Screening of a virtual mirror-image library of natural products

Article abstract of DOI:10.1039/c6cc03114e

We established a facile access to an unexplored mirror-image library of chiral natural product derivatives using d-protein technology. In this process, two chemical syntheses of mirror-image substances including a target protein and hit compound(s) allow the lead discovery from a virtual mirror-image library without the synthesis of numerous mirror-image compounds.

Full text of DOI:10.1039/c6cc03114e

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Screening of a virtual mirror-image library of
natural products†
Cite this:DOI: 10.1039/c6cc03114e
Taro Noguchi,a Shinya Oishi,*a Kaori Honda,b Yasumitsu Kondoh,b Tamio Saito,b
Hiroaki Ohno,a Hiroyuki Osadab and Nobutaka Fujii*a
Received 14th April 2016,
Accepted 11th May 2016
DOI: 10.1039/c6cc03114e
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We established a facile access to an unexplored mirror-image library of metabolite products.8 Epigenetic remodeling via activation of
of chiral natural product derivatives using D-protein technology. In this silent gene cluster(s) also leads to the production of unprecedented
process, two chemical syntheses of mirror-image substances including natural products from fungi.9 However, the stereochemical out-
a target protein and hit compound(s) allow the lead discovery from a comes of the newly obtained scaffolds from these approaches are
virtual mirror-image library without the synthesis of numerous highly dependent on those of the parent substrates. To improve
mirror-image compounds.
the access to mirror-image natural products and their derivatives
with potentially desirable bioactivities as valuable resources in
Natural products and their derivatives have been valuable resources drug discovery, a complementary approach needs to be developed.
for drug discovery.1 A number of natural products and their Herein, we report a novel strategy to expand the structural diversity
derivatives are used in clinical practice as anticancer, antibacterial of chiral natural products and their derivatives by assessment of
and immunosuppressive agents; these compounds were originally mirror-image forms.
produced by plants, fungi, bacteria, or others as secondary
We designed a new screening strategy for a virtual mirror-
metabolites for defensive and reproductive purposes.2 The image library of chiral natural products with favorable drug-like
desirable biological activities are attributed to the unique, complex, properties, taking advantage of the mirror-image protein technology.
and sp3-carbon-rich scaffolds. While many natural products are The mirror-image proteins have been used for understanding a
produced as a single enantiomer when bearing a chiral structure(s), variety of biological phenomena.10 Mirror-image phage display
in some limited cases, two enantiomeric forms of chiral natural has also led to the identification of antiviral and anticancer
products are produced by a single or various species.3 The two D-peptides, which have resistance against peptidase-mediated
mirror-image isomers with identical chemical and physicochemical degradation.11 By using D-proteins, the interactions with compounds
properties often exhibit different functions and/or biological are diastereomeric to those afforded by the same compounds with
activities.4 The underlying chiral interactions between natural native proteins (L-proteins).12 In this sense, chiral compounds could
products and the target biomolecules are fundamental to the effectively bind in entirely new ways. We anticipated that screening
remarkable stereospecificity of these substances.
performed using mirror-image proteins could potentially double the
Recent reports have described attempts to expand the diversity chiral natural product resources available for drug discovery (Fig. 1).
of natural-product-like scaffolds.5 Using complex natural product This process involves the following: (1) initially, the D-protein
structures as starting materials, a variety of natural-product-like form of the target protein molecule is prepared by chemical
scaffolds were prepared via a ring-distortion strategy using protein synthesis; (2) using the synthetic D-protein, chiral natural
chemical modifications.6 Similarly, the component sesquiterpenes products are screened to identify hit compound(s); (3) the mirror-
in the extract of Curcuma zedoaria were converted into a variety image structure(s) of the hit compound(s) is synthesized and (4)
of derivatives with unprecedented scaffolds by treatment with the biological activities for the target L-protein could subsequently
oxidizing agents.7 Manipulation of the biosynthetic pathway by be assessed.
modification of culture conditions increases the chemical diversity
MDM2 is a negative regulator of tumor-suppressor protein
p53.13 We chose the p53-binding domain of MDM2 (MDM225–109
)
as the target, because a number of chiral inhibitors against
MDM2–p53 interaction have been identified as potential anti-
cancer agents.14 For example, nutlin-3a potently binds to MDM2,
while nutlin-3b (enantiomer of nutlin-3a) shows significantly less
a Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan. E-mail: soishi@pharm.kyoto-u.ac.jp, nfujii@pharm.kyoto-u.ac.jp;
Fax: +81 75-753-4570
b RIKEN CSRS, Wako, Saitama 351-0198, Japan
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6cc03114e activity.15 Initially, we synthesized D-MDM225–109 by standard
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Fig. 2 Two-step screening process using D-MDM2. Initially, chemical
array screening was carried out using TMR-labeled MDM2 proteins
Fig. 1 Concept of the screening strategy for a virtual mirror-image library of
natural products. Screening of chiral natural products using D-protein corre-
sponds to that of mirror-image natural products using L-protein in a mirror.
