Novel Staphyloxanthin Inhibitors with Improved Potency against Multidrug Resistant Staphylococcus aureus

Article abstract of DOI:10.1021/acsmedchemlett.7b00501

Diapophytoene desaturase (CrtN) is a potential novel target for intervening in the biosynthesis of the virulence factor staphyloxanthin. In this study, 38 1,4-benzodioxan-derived CrtN inhibitors were designed and synthesized to overwhelm the defects of leading compound 4a. Derivative 47 displayed superior pigment inhibitory activity, better hERG inhibitory properties and water solubility, and significantly sensitized MRSA strains to immune clearance in vitro. Notably, 47 displayed excellent efficacy against pigmented S. aureus Newman, Mu50 (vancomycin-intermediate MRSA, VISA), and NRS271 (linezolid-resistant MRSA, LRSA) comparable to that of linezolid and vancomycin in vivo.

Full text of DOI:10.1021/acsmedchemlett.7b00501

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Letter
Novel Staphyloxanthin Inhibitors with Improved Potency
Against Multidrug Resistant Staphylococcus aureus
Shuaishuai Ni, Baoli Li, Feifei Chen, Hanwen Wei, Fei Mao, Yifu Liu, Yixiang Xu, Xiaoxia Qiu, Xiaokang
Li, Wenwen Liu, Linghao Hu, Dazheng Ling, Manjiong Wang, Xinyu Zheng, Jin Zhu, Lefu Lan, and Jian Li
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00501 • Publication Date (Web): 22 Feb 2018
Downloaded from http://pubs.acs.org on February 23, 2018
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Novel Staphyloxanthin Inhibitors with Improved Potency Against
Multidrug Resistant Staphylococcus aureus
Shuaishuai Nia, †, Baoli Lia, †, Feifei Chenb, †, Hanwen Weia, Fei Maoa, Yifu Liua, Yixiang Xua, Xiaoxia Qiua,
Xiaokang Lia, Wenwen Liua, Linghao Hua, Dazheng Linga, Manjiong Wanga, Xinyu Zhenga, Jin Zhua, *, Lefu
Lanb, *, Jian Lia, *.
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aShanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai
200237, China
bState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China
†These authors contributed equally to this work.
*To whom correspondence should be addressed: jianli@ecust.edu.cn, llan@simm.ac.cn, jinz@ecust.edu.cn.
KEYWORDS: virulence factor, pigment, multidrug resistant MRSA, CrtN inhibitor
ABSTRACT: Diapophytoene desaturase (CrtN), is a potential novel target for intervening in the biosynthesis of the virulence factor
staphyloxanthin. In this study, 38 1, 4-benzodioxan-derived CrtN inhibitors were designed and synthesized to overwhelm the defects of
leading compound 4a. Derivative 47 displayed superior pigment inhibitory activity, better hERG inhibitory properties and water
solubility, and significantly sensitized MRSA strains to immune clearance in vitro. Notably, 47 displayed excellent efficacy against
pigmented S. aureus Newman, Mu50 (vancomycin-intermediate MRSA, VISA) and NRS271 (linezolid-resistant MRSA, LRSA)
comparable to that of linezolid and vancomycin in vivo.
Staphylococcus aureus (S. aureus), a type of Gram-positive
obtained (Figure 1).20-21 Moreover, analogue 4a displayed
excellent potency in vitro (pigment inhibition: IC50 = 1.9 nM,
Figure 1), but was still limited by its strong inhibition of human
Ether-a-go-go Related Gene (hERG), high required dosage and
moderate water solubility in subsequent investigations.21
Therefore, in this study, a novel compound was designed and
synthesized to overcome the defects of lead compound 4a. In
addition, variable administrations and dosages were introduced
for an actual evaluation of the efficiency of our new compounds
against multidrug resistant S. aureus in vivo.
bacteria, causes a series of infections, including skin and soft
tissue
infections
and
fatal
systemic
infections.1-2
Methicillin-resistant S. aureus (MRSA) has been recognized as a
major pathogen that mutates rapidly under exposure to
antibiotics.3-6 In recent years, more multidrug resistant MRSA
strains have been identified, including linezolid-resistant MRSA
(LRSA) and vancomycin-intermediate and -resistant MRSA
(VISA and VRSA),7 which have almost destroyed the entire
"antibio-defense".8 The World Health Organization (WHO)
published its first ever list of antibiotic-resistant "priority
pathogens" in 2016, and MRSA, VISA and VRSA were graded
and classified as high priority.9 We urgently need novel strategies
against multidrug resistant S. aureus in this post-antibiotic era.10
Virulence factors are products of the pathogen, which affect
the host-pathogen relationship by damaging the host immune
system.11-12 The golden pigment staphyloxanthin (STX) is a
carotenoid virulence factor of S. aureus, and defends the strains
against reactive oxygen substances (ROS) produced by
neutrophils.13-14 Therefore, it is a viable avenue to treat MRSA
infections by inhibiting STX biosynthesis. The STX biosynthesis
genes are organized in the crtOPQMN operon, and biosynthesis
begins with the condensation of farnesyl diphosphate catalyzed
Figure 1 Structures of representative pigment inhibitors reported
in the literature and those derived from lead compound 4a.
