Elucidating the Structural Requirement of Uridylpeptide Antibiotics for Antibacterial Activity

Article abstract of DOI:10.1021/acs.jmedchem.0c00973

The synthesis and biological evaluation of analogues of uridylpeptide antibiotics were described, and the molecular interaction between the 3′-hydroxy analogue of mureidomycin A (3′-hydroxymureidomycin A) and its target enzyme, phospho-MurNAc-pentapeptide

Full text of DOI:10.1021/acs.jmedchem.0c00973

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Article
Elucidating the Structural Requirement of
Uridylpeptide Antibiotics for Antibacterial Activity
Yuma Terasawa, Chisato Sataka, Toyotaka Sato, Kazuki Yamamoto, Yukari
Fukushima, Chie Nakajima, Yasuhiko Suzuki, Akira Katsuyama, Takanori
Matsumaru, Fumika Yakushiji, Shin-ichi Yokota, and Satoshi Ichikawa
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c00973 • Publication Date (Web): 28 Jul 2020
Downloaded from pubs.acs.org on July 28, 2020
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Elucidating the Structural Requirement of
Uridylpeptide Antibiotics for Antibacterial Activity
a
a
b
a
Yuma Terasawa, Chisato Sataka, Toyotaka Sato, Kazuki Yamamoto, Yukari
c
c,d
c,d
a,e
Fukushima, Chie Nakajima, Yasuhiko Suzuki, Akira Katsuyama, Takanori
Matsumaru,a,e,† Fumika Yakushiji, Shin-ichi Yokota, and Satoshi Ichikawa
a,e
b
a,e,f*
aFaculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, Japan
bDepartment of Microbiology, Sapporo Medical University School of Medicine, South-1,
West-17, Chuo-ku, Sapporo, 060-8556, Japan
cDivision of Bioresources, Hokkaido University Research Center for Zoonosis Control,
Sapporo, 001-0020, Japan
dGlobal Station for Zoonosis Control, Global Institution for Collaborative Research and
Education (GI-CoRE), Hokkaido University, Kita-20, Nishi-10, Kita-ku, Sapporo, 001-
0
020, Japan
eCenter for Research and Education on Drug Discovery, Faculty of Pharmaceutical
Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
fGlobal Station for Biosurfaces and Drug Discovery, Global Institution for Collaborative
Research and Education (GI-CoRE), Hokkaido University, Kita-12, Nishi-6, Kita-ku,
Sapporo 060-0812, Sapporo, Japan
†Current affiliation: Department of Chemistry, Faculty of Science and Technology, Keio
University, Hiyoshi 3-14-1, Minatokita-ku, Yokohama, 223-8522, Japan
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Abstract
The synthesis and biological evaluation of analogues of uridylpeptide antibiotics were
described, and the molecular interaction between the 3′-hydroxy analogue of
mureidomycin A (3′-hydroxymureidomycin A) and its target enzyme, MraY, was
analyzed in detail. The structure-activity relationship involving MraY inhibition suggests
that the side chain at the urea-dipeptide moiety does not affect the MraY inhibition.
However, the anti-Pseudomonas aeruginosa activity is in great contrast and the urea-
dipeptide motif is a key contributor. It is also suggested that the nucleoside peptide
permease NppA1A2BCD is responsible for the transport of 3′-hydroxymureidomycin A
into the cytoplasm A systematic structure-activity relationship analysis of the urea-
dipeptide moiety of 3′-hydroxymureidomycin A was further conducted and determined
that the antibacterial activity. This study provides a guide for the rational design of
analogues based on uridylpeptide antibiotics.
