Novel hybrid-type antimicrobial agents targeting the switch region of bacterial RNA polymerase

Article abstract of DOI:10.1021/ml300350p

The bacterial RNA polymerase (RNAP) is an ideal target for the development of antimicrobial agents against drug-resistant bacteria. Especially, the switch region within RNAP has been considered as an attractive binding site for drug discovery. Here, we designed and synthesized a series of novel hybrid-type inhibitors of bacterial RNAP. The antimicrobial activities were evaluated using a paper disk diffusion assay, and selected derivatives were tested to determine their MIC values. The hybrid-type antimicrobial agent 29 showed inhibitory activity against Escherichia coli RNAP. The molecular docking study suggested that the RNAP switch region would be the binding site of 29.

Full text of DOI:10.1021/ml300350p

Letter
pubs.acs.org/acsmedchemlett
Novel Hybrid-Type Antimicrobial Agents Targeting the Switch
Region of Bacterial RNA Polymerase
Fumika Yakushiji,*,† Yuko Miyamoto,† Yuki Kunoh,† Reiko Okamoto,† Hidemasa Nakaminami,‡
Yuri Yamazaki,† Norihisa Noguchi,‡ and Yoshio Hayashi*,†
†
‡
Departments of Medicinal Chemistry and Microbiology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences,
1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
S
* Supporting Information
ABSTRACT: The bacterial RNA polymerase (RNAP) is an ideal target for the
development of antimicrobial agents against drug-resistant bacteria. Especially, the
switch region within RNAP has been considered as an attractive binding site for drug
discovery. Here, we designed and synthesized a series of novel hybrid-type inhibitors
of bacterial RNAP. The antimicrobial activities were evaluated using a paper disk
diffusion assay, and selected derivatives were tested to determine their MIC values.
The hybrid-type antimicrobial agent 29 showed inhibitory activity against Escherichia
coli RNAP. The molecular docking study suggested that the RNAP switch region would be the binding site of 29.
KEYWORDS: bacterial RNA polymerase, switch region, antimicrobial agent, bacterial RNA polymerase inhibitor, hybrid
site in RNAP has attracted the attention of medicinal chemists
to develop new antimicrobial agents.
ifamycins are a class of bacterial RNA polymerase
R(RNAP) inhibitors commonly used in clinical practice.
The potency and broad-spectrum nature of rifamycins have
improved the treatment of infectious diseases, including
tuberculosis. Nevertheless, the emergence of drug-resistant
bacteria poses an ongoing threat to the therapeutic index of
rifamycins. For this reason, the development of a novel RNAP
inhibitor is in demand. In 2008, a research team led by Arnold
and Ebright elucidated the inhibitory mechanism of myxopyr-
onin A (1) (Figure 1),1 which was targeting the switch region
of bacterial RNAP, and an X-ray crystal structure of 1 in
complex with T. thermophilus RNAP was reported.2,3
Corallopyronin (3) and ripostatins also appeared to bind to
the RNAP switch region.2 Hence, this newly identified binding
Before now, the research group of Simonsen and Xiang
attempted a direct structure−activity relationship study of
myxopyronin.4,5 In addition, Fishwick and co-workers tried
structure-based ligand design at the myxopyronin binding site.6
On the other hand, Moy and co-workers reported that
myxopyronin was an unsuitable lead compound for medicinal
development because of the high binding to serum albumin, the
low stability, and the occurrence of resistant strains.7
In this report, we disclose our effort to develop novel
antimicrobial agents with a hybrid strategy8,9 for myxopyronin
derivatives. To enhance the antimicrobial activity, reduce the
lipophilicity, and improve the instability, the antibiotic
holomycin (4)10 was incorporated into the mother skeleton
of the myxopyronin-type RNAP inhibitors.