Our previous study had demonstrated that the strategy of
inserting a hydrophilic oxygen into the cycloalkane of lead
by dehydrosqualene synthase (CrtM) and
a downstream
termination region of diapophytoene desaturases (CrtN).15 In
2008, Eric Oldfield and colleagues first reported that cholesterol
biosynthesis inhibitor BPH-652 (1, Figure 1) had an inhibitory
effect on pigment synthesis by targeting CtrM.16-18 Subsequently,
our research group revealed that Naftifine hydrochloride (NTF,
2, Figure 1), an FDA-approved antifungal agent, could block the
STX biosynthesis pathway by targeting CrtN, revealing a novel
strategy for anti-virulence treatments against MRSA infections.19
On the basis of the results above, we further designed and
synthesized a series of novel benzofuran and benzocycloalkane
CrtN inhibitors, from which the efficient derivatives 3 and 4 were
compound 4a was
a feasible means of improving water
solubility.22 As an extension to our previous work, we speculated
that adding more oxygen atoms into the skeleton of 4a was a
rational approach to relieve the stress of solubility brought by
possible lipophilic substituents.22 1, 4-benzodioxan derivative 16
was synthesized in accordance with these hypotheses, and it had
superior solubility in contrast to 4a (13.5 mg/mL vs 0.69
mg/mL), along with the maintenance of pigment inhibitory
activity (IC50 = 1.9 nM by 4a vs IC50 = 2.9 nM by 16). Building on
these data, we next aimed at improving the pharmacologic
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activity and hERG inhibition properties by rational molecular
properties. Hence, systematic modifications in this study were
investigated directly toward diversifying substituents with R1 and
inserting more olefins into linker.
1
2
3
modification. In addition, our previous work had revealed that
the N-methyl group and unsubstituted alkenyl linker were critical
for improving pigment inhibitory activity and hERG inhibition
4
5
Table 1 Chemical Structures of Derivatives 11-48 and Their Pigment Inhibitory Activities Against S. aureus Newman
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7
8
9
Compd.
11
R1
n
1
1
IC50 (nM)a
370.9±35.4
20.1±0.9
Compd.
30
R1
n
1
1
IC50 (nM)a
>1000
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phenyl
3-fluorophenyl
3-chlorophenyl
12
4-fluorophenyl
31
111.6±4.1
13
14
15
4-chlorophenyl
4-bromophenyl
4-methylphenyl
1
1
1
6.7±0.3
8.2±0.3
3.2±0.2
32
33
34
3-methylphenyl
3-trifluorophenyl
3-nitrophenyl
1
1
1
43.4±4.8
>1000
>1000
16
17
18
19
20
21
4-trifluorophenyl
4-t-butylphenyl
4-methoxylphenyl
4-ethoxylphenyl
4-nitrophenyl
1
1
1
1
1
1
2.9±0.1
156.0±9.3
48.6±7.4
8.2±0.8
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3-methoxylphenyl
2,4-dichlorophenyl
2-fluoro-4-trifluorophenyl
3-fluoro-4-trifluorophenyl
2-fluoro-4-methoxylphenyl
3-fluoro-4-methoxylphenyl
1
1
1
1
1
1
>1000
15.0±3.7
8.8±0.1
13.7±0.1
43.5±1.2
57.9±3.0
18.9±0.5
19.2±8.0
4-cyanophenyl
4-methyl
formatephenyl
22
1
6.5±0.3
41
methyl
1
>1000
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28
29
4
4-biphenyl
1
1
1
1
1
1
1
4.1±0.2
>1000
90.3±25.7
>1000
>1000
>1000
>1000
4.1
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47
48
4a
cyclopentyl
cyclohexyl
1
1
1
1
1
2
3
320.8±4.7
>1000
2-fluorophenyl
2-chlorophenyl
2-methylphenyl
2-trifluorophenyl
2-nitrophenyl
1-furyl
227.7±4.5
220.6±8.3
>1000
1-thienyl
1-naphthyl
4-trifluorophenyl
4-trifluorophenyl
2.2±0.1
1.6±0.2
1.9
2-methoxylphenyl
aValues given are the IC50 values for pigment inhibition in S. aureus Newman. Values are reported as the average ± S.D.