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Introduction
Pseudomonas aeruginosa, which is classified as a Gram-negative bacteria, is a
common nosocomial pathogen; healthy individuals are rarely infected by this pathogen
because of its low pathogenicity. However, opportunistic and nosocomial infections in
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newborns and patients with weakened immunity have become problematic. P. aeruginosa
also locally infects the urethra, airways and wounds following medical procedures such
as surgery and catheterization, in addition to burns, trauma and purulent sites; moreover,
infection of patients with cystic fibrosis and chronic respiratory diseases by P. aeruginosa
can cause increased patient mortality.1-4 In addition, local infection is followed by
systemic infections, such as pneumonia and peritonitis, and it is known that the fatality
rate of sepsis due to P. aeruginosa in the blood is as high as 80%. Thus, P. aeruginosa is
among pathogens that must be carefully treated because it worsens the prognosis and
mortality of patients. Compared to Gram-positive pathogens, P. aeruginosa is inherently
less susceptible to antibacterial drugs currently used in the clinic because it possesses an
outer membrane that acts as a barrier to prevent their permeation. Furthermore, multi-
drug-resistant P. aeruginosa, which is resistant to all carbapenems, aminoglycosides, and
fluoroquinolones used for the treatment of P. aeruginosa infection, has emerged in the
clinic. The fatality rate caused by its infection is high because of the very limited number
of effective drugs and is the cause of a severe global health problem. Consequently,
development of new drugs for multi-drug-resistant P. aeruginosa is an urgent task for
worldwide health care.5-7 In choosing novel antibacterial agents, the target must be
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essential for their growth, and the agent’s mechanism of action should be different from
those of existing drugs.8-12
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Uridylpeptide antibiotics are nucleoside natural products sharing a common
structural feature; a 3′-deoxyuridine with a Z-enamide linkage is attached to a tetrapeptide
moiety with a urea linkage reversing the direction of the N- and C-termini, with ,-
diaminobutylic acid playing a pivotal role by connecting the N-terminal amino acid, the
urea-dipeptide, and the 3′-deoxyuridine moieties (Figure 1).13-14 The class includes
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mureidomycins (MRDs), napsamycins, pacidamycins (PCDs), and sansanmycins.
Uridylpeptide antibiotics are a growing class of nucleoside natural products, and their
chemical structures vary with various amino acid residues. Among the class of
uridylpeptide antibiotics, the MRDs, isolated from Streptomyces flavidoviridens SANK
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0486, have been reported to show the most potent antibacterial activity against strains
of Pseudomonas (minimum inhibitory concentration (MIC) 1.5–12.5 g/mL) in vitro and
can also protect mice against P. aeruginosa infection.15b,c The MRDs and PCDs have
been reported to inhibit the formation of lipid II and peptidoglycan and are strong
inhibitors of the phospho-MurNAc-pentapeptide transferase (MraY),19,20 which is
responsible for the formation of lipid I in the peptidoglycan biosynthesis pathway.19-24
Since MraY is an essential enzyme in bacteria, it is a potential target for the development
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of general antibacterial agents. A narrow antibacterial spectrum is characteristic of
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uridylpeptide antibiotics.15b,c They exhibit highly selective antibacterial activity against
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P. aeruginosa but not against other Gram-negative and Gram-positive bacterial pathogens.
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Different from Gram-positive bacteria, Gram-negative bacteria possess an outer
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membrane, which acts as a physiological barrier preventing antibacterial drugs from
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reaching the periplasm. Only limited nutrients and ions necessary for bacterial growth
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OH
N
OH
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NH2
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H
NMe
H
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NMe
H
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NH
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HO C
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HO C
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H
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HO
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HO
O
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OH
OH
SMe
mureidomycin A (MRD A)
NH
napsamycin B
O
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O
NH2
NH2
NMe
O
NMe
NH
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NH
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H
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H
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H
N
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HO C
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O
HO C
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N
N
O
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N
H
N
H
HO
O
O
O
O
OH
HN
OH
SMe
sansanmycin A
Figure 1. Structures of Uridylpeptide Natural Products
pacidamycin D (PCD D)
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can be incorporated through specific transporters. The observed narrow spectrum of
uridylpeptides indicates that they utilize a transporter specifically expressed on P.
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aeruginosa. We have recently investigated the MraY inhibitory and anti-P.aeruginosa
activities of synthetic pacidamycin D (PCD D) and its 3′-hydroxy analogue 1 (3′-hPCD
D, Figure 2).27,28 They exhibited potent MraY inhibitory activity with IC values of 22
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nM for PCD D and 42 nM for 1 and moderate antibacterial activity against P. aeruginosa.
