Total Synthesis of Xanthoangelol B and Its Various Fragments: Toward Inhibition of Virulence Factor Production of Staphylococcus aureus

Article abstract of DOI:10.1021/acs.jmedchem.8b01012

As an alternative strategy to fight antibiotic resistance, two-component systems (TCSs) have emerged as novel targets. Among TCSs, master virulence regulators that control the expression of multiple virulence factors are considered as excellent antivirulence targets. In Staphylococcus aureus, virulence factor expression is tightly regulated by a few master regulators, including the SaeRS TCS. In this study, we used a SaeRS GFP-reporter system to screen natural compound inhibitors of SaeRS, and identified xanthoangelol B 1, a prenylated chalcone from Angelica keiskei as a hit. We have synthesized 1 and its derivative PM-56 and shown that 1 and PM-56 both had excellent inhibitory potency against the SaeRS TCS, as demonstrated by various in vitro and in vivo experiments. As a mode of action, 1 and PM-56 were shown to bind directly to SaeS and inhibit its histidine kinase activity, which suggests a possibility of a broad spectrum inhibitor of histidine kinases.

Full text of DOI:10.1021/acs.jmedchem.8b01012

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Article
Total synthesis of Xanthoangelol B and its various fragments: towards
inhibition of virulence factor production of Staphylococcus aureus
Pushpak Mizar, Rekha Arya, Truc Kim, Soyoung Cha, Kyoung-Seok Ryu, Won-Sik
Yeo, Taeok Bae, Dae Wook Kim, Ki Hun Park, Kyeong Kyu Kim, and Seung Seo Lee
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01012 • Publication Date (Web): 02 Nov 2018
Downloaded from http://pubs.acs.org on November 3, 2018
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Total Synthesis of Xanthoangelol B and Its Various Fragments:
Towards Inhibition of Virulence Factor Production of Staphylococcus
aureus
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Pushpak Mizar,† Rekha Arya,‡ Truc Kim, ‡ Soyoung Cha§, Kyoung-Seok Ryu§, Won-sik
Yeo,¶ Taeok Bae, ¶ Dae Wook Kim, ┴ Ki Hun Park, ┴ Kyeong Kyu Kim,*‡ and Seung Seo Lee*
†
†Chemistry, Highfield Campus, University of Southampton, Southampton, SO17 1BJ, UK.
‡Department of Molecular Cell Biology, Institute for Antimicrobial Resistance and
Therapeutics, Samsung Medical Center, Sungkyunkwan University School of Medicine,
Suwon 16419, Republic of Korea.
§Protein structure research group, Korea Basic Science Institute, 162 Yeongudanji-Ro,
Ochang-Eup, Cheongju-Si, Chungcheongbuk-Do 28119, Republic of Korea.
¶Department of Microbiology and Immunology, Indiana University - School of Medicine-
Northwest, Gary, IN 46408 US
┴Division of Applied Life Science (BK21 Plus), IALS, Gyeongsang National University, Jinju
52828, Republic of Korea
 These authors contributed equally to this work.
* Corresponding authors: kyeongkyu@skku.edu & s.s.lee@soton.ac.uk
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Abstract
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As an alternative strategy to fight antibiotic resistance, two-component systems (TCSs) have
emerged as novel targets. Among TCSs, master virulence regulators that control the expression
of multiple virulence factors are considered as excellent anti-virulence targets. In
Staphylococcus aureus, virulence factor expression is tightly regulated by a few master
regulators, including the SaeRS TCS. In this study, we used a SaeRS GFP-reporter system to
screen natural compound inhibitors of SaeRS, and identified xanthoangelol B 1, a prenylated
chalcone from Angelica keiskei as a hit. We have synthesized 1 and its derivative PM-56, and
shown that 1 and PM-56 both had excellent inhibitory potency against the SaeRS TCS, as
demonstrated by various in vitro and in vivo experiments. As a mode of action, 1 and PM-56
were shown to bind directly to SaeS and inhibit its histidine kinase activity, which suggests a
possibility of a broad spectrum inhibitor of histidine kinases.
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INTRODUCTION
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Antibiotic resistance has arisen as an extremely serious health problem in all parts of the world.
New resistance mechanisms are emerging and spreading globally, threatening our ability to
treat common infectious diseases. The list of resistant infections – such as pneumonia,
tuberculosis, blood poisoning, gonorrhea, and foodborne diseases – is growing longer and
sometimes these are impossible to treat as antibiotics become less effective.1,2 It is estimated
that, by 2050, worldwide drug-resistant infections will claim 10 million deaths annually. One
particularly problematic pathogen is methicillin-resistant Staphylococcus aureus (MRSA).
MRSA is a nosocomial and communal menace that is resistant to many antibacterial drugs and
antiseptics.3-5 The U.S. Centers for Disease Control and Prevention recently issued a report to
various governments and organizations outlining the concern about MRSA. This report
referenced the New Drugs for Bad Bugs (ND4BB) project initiated by the European Union,
the United States military, and the World Health Organization (WHO).6,7 The Antimicrobial
Resistance Global Report on Surveillance released by the WHO in 2014 outlined a global
initiative to tackle the MRSA superbug and rekindle a campaign for new, effective, and safe
drugs.8-10 Since they suppress bacterial growth, conventional antibiotics are confined to a
limited lifespan. It is urgent to establish a new path forward for medicinal chemists to address
successfully this problem.
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Among many new approaches, the inhibition of virulence factors has shown promising
outcomes. This strategy bypasses the route of killing pathogens and thereby has the potential
to suppress the development of resistance. Many such concepts have been tested.11-13 Among
these, two-component systems (TCSs) that control virulence factors have been of particular
interest.14-17 Some TCSs control multiple virulence factors in pathogens and are thus called
master virulence regulators.18 These TCSs are attractive targets for novel anti-virulence agents.