Holomycin (4) also possessed antibacterial properties and
provided moderate but broad antibiotic spectrum against
Gram-positive and -negative bacteria.11,12 In addition, 4 showed
the inhibition of RNA synthesis,11 and the calculate log P
(ClogP) did not exceed 2.0.13 These properties fascinated us to
hybridize myxopyronins with the holothin molecule to expect
the efficacy beyond our anticipations.
Our hybrid strategy is shown in Figure 2. We designed two
types of hybrid derivatives based on the reported X-ray crystal
structure of RNAP with 1 (PDB: 3DXJ).2,3 One was the “right-
hand amide derivative (6)” with the core α-pyrone at the center
and the holothin component positioned on the right-hand side.
Toward the right side of the X-ray crystal structure, as shown in
the blue circle in Figure 2, there is an intermolecular hydrogen
Received: October 17, 2012
Accepted: December 30, 2012
Published: January 11, 2013
Figure 1. Structures of antibiotics myxopyronins A (1) and B (2),
corallopyronin (3), and holomycin (4).
© 2013 American Chemical Society
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ACS Medicinal Chemistry Letters
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bond network involving a water molecule. The holothin (5),
which included several heteroatoms, was incorporated into the
right-hand moiety to form the hydrogen bonds. The other is
the “left-hand amide derivative (7)”. The holothin was
introduced at the left side of the molecule relative to the C3
side chain. At the left side of the myxopyronin binding site, as
shown in the red circle in Figure 2, there is a useful space
around the C20 hydrocarbon chain. This interspace was
advantageous for accommodating the holothin moiety to
increase the interaction with RNAP.
We first synthesized right-hand amide derivatives (Scheme
1). The C3-acylation of starting material 8 proceeded in the
presence of propionic acid via condensation and rearrangement
to obtain 9.14 The pyrone 9 was alkylated with 10,15 then the
silyl protecting group was removed, and two-step oxidation
produced the carboxylic acid 11.4 Compound 11 was treated
with oxalyl chloride and condensed with holothin hydro-
chloride 1216 under basic conditions to yield the simplified
hybrid derivative 13. On the other hand, 17 was synthesized to
ascertain the importance of the left-hand portion of the hybrid
compound. Compound 8 was acylated with nonanoic acid, and
the resultant pyrone 14 was converted to a carboxylic acid 16
through the same four steps. Compound 16 was condensed
with the holothin salt 12 to construct the desired 17. We
attempted to prepare a series of right-hand derivatives with
unsaturated carbon side chains; however, the final products
could not be isolated due to decomposition at the purification
step. These observations suggested that the unsaturated side
chain of 1 was one factor for the reported instability.7
We next attempted to synthesize the left-hand amide
derivatives. As shown in Scheme 2A, 8 and different lengths
of the hydrocarbon linkers 18−20 were treated with N,N′-
diisopropylcarbodiimide (DIC) and DMAP for the C3-
acylation.14 The methyl ester groups of the obtained 21−23
were then hydrolyzed to the carboxylic acids 24−26, followed
by the condensation of 24−26 with 12 to generate 27−29.
Compound 28 was subsequently converted to the sulfone 30
using oxone as the oxidizing reagent.17,18
Figure 2. Design of hybrid-type derivatives including core α-pyrone
and holothin (5) based on the X-ray crystallography of 1 within
bacterial RNAP from T. thermophilus (PDB: 3DXJ; green stick,
myxopyronin A; gray stick and ribbon, bacterial RNAP; gray mesh,
myxopyronin binding site; W, water molecule; blue circle,
intermolecular hydrogen bond network at the right-hand side; and
red circle, space at the left-hand side).
