Development of biotin-retinoid conjugates as chemical probes for analysis of retinoid function

Article abstract of DOI:10.1016/j.bmcl.2018.06.011

Herein, we report the rational design, synthesis and biological evaluation of conjugates consisting of the synthetic retinoid Am580 and biotin connected via a linker moiety. We found that the linking substructure between the retinoid part and the biotin part is critical for retaining the biological activity. Conjugate 4 with a shorter linker showed similar potency to endogenous retinoid ATRA (1) and the parent compound Am580 (2) for neural differentiation of mouse embryotic carcinoma P19 cells, and showed the same pattern of induction of gene expression. It is expected to be useful as a probe for investigations of retinoid function. The design rationale and structure-activity relationship of the linker moiety are expected to be helpful for developing biotin conjugates of other nuclear receptor ligands.

Full text of DOI:10.1016/j.bmcl.2018.06.011

Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of biotin-retinoid conjugates as chemical probes for analysis of
retinoid function
⁎
Shinya Fujiia,b, , Shuichi Morib, Hiroyuki Kagechikab, Marco Antonio Mendoza Parrac,⁎
,
Hinrich Gronemeyerc
Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan
Department of Functional Genomics and Cancer, Institute of Genetics and Molecular and Cellular Biology, (IGBMC), Centre National de la Recherche Scientifique
a
b
c
UMR7104, Institut National de la Santé et de la Recherche Médicale U964, Université de Strasbourg, BP 10142, Illkirch Cedex 67404, France
A R T I C L E I N F O
A B S T R A C T
Keywords:
Retinoid
Biotin conjugate
P19 cells
Neural differentiation
Chem-seq
Herein, we report the rational design, synthesis and biological evaluation of conjugates consisting of the syn-
thetic retinoid Am580 and biotin connected via a linker moiety. We found that the linking substructure between
the retinoid part and the biotin part is critical for retaining the biological activity. Conjugate 4 with a shorter
linker showed similar potency to endogenous retinoid ATRA (1) and the parent compound Am580 (2) for neural
differentiation of mouse embryotic carcinoma P19 cells, and showed the same pattern of induction of gene
expression. It is expected to be useful as a probe for investigations of retinoid function. The design rationale and
structure-activity relationship of the linker moiety are expected to be helpful for developing biotin conjugates of
other nuclear receptor ligands.
The tight interaction between biotin and streptavidin has been
widely used in biochemistry and chemical biology,1 and many kinds of
biotin conjugates have been developed for target identification, la-
beling or functionalization of proteins of interest.2 Biotin conjugates are
also employed in the developing technology of genome-wide analysis.
For example, ChIP-sequencing (ChIP-seq) combines chromatin im-
munoprecipitation (ChIP) with massively parallel sequencing to iden-
tify the binding sites of DNA-binding proteins in a genome-wide
manner,3,4 but chemical affinity capture, such as biotin-avidin inter-
action, can also be used as an alternative to the antigen-antibody in-
teraction. Such chemical affinity-based sequencing (Chem-seq) is a
useful technique to identify genomic sites where biologically active
compounds and their target proteins interact.5,6 The Chem-seq method
was first employed in 2014 to investigate bromodomain (BRDs), CDK9
and DNA intercalator, based on the use of BRDs inhibitor JQ1, CDK
inhibitor AT7519 and the intercalator psoralen, respectively,7 and
further work been reported since then.8 Though the Chem-seq method
is in principle a powerful tool for identifying DNA target sites, its
practical application has been limited, probably due to the difficulty of
developing suitable biotin conjugates as Chem-seq probes, because in-
troduction of a biotinylated moiety is likely to cause loss of ligand ac-
tivity. Therefore, development of novel biotin conjugates of transcrip-
tion-modulating compounds that retain the biological activity is
necessary. Here, we report the rational design, synthesis, and biological
evaluation of biotin conjugates of the synthetic retinoid Am580.9,10
Retinoids, which are ligands of retinoic acid receptors (RARs), regulate
various key physiological processes, including cell fate decision, by
regulating expression of their target genes.11 To date, a number of re-
tinoid analogues have been developed as drug candidates, and several
natural and synthetic retinoids are in clinical use as therapeutic
agents.12,13 The development of retinoid-biotin conjugates as chemical
probes would be useful for genome-wide analysis of their action me-
chanisms.
As a model to guide the development of the retinoid probes, we
focused on the retinoid-induced differentiation of mouse embryotic
carcinoma (EC) cell line P19. Activation by all-trans retinoic acid
(ATRA: 1) of the cognate gene regulatory network in P19 cells induces
differentiation into neuronal precursor cells.14,15 We first confirmed
that a synthetic retinoid could induce the neural-differentiating signals
in P19 in the same manner as the endogenous RARs ligand, ATRA.
Indeed, we found that synthetic retinoid Am580 (2) induced the ex-
pression of neural differentiation markers (vide infra). Based on this
finding, we selected Am580 as the retinoid moiety of the desired con-
jugates because of its ready availability and high chemical stability in
comparison to ATRA.
In order to design the Am580-biotin conjugates, we focused on the
⁎
Corresponding authors at: Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan (Shinya Fujii).
E-mail addresses: fujiis@iam.u-tokyo.ac.jp (S. Fujii), marco@igbmc.fr (M.A. Mendoza Parra).
https://doi.org/10.1016/j.bmcl.2018.06.011
Received 27 April 2018; Received in revised form 4 June 2018; Accepted 5 June 2018
0960-894X/©2018ElsevierLtd.Allrightsreserved.
Pleasecitethisarticleas:Fujii,S.,Bioorganic&MedicinalChemistryLetters(2018),https://doi.org/10.1016/j.bmcl.2018.06.011