Next, 38 novel 1, 4-benzodioxan derivatives 11-48 were
synthesized and assayed for pigment inhibitory activities against
S. aureus Newman. The results are outlined in Table 1. First, the
replacement of phenyl with methyl, cycloalkyls, heteroaryls or
naphthyl groups reduced the pigment inhibitory activity. The
results further confirmed that there was no significant
relationship between the electron effect and activity, as
previously reported.21 Unexpectedly, substitutions at the
para-position showed more potency than those at the ortho- or
meta-position (12-23 vs 24-29 vs 30-35). Moreover, the suitable
bulk of substituents could significantly improve inhibitory
activities (12 vs 13 vs 14 and 15 vs 16 vs 17). Among these
derivatives, both 15 and 16 displayed excellent pigment
inhibitory activities (IC50 = 2.9 ± 0.1 nM, IC50 = 3.2±0.2 nM,
respectively), but derivative 16 was selected for further
optimization due to its better solubility (~2-fold higher than 15,
Table 2). Next, more unsubstituted multiconjugated polyene
groups were introduced into the structure of 16 to yield
derivatives 47 and 48, which displayed comparable activities to
16 (IC50 = 2.2 nM by 47 and IC50 = 1.6 nM by 48).
Table 2 Enzyme (CrtN, IC50), Pigment (Mu50, NRS271, USA400 MW2 and USA300 LAC, IC50), hERG and Water Solubility Results of
Representative Derivatives.
USA400
MW2
Water
CrtN
Mu50
NRS271
USA300 LAC
IC50(nM)b
hERG
compd
Solubility
IC50 (nM)a IC50 (nM)b IC50(nM)b
IC50(μM)c
IC50(nM)b
(mg/mL)d
16
47
48
4
4a
15
340.7±65.5
270.4±43.8
258.4±57.9
320
1.2±0.3
< 0.1
1.2±0.5
0.5
1.1±0.2
0.6±0.2
0.4±0.3
6.5±0.3
6.5±0.1
5.3±0.1
7.1
5.9±0.2
5.5±0.5
3.7±0.2
7.7
3.9
12.1
8.7
3.2
1.4
13.5
7.9
3.3
4.2
0.69
6.5
4.6
aValues given are the IC50 values for CrtN enzyme inhibition in S. aureus Newman. The values are reported as the average ± S.D., in nM.
bValues given are the IC50 values for pigment inhibition in Mu50, NRS271, USA400 MW2 and USA300 LAC, in nM. Values given are
c
the IC50 values for hERG inhibition, in μM. dValues given are the solubility in water, in mg/mL.
Further in vitro investigations results are shown in Table 2.
First, three potency derivatives, 16, 47, and 48 were, bioassayed
for CrtN enzymatic inhibitory activity, and all of the selected
derivatives had hundred-nanomolar inhibitory activity ranges,
which were comparable with 4. Next, two multidrug resistant
MRSA strains (Mu50 as VISA and NRS271 as LRSA)23-24 and
two community-acquired MRSA strains (USA300 LAC and
USA400 MW)25-26 were introduced to test the pigment inhibitory
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activities of selected derivatives. All of the compounds displayed
mark the limits of detection; Statistical significance was
determined by the Mann-Whitney test (two-tailed): * p < 0.05, **
p < 0.01, *** p < 0.001, ns indicates no significant difference.
1
2
3
4
5
6
7
8
potent inhibitory activities in the single-nanomolar range against
the four MRSAs above (IC50 = 0.1 ~ 6.5 nM, Table 2). In this
study, both hERG profiles and solubility were important
properties to investigate with regards to the drugability of
compounds. Regrettably, we found conflicts in these derivatives
relating to their solubility and hERG inhibition, as shown in
Table 2. Appropriate olefins significantly ameliorated the hERG
inhibitory activities of 47 and 48, but decreased their solubility.
In light of the balance between hERG safety profiles and
solubility, derivative 47 was selected to further survey, due to its
significant improvement of hERG inhibition (IC50 = 12.1 μM by
47 vs 1.4 μM by 4a, ~ 8-fold increase) and good water solubility
(7.9 mg/mL). In contrast with traditional antibiotics, derivative
47, which originated from antifungal agent 2, not only did not
affect the growth of MRSAs at 0.2 mM (Newman and three
MRSA strains, Figure S1, Supporting Information) but also lost
its antifungal capacity (Table S1, Supporting Information).