We identified one such TCS, the SaeRS system of S. aureus, as an excellent anti-virulence
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target that is less likely to evolve resistance.19 We developed a GFP reporter system that can
be applied to a high-throughput assay format. Using this assay, we discovered certain FDA-
approved drugs that exert anti-virulence effects on S. aureus.20 In continuing such studies, we
identified natural compound inhibitors of the SaeRS TCS by screening plant-derived products21
using the same assay. Of the identified natural products that showed promising inhibitory
activity, we chose xanthoangelol B for further study including the synthesis of complete
xanthoangelol B, fragments and a derivative, to test for anti-virulence activities.
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Xanthoangelol B is a prenylated chalcone isolated from Ashitaba (Angelica keiskei Koidzumi,
Apiaceae). The Japanese herb Ashitaba is a perennial plant found along the Pacific coast of
Japan. Ashitaba is enriched with numerous active compounds such as coumarins, flavanones,
and chalcones.22-25 Xanthoangelol B is relatively scarce among these compounds, although it
is found in all parts of the plant.26 It shows various therapeutic properties, such as anti-oxidative
activity, anti-hypertensive activity, anti-inflammatory activity, and anti-viral activity.27
However, although its related chalcones, xanthoangelol and 4-hydroxyderricin were shown to
have an antibacterial activity against gram positive bacteria, 28 its anti-bacterial activity has not
been established. Thus, given our intriguing screening results, we set out for an in-depth
structure-activity study of xanthoangelol B. We were particularly interested in its effect on the
virulence of S. aureus and its feasibility as an anti-virulence agent.
Xanthoangelol B has a modular structure composed of chalcone and isoprene moieties. It was
deemed important to analyze the biological activity of its fragments to establish any structure-
activity relationships. Thus, we synthesized fragments of xanthoangelol B along with the parent
compound and tested their activities. During the synthesis, we obtained a derivative of
xanthoangelol B (compound PM-56) that had a comparable activity, and tested its activities.
Here, we report these processes and describe the anti-virulence activities of the compounds that
we synthesized.
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RESULTS AND DISCUSSION
Chemistry
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The synthesis of biologically relevant geranylated flavanones and geranylated chalcones (such
as (±)-prostratol F, (±)-6-geranyl-5,7-dihydroxy-3’,4’-dimethoxyflavanone, xanthoangelol, 3-
geranyl-2,3’,4,4’-tetrahydroxychalcone, and (±)-lespeol) has been reported.29-31 With these
precedents, we designed our syntheses using a retrosynthetic analysis, as outlined in Scheme
1. Xanthoangelol B 1 can be disconnected to two main fragments: fragment 3 and 4-
hydroxybenzaldehyde 2. Fragment 3 can be obtained from 2’,4’-dihydroxyacetophenone 4,
which is commercially available. There are two ways to synthesize this fragment 3 (Scheme
1). A synthetic route to 1 was also designed to generate various fragments that could be
evaluated for their biological activity. The fragments shown in Scheme 1 can provide insight
into the structure-activity relationship of 1 that may help to curtail or enhance its activity.
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OH
O
OH
OH
HO
1
O
OH
OH
OHC
OH
O
+
4
OH
OH
OH
2
+
3
Br
O
5
OH
O
+
O
OH
8
7
Scheme 1. Retrosynthetic analysis of xanthoangelol B 1
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Our synthetic design involved the limited use of protecting groups (Scheme 2). Our initial
approach was to introduce 2,6-dimethylhepta-1,6-dien-3-ol at the end of the synthesis to reduce
complications arising from the chiral aliphatic hydroxyl group. The 4’-hydroxyl position of
2′,4′-dihydroxyacetophenone was alkylated with allyl bromide that was heated at 220 oC in N,
N-diethylaniline for 24 h. This allowed for Claisen rearrangement, giving a 3,3-sigmatropic-
shifted major product 10 with a 76% yield. The minor product was 5-allyl-2,4-dihydroxy
acetophenone. The regioselectivity of Claisen rearrangement giving a 3-allylated major
product is well documented.32 10 was then subjected to aldol condensation by refluxing for 18
h in KF-alumina (40%), a solid-support catalyst in toluene. 11 was obtained as the major
product, with a yield of approximately 60%. This yield was slightly lower than that of most
aldol condensation reactions owing to the two 2’,4’-OH groups. A reaction longer than 18
hours resulted in the formation of a non-polar mixture of spots observed by TLC analysis. The
nature of the solvent also drastically influenced the yield of the reaction. 2,6-Dimethylhepta-
1,6-dien-3-ol was introduced later by initially synthesizing 2,2-dimethyl-3-(3-methylbut-3-en-
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o
1-yl)oxirane 8 from citral by subjecting it to decarbonylation via heating at 130 C in the
presence of catalytic amounts of palladium acetate (10 mol%) in a sealed tube for 24 h. This
produced 2,6-dimethylhepta-1,5-diene 12, with a yield of ca 62%.33 Dimethylhepta-1,5-diene
was subjected to selective oxidation using m-chloroperbenzoic acid (mCPBA) to obtain an
epoxide 8 with a 45% yield.34 8 was then subjected to cross-olefin metathesis with 11, using a
Hoveyda-Grubbs second-generation catalyst to yield 70% of an E-product 13.35 The epoxide
ring was subsequently subjected to selective ring opening reactions or isomerization using in
situ-generated diethylaluminum 2,2,6,6-tetramethyl-piperidide to yield the desired product
xanthoangelol B 1 at a 69% yield.36
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NEt2
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O
OH
Br
O
OH
O
OH
(1.1 equiv.)