(L-MDM2TMR or D-MDM2TMR
) for 22 293 compounds. Among 43 hit
compounds with selective binding affinity toward L-MDM2 or D-MDM2
(Table S1, ESI†), four compounds were identified as L-MDM2–L-p53
and/or D-MDM2–D-p53 interaction inhibitors by a competitive binding
experiment in the FP assay. Abbreviation: FAM = carboxyfluorescein.
Fmoc-based solid-phase peptide synthesis according to the protocol
used in our previous study.16 The tetramethylrhodamine (TMR)-
labeled protein (D-MDM2TMR) was also prepared by conjugation
of TMR-(PEG)3-azide onto the N-terminal alkyne tag of
D-MDM225–109. The folded L-MDM225–109 and D-MDM225–109
had symmetric circular dichroism spectra, supporting the presence
of mirror-image a-helices (Fig. S3, ESI†).11f To verify the biological
activity of the synthetic MDM2 proteins, the binding affinities
toward p53 peptides (biotinyl-aminocaproyl-GSGSSQETFSDLW
KLLPEN-NH2) were evaluated by surface plasmon resonance
(SPR) analysis (Fig. S4, ESI†). The high-affinity bindings of the
L-MDM2–L-p53 peptide and the D-MDM2–D-p53 peptide were
observed [KD (L-MDM2–L-p53): 0.75 Æ 0.04 mM; KD (D-MDM2–D-
p53): 0.51 Æ 0.02 mM], while the binding activities of the L-MDM2–
Fig. 3 Structure and biological activity of NP843. IC50 values were measured
by the FP assay using a 0.5 nM fluorescein-labeled p53-like peptide (P4
peptide)18 and 10 nM MDM2 protein.
D-p53 peptide and the D-MDM2–L-p53 peptide were practically nil, with 1 N NaOH for hydrolysis of the formyl ester derived from 4.
suggesting high enantiomeric recognition of MDM2 toward p53. C1 elongation of 4 was then performed via tosylation and
Chemical array screening of 22 293 compounds including cyanylation followed by reduction to give an alcohol 6, which
natural products and their derivatives from RIKEN NPDepo was was converted into phosphonium salt 8. (R)-Trolox-derived
carried out using L-MDM2TMR and D-MDM2TMR 16,17
Among 43 aldehyde 9 was employed as a chiral chromane component
.
initial hit compounds (Table S1, ESI†), four compounds showed for the Wittig reaction. The subsequent two-step catalytic
inhibitory activity against L-MDM2–L-p53 and/or D-MDM2–D-p53 reduction including PtO2-mediated hydrogenation of olefins
interactions in the competitive binding inhibition assay by and subsequent Pd/C-mediated hydrogenolysis of the benzyl
fluorescence polarization (FP) (Fig. 2 and Fig. S5, ESI†). Of group provided the L-a-tocopherol (10b) in 84% yield. Alkylation
these, a chiral a-tocopherol derivative NP843 (1a) was identified of 10b with bromide 11 followed by deprotection gave ketone
to be a selective inhibitor against D-MDM2–D-p53 interaction (Fig. 3). 12b. Construction of the left-part of the chromone substructure
The next step was to verify the bioactivity of the enantio- by treatment of 12b with N,N-dimethylformamide dimethyl
meric form of 1a (ent-NP843, 1b) toward L-MDM2–L-p53 inter- acetal (DMF-DMA) afforded the mirror-image compound 1b.
