Development of Chemical Probes for Functional Analysis of Anticancer Saponin OSW-1

Article abstract of DOI:10.1002/tcr.201900042

Chemical probe-based approaches have proven powerful in recent years in the target identification studies of natural products. OSW-1 is a saponin class of natural products with highly potent and selective cytotoxicity against various cancer cell lines. Un

Full text of DOI:10.1002/tcr.201900042

Personal Account
DOI: 10.1002/tcr.201900042
Development of Chemical Probes for
Functional Analysis of Anticancer
Saponin OSW-1
T H E
C H E M I C A L
R E C O R D
Rina Komatsu and Kaori Sakurai*[a]
Chem. Rec. 2019, 19, 1–9
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Abstract: Chemical probe-based approaches have proven powerful in recent years in the target
identification studies of natural products. OSW-1 is a saponin class of natural products with
highly potent and selective cytotoxicity against various cancer cell lines. Understanding its
mechanism of action is important for the development of anticancer drugs with potentially novel
target pathways. This account reviews recent progress in the development of OSW-1 derived
probes for exploring the mechanism of its action. The key to the probe development is a
judicious choice of functionalization sites and a selective functionalization strategy. The types of
probes include fluorescent probes for cellular imaging analysis and affinity probes for target
identification analysis.
Keywords: OSW-1, anticancer natural products, saponin, chemical probes, mechanism of
action
in the cellular proteome.[7] Here we referred to chemical probes
as functional analogues of compounds of interest, which are
1. Introduction
OSW-1 is a steroidal saponin isolated from the bulbs of derivatized with functionalities required for mechanistic
Ornithogalum saundersiae by Sashida et al. more than 20 years analysis. For example, fluorescently derivatized probes can
ago.[1] It is structurally unique among the saponins having reveal cell internalization and intracellular localization proper-
glycosylated at the D-ring of the sterol core and the acylation ties of the compound whereas affinity probes with an affinity
at each ring of the carbohydrate moiety.[2] It was initially tag such as biotin moiety enables the binding analysis between
identified as an active agent in a screening for cyclic AMP the compound and the target proteins and the affinity
phosphodiesterase inhibitors. Subsequently in the National purification of target proteins for their subsequent sequence
Cancer Institute 60-cell in vitro screen, it was revealed that identification by mass spectrometry. The major advantage of
OSW-1 displays not only a highly potent anticancer activity the chemical probe-based approaches to target identification of
with an average GI50 of 0.78 nM but also a 10–100 fold natural products lies in their ability to directly determine the
selective cytotoxicity against cancer cell lines over non- target proteins. The discovery of target proteins should provide
malignant cells.[3] The bioinformatics analysis on the panel the first critical step toward deciphering the target cellular
assay data predicted that it exerts cytotoxicity by a potentially pathway leading to the phenotypes of interest.
novel mechanism, which is distinct from other known
Because of the biomedical interest, several groups have
anticancer agents. In 2010, Shair et al. reported that OSBP achieved total synthesis of OSW-1 and a number of its
and ORP4 L, two members of the oxysterol binding protein analogues have been synthesized to decipher the structure-
(ORP) superfamily implicated in lipid/sterol transport and activity relationship (SAR) and to explore more potent
sensing are the major protein targets of OSW-1.[4,5] Using analogues.[8] In contrast, a relatively few studies have been
competitive binding assays based on recombinant OSBP and reported on the development of chemical probes of OSW-1
ORP4 L and [3H]-25-HC, their endogenous ligand, OSW-1 for investigation of its biological role.[4,9,10] Here, we summarize
exhibits high affinity toward these proteins (Ki =26 nM for our recent efforts in the synthesis, biological properties of
OSBP and 54 nM for ORP4 L). However, the link between probes derived from naturally occurring OSW-1 and their
the inhibition of OSBP and ORP4 L and apoptotic induction application to the analysis of intracellular localization and the
has remained unclear. As a consequence, the molecular basis of interaction with known target proteins.
the cancer selective activity of OSW-1 is not well understood.