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9
220 oC,
O
K2CO3 (1.5 equiv.)
acetone, 14h, reflux
OH
OH
76%
9
70%
Heat, 24h
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O
OH
KF-Alumina (40%)
(3 equiv.)
10
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HO
OH
toluene, reflux, 18h
60%
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Pd(OAc)2 (8 mol%)
mCPBA
(80% in ether)
CHO
4Ao
CHCl3, ehter
O
Cyclohexane
130 oC, sealed tube
62%
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45%
8
O
OH
Hoveyda-Grubbs
2nd Gen. (0.05 equiv.)
11
8
+
O
DCM, 40 oC
HO
OH
70%
13
DATMP, 0 oC
Toluene
1
13
69%
Scheme 2. Synthesis of xanthoangelol B 1
There were a few interesting observations during the synthesis of 1 (Scheme S1). As an
alternative route to xanthoangelol B, the introduction of 2,6-dimethylhepta-1,6-dien-3-ol to the
3’-position of 2,4-dihydroxyacetophenone resulted in decreased reactivity toward
condensation reactions. Use of a solid-support reagent (such as KF-alumina) or use of highly
basic conditions yielded no product or a complex mixture of products. Heating the reaction in
the presence of a high boiling-point solvent (either in highly basic or acidic conditions) yielded
no desired product. Finally, the use of L-proline as an organic catalyst37-38 resulted in an aldol
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addition product 15. However, this product did not react further (to elimination) to yield 1 in
any of the acidic or basic conditions attempted. Thus, 15 was subjected to selective epoxide
isomerization, and the resulting compound PM-56 was tested for anti-virulence activities as a
derivative of 1.
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HO
OH
CHO
OH
O
O
60% KOH/Ethanol
reflux, 24h
+
HO
OH
OH
80%
17
+
mCPBA
Br
Br
CHCl3, 0 oC-rt
O
18
DATMP, 0 oC
toluene, 2h
KOH,
reflux,
13
34
1
17 18
+
methanol
%
80
%
Ph
HN
O
nBu
TCCA, CHCl3, -40 oC
enationmerically pure
1
34
%
Scheme 3. Alternative route for the synthesis of xanthoangelol B 1. No metal catalysts involved.
Synthesized xanthoangelol B 1 was structurally identical to the natural product, except for the
chiral center. Selective epoxidation and subsequent epoxide ring opening resulted in an
enantiomeric mixture. We speculated that the number of required steps to synthesize 1 could
be reduced by using a longer chain (i.e., geranyl bromide) and carrying out subsequent selective
epoxidations, which could be reacted further to yield 1. This would provide us with an
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additional fragment for structure-activity tests and allow us to incorporate the metal-free
chemistry. Thus, we devised a shorter synthetic route (without using metal catalysts) by
minimizing the use of protecting groups. Specifically, 2,4-dihydroxy acetophenone was
subjected to aldol condensation to give an intermediate that was reacted with 1,2-epoxy geranyl
bromide. This generated 13, which underwent selective epoxy-isomerization to give 1 as an
enantiomeric mixture (Scheme 3). Then, we attempted an optical resolution of synthesized
compound 1. We chose a method of enantioselective organocatalyst oxidative kinetic
resolution using the 7-Bn-3-n-Bu-4-oxa-5-azahomoadamantane/trichloroisocyanuric acid
(TCCA) system to yield a resolved 1.39 The 1H and 13C NMR spectra of the resolved compound
1 were in agreement with spectra previously reported.21 The mass spectrum showed a peak at
409.19 corresponding to [M+H]+, consistent with the calculated mass of the desired product.
In a direct comparison using HPLC, the synthetic and resolved 1 was shown to elute at exactly
the same retention time as the natural product (Figure S1). The optical rotation for kinetically
resolved 1 was D= +13.5 (MeOH, c=0.5). This is consistent with the data reported for
naturally-extracted 1,23 which indicates the authenticity of the kinetically resolved synthetic
xanthoangelol B 1.
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Biological evaluation of xanthoangelol B, its fragments, and its derivative
Previously, we constructed a SaeRS GFP reporter strain based on the S. aureus USA300 isolate,
and found promising leads among FDA-approved drugs through a high throughput screening.20
The same SaeRS GFP reporter strain was constructed for screening a natural product library in
this study. Briefly, as the α-hemolysin promoter (Phla) is a well-characterized target of
theꢀSaeRS TCS, we generated a minimal Phla, termed Phlam, which contains only the SaeR
binding sites and the -10/-35 promoter sequences of the hemolysin gene (hla). Phlam was fused
to a green fluorescence protein (gfp) gene instead of hla in the single-copy integration plasmid
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pCL55. The resulting plasmid (pCL-Phlam-gfp) was inserted into S. aureus USA300. Hence,
if the expression of GFP in a sample was lower than the control sample, it meant that the SaeRS
TCS was inhibited. As the complete inhibition of Phlam requires a complete shutdown of the
SaeRS TCS (such as a deletion of saeS), Phlam will detect only potent inhibitors of the SaeRS
reporter. Xanthoangelol B 1 was one of hits among the natural products screened with this
method. Thus, we assayed 1, its fragments, and a derivative (PM-56). The structures of all the
assayed compounds and fragments are presented in Scheme 4 and Scheme S2.
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Structure-activity studies of xanthoangelol B and its inhibitory effect on the SaeRS TCS
On initial screening, 1 and PM-56 showed an excellent inhibitory activity against SaeRS. These
compounds repressed GFP expression while allowing bacterial growth, making them ideal anti-
virulence candidates. In contrast, all of the fragments (Scheme S2) failed to inhibit the SaeRS
TCS, even at 20 µM. This indicates that the whole structure is needed for the inhibitory activity.