Furthermore, the identification of CrtN targeting demonstrated
that derivative 47 indeed inhibited the function of CrtN (Figure
S2, Supporting Information).
The results above indicated that candidate 47 could
sensitize MRSAs to immune clearance in vitro. Next, murine
abscess formation models were established to assess the
bioactivity of 47 in vivo. Candidate 47 with two administrations
timings (normal treatment or pretreatment) at two dosages (0.4
mg b.i.d. or 0.1 mg b.i.d.) were used to imitate authentic
infections, along with vancomycin (VAN) and linezolid (LZD)
as positive controls. Bacterial survival was then measured in mice
hearts and livers. First, as shown in Figure 3, in contrast with the
non-treatment groups by a 6.34 log10 CFU in hearts or by a 6.41
log10 CFU in livers, 47-treatment groups displayed significant
activities against Newman strains (at least a 0.96 log10 CFU
reduction in the hearts, or an 89.01% decrease in surviving
bacteria, and 0.87 log10 CFU reduction in the livers, an 86.70%
decrease in surviving bacteria). Although 47-treatment appeared
less efficacious than the two positive controls in the hearts, it was
still comparable with the positive controls in the livers. In
particular, the dosage of 0.1 mg/bid exhibited an excellent
performance in the livers of the normal treatment groups (3.31
log10 CFU, a ~ 99% decrease in surviving bacteria), even better
than positive controls.
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ꢀ
Figure 2 Effects of derivative 47 on susceptibility of S. aureus
Newman, Mu50, USA300 LAC, and USA400 MW2 to hydrogen
peroxide and human whole blood killing assays in vitro.
STX could protect S. aureus from the oxidative stress by
ROS, and nonpigmented MRSAs were vulnerable to immune
clearance.19-22 Based on this, a hydrogen peroxide (H2O2) assay
and human whole blood killing assay were used to imitate ROS in
vitro to verify the efficiency of our compounds. As shown in
Figure 2, three groups, including mock-treated (yellow),
47-treated (blue) and N-acetylcysteine (NAC, known as an
antioxidant substance)-treated (red), were incubated with S.
aureus Newman or the other three MRSAs (Mu50, USA300
LAC and USA400 MW). In the H2O2 killing assay, the survival of
all 47-treated groups was obviously lower than that of the
mock-treated and NAC-treated groups, which demonstrated that
the depigmented MRSAs were vulnerable to H2O2 after
treatment with our compound. In addition, antioxidant
NAC-treated groups gave the worst performance due to
counteracting H2O2. Similarly, in the whole blood killing model,
47-treated S. aureus groups (in blue) were more susceptible to
being killed by blood (survival, < 0.5% in general).
ꢀ
Figure 4 Effects of derivative 47 (in red) on S. aureus bacteria
survival in the hearts and livers of mice (n=10 for each group),
challenged with 1.1 × 109 CFU of Mu50 and 1.8 × 108 CFU of
NRS271 in vivo section, respectively; P = pretreatment, drugs or
compounds were intraperitoneally administered 24 hours before
infection in contrast with the normal treatment (in which drugs
or compounds were administered 6 hours after infection); Each
symbol represents the value for an individual mouse; Horizontal
bars indicate the observation means, and dashed lines mark the
limits of detection; Statistical significance was determined by the
Mann-Whitney test (two-tailed): * p < 0.05, ** p < 0.01, *** p <
0.001, ns indicates no significant difference.
We had demonstrated that CrtN inhibitors 47 could
efficaciously ameliorate the infections of Newman strains. Next,
two multidrug resistant MRSAs, Mu50 (VISA) and NRS271
(LRSA), were introduced for evaluating the inhibitory effects of
47 against multidrug resistant MRSA infections. In general, most
of the 47-treated cases in either Mu50 or NRS271 showed
significant efficacy in contrast with the mock treatments, as
shown in Figure 4. On one hand, as shown in the heart section in
Mu50, the staphylococcal loads of groups that received the
normal treatment of compound 47 were decreased significantly,
as measured by 3.81 log10 CFU with 0.4 mg/bid and 3.67 log10
CFU with 0.1 mg/bid, corresponding to a ~99% decrease in
surviving bacteria, compared with positive controls, while the two
ꢀ
Figure 3 Effects of derivative 47 (in red) on S. aureus bacteria
survival in the hearts and livers of mice (n=10 for each group)
challenged with 2.2 × 107 CFU of Newman; P = pretreatment,
drugs or compounds were intraperitoneally administered 24
hours before infection in contrast to normal treatment (in which
drugs or compounds were administered 6 hours after infection);
Each symbol represents the value for an individual mouse;
Horizontal bars indicate the observation means, and dashed lines
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pretreatment groups produced less benefit. In parallel, as shown
Shanghai 200237, China
bState Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203,
China
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2
3
4
5
6
7
8
in the liver section Mu50, most of the 47-treated groups
displayed good resistance against encroachments, which was
comparable with positive control groups. On the other hand, as
shown in the NRS271 infectious model in Figure 4, in general,
compared with the VAN-treatment groups and most of the
47-treatment groups in the hearts and livers, both mock and
LZD-treatment groups displayed inferior inhibitory activities.