Elucidation of the mechanism of action of natural products
entails a complex challenge, having to identify the cellular site
2. Synthesis of Fluorescent Probe by Site-Selective
of action, the target proteins and the target cellular pathways.[6]
Acylation of OSW-1
Recently, chemical probe-based approaches have emerged as
powerful methods for mechanistic studies of natural products As a first step toward understanding the cellular effects of
OSW-1, we aimed to elucidate its site of action and its cellular
targets. It was then unknown whether OSW-1 translocates
[a] R. Komatsu, Prof. K. Sakurai
plasma membrane and has a specific distribution in cells. To
investigate the cellular localization of OSW-1, we thus planned
to develop a fluorescent probe based on naturally occurring
OSW-1. We chose to employ a chemical modification
approach to develop chemical probes based on naturally
Department of Biotechnology and Life Science
Faculty of Engineering, Tokyo University of Agriculture and
Technology
2-28-16, Nakacho, Koganei-shi, Tokyo, 184-8588, Japan
E-mail: sakuraik@cc.tuat.ac.jp
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occurring OSW-1 for two reasons. First, OSW-1 is not
commercially available nor is readily available through syn-
thesis but could be prepared from natural sources. Secondly,
several congeners with varying degrees of activity have been
identified from the same plant source,[1] which could be used
as negative control compounds in the biological studies of
OSW-1. For example, congener 1 displays a cinnamoyl group
instead of p-methoxybenzoyl (MBz) group at C2’’ position of
the xylose residue, which has been found with a 4-fold higher
activity than OSW-1. Of particular interest are two deacylated
congeners: 2 lacks MBz group at C2’’ of the xylose residue
with 10-fold decreased anticancer activity whereas 3 lacks both
MBz group and acetyl (Ac) group at C2’ of the arabinose
residue with 100-fold decreased activity.[1,3]
Figure 1. Structures of OSW-1, its naturally occurring congeners (1–3),
crystal structure of 1.[11]
Successful design of chemical probes for natural products
needs to meet the following criteria: 1) the derivatized
The synthesis of the fluorescent probe of OSW-1 (4) is
analogue retains the activity of the parent natural product, 2) shown in Scheme 1a. We employed 4-(N,N-dimethyl amino-
functional groups should be appropriately positioned to serve sulfonyl)-2,1,3-benzoxadiazole (DBD) group as a fluorescent
their purposes. In the case with fluorescent probes, a tag with an expectation that its small size should have minimal
fluorophore should be introduced distal to the pharmacophore structural perturbation. Our initial effort to synthesize DBD-
of the parent molecule to avoid imposing steric hindrance. To probe by standard acylation method proved low yielding.[12]
fulfill these requirements, prior knowledge on the SAR of Because naturally occurring OSW-1 is limited in supply, we
natural products is critical. The early studies on SAR of OSW- subsequently developed an efficient site-selective functionaliza-
1 indicated that both the sterol aglycone and the sugar moiety tion strategy to modify OSW-1.[13] Based on the report by
are required and their relative orientation to each other are Onomura et al.,[14] we developed a method to site-selectively
important for its extremely potent cytotoxicity.[1,3,8] In partic- acylate C4’-OH of the monomeric xyloside derivative with a
ular, the sterol side chain at C21 and the acyl groups of the variety of substituents in the presence of Me2SnCl2. Me2SnCl2
disaccharide residue were found essential. Our structural serves to activate 1,2-diols and with the use of a hindered base
analysis on OSW-1 and the cinnamoyl analogue by NMR and such as N,N-diisopropylethylamine (DIPEA), and allows
the X-ray crystallography showed that the C21-sterol side acylation to occur at the less sterically hindered hydroxyl
chain, C2’-Ac group and C2’’-MBz group form a hydrophobic group. It was found that catalytic amounts of Me2SnCl2 are
cluster, leading us to propose it as a protein binding site inefficient in acylating the xyloside with alkyl acid chlorides
(Figure 1).[11] Based on these findings, C3’’ or C4’’-hydroxyl such as DBD-COCl and hexynoyl chloride. Screening of
groups at the terminal xylose residue was selected as the molar equivalents of the catalyst, acylating reagents and
suitable site of functionalization.[12,13]
substrate concentration showed that 2 to 4-fold excess amounts
Rina Komatsu is a native of Hokkaido,
Kaori Sakurai received her Ph. D. in
chemistry from Princeton University in
2002. From 2003 to 2006, she studied as
a postdoctoral fellow at the Department of
Chemistry and Chemical Biology, Harvard
University. She started her independent
research group at the Department of
Biotechnology and Life Science, Tokyo
University of Agriculture and Technology
in 2006, where she is currently an associate
professor. Her research interests are fo-
cused on the development and application
of new chemical probes for identification
and analysis of protein targets of natural
products and bioactive small-molecules.