Furthermore, xanthoangelol and xanthoangelol F (Scheme 4) did not show inhibitory activity
at 20 µM.
O
OH
OH
O
OH
OH
OH
HO
HO
OH
Xanthoangelol B 1
OH
PM-56
O
OH
O
OH
HO
HO
OCH3
Xanthoangelol F
OH
Xanthoangelol
Scheme 4. Derivatives of xanthoangelol B
These two analogs are also natural prenylated chalcones from Ashitaba, but they lack a
hydroxyl group in the terminal isoprene unit. This hydroxyl group appears to be crucial for the
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anti-virulence activity of this class of compounds. Xanthoangelol with an identical structure to
that of 1, except for this hydroxyl group, did not have any activity (although methoxylation in
the central phenol hydroxyl group of xanthoangelol F may still have an impact). IC50 values
against the SaeRS GFP reporter were measured to be 2.1 µM for 1 and 4.3 µM for PM-56. In
these experiments (at up to 8 and 16 µM), 1 and PM-56 did not disrupt bacterial growth (Figure
1A and C). To elaborate the inhibitory effect of both compounds on the growth of S. aureus,
we measured the growth of S. aureus in the presence of 1 and PM-56 at higher concentrations
up to 20 h. From 16 M, 1 started showing growth inhibition, and at 64 M of 1, the growth
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of S. aureus was severely inhibited (Figure S2). On the other hand, up to 64 M of PM-56, S.
aureus still showed comparable growth to the control (Figure S2). The trends shown at 8 h did
not change up to 20 hours. This indicates that the rigidity conferred to the chalcone structure
by the conjugated double bond is not essential for anti-SaeRS activity, but may lead to higher
toxicity (which affects bacterial growth). Together, these data strongly indicate that the
isoprene and chalcone moieties are necessary in their entirety for the inhibitory activity.
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B
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D
C
Figure 1. Various concentrations of compounds were used to treat the USA300-Phlam GFP strain,
and GFP expression was analyzed at 8 h. A, Measurement of bacterial growth in the presence
of xanthoangelol B 1; B, measurement of fluorescence in the presence of xanthoangelol B 1;
C, measurement of bacterial growth in the presence of PM-56; D, measurement of fluorescence
in the presence of PM-56. All the experiments were performed in triplicates and significance
was compared with control (no treatment) using one way ANOVA.
Effects of inhibitors on downstream virulence factors
Staphylococcal α-hemolysin, which causes lysis of erythrocytes, is the target virulence factor
of the SaeRS TCS. Therefore, if the SaeRS TCS is inhibited, the expression of α-hemolysin
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should decrease to a certain degree. When 1 and PM-56 were incubated with erythrocytes in
the presence of methicillin-resistant S. aureus USA300, notable protection from hemolysis was
observed (Figure 2 and Figure S3). At only 4 µM, both compounds showed visible protection.
At 8 µM, a comparable level of protection to that of saeS knockout mutants (SaeS:Tn551) was
observed. The hemolysis activity of this saeS mutant was reduced to 82.6% in comparison to
the wild type S. aureus. To confirm the relevance of the protection effect observed with 1 and
PM-56 to the SaeRS pathway, we also tested 1 and PM-56 against the saeS knockout mutant.
This experiment conducted at 8 M of 1 and 15 M of PM-56 showed the reduction in
hemolysis by 86.9% and 87.4%, respectively, compared to hemolysis by the wild type (Figure
S4). Although apparent further inhibition appeared to exist, the differences were not
statistically significant, which thus suggests that the protection against hemolysis by 1
and PM-56 is highly likely attributed to inhibition of the SaeRS pathway. The IC50 values
of 1 and PM-56 for the inhibition of erythrocyte hemolysis were then measured to be 4.6 and
4.0 µM, respectively. These values were consistent with the IC50 values of both compounds for
the SaeRS TCS GFP reporter. Therefore, these strongly suggests that the protection of
erythrocytes from hemolysis in the presence of MRSA can be attributed to inhibition of the
SaeRS TCS.
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Figure 2. Percentage hemolysis values were determined after treating samples with compounds
1 and PM-56 after an 8 h incubation. A, 1: various concentrations were used to treat human
RBCs, and percentage hemolysis values were calculated relative to the control sample; B, PM-
56: various concentrations were used to treat human RBCs, and percentage hemolysis values
were calculated relative to the control sample. All the experiments were performed in triplicates
and the data calculated by one way ANOVA. CT: positive control, SaeS:Tn551: saeS knockout.
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Figure 3. Effects of 1 and PM-56 on the virulence gene expression were analyzed. A, hla; B,
aureolysin (aur); C, ϒ-hemolysin; D, staphylokinase. 16S rRNA was used as a reference
control gene. All the experiments were performed in triplicates and the data compared with no
treatment control and calculated by one way ANOVA.