action. Compound 1b was prepared by chiral pool synthesis
The inhibitory activity of mirror-image compound 1b against
from three commercially available optically pure components the interaction between MDM2 and the p53 peptide was evaluated
(Scheme 1). Initially, (R)-Roche ester-derived tosylate 2 was by an FP assay (Table 1 and Fig. S6, ESI†). Compound 1b inhibited
reacted with Grignard reagent 3, prepared from (S)-citronellyl L-MDM2–L-p53 interaction [IC50 (1b) = 7.6 Æ 1.9 mM for L-MDM2–L-
bromide, in the presence of a copper catalyst. The subsequent p53], while no inhibition was observed against D-MDM2–D-p53
deprotection of the trityl-group gave alcohol 4, after treatment interaction at 30 mM of 1b. This corresponded with the inhibitory
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for D-MDM2–D-p53].19 These data identified the mirror-image
a-tocopherol derivative 1b (ent-NP843) as a selective inhibitor of
the L-MDM2–L-p53 interaction.20
Further structure–activity study of compound 1b and its
derivatives revealed the structural requirements for inhibition
of L-MDM2–L-p53 interaction (Table 1 and Fig. S6, ESI†). We
designed two diastereomers of compound 1b: epimer 1c at the
tetrasubstituted carbon on the chromane scaffold, and diastereomer
1d with two epimeric methyl groups on the right-hand alkyl
chain (mirror-image of 1c) (Fig. 4). Compound 1d, having a
tetrasubstituted carbon with (S)-configuration on the chromane
skeleton (identical to that of compound 1b), reproduced the
bioactivity of 1b against L-MDM2–L-p53 interaction [IC50 (1d) =
6.9 Æ 1.9 mM for L-MDM2–L-p53], while compound 1c with
(R)-configuration (identical to that of compound 1a) showed
no inhibition. However, for D-MDM2–D-p53 inhibition, compound
1c was potent [IC50 (1c) = 7.8 Æ 2.0 mM for D-MDM2–D-p53]. These
results indicate that the stereochemistry of the tetrasubstituted
carbon on the chromane skeleton is dominant in the chiral
recognition by L-MDM2 protein when compared with the stereo-
centers on the extended alkyl chain. To demonstrate the significance
of the aliphatic chain length of 1b, we also designed derivatives 13a,b
and 14a,b with shorter isoprene units (Fig. 4). Derivatives 13a,b and
Scheme 1 Synthetic scheme for ent-NP843. Reagents and conditions:
(a) Li2CuCl4, THF, 0 1C; (b) HCO2H, Et2O, rt; (c) NaOH, MeOH/H2O, rt;
(d) TsCl, pyridine, 0 1C; (e) NaCN, DMSO, rt; (f) DIBAL-H, THF/CH2Cl2,
À78 1C, then H3O+; (g) DIBAL-H, THF/CH2Cl2, À78 1C; (h) TsCl, pyridine,
0 1C; (i) LiBr, acetone, reflux; (j) PPh3, neat, 100 1C; (k) LiHMDS, 9, THF,
À40 1C to rt; (l) H2, PtO2, TBME, rt; (m) H2, Pd/C, MeOH, rt; (n) 11, K2CO3,
DMF, rt; (o) H2, Pd/C, EtOAc, rt; (p) DMF-DMA, THF, reflux.
Table 1 Biological activity of NP843 and their derivatives
a
IC50 (mM)
Compound
L-MDM2
D-MDM2
NP843 (1a)
ent-NP843 (1b)
1c
1d
13a
13b
14a
14b
430
6.5 Æ 0.5
430
7.6 Æ 1.9
430
7.8 Æ 2.0
6.9 Æ 1.9
430
430
430
430
430
430
430
430
430
a
IC50 values for L/D-MDM2 were measured by the FP assay using 1.0 nM
fluorescein-labeled P4 peptide and 10 nM MDM2.
activities of the original hit 1a against D-MDM2–D-p53 and
Fig. 4 Structures of NP843 derivatives. The methyl group in blue indicates
L-MDM2–L-p53 interactions, respectively [IC50 (1a) = 6.5 Æ 0.5 mM the identical stereochemical configuration to that in ent-NP843 (1b).
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14a,b showed no inhibitory activity against L-MDM2–L-p53 inter-
This work was supported by Grants-in-Aid for Scientific
action (nor against D-MDM2–D-p53 interaction). These results Research from JSPS, Japan; the Targeted Protein Research
suggest that the chain length of three isoprene units is indispensable Program; and the Platform for Drug Discovery, Informatics,
for the biological activity of compound 1b.
and Structural Life Science from MEXT, Japan. T. N. is grateful
In this study, isolated natural products and derivatives, which for Research Fellowships for Young Scientists from JSPS, Japan.
were immobilized on microarray slides, were used as screening
resources. This approach would be further applicable to the
mirror-image screening of mixture samples from natural
resources including crude plant extracts and fermentation broth
extracts, which could provide an unprecedented opportunity to
Notes and references
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structures of chiral ingredients and native proteins. Once the hit
extract(s) is identified, structural determination of the active
ingredient(s) and synthesis of its enantiomer(s) would be
needed. In this way, a novel hit compound(s) with ‘‘unnatural’’
chirality would be obtained with a high probability. It is our
expectation that future analysis of screening data sets against a
number of target molecules may give some insights into the
value of mirror-image natural resources for drug discovery. In
this strategy, two chemical syntheses of mirror-image substances
(target protein and initial hit compound), and chemical array
technology using synthetic D-protein facilitated access to a variety
of virtual mirror-image natural products. For further application to
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enantiomeric compound (1b) enabled the validation of the
inhibitory activity of 1b against L-MDM2–L-p53 interaction. The
selective recognition by MDM2 was attributed to the stereochemistry
of the tetrasubstituted carbon on the chromane skeleton of 1b. The
aliphatic side chain of three isoprene units was also needed for
inhibition. In contrast to conventional screenings, this process could
identify hit compounds from unavailable mirror-image chiral
natural products, thus providing unprecedented lead compounds
for drug discovery. To our knowledge, this is the first application of
mirror-image protein technology in the screening of chiral small
molecules including natural products.
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cells were observed at 30 mM (Fig. S7, ESI†).
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