The existence of a hydroxyl group at the 3′-position of the uridine moiety does not
interfere with either the MraY inhibition or the antibacterial activity. The 3′-hydroxy
analogues of uridylpeptide antibiotics are a good starting point to investigate a structure-
activity relationship (SAR) of uridylpeptide antibiotics since the 3′-hydroxy analogues
are more accessible from a chemical synthesis point of view. Judging from the data
O
O
NH2
5
NMe
H
N
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NH 3
O
O
H
H
N
HO C
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5' 4' O
HO
2
1
N
1'
H
O
O
HN
OH
3'-hydroxypacidamycin D (3'-hPCD D, 1)
previous study
OH
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NH2
NMe
O
NH
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H
H
H
N
HO C
N
N
N
O
2
N
H
HO
O
O
HO
OH
SMe
3'-hydroxymureidomycin A (3'-hMRD A, 2)
target in this study
Figure 2. Structures of 3'-hydroxyuridylpeptide analogues
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reported in the literature, among the uridylpeptide antibiotics shown in Figure 1, those
possessing the L-m-tyrosine (L-mTyr) on both N- and C-termini, namely, the MDRs, seem
to exhibit the strongest antibacterial activity against P. aeruginosa. Although SAR studies
of antibacterial activity of this class of natural products have independently been
conducted,29-39 the strains tested were not uniform. Therefore, a side-by-side comparison
of the impact of each amino acid residue on both MraY inhibition and antibacterial
activity must be performed to understand the molecular basis of uridylpeptide antibiotics.
Very recently, we reported a complex structure of 3′-hydroxymureidomycin A (3′-hMRD
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A, 2, Figure 2) bound to MraY from Aquifex aeolicus (MraY ). Here, we describe the
AA
detailed synthesis, biological evaluation and further SAR of 2. In addition, a transporter
responsible for uptake of 2 is also proposed. The aim of this study is to elucidate the
structural requirements of 2 for both MraY inhibition and antibacterial activity against P.
aeruginosa to gain insight into future drug design based on uridylpeptide antibiotics.
Results and discussion
Synthesis of 3′-hydroxymureidomycin A. We have previously accomplished the total
synthesis of PCD D and 1 featuring a stereoselective construction of the Z-oxy-acyl
enamide architecture by copper-catalyzed C-N cross-coupling.27,28 We have also
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developed the second-generation synthesis of 1 via a Ugi four-component reaction. This
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Scheme 1
3
'-hMRD A (2)
sequential peptide coupling
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H
t-BuO C
N
CO H
2
2
NMe
NH
O
OTIPS
H
N
+
N
+
TIPSO
O
O
H N
2
HO C
NHBoc
2
SMe
O
TBSO
OTBS
5
4
3
Cu(I)-mediated
C-N cross coupling
O
NBOM
O
NHMe
CONH2
N
I
O
+
BocHN
6
TBSO
OTBS
7
second-generation synthesis is a convergent method to provide a range of analogues.