We next analyzed the transcription of virulence genes in the presence of 1 and PM-56 (Figure
3). We used concentrations of 8 µM (1) and 10 µM (PM-56), corresponding to an IC90 value
for the SaeRS GFP reporter. We observed significant transcriptional suppression of the
following genes: α-hemolysin (hla), aureolysin (aur), γ-hemolysin, and staphylokinase. These
four genes have a direct SaeR binding site in their promoter regions and are tightly regulated
by the SaeRS TCS.40 While it has been reported that hla is regulated by an accessory gene
regulator (agr) as well as by sarA, a direct connection between these regulatory systems and
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the expression of hla remains under debate.41 Their suppression likely reflects the inhibitory
effects of both compounds on the SaeRS TCS. Then, we analyzed the transcription of a gene
encoding Staphylococcal accessory regulator (SarA). SarA is responsible for the biofilm
formation and not regulated by saeS. Thus, it was considered a good negative control, and its
transcription analyzed post treatment of xanthoangelol B 1 and PM-56 (Figure S5). As
anticipated, it can be seen that the level of sarA transcription was not significantly affected by
the two compounds. These results imply that 1 and PM-56 indeed inhibited the SaeRS pathway
with specificity, and thus are promising candidates for future development of anti-virulence
agents or chemical probes specific for the SaeRS two-component system.
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Cytotoxicity
In order to explore the potential of xanthoangelol B 1 and PM-56 as initial leads for further
translation, we have examined the cytotoxicity of both compounds on Hela cells, quantitatively
and qualitatively. Firstly, LDH release assay was performed, in which we have treated Hela
cells with 1 and PM-56 at various concentrations (2 M - 128 M) for 72 h and measured the
optical density at 450 nm. It was shown that IC50 values for Hela cell survivals were 19.70 M
and 28.94 M with 1 and PM-56, respectively (Figure 4 A & B). These values were 10 fold
and 7 fold higher than IC50 values for the GFP-reporter inhibition by 1 and PM-56,
respectively. Hence, the toxicity/efficacy ratio is reasonable for the initial leads. Next, the
live/dead imaging experiment has been performed. The Hela cells were seeded in 96 well plates
for 24 h, and after attachment of cells, the culture media (DMEM, 10% FBS, 1%
penicillin/streptomycin) were changed, followed by treatment with 1 and PM-56 at various
concentrations (from 2 μM to 64 μM) for 72 h. The Hela cells stained with fluorescein diacetate
dye (live cell staining, green color) and propidium iodide dye (dead cell staining, red color)
were observed using the confocal microscopy (Figure 4 C & D).
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Figure 4. Cytotoxicity of xanthoangelol B and PM-56 on Hela cells. A (xanthoangelol B), B
(PM-56): LDH release assay. Hela cells were seeded in 5x105 cfu and treated with
xanthoangelol B 1 and PM-56 from 2 μM to 128 μM and incubated for 72 h. The LDH release
was analyzed at OD450nm. All the experiments were performed in three biological replicates and
data are presented as a mean ± SEM. C (xanthoangelol B), D (PM-56), live/dead cell staining.
Hela cells were seeded 5x105 cfu in a 96 well plate and incubated 24 h for attachment. The
various concentrations of 1 and PM-56 (2 μM to 64 μM) were tested for 72 h. Then, the cells
were stained with FDA and PI and imaged under the confocal microscope. Green, live; red,
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dead, (i) vehicle control; (ii) 2 μM; (iii) 4 μM; (iv) 8 μM; (v) 16 μM; (vi) 32 μM; (vii) 64 μM.
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Confocal images were in accordance with the quantitative results as mentioned earlier. Both
compounds at their IC90 (approximately 8 M) for the GFP-reporter did not produce
substantially visible toxicity to the Hela cells while a reduction in viability was observed only
when the cells were treated with concentrations higher than IC50 for cytotoxicity. Cytotoxicity
tests demonstrated that 1 and PM-56 indeed had the promising traits as initial leads.
Validation of the anti-virulence activity of compounds 1 and PM56 using a worm infection
model
With the encouraging in vitro data, we further validated the anti-virulence activity of 1 and
PM-56 in a Galleria mellonella infection model. G. mellonella worms possess both a humoral
and an innate immune system. The worms provide an attractive, simple, and easy-to-handle
infection model with few biosafety or ethical issues. The G. mellonella infection model has
previously been used with several human pathogens to test the in vivo efficacy of antibacterial
and anti-virulence drugs.42 Thus, G. mellonella larvae was challenged with 5x106 colony
forming units (CFUs), a load designed to allow only 20% survival after 72 hours. The larvae
was post-treated with various doses of either 1 or PM-56 at 12 h intervals, up to 72 h. Larvae
health indices were strictly followed and recorded. A dose-dependent protection from virulence
by both compounds (1 and PM-56) was observed in the larvae (Figure 5). Compared to the
vehicle control, 1 and PM-56 generated a 3.5-fold higher survival at doses as low as 2.0 and
2.1 mg/kg, respectively. 1 showed 90% survival at a dose of 3.1 mg/kg and complete survival
at 4.1 mg/kg. PM-56 showed 90% survival at 3.2 mg/kg and complete survival at 4.3 mg/kg.
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These survival rates were comparable to or slightly better than survival rates observed in worms
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(a)
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Xanthoangelol B Concentration (mg/kg body weight)
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Figure 5. The ability of compounds 1 (A) and PM-56 (B) to protect G. mellonella from lethal
S. aureus infection. Kaplan-Meier survival curves for G. mellonella challenged with 5x106
CFU S. aureus and treated with compounds 1 and PM-56 at 2 h post-infection. The compounds
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were injected every 12 h for 72 h. The experiments were performed using biological replicate
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Next, we measured bacterial burden in the host worms. As shown in Figure S4, the inactivation
of saeS resulted in a decline in the bacterial burden in the host worms (up to 1.7 log10
CFU/worm). Treatment with 1 or PM-56 significantly lowered the bacterial burden in worms
in a dose-dependent manner. Compared to the vehicle control, a dose of approximately 1 mg/kg
allowed a sharp decrease in the bacterial burden. At concentrations of 4.1 and 4.3 mg/kg,
respectively, 1 and PM-56 generated a decline of 1.72 log10 and 1.84 log10 CFU/worm (Figure
S6). Both compounds again showed comparable efficacy to that of seaS knockout. These
results are highly consistent with survival tests. Then, to evaluate whether the activities of 1
and PM-56 were based on the saeS dependent pathway, we have measured the effects of both
compounds on G. mellonella infected with the saeS knockout mutant under the same conditions
as above. G. mellonella were challenged with 5x106 cfu of the wild-type and the saeS knockout
S. aureus, which were subsequently treated with 1 (2.042mg/kg body weight) or PM-56
(2.131mg/kg body weight) for every 12 h interval for 72 h. These concentrations were
corresponding to IC90 for the SaeRS GFP reporter. The survival rates of G. mellonella infected
with the knockout strain treated with 1 and PM-56 were 70% and 90%, respectively, while
they were 20% for the wild type-infected larvae and 80% for the knockout strain-infected
larvae without compounds (Figure S7 A), which showed protection effects were comparable.