Japan. She graduated from the Depart-
ment of Biotechnology and Life Science at
Tokyo University of Agriculture and
Technology in 2015 with B. Eng., where
she is currently enrolled as a doctorate
student. Her research involves the develop-
ment and application of new chemical
probes for target identification studies of
anticancer saponin OSW-1.
Chem. Rec. 2019, 19, 1–9
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Scheme 1. Synthesis of fluorescent probes (4–6) derived from (a) OSW-1 and (b) its congeners 2–3 by Me2SnCl2-mediated site-selective monoacylation strategy.
of the catalyst are required to achieve improve acylation yields. of the parent natural products in live cell studies. We thus
Since OSW-1 was available in minute quantity, we further successfully prepared a series of related fluorescent probes from
optimized the reaction conditions to efficiently functionalize it OSW-1 and its congeners in one step in good yields and in a
with acyl groups under a dilute substrate concentration. Upon site-selective fashion by using Me2SnCl2.
treatment of OSW-1 at 0.01 M in CH2Cl2 with 4 equivalents
With the appropriate fluorescent probes in hand, we then
of the Me2SnCl2 and DBD-COCl, the desired 4’’-O-acylated applied them to the cell-imaging studies to investigate cell
product 4 was obtained in 55% yield (Scheme 1a) with the internalization and intracellular distribution of OSW-1 and its
3’’-O-acylated product in 6%, while no bis-acylated product congeners.[16] All three fluorescent probes 4–6 were rapidly
was observed.[13] It was thus demonstrated Me2SnCl2 can untaken up by cells and co-localized with specific markers of
efficiently promote site-selective acylation of a polyol natural endoplasmic reticulum (ER) and Golgi apparatus (Figure 2).
products.
We thus showed for the first time that OSW-1 is internalized
To study the structure-cellular localization property into cells and that it potentially targets these subcellular
relationship, the fluorescent probes of the deacylated congeners organelles. Comparison of the data for 4–6 suggested that
of OSW-1 were also synthesized (5-6, Scheme 1b). The differences in cytotoxicity of these compounds are not due to
Me2SnCl2-mediated site-selective acylation method was applied differences in their cell permeability nor subcellular localization
to synthesize fluorescent probes 5 using the deacylated OSW-1 but more likely due to differences in the direct interaction
2 with six hydroxyl group and the acetylated OSW-1 6 using with target proteins. They also suggested that the sterol moiety
the dideacylated OSW-1 3 with seven hydroxyl groups. is responsible for transporting OSW-1 and the congeners to
Deacetylated congeners were successfully acylated at C4’’-OH ER and Golgi apparatus. In contrast, Ac and MBz groups on
to give 5 in a 50% yield (62%, based on the recovered starting the sugar residues do not affect their cell localization property
material, brsm)[15] and 6 in 48% (58%, brsm).[16]
but are likely involved in target protein binding. More
The fluorescent probes 4–6 were tested using XTT assay recently, we have demonstrated by using high-resolution
for their antiproliferative activity in HeLa cells.[16] The IC50 microscopy that fluorescent OSW-1 4 selectively targets Golgi
values of the probes 6 (4: 1.4 nM, 5: 38 nM, 6: 3300 nM) apparatus. The study furthermore led us to reveal that OSW-1
were similar to these of the parent compounds (1: 0.46 nM, 2: induces the Golgi stress response pathways leading to cell
83 nM, 3: 5200 nM). These results strongly suggested that the death.[17]
fluorescently-tagged analogues serve as valid functional mimics
Chem. Rec. 2019, 19, 1–9
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experiments, we designed a biotinylated derivative of OSW-1
(9). A biotin tag has been widely utilized in affinity
purification of target proteins using avidin-immolized resins by
virtue of the strong biotin-avidin interaction. It has been
shown that the linker length for appending the biotin tag has a
strong impact on the efficiency of affinity purification.[19] We
included tetraethylene glycol as a hydrophilic linker between a
biotin tag and OSW-1 so that the steric hindrance would be
minimal.[20] As in the case with a fluorescent tag, a biotin tag
was to be attached at C4’’-OH to avoid the putative
pharmacophore of OSW-1 and to facilitate ready synthesis.