However, it also provided an undesired and inactive diastereomer at the 2,3-
diaminobutanoic acid residue. Therefore, we developed a more efficient third-generation
synthesis to investigate the systematic SAR of uridylpeptide antibiotics. Our
retrosynthetic analysis of 2 is shown in Scheme 1. The 2,3-diaminobutanoic acid residue
linking to the uridine moiety with a Z-enamide linkage plays a role as a branching scaffold
and both the urea-dipeptide and the amino acid are linked to two nitrogen atoms by
forming primary and secondary carboxamide linkages. Thereby, 2 was disconnected from
these components, 3-5. Thus, a range of analogues suitable for investigating the SAR
regarding the urea-dipeptide and the amino acids on both termini could be attained in the
last stage of the synthesis. A uridine derivative connected to diaminobutanoic acid via an
1
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
enamide linkage 3 was disconnected from the 2,3-diaminobutancarboxamide 6 and the Z-
vinyl iodide 7 by copper-catalyzed C-N cross-coupling.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 2
1
) Boc O, NaHCO3
2
aq. THF
2
) TIPSCl, imidazole
NH
HO C
NH2
CH Cl2
t-BuO C
NHBoc
2
2
2
t-BuOCCCl3
CH Cl2
3) K CO3
2
THF-MeOH-H O
2
2
5
91% over
95%
3 steps
OH
OTIPS
L-m-Tyr-OH (mTyr, 8)
9
TFA
t-BuO C
NH2
2
CH Cl2
2
0
°C
97%
OTIPS
10
Suitably protected L-mTyr derivatives 5 and 10 were prepared starting from
commercially available L-mTyr as shown in Scheme 2. Solution phase synthesis has thus
far been used for synthesis of urea-dipeptide; however, it sometimes causes a decrease in
the yield of the desired urea-dipeptide due to homodimer formation. Therefore, solid
phase synthesis was applied, in which an asymmetric urea-dipeptide can be selectively
4
1
and easily synthesized a simple purification procedure (Scheme 3). Fmoc-L-methionine
immobilized on 2-chlorotrityl resin was treated with piperidine, and the resulting amine
was converted to the corresponding carbonylimidazolidate, which was reacted with 10 in
the presence of i-Pr NEt to yield urea 12. Finally, removal of the urea-dipeptide from the
2
resin by treatment with 1% TFA in CH Cl cleanly provided 4 with a 78% yield over four
2
2
1
4
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steps from 11. This method is suitable for the comprehensive preparation of a variety of
urea-dipeptides.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
) piperidine/DMF
Scheme 3
2) 1,1'-carbonyldiimidazole
CH Cl2
2
3) 10, i-Pr NEt
2
O
O
DMF
H
H
4) TFA/CH Cl
2 2
FmocHN
t-BuO2C
N
N
O
O
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
78% over
O
4 steps
SMe
SMe
: 2-chlorotrityl resin OTIPS
12
11
With 4 and 5 in hand, synthesis of 2 was investigated as shown in Scheme 4. The
1
6
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carboxylic acid 13, which was readily prepared from L-threonine in six steps in large
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
3
1
quantity by using a previous procedure, was converted to the carboxamide 14 in 81%
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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yield (NH Cl, HATU, i-Pr NEt, DMF). After cleavage of the N-O bond of 14 by catalytic
4
2
hydrogenolysis (H , Pd(OH) , MeOH), the liberated amine 6 was directly coupled with
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 4
NH Cl, *HATU
4
OBn
OBn H , Pd(OH)
2 2
i-Pr NEt, DMF
2
NMe rt. 1 h
NMe
MeOH, rt. 12 h
L-Thr
6
81%
NH2
6
steps
BocHN
CO H
BocHN
2
(ref 28)
O
7, CuI,
13
14
Me NCH CH NMe
2
2
2
2
Cs CO , THF
2
3
70 °C, 7 h
67% over 2 steps
O
O
NMe
O
O
AllocCl
i-Pr NEt
2
NHMe
H
N
NBOM
O
NBOM
CH Cl
H
2
2
N
N
N
O
O
BocHN
BocHN
O
93%
O
O
TBSO
16
OTBS
TBSO
OTBS
15
BCl , CH Cl
2
3
2
53%

78 °C, 1 h
3
*HBTU
4, *HBTU
i-Pr NEt, DMF
84%
N
2
N
O
O
O
N
NMe
H
N
N

NH
O
PF6
O
H
H
N
O
N
t-BuO C
N
N
O
2
N
H
O
O
*HATU
TBSO
OTBS
SMe
N
N
17
OTIPS
N
N
N
PF6
1
) Pd(PPh3)
4
morpholine
THF
44% over
N
2
steps
OTIPS
O
2
) 5, HATU
O
O
NHBoc
i-Pr NEt, DMF
2
NMe
H
N
NH
O
O
H
H
N
t-BuO C
N
N
O
2
N
H
O
O
TBSO
OTBS
SMe
18
OTIPS
1
) 3HF·Et N