Then, we counted CFU from G. mellonella of each group after a 72 hour infection. It was
shown that CFU was significantly decreased in the saeS knockout group as compared to the
wild-type infection group by up to 74.5%. 1 and PM-56 treated groups also showed comparable
reduction in CFU by 76.8% and 83.3%, respectively (Figure S7 B). Together, these results
suggest that the protection effects initiated by the saeS knockout were not drastically improved
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by the treatment with 1 and PM-56, and subsequently, the activities of both compounds on G.
mellonela were relevant to the saeS dependent pathway although we cannot rule out an extra
activity of PM-56 as the effects of PM-56 consistently showed a slight but non-significant
improvement over the saeS knockout.
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Overall, the superb protection given by 1 and PM-56 and the sharp decline in the bacterial
burden following their use allowed us to draw comparisons with saeS knockout experiments.
This hinted at a mode of action for these compounds. Thus, we next tested 1 and PM-56 for
their ability to inhibit the histidine kinase or phosphotransferase activities of SaeS.
Effects of compounds 1 and PM-56 on the histidine kinase and phosphotransferase
activities of SaeS
The sensor histidine kinase (HK) of a bacterial two-component system represents a promising
antibacterial target, as the catalytic and ATP-binding (CA) domain of the sensor HK is well
conserved across bacterial species. Thus, an inhibitor binding this domain has the potential to
be a broad-spectrum antibacterial agent.15 Indeed, many efforts have been made to discover
such a class of inhibitors, with some degree of success.15, 43-44 1 and PM-56 displayed excellent
SaeRS-related anti-virulence activity in vitro and in vivo. These results were comparable to the
effects seen in seaS knockout studies. Therefore, it was of great interest to determine whether
these compounds inhibited the sensor HK SaeS. We tested the two compounds against the
purified minimal kinase domain of SaeS. The minimal kinase domain contains a dimerization
and histidine phosphotransfer (DHp) domain and a CA domain (amino acid residues 118–351).
Measurements were conducted using a radioisotope method with SDS-PAGE autoradiography.
Both compounds clearly inhibited the autophosphorylation of SaeS (Figure 6), with IC50 values
of 220 and 160 µM for 1 and PM-56, respectively. This was generally consistent with their
observed activities comparable to the saeS knockout. In contrast, when we measured the
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inhibition of phosphotransferase (PT) activity using radioactively phosphorylated SaeS in the
presence of SaeR, no inhibition was observed (Figure S8). This implies that only the HK
activity (and not the PT activity) of SaeS is relevant to the inhibitory activity of 1 and PM-56.
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Figure 6. Inhibitory effects of xanthoangelol B 1 and PM-56 on the in vitro autokinase activities
of SaeS and AgrC. Purified recombinant minimal kinase domains of SaeS and AgrC (5 µM
each) were incubated at various concentrations (0–1 mM) of 1 (A) or PM-56 (B) for 15 min
on ice before addition of ATP to initiate the autophosphorylation reactions. After SDS-PAGE
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analysis, total and P-labeled proteins were visualized by Coomassie-Brilliant Blue staining
and autoradiography (32P), respectively. Each lane contains approximately 0.5 µg purified SaeS
or AgrC. The 32P-labeled protein bands were quantified by densitometric analysis and the
autokinase activities were calculated as the percentages of 32P-labeled proteins in the samples
treated with inhibitory chemicals compared to those in the control samples treated with 10%
DMSO. Dose-response curves were generated by plotting the percent autokinase activity
against the chemical concentration. The experiments were conducted in duplicate.
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To probe a possibility of 1 and PM-56 as broader HK inhibitors, we also tested both compounds
on AgrC, a component of another master virulence regulator and the most well characterized
TCS in S. aureus.45 Thus, it was shown that 1 and PM-56 inhibited AgrC with IC50 values of
339 µM and 140 µM, respectively (Figure 6). PM-56 showed a comparable activity to that for
SaeS while 1 showed 1.5 fold lower activity compared to that for SaeS. This extra activity of
PM-56 may explain slightly higher but non-significant improvement in the protection effect of
PM-56 over the saeS knockout. Furthermore, PM-56 was 1.4 fold higher activity than 1 for
SaeS while this difference expanded to 2.4 fold for AgrC. Thus, this suggests a potential of the
xanthoangelol B scaffold for further development of broad-spectrum or specific inhibitor of
bacterial histidine kinases.