The synthetic route B (Scheme 2B) showed the synthesis of
the derivative 32 including the right-hand alkyl chain and the
left-hand amide part. Compound 8 was alkylated using 4-
a
Scheme 1. Syntheses of the Right-Hand Amide Derivatives 13 and 17
a
Reagents and conditions: (a) Propionic acid or nonanoic acid, DIC, DMAP, toluene, 100 °C, 5 or 14 h, 72 or 74%, respectively. (b) Compound 10
or 15, LDA, HMPA, THF, −78 °C, 1 h, 75%. (c) AcOH/THF/H2O (3/1/1), rt, 26 h, 66−89%. (d) PCC, CH2Cl2, rt, 30 min. (e) NaClO2,
NaH2PO4, 2-methyl-2-butene, tBuOH/THF/H2O (1/1/1), rt, 4 h, 63−84%. (f) (COCl)2, toluene, rt, 1 h. (g) Compound 12, Et3N, THF/toluene,
rt, 30 min, 61−65%.
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ACS Medicinal Chemistry Letters
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a
Scheme 2. Syntheses of the Left-Hand Amide Derivatives 27−29, 30, 32, and 35
a
Reagents and conditions: (a) Compound 18, 19, or 20, DIC, DMAP, toluene, 100 °C, 6−14 h, 64−93%. (b) 1 M LiOH aq/THF (1/4), rt, 14 h.
(c) (COCl)2, toluene, rt, 1 h. (d) Compound 12, Et3N, THF/toluene, rt, 30 min, 38−66%. (e) Oxone, acetone/H2O, 0 °C, 30 min, 40%. (f) 4-
Bromo-1-butene, LDA, HMPA, THF, rt, 14 h, 85%. (g) Ba(OH)2, H2O/THF, 73 °C, 2 h.
Figure 3. Structures of the synthesized derivatives 36−38.
bromo-1-butene, and the C3 position of the obtained pyrone
was acylated with 19. After hydrolysis, the resultant carboxylic
acid 31 was condensed with 12 to obtain 32.
The synthetic route C (Scheme 2C) describes the
preparation of a C3 amide derivative. The amide ester analogue
33, prepared from 4-hydroxy-6-methyl-2-oxo-2H-pyran-3-car-
boxylic acid19 and methyl 7-aminoheptanoate, was mildly
hydrolyzed to 34, and then, the carboxylic acid was condensed
with 12 to synthesize the product 35.
As other derivatives, three compounds 36−38 were also
synthesized (Figure 3).20 Compound 36 was constructed to
ascertain the significance of the length of the left-hand
hydrocarbon linker. The piperidine-containing derivative 37
was synthesized to examine the effect of the holothin
component. The holothin derivative 3821 was also synthesized
to observe the efficacy of a hybrid structure including α-pyrone.
The synthesized derivatives were evaluated as an antimicro-
bial agent using a simple paper disk diffusion assay against
Bacillus subtilis (Table 1).22 The right-hand amide derivatives
were tested first. The synthetic intermediate 9 did not exhibit a
zone of inhibition. The right-hand amide derivative 13 also
showed no antimicrobial activity. By contrast, 14, 16, and 17,
Table 1. Disk Diffusion Assay against B. subtilis
a
b
a
b
compd
inhibition zone (mm)
compd
inhibition zone (mm)
9
13
14
16
17
25
27
28
29
30
no zone
no zone
12
31
no zone
10
32
34
no zone
slight
9
slight
9
35
36
no zone
14
37
no zone
9
38
11
RFP
( )-2
25
15
slight
no zone
no zone
MeOH
a
All compounds were dissolved in MeOH and evaluated at a dose of
b
30 μg/disk. Diameter of zones with complete growth inhibition.
which included left-hand side chains, produced inhibition
zones. These results suggested that the left-hand side chain
played an important role in the antimicrobial activity.
Furthermore, it became clear that the antimicrobial properties
of this series did not depend on the holothin moiety.
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ACS Medicinal Chemistry Letters
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Next, left-hand amide derivatives were evaluated. As
compared with the carboxylic acid 25 and the hybrid derivatives
27−29, the hybrid-type derivatives showed zones of inhibition.
These results suggested that the presence of the holothin
moiety enhanced the activity. Compound 30, including a
sulfone group, and 31 and 32 with right-hand hydrocarbon
chains were compared with 28; however, these derivatives did
not show significant validity, suggesting that the sulfone group
and the right-hand chains did not contribute to the activity.
