydrogen bond with Trp1038 (with a distance of 3.09 Å).
In addition, the carbonyl group formed two hydrogen bonds with SER1084 and Lys621 with distance of 3.17 Å and
2.76 Å, respectively. The methoxy group in the aromatic ring of compound 6e also interacted with LYS1463,
Asp1100 and Trp1038 via a conserved water molecule (1539) within the target, which acts as a bridging molecule
(Figure 4A, C). In addition, hydrophobic interactions between compound 6e and Val1037, Ile1467, Val1466,
Leu1053, Leu619, Gly1033 were also observed (Figure 3C). The results indicate that compound 6e has an important
common binding mode that is known for currently reported inhibitors of the switch region of bacterial RNAP (Mosaei
and Harbottle, 2019; Sahne et al., 2013; Mukhopadhyay et al., 2008). Based on inspection of our AutoDock4.2 model,
myxopyronin A was observed to have a binding energy of –8.76 kcal/mol. Rifampin binds to a site on bacterial RNAP
β subunit (Irschik et al., 1983) which has no interactions with the switch region of the bacterial RNAP (binding energy
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only is –2.8 kcal/mol ) (Table 5). Myxopyronin A is the potent bacterial RNAP inhibitor, and adopts a U-shaped
conformation that fully occupies the switch region binding pocket of RNAP (Sahner et al., 2013; Fruth et al., 2014)
(Figure 4B, D). Similar to myxopyronin A, compound 6e adopted a similar conformation in the switch region of
RNAP (Figure 4B). These results suggest that compound 6e might interact with the switch region of bacterial RNAP.
Table 6. Docking results for selected compounds into the switch region of bacterial
RNAP (PDB ID: 3DXJ).
Compd.
Binding energy (kcal/mol)a
2g
–7.15
–7.69
–8.44
–8.92
–8.22
–6.20
–2.80
–8.76
2h
2k
6e
6f
6i
Rifampin
Myxopyronin Ab
a Binding energy, indicating binding affinity and capacity for the bacterial RNAP.
b Myxopyronin A was used as the positive control ligand for the bacterial RNAP.
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Figure 4. (A) Proposed binding mode of 6e (green) in the switch region of bacterial RNAP; (B) Comparison of the conformation of
myxopyronin A (magenta) and compound 6e (green). W (cyan, water molecule 1539); 2D Ligand interaction diagram of compound
6e (C) and myxopyronin A (D). Dashed lines indicate hydrogen bonds. Carbons in black, nitrogens in blue, and oxygens in red and
water molecule in cyan. Figures were generated by PyMOL1.7.4 (Schrödinger, LLC, Cambridge, MA, USA) and Ligplot+v1.4.5
(European Bioinformatics Institute, London, England).
To further investigate the binding modes of the synthesized compounds with the switch region of the bacterial
RNAP, molecular docking simulations of compounds 2h and 6f with different substituents at the N-4 position were
also conducted. As illustrated in Figure 5A, the 2-methylphenyl group of compound 2h does not form interactions
with key amino acids, such as Trp1038, Asp1100 and Lys1463. Because of the absence of the polar 2-methoxy group
in the aromatic ring, the docked conformation of compound 6f in the bacterial RNAP is also different from that of
compound 6e (Figure 5B). This structural observation indicates that the 2-methoxy group in the aromatic ring allows
the compound to properly orient within the binding site. In addition, compound 2k with 2,4-dimethoxylphenyl group
at the N-4 position and a different substituent at the side chain also displayed a low negative binding energy that was
similar to that of compound 6e (Table 6). All these data from molecular docking suggest that 2,4-dimethoxylphenyl
group at the N-4 position of the pyrrothine scaffold is crucial for high affinity binding.
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Figure 5. Proposed binding mode of 2h (A, magenta) and 6f (B, orange) in the switch of bacterial RNAP, W (cyan, water molecule
1539). Figures were generated by PyMOL1.7.4 (Schrödinger, LLC, Cambridge, MA, USA).
3 CONCLUSIONS
In this study, twenty N-aryl pyrrothine derivatives were designed, synthesized, and evaluated for the antibacterial
activity. The results showed that some N-aryl pyrrothine derivatives have potent antimicrobial activity. Compound 6e
exhibited potent antibacterial activity against the clinical isolates of rifampin-resistant S. aureus and the strongest
inhibitory activity against E. coli RNAP as well. Molecular docking studies further suggested compound 6e interacts
with the switch region of the bacterial RNAP, but further studies are required to confirm the exact binding mode. In
conclusion, the N-aryl pyrrothine scaffold is a promising lead for antibacterial drug development.
ACKNOWLEDGMENTS
The work was funded by National Natural Science Foundation of China (No. 81660571, 81460528, 21762010),
Guangxi Natural Science Foundation of China (No. 2018GXNSFAA281114, 2018GXNSFAA281135).
DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the supporting information of this article.
CONFLICT OF INTEREST
The authors disclose that there is no potential conflict of interest.
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