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Next, we attempted to measure the direct binding of 1 and PM-56 with SaeS using the intrinsic
fluorescence quenching assay. Since SaeS contains seven Tyr (and zero Trp) residues, the Tyr
fluorescence at 306 nm was monitored before and after binding of 1 and PM-56. The reduction
of fluorescence upon ligand binding was expressed as the percent fluorescence quenching and
the Kd value was calculated by non-linear regression fitting (Figure S9). The quenching assay
revealed that 1 and PM-56 bound to SaeS with the Kd values of 40 µM and 6.8 µM,
respectively. These Kd values are smaller than IC50 values. It is not rare that IC50 values are
measured to be much higher than Kd values46-48 and the data are clear indication that both
compounds directly bind to SaeS with consistent trends of stronger interaction of SaeS with
PM-56 than with 1.
Since the binding of PM-56 to SaeS was stronger than that of 1 as revealed in the inhibition
and fluorescence quenching studies, we selected PM-56 for further dissection of the inhibitor
binding to SaeS. To identify the binding of PM-56 to SaeS, 1-dimensional transverse relaxation
filtered experiment with a Carr–Purcell–Meiboom–Gill pulse trains (1D CPMG) and 1D
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saturation transfer difference experiment (1D STD) were performed in the presence and
absence of the minimal kinase domain of SaeS.49 1D CPMG experiment is able to detect the
reduced T2-relaxation time of the PM-56 peaks caused by the interaction with SaeS, which
results in the peak broadening and decreasing. The STD NMR spectra obtained with the
minimal kinase domain of SaeS were virtually identical to those with the kinase domain of
SaeS fused to maltose binding protein (MBP-SaeS) (Figure 7 and S10), indicating that the
presence of MBP does not influence the binding. Therefore, MBP-SaeS was used for
interpretation of the 1D STD experiment to enhance the efficiency of saturation transfer from
the protein to PM-56. The transferred saturation from the protein to the ligand is amplified by
the binding exchange process and is efficient for target proteins with large molecular weight.50
PM-56 clearly displayed the binding exchange process to SaeS. Simple 1D spectra also showed
that the peaks of PM-56 were specifically broadened and shifted by SaeS, as compared to those
of free PM-56 (Figure 7A). Although the SaeS and MBP-SaeS proteins were dialyzed in the
NMR-buffer before the NMR experiments, the HEPES molecule included in the purified
proteins was not completely removed. Nevertheless, the spectra of the 1D CPMG (Figure 7B)
and 1D STD (Figure 5C) clearly show that PM-56 specifically binds to SaeS, but HEPES did
not. Furthermore, the similar peak intensities between the 1D spectrum (Figure 7A, red
spectrum) and the 1D STD spectrum (Figure 7C) showed putative interactions involving most
part of PM-56. Hence the entire molecular structure appears to be needed to exert a meaningful
inhibition, which is consistent with the test of fragments. The presence of binding exchange
process accompanied by the clear peak broadening of PM-56 indicates that the binding affinity
between SaeS and PM-56 is likely not too strong (i.e. nano-molar Kd) or too weak (i.e. mili-
molar Kd), which is in agreement with inhibition and binding measurements.
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Figure 7. 1D NMR analysis of the PM-56 binding to SaeS. (A) 1D spectrum of PM-56 alone
is used as a blank reference (blue). The several peaks of PM-56 are not shown in the spectrum
due to an overlap with the huge water peak (marked with an asterisk). The presence of 0.1 mM
of the minimal kinase domain of SaeSc apparently decreases and broadens the peaks of PM-
56 (red), and the peak shift of aromatic and olefin protons is also noted (inset). (B) The
transverse relaxation filter using 200 msec CPMG pulse trains completely eliminated the peaks
of PM-56 in the presence of 0.1 mM of the minimal kinase domain of SaeS (red). On the other
hand, the reduction of the PM-56 peaks by the CPMG filter is not apparent in the absence of
SaeS (blue). (C) The presence of a binding exchange process between PM-56 and MBP-SaeS
is clearly confirmed by 1D STD experiment. The PM-56 peaks are specifically identified in
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1D STD spectrum in the presence of 30 μM MBP-SaeS. Although the peak spikes of the
remaining buffer (HEPES) are identified, their integrations are zero; slightly different peak
shapes between on- and off-saturation at 0.5 and 30 ppm, respectively, could result in these
peak spikes.
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The IC50 values for HK activity were higher than expected considering the IC50 values for the
SaeRS TCS GFP reporter in intact bacteria cells. Nevertheless, an observation of substantially
lower activity for purified enzymes than enzymes in intact cells is not rare.51-52 This could be
attributed to the different states of purified target proteins compared to proteins in intact cells.
There are several factors that may affect protein activity in intact cells, such as the intracellular
distribution of compounds, processing of target proteins, and presence of interacting proteins.52
In our case, the purified SaeS comprised the minimal kinase domain, which contains only the
DHp and CA domains (amino acid residues 118–351). However, native SaeS is a modular
protein containing transmembrane domains, a linker, and cytoplasmic domains. The presence
of the linker has a significant impact on the kinase activity.53 Therefore, it is possible that
integrity of the whole protein structure is crucial for its kinase activity. This would also affect
its binding affinity to small molecules. Subsequently, the two inhibitors may appear to exert a
lower inhibitory activity on the purified SaeS minimal kinase domain. Nevertheless, inhibition
of the kinase activity of SaeS by 1 and PM-56, supported by STD NMR and the fluorescence
quenching assay, is a significant indicator of their mechanism of action.
We cannot rule out possible contributions to inhibition by other mechanisms, such as SaeS
sensing or SaeR binding to the promoter regions of virulence genes. Although a majority of the
inhibitors of TCSs reported thus far54 have been HK inhibitors, a few have shown separate
activities against the sensor16 or the response regulator.15 The evaluation of these possibilities
is complicated, particularly for inhibitors of sensing activity, because SaeS does not have a
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sensing domain and likely requires an unknown auxiliary protein.53 Nevertheless, these studies
are currently underway and will be reported in due course. Given that 1 and PM-56 did not
suppress bacterial growth at IC90 (while inhibiting the SaeRS TCS), these compounds are
excellent candidates for specific action against the virulence of S. aureus. In light of this, the
necessity of the whole xanthoangelol B molecule for optimal activity is an interesting finding.