MeCN
3
OH
O
O
2) 80% aq. TFA
NH2
NMe
H
N
NH
O
O
H
H
N
+
O
3'-hMRD A (2)
HO C
N
N
2
N
H
45% over 2 steps
O
O
HO
OH
S(O)Me
19 (35%)
OH
1
9
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1
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1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
the iodide 727,28 under the conditions using CuI, MeNHCH CH NHMe, and Cs CO in
2
2
2
3
THF at 70 °C to give the desired Z-enamide 15 in 67% yield. Cross coupling of 7 and 14
also proceeded to produce the corresponding Z-enamide in good yield, however the
following N-O bond cleavage was unsuccessful. After protection of the N-methylamine
of the carboxamide 6 with an allyloxycarbonyl (Alloc) group, the corresponding N-Alloc
carboxamide was used for the coupling. In this case, an unknown byproduct was mainly
generated, and the desired 16 was not obtained at all. This problem was solved by simply
changing the order of the cross coupling and Alloc protection reactions. After the N-
methyamino group of 15 was protected by the Alloc group to give 16 in 93% yield,
simultaneous deprotection of the benzyloxymethyl (BOM) and Boc groups was achieved
with BCl in CH Cl at 78 °C to afford 3 in 53% yield. The primary amine 3 was
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
2
2
condensed with the urea-dipeptide 4 (HBTU and i-Pr NEt in DMF) to give 17 in 84%
2
yield. Removal of the Alloc group at the N-terminus of 17 was conducted by the use of
conventional conditions (Pd(PPh ) , morphorine, THF), and the resulting N-methylamine
3
4
was condensed with 5 in the presence of HATU and i-Pr NEt in DMF to afford the fully
2
protected 3′-hMRD A 18 in 44% yield over two steps. Finally, deprotection of the TBS
groups (3HF ∙ Et N, MeCN) followed by the Boc and t-Bu groups (80% aq. TFA)
3
successfully provided 3′-hMRD A (2) in 45% yield over two steps. During 80% aq. TFA
2
0
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treatment, the methythio group was oxidized, and the corresponding sulfoxide 19 was
also obtained in 35% yield, which was easily separated by reverse-phase column
chromatography.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
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8
9
0
2
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1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Biological activity of 2. The MraY inhibition of 2 was evaluated with fluorescence-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
1
2
3
4
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8
9
0
3'-hPCD D (1)
3'-hMRD A (2)
tunicamycin V
Figure 3. MraY inhibitory activity of analogues. aThe inhibitory
activities of the compounds against purified MraY from S. aureus.
Reaction was conducted with 50 mM Tris-HCl (pH 7.6), 50 mM KCl, 25
mM MgCl , 0.2% Triton X-100, 8% glycerol, 50 M C55-P, 10 M
2
UDP-MurNAc-dansylpentapeptide, and MraY enzyme (55 ng/5 L/well).
After 3-4 h incubation at room temperature, the formation of dansylated
lipid I was monitored by fluorescence enhancement (excitation at 355
nm, emission at 535 nm). The data were obtained from three independent
experiments.
2
2
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based MraY inhibitory assay using the purified MraY enzyme (S. aureus, MraY ) and
SA
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
dansylated Park’s nucleotide that was prepared by dansylation of Park’s nucleotide
0
1
2
3
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5
6
7
8
9
0
1
2
3
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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0
1
2
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chemically synthesized with our method.43 The data were obtained from three
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
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1
1
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
6
independent experiments. As shown in Figure 3 and Table 1, 2 exhibits a strong MraYSA
inhibitory activity, with an IC value of 1.3±0.2 nM, and is 34-fold more potent than 1.