Furthermore, 34 did not show any activity, although 35
exhibited a slight zone of inhibition. The α-pyrone 36 and the
holothin derivative 38 showed weak activities, whereas 37
produced no zone of inhibition. The synthesized ( )-myx-
opyronin B15 was also tested; however, ( )-2 produced only a
slight zone of inhibition in our assay system. Therefore, the
authentic rifampicin (RFP) was used as a standard antibiotic in
all assays.
Table 3. In Vitro Inhibitory Activity against E. coli RNA
Polymerase
a,b
compd
IC50 (μM)
14
>2 mM
>2 mM
14 1.7
27
29
RFP
0.08 0.01
a
IC50 values are means
SEMs from three independent dose−
response curves, in which each measurement was done in duplicate.
b
The reported IC50 values of myxopyronin B were 46.56 and 247 μM,
respectively.
the results in Table 3 suggested that the target of 29 was the
bacterial RNAP.
From the above, the expected binding site of 29 in RNAP
was considered in a molecular docking study based on X-ray
crystal data of myxopyronin A and RNAP complex (PDB:
3DXJ).24 As shown in Figure 4, 29 showed good conformation
The results in Table 1 indicated that the C3 position of the
α-pyrone should be substituted with a long hydrocarbon moiety
for the expression of the antimicrobial activity. In addition, the
hybrid derivatives containing α-pyrone and the holothin moiety
exhibited the enhanced activity.
On the basis of results of the paper disk assay, we selected
the effective derivatives 14, 27, and 29 for the next evaluations
of higher order assessments. These simplified derivatives
presented larger zones of inhibition, small molecular weights,
and low lipophilicities, as assessed by ClogP.13
The minimal inhibitory concentration (MIC) was evaluated
to characterize the antimicrobial activity of 14, 27, and 29
against eight kinds of Gram-positive and -negative bacteria and
fungi (Table 2). After 24 h of incubation, α-pyrone-type
Table 2. MIC Values for Selected Derivatives 14, 27, and 29
MIC (μg/mL)
organism
14
27
29
RFP
Figure 4. Molecular docking study of 29 in the switch region of T.
thermophilus RNAP (PDB: 3DXJ). Binding energy, −8.3 kcal/mol
(blue stick, 29; green stick, myxopyronin A; gray stick and ribbon,
bacterial RNAP; gray mesh, myxopyronin binding site; and W, water
molecule).
Staphylococcus aureus ATCC29213
Enterococcus faecalis ATCC29212
M. luteus ATCC9341
8
64
16
4
4
1
0.5
1
4
2
8
≤0.125
≤0.125
8
B. subtilis ATCC6633
8
64
16
≥256
128
E. coli ATCC25922
128
128
64
Pseudomonas aeruginosa ATCC27853 128
32
in the hydrophobic pocket of the switch region with a
minimum binding energy of −8.3 kcal/mol. Other predicted
conformations of 29 within RNAP were also examined;
however, these provided high binding energies over 13 kcal/
mol at least. Consequently, the expected binding site of 29
within RNAP would be regarded as the switch region.
Serratia marcescens ATCC13880
Candida albicans ATCC10231
128
64
128
128
≥256
≥256
32
≥256
derivative 1423 and the hybrid-type derivative 29 showed good
antimicrobial activity against Gram-positive bacteria, although
the activity against Gram-negative bacteria and fungi was weak.
The extension of the left-hand chain was effective for the
inhibition against the Gram-positive bacteria. Furthermore, 14,
27, and 29 gave the strongest activity against Micrococcus luteus.
The selective inhibitory activity against bacteria would depend
on our hybrid derivatives.