Neither the isoprenyl nor the chalcone unit alone displayed activity. Moreover, an overlapping
fragment also failed to show activity. 1 and PM-56 are modular compounds composed of
isoprene and chalcone. This result therefore deviates slightly from the logic of a fragment-
based approach.55 This demonstrates that great caution is needed with fragment-based
approaches (although these are still powerful methods of drug development). The major
difference between 1 and PM-56 is the conversion of the α,β-unsaturated carbonyl in the
chalcone moiety of 1 to the β-hydroxyl carbonyl in compound PM-56. This subtle change
appears to confer a different activity profile. This will be valuable information in the further
development of anti-virulence agents. From the perspective of drug development, it may be
beneficial to think about the route of administration. It was shown that orally administered
xanthoangelol, a structurally related compound, was absorbed and distributed rapidly in mice
in a single dose study,56 which allows speculation that 1 and PM-56 may behave similarly.
However, in the same study, the absorption efficiency of the chalcone was evaluated to be
low,56 potentially indicative of low oral bioavailability. The authors stated the limitation of a
single dose study and the major difference between the results of bioavailability studies in
animals and humans for possible reasons. Hence, this suggests that many aspects need to be
taken into consideration, and it is considered that it is too early at the current stage to speculate
the route of administration. Regardless, our data provide a strong starting point for further
optimization.
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In conclusion, we found a hit (xanthoangelol B 1) in a screening with inhibitory activity against
the SaeRS TCS of S. aureus. A prenylated chalcone, xanthoangelol B 1, was synthesized in the
racemic form and then resolved into an enantiomeric form. Synthesized xanthoangelol B 1
showed identical spectroscopic and optical properties to those of xanthoangelol B 1 extracted
from a natural source. We also synthesized PM-56, a derivative of 1 with a hydrated chalcone
double bond. The two compounds displayed similarly good inhibitory activities against the
SaeRS TCS GFP reporter. They also inhibited the expression of downstream virulence factors,
reducing hemolysis. PM-56 displayed a better tolerance for bacterial growth, which is desirable
for anti-virulence agents. Interestingly, fragments of the two compounds did not show any
inhibitory activity. Thus, these compounds are required in their entirety for activity. 1 and PM-
56 displayed excellent survival rates and low bacterial burdens in the G. mellonella infection
model. These results were comparable to the effect of saeS knockout. The in vivo data
demonstrated a good correlation with the in vitro data. Indeed, 1 and PM-56 showed substantial
inhibition of the minimal kinase domain of SaeS although measured IC50 values were higher
than expected from the in vitro (bacteria) and in vivo data. However, the purified minimal
kinase domain is not likely to have the same kinase activity as the full-length membrane protein
SaeS. This might explain the lower potency of 1 and PM-56 against the purified minimal SaeS.
Fluorescence quenching assay and NMR data support the direct binding of PM-56 with the
minimal kinase domain of SaeS. This study demonstrates that natural products are still a
valuable source of compounds for therapeutic agents. Efforts toward the partial or total
chemical synthesis or modification of natural products can help diversify a candidate’s
structure. With the expertise and insight gained from our synthesis of xanthoangelol B 1, we
are now in a position to further develop inhibitors of the SaeRS TCS.
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EXPERIMENTAL SECTION
General Methods
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All reactions were carried out under open flask unless otherwise stated. THF, toluene, diethyl
ether and CH2Cl2 were procured from Sigma Aldrich. Reagents were purified prior to use
unless otherwise stated. Purification of reaction products was carried out by flash
chromatography using Fisher silica gel (35-70 mesh). NMR spectra were recorded on, Bruker
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1
DPX 400. H NMR spectra were measured at 400 and 500 MHz. 13C NMR spectra were
measured at 126 and 150 MHz using CDCl3, acetone-D6 or DMSO-d6 as a solvent and internal
reference. Coupling constants J’s are given in Hz. Multiplicity as follows: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, br = broad signal. Mass spectrometric data were
obtained on a Waters Acquity UPLC and UPC2 LC systems. Agilent 1260 infinity was used as
an HPLC system to analyze and purify compounds. Synthesized xanthoangelol B 1 and PM-
56 were screened for PAINS chemotypes using two online tools, PATTERNS
(http://zinc15.docking.org/patterns/home) and FAFDrug4 (http://fafdrugs3.mti.univ-paris-
diderot.fr/), and the results were negative or PASS.
Xanthoangelol B (1)
Diethylaluminum 2,2,6,6-Tetramethylpiperidide (DATMP) was prepared in-situ by reacting
diethylaluminum chloride (6.79 mmol) and lithium 2,2,6,6-tetramethyl piperidide (6.79 ml) in
dry toluene (10 ml) under Argon atmosphere at 0 oC for 1h. The DATMP prepared was used
immediately.36 To a stirred mixture of DATMP (4 mmol) and dry toluene (10ml), a solution
of oxirane 13 (1 mmol) in toluene (3ml) was added dropwise at 0 oC over a period of 5-10 min.
The reaction mixture was stirred at 0 oC until the starting material was not detected by TLC.
The reaction upon completion was quenched with ice-cold 1M HCl and the resulting organic
phase was separated. The aqueous layer was extracted with diethyl ether (2 x 10 ml). The
organic layer was combined, washed with brine, dried over anhydrous MgSO4, and
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