5
0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The antibacterial activity of 2 against a range of P. aeruginosa strain, including a variety
Table 1. Biological activity of 1 and 2
1
2
IC50 for MraYSA (nM)a
44 ± 4
>100
1.3 ± 0.2
>100
IC50 for HepG2 (M)b
MIC (g/mL)a
strains
PAO1
ATCC27853
MDRP62
16-32
32-128
>128
4-16
4-16
16-32
8-32
multi-drug resistant
clinical isolate
clinical isolate
clinical isolate
clinical isolate
clinical isolate
serotype A
1483
1485
2867
2931
9728b
>128
16-64
32-128
>128
4-8
8-32
16
>128
32
IID1001
>128
64-128
IID1002
IID1007
serotype B
serotype B
>128
>128
16
32-128
IID1013
IID5004
IID1021
IID1004
IID1130
IID1006
IID1020
IID1009
IID1010
serotype B
serotype B
serotype C
serotype D
serotype E
serotype F
serotype G
serotype H
serotype I
serotype J
serotype K
serotype L
serotype M
serotype M
>128
64-128
16-64
>128
>128
>128
>128
8-16
8-32
8-16
16-32
>128
8-16
>128
32-128
>128
8-32
32-128
IID1011
IID1012
IID5141
IID1015
IID5018
>128
64-128
32
16-32
16-64
4-16
>128
>128
16-32
>128
aThe inhibitory activities of the compounds against purified MraY from S. aureus was
b
determined in a manner similar to the experiments shown in Figure 3. HepG2 cells were
5
seeded in 96-well tissue culture plates at a 1x10 cells/well. After 24 h, cells were treated
with varying concentrations of compounds for 48 h. After the treatment, the cell viability
was measured by WST-8 assay. In there experiments, tunicamycin exhibited cytotoxicity
with the IC50 value of 38.6 M. cMICs were determined by a microdilution broth
method as recommended by the CLSI. The data were obtained from three independent
experiments described as a range of the values.
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of clinical isolates and serotypes was then investigated. The data were obtained from three
independent experiments, and MIC values for each compound are within four-fold, which
are described as a range of these values. 3′-Hydroxymureidomycin (2) exhibits a potent
antibacterial activity over a wide range of P. aeruginosa strains with MIC values ranging
from 4-128 g/mL, which are lower than those of 1. It is noteworthy that 2 is effective
against multi-drug-resistant P. aeruginosa (MDRP62) with an MIC value of 16-32
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g/mL. Improvement of the MraY inhibitory activity of 2 over 1 is correlated with the
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anti-P. aeruginosa activity, and it was revealed that 2 possesses superior properties in
terms of both MraYSA inhibitory activity and anti-P. aeruginosa activity. Overall, the
replacement of amino acid resides at both C- and N-termini with L-mTyrs improved the
biological activity, confirming 2 as a superior candidate, as expected from the literature
review.13-20 Selective toxicity is an important issue in chemotherapy for infectious
disease. Accordingly, the cytotoxicity of 2 against human hepatocellular carcinoma
(HepG2) cells was then evaluated. Tunicamycins were also tested as a positive control
and showed evidence of cytotoxicity, with an IC of 38.6 μM. Under these conditions, 2
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exhibited no cytotoxicity (IC >100 μM). This result indicates that 2 showed selective
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toxicity against bacterial strains. The observed high therapeutic index is a desirable
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property for antibacterial agents. Thus, 2 was confirmed to be a selective anti-P.
aeruginosa agent.
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Elucidating the interaction between MraY and 2. The X-ray crystal structure of a
complex of muraymycin D2 (MRY D2), other class of nucleoside natural product and a
strong MraY inhibitor, bound to MraY from Aquifex aeolicus (MraY ) has been
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elucidated (PDB code 5CKR, 2.95 Å). It is also revealed that MraY undergoes a large
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conformational change upon binding to MRY D2 by comparing the structure with the
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apoMraYAA structure (PDB code 4J72) (Figure 4b, d, f). With 2 possessing better
properties, the X-ray crystal structure of a complex of 2 bound to MraYAA (PDB code
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OZ6, 3.832–3.70 Å) was solved by our collaborative study to understand the molecular
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basis of the interaction of 2 and MraYAA (Figure 4a, c, e). The complex of 2 bound to
MraYAA is similar to that of MRY D2. Using the alignment of the sequence of MraYSA
and MraY from P. aeruginosa (MraY ) (Figure 5, for larger image, see Supporting
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Information) on the crystal structure of MraYAA bound to 2 as a template, in this study,
we developed homology models of MraYSA and MraYPA (Figure 6) using SwissModel46
to predict the molecular basis of 2 interacting with MraY , which is used for enzymatic
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assays, and MraY expressed in P. aeruginosa, the target bacteria. These models reveal
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that overall, the three-dimensional structures of the MraY and MraY models are quite
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similar to that of MraY , and the amino acid residues and their interacting modes are
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