Besides, 14, 27, and 29 were evaluated for their in vitro
inhibitory activity against Escherichia coli. RNAP (Table 3). The
derivative 29 inhibited E. coli RNAP, and the IC50 value was
determined to be 14 1.7 μM. Unfortunately, 14 and 27 did
not exhibit inhibitory activity. The IC50 value of 29 was higher
than that of RFP, but the difference of activities might be a
result of the binding site recognition.3 In addition, the
ineffectiveness of 29 against E. coli in MIC might be due to
the low penetration ability in Gram-negative bacteria. However,
In summary, we designed and synthesized hybrid-type
antimicrobial derivatives including the core α-pyrone and the
holothin moiety toward the development of novel bacterial
RNAP inhibitors. The simplified derivative 29, in particular,
showed reliable antimicrobial activity against Gram-positive
bacteria. Furthermore, 29 inhibited E. coli RNAP, and the
molecular docking study suggested that the targeted switch
region would be the binding site. Our hybrid strategy revealed
that the left-hand moiety of the α-pyrone component had a
great effect on the antimicrobial activity, and the inserted
holothin part enhanced the ability. In addition, a derivative with
low lipophilicity and inhibitory activity against bacterial RNAP
was identified. Significantly, selectivity of antimicrobial action
and profiles of our hybrid-type derivatives indicated the
unexpected validity of hybridization. Hence, the hybrid strategy
would be one of the effective ways to explore novel drug
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candidates. The structure−activity relationship study of the
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the cocrystal (29 and bacterial RNAP) are in progress.
ASSOCIATED CONTENT
■
S
* Supporting Information
General information, details of the compound syntheses and
characterization, and the biological assay protocols. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(13) The value of calculate Log P (ClogP) was calculated by CS
ChemBioDraw Ultra 12.0 (Cambridgesoft.com).
(14) Mori, T.; Ujihara, K.; Matsumoto, O.; Yanagi, K.; Matsuo, N.
Synthetic studies of fluorine-containing compounds for household
insecticides. J. Fluorine Chem. 2007, 128, 1174−1181.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 81-42-676-3279. Fax: 81-42-676-4475. E-mail: fyakushi@
toyaku.ac.jp. E-mail: yhayashi@toyaku.ac.jp.
(15) Hu, T.; Schaus, J. V.; Lam, K.; Palfreyman, M. G.; Wuonola, M.;
Gustafson, G.; Panek, J. S. Total synthesis and preliminary antibacterial
evaluation of the RNA polymerase inhibitors ( )-myxopyronin A and
B. J. Org. Chem. 1998, 63, 2401−2406.
(16) Compound 12 was synthesized according to the following
manuscript. Hjelmgaard, T.; Givskov, M.; Nielsen, J. Expedient total
synthesis of pyrrothine natural products and analogs. Org. Biomol.
Chem. 2007, 5, 344−348.
(17) Adam, W.; Chan, Y.-Y.; Cremer, D.; Gauss, J.; Scheutzow, D.;
Schindler, M. Spectral and chemical properties of dimethyldioxirane as
determined by experiment and ab initio calculations. J. Org. Chem.
1987, 52, 2800−2803.
Funding
This research was supported by grants from MEXT (the
Ministry of Education, Culture, Sports, Science and Technol-
ogy), Japan, including the Platform for Drug Discovery,
Informatics, and Structural Life Science, the Grant-in-Aid for
Research Activity Start-up (No. 21890262), and the Grant-in-
Aid for Young Scientists (B) (No. 23790142).
Notes
The authors declare no competing financial interest.
(18) Yu, B.; Liu, A.-H.; He, L.-N.; Li, B.; Diao, Z.-F.; Li, Y.-N.
Catalyst-free approach for solvent-dependent selective oxidation of
organic sulfides with oxone. Green Chem. 2012, 14, 957−962.
(19) Suzuki, E.; Sekizaki, H.; Inoue, S. A new and simple synthesis of
triacetic acid lactone-3-carboxylic acid (3-carboxy-4-hydroxy-6-methyl-
2-pyrone). Synthesis 1975, 652−653.
ACKNOWLEDGMENTS
■
We thank C. Sakuma of Tokyo University of Pharmacy and Life
Sciences for NMR and mass spectroscopy measurements.
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