To synthesize the biotin probe 9, alkyne group was first
introduced to C4’’-OH of OSW-1 by Me2SnCl2 promoted
site-selective acylation using hexynoyl chloride to give 7 in
55% yield (Scheme 2). Subsequently, biotin-PEG-azide 8 was
conjugated to alkyne-tagged OSW-1 (7) by click chemistry
under copper-promoted alkyneÀ azide cycloaddition condition
in 83% yield.[13]
We next designed photoaffinity probes 13 (Scheme 3) and
16 (Scheme 4) involving a photoreactive group to crosslink
target proteins and a reporter tag for photoaffinity labeling
(PAL), which have proven powerful in recent years in the
chemical probe-based approaches to target identification
studies.[7b,21] They are particularly useful in detecting and
analyzing target proteins in live cells, which is not possible
with the affinity chromatography based approaches. Alkyl
diazirine group[22] was included in probe 13 as a photoreactive
group to crosslink the target proteins based on its small size
Figure 2. Intracellular localization of fluorescent probes 4–6 shown in green.
HeLa cells were incubated with probes (1 μM) for 1 h and co-stained for
mitochondria by tetramethylrhodamine etheyl ester, the Golgi apparatus by
BODIPY-TRX ceramide, endoplasmic reticulum by ER Tracker Red and
lysosome by Lysosome Tracker Red (shown in red). Scale bars: 10 μM.
3. Development of Affinity Probes for Target
Protein Analysis
We next developed a set of affinity probes toward investigation and selective reactivity. Our previous studies comparing the
of cellular targets of OSW-1.[13,18] For simple target enrichment major photoreactive groups (benzophenone, phenylazide and
Scheme 2. Synthesis of biotinylated OSW-1 9 as an affinity probe.
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Scheme 3. Two-step synthesis of OSW-1-based clickable photoaffinity probe 13.
Scheme 4. Synthesis of OSW-1-based fluorescent photoaffinity probe 16 by sequential site-selective acylation reaction using Me2SnCl2.
alkyl diazirine) found that alkyl diazirine provides the most residues to achieve maximal crosslinking efficiency without
selective photoreactive group in capturing low affinity compromising the bioactivity and effective post-labeling
carbohydrate-protein interactions.[23] Moreover, the terminal analysis. We designed another type of a photoaffinity probe, in
alkyne group was employed for 13 as a small and clickable which a photoreactive group is positioned at C2’’-OH, a
chemical handle, which facilitates conjugation of a reporter putative protein binding site of OSW-1. It has been known
group of choice such as a fluorophore or biotin.[24] We planned that various aromatic carboxylic acid ester groups can
a two-step approach to capturing target proteins, where PAL substitute C4’’-OH of OSW-1 without loss of activity.[1,3,25]
and click chemistry are sequentially conducted before the We thus employed trifluorophenyl diazirine (TPD) group[26] as
affinity purification step (Figure 3a). We employed a two-step an aromatic photoreactive group to replace MBz group of
synthesis where we first site-selectively derivatized C4’’-OH OSW-1. To simplify the synthetic scheme, a fluorescent tag
with a glutaryl linker 10 to generate an N-hydroxysuccinimide was introduced to C4’’-OH of OSW-1. We envisioned that
ester 11, which was then coupled with a photoaffinity label 12 probe 16 could be synthesized by sequential acylation of a
to give the desired alkyne-tagged photoaffinity probe 13 in naturally occurring deacylated OSW-1 (2, Scheme 4) at C4’’-
61% yield (Scheme 3).[18]
OH and then at C2’’-OH by using Me2SnCl2.[15] Treatment of
For the design of photoaffinity probes, a photoreactive 5 under the Me2SnCl2-mediated acylation conditions using
group should ideally be placed at the protein binding site while acid chloride 15 gave the desired C2’’-OH acylated product in
a detection group at any site distant from the protein binding 50% (62% brsm). While extensive structure–activity relation-
Chem. Rec. 2019, 19, 1–9
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Figure 3. (a) Photoaffinity labeling-click chemistry approach to capture and detection of binding proteins by using a clickable photoaffinity probe. (b)
Photoaffinity labeling of BSA by using clickable photoaffinity probe 13. Rhodamine-PEG azide 17 was used to detect the probe-crosslinked BSA. Fl: fluorescently
scanned image; CBB: Coomassie Brilliant Blue stained image. Cholesterol was used as a competitive ligand. (c) Dose-dependence of photoaffinity labelling by
probe 13 in comparison with a control probe 14.