Furthermore, the surviving bacteria of normal administration
47-treatment groups decreased over 99.0%, was comparable to
that of the VAN-treated groups in the hearts. However, although
most of the 47-treated groups were reduced by at least 0.75 log10
CFU in the livers, and these groups were inferior to the VAN
group. Consequently, all of the pharmacodynamical results
showed that: (1) most of 47-treatment groups had more
beneficial effects on lowering abscess formation than mock
controls. (2) 47 in the low-dosage by normal treatment displayed
comparable effects with the two positive groups (VAN and LZD)
in the hearts in Mu50 and NRS271 and was even more effective
than the two positive groups in the livers in Newman. In view of
this anomaly, we suggest a preliminary hypothesis: (i) as shown
in liver microsomes in Table3, a moderate stability (t1/2 = 7.9 min)
of 47 caused the pretreatment to fail to proactively exploit its
advantage against MRSA infections. (ii) as a superactive CrtN
inhibitor, derivative 47 still had enough ability to be efficacious
against MRSAs at the low dosage. On the other hand, a potential
cytotoxicity of 47 possibly reduced the immunity (Table 3),
which constrained the potency of the high dosage groups.
ORCID
Feifei Chen: 0000-0002-7251-1956
Fei Mao: 0000-0001-5871-6878
Jin Zhu: 0000-0003-3934-0312
Lefu Lan: 0000-0002-5551-5496
Jian Li: 0000-0002-7521-8798
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Author Contributions
†These authors contributed equally to this work.
Funding Sources
Financial support for this research was provided by the National
Key R&D Program of China (Grant 2017YFB0202600), the
National Natural Science Foundation of China (Grant
21672064), the “Shu Guang” project supported by the Shanghai
Municipal Education Commission and Shanghai Education
Development Foundation (Grant 14SG28). The Fundamental
Research Funds for the Central Universities are gratefully
acknowledged.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS
S. aureus, Staphylococcus aureus; MRSA, methicillin-resistant
Staphylococcus aureus; STX, staphyloxanthin; NTF, naftifine
hydrochloride; CrtM, dehydrosqualene synthase; CrtN,
diapophytoene desaturases; ROS, reactive oxygen species; SAR,
structure-activity relationship; IC50, half maximal inhibitory
concentration; CFU, colony-forming unit.
Table 3 Liver Microsomes and Cytotoxicity of Derivative 47
Liver
Cytotoxicity CC50 (μM)
microsomes
compd
t1/2
HEK-293
T
k
HepG2
(min)
REFERENCES
47
Midazolam
Amphotericin
B
0.089
0.165
7.9
4.2
50.33±2.7
67.76±3.5
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27.46±5.2
37.81±4.6
In summary, to improve the water solubility, hERG
inhibition and dosage of leading compound 4a, 38 derivatives
were synthesized for a systematic study of structure and activity
relationships, along with an investigation of pigment inhibitory
activities against S. aureus Newman in vitro. Candidate 47 was
selected for further evaluation of its potency in vivo against
multidrug resistant MRSA infections, including S. aureus
Newman, Mu50 (VISA/MRSA) and NRS271 (LRSA/MRSA).
Fortunately, normal 47-treatment in the low-dosage group had a
comparable effect to the positive groups (VAN and LZD).
Altogether, these results demonstrate that 47 is a good candidate
for combating MRSA, VISA and LRSA infections.
ASSOCIATED CONTENT
Supporting Information
Purity, chemical synthesis, structural characterization, and solubility
of the target compounds; protocols of biological assays. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Email: jianli@ecust.edu.cn
*Email: llan@simm.ac.cn
*Email: jinz@ecust.edu.cn.
Present Addresses
aShanghai Key Laboratory of New Drug Design, School of
Pharmacy, East China University of Science and Technology,
staphylococcal bacteraemia:
study in Italy. Int. J. Antimicrob. Agents 2015, 45 (3), 255-261.
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