ship studies have been conducted for OSW-1, probe 16 These results demonstrated that 13 can photocrosslink a
represented the first example in the synthetic modification of protein in a specific ligand-dependent fashion and that the
the biologically essential C2’’-moiety. It thus demonstrated the protein crosslinked with probe 13 can be detected either by a
versatility and straightforward utility of our Me2SnCl2-medi- fluorophore or biotin label through click chemistry. To
ated site-selective modification method, which can be used to demonstrate the affinity dependent reactivity, we reacted BSA
access not only the C4’’-substituted analogs of OSW-1 but with varied concentrations of probe 13 and subsequently with
also the C2’’-analogues.
rhodamine-azide 17 by click chemistry (Figure 3c).[18] Probe
The antiproliferative activity of probe 13 and 16 were 14 presents a negative control displaying a substructure of
evaluated by XTT assay.[13,18] The IC50 of the probes were OSW-1 lacking the sterol moiety, which should not specifi-
found to be comparable (13: 1.1 nM; 16: 3.2 nM) to that of cally bind BSA. A titration curve for probe 13 in Figure 3c
OSW-1 (0.46 nM). It thus suggested that they serve as suitable shows that BSA was crosslinked by 13 in a dose-dependent
cell permeable probes to explore OSW-1 binding proteins in fashion with the EC50 value of 14 μM. On the other hand,
live cell studies.
probe 14 showed marginal reactivity toward BSA even at
To first assess the reactivity of the photoaffinity probe 13, 100 μM. Taken together, we have shown that probe 13 retains
PAL and click chemistry were sequentially performed using potent anticancer activity of the parent natural product and is
bovine serum albumin (BSA) as a model sterol binding protein capable of effectively photocrosslink specific binding proteins.
(Figure 3a).[18] PAL experiments were typically conducted by
incubating a photoaffinity probe with the protein solution at
°
°
4 C and then were irradiated at 365 nm at 0 C for 10 min.
The reaction mixture was precipitated by cold acetone to
4. Summary and Outlook
remove the unreacted probe. The probe-crosslinked protein In this review, we have described the design and synthesis of
was conjugated to rhodamine-PEG azide 17 (Figure 3) by click our chemical probes derived from naturally occurring OSW-1.
chemistry. The resultant rhodamine-labeled protein was Based on the previous structural analysis, C4’’-OH was chosen
resolved by SDS-PAGE, which was then quantitated by in-gel as a functionalization site and the site-selective monoacylation
fluorescence imaging. We confirmed that probe 13 reacted strategy was successfully developed using Me2SnCl2 to access
with BSA only under the reaction condition involving UV various OSW-1 analogues bearing a fluorescent tag, an alkyne
irradiation (Figure 3b, lane 1 and 2). To validate the specific tag, a biotin tag or a bifunctional photoaffinity label. In
reactivity of probe 13, we also conducted a PAL reaction of addition to the fluorescent-tagged OSW-1, less active con-
BSA and 13 in the presence of cholesterol as a competitive geners of OSW-1 lacking acyl groups were modified with a
ligand in 400-fold excess of 13 (Figure 3b, lane 1 and 3). fluorophore at C4’’ position using the Me2SnCl2-mediated
Chem. Rec. 2019, 19, 1–9
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PERSONAL ACCOUNT
OSW-1 is an extremely potent and
selective anticancer saponin with a po-
tentially novel mechanism. This account
reviews recent progress in the develop-
ment of OSW-1 derived probes for
exploring its mechanism of action.
R. Komatsu, Prof. K. Sakurai*
1 – 9
Development of Chemical Probes for
Functional Analysis of Anticancer
Saponin OSW-1