S. Fujii et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
The biological potency of the synthesized retinoid-biotin conjugates
as retinoids was assessed in terms of the neural differentiating activity
toward P19 cells. We investigated the expression levels of retinoid-re-
sponsive genes, including neuronal differentiation markers Ascl120,21
and Brn2 (Pou3f2),22,23 by quantitative RT-PCR (RT-qPCR). As shown in
Fig. 2, treatment with ATRA (1) for 72 h significantly induced the ex-
pression of Rarb, Cyp26a1, Ascl1 and Brn2, and Am580 (2) exhibited
similar gene expression-inducing potency, as mentioned above. As for
the biotin conjugates, we found that Am580-biotin-1 (4) exhibited
significant gene expression-inducing activity. Conjugate 4 induced Rarb
expression in a dose-dependent manner, and the response to 10 μM 4
was similar to that in the case of 1 μM 1 or 2. Regarding the three other
genes, conjugate 4 also significantly induced Cyp26a1, Ascl1 and Brn2
gene expression, and 1 μM or 10 μM 4 induced similar responses to
those seen with 1 μM 1 or 2. On the other hand, in contrast to conjugate
4, the gene expression-inducing activity of conjugate 5 was quite weak.
At 10 μM concentration, 5 induced expression of Rarb and Cyp26a1 to
some extent, but the mRNA levels were significantly lower than those
induced by 1, 2, and 4. No significant increase of Ascl1 and Brn2 ex-
pression was observed (Fig. 2).
In the course of the neural differentiation of P19 cells, the expres-
sion levels of retinoid-responsive genes exhibit markedly different time
courses. For example, Rarb and Cyp26a1 increase rapidly in response to
retinoid, while other genes including Ascl1 and Brn2 show a slower
increase of expression level.24 Based on this background, we in-
vestigated the time courses of Rarb, Cyp26a1, Ascl1 and Brn2 expression
in P19 cells induced by these compounds. Fig. 3 shows the expression
levels of these genes after 6, 24 and 72 h treatment with each com-
pound. ATRA (1) induced a rapid increase of Rarb and Cyp26a1 mRNAs,
followed by a decrease at the time point of 72 h. On the other hand, the
expression of Ascl1 was only weakly induced at 6 h, and then continued
to increase up to 72 h. Similarly, induction of Brn2 expression was not
observed at 6 h, but was observed at 24 and 72 h. Am580 (2) induced
essentially the same gene expression patterns as 1. Conjugate 4 also
exhibited similar gene expression patterns, namely, a rapid increase and
then decay of Rarb and Cyp26a1 mRNAs, and a relatively slow increase
of Ascl1 and Brn2 mRNAs. On the other hand, conjugate 5 induced only
modest increases of Rarb and Cyp26a1 mRNAs, and did not induce
expression of Ascl1 and Brn2 (Fig. 3).
The above results show that conjugate 4 retains neural differentia-
tion-inducing potency toward P19 cells, and thus could serve as a probe
to investigate retinoid function. Thus, connecting the biotinylated side
chain at the proximal position of the carboxylic acid appears to be a
reasonable design strategy for functional retinoid probes. On the other
hand, conjugate 5 exhibited significantly weaker biological activity
than 4. It is interesting that the biotin conjugate with a longer linking
substructure exhibited weaker potency than the conjugate bearing a
shorter linking substructure. A possible explanation is that the protein
surface of RAR, including the tunnel moiety, contains many hydrophilic
amino acid residues, so that the relatively hydrophobic character of the
linking substructure adjacent to the retinoid part of conjugate 5 in
comparison with 4 may be disadvantageous for binding to the receptor.
Also, the relatively rigid nature of the two serial amide moieties of 5
might inhibit proper folding of the receptor. Nuclear receptors share the
common domain structure and high sequence similarity in the LBD.
Therefore, this structure-activity relationship could represent a useful
clue for designing chemical probes for other nuclear receptors.
In summary, we rationally designed and synthesized Am580-biotin
conjugates, and found that conjugate 4 retains the biological activity of
the retinoid, at least as regards potency for induction of P19 cell dif-
ferentiation. Conjugate 4 exhibited the same gene induction pattern as
retinoids 1 and 2, and should be available as a probe for investigation of
retinoid function. The linking substructure proved to be critical for
retaining the biological activity. This design rationale and the structure-
activity relationship could also be helpful for developing biotin-con-
jugates of other nuclear receptor ligands. We are planning to conduct
Fig. 1. A) Structures of ATRA (1), Am580 (2) and the model compound 3 used
in the docking simulation. B) Docking model of 3 with hRARα LBD (PDB ID:
3KMR) obtained with AutoDock.17 The hRARα LBD complex with 2 (gray) is
superimposed on the docking model of 3 (green). (Left) The protein surface of
the ligand-binding pocket is indicated as a blue mesh. Note that the side chain
moiety of 3 extends outside the protein. (Right) The protein surface is indicated
as a blue solid. C) Structures of the designed Am580-biotin conjugates.
X-ray co-crystal structure of Am580 bound to RARα.16 The co-crystal
exhibits a tunnel structure with conserved water molecules, directed
from the receptor surface to the ligand-binding pocket in close proxi-
mity to the carboxylic acid of 2. Based on these considerations, we
conducted docking simulation of model compound 3 bearing a long
side-chain substructure. In the docked structure, the side chain moiety
of 3 accesses the receptor surface, suggesting that a linker moiety po-
sitioned at the ortho-position of the benzoic acid would be acceptable.
This is consistent with the structures of previously developed RAR-
targeting retinoid conjugates such as RAR-degradation inducers18
(SNIPERs: Specific and Nongenetic IAPs-dependent Protein ERasers).19
Based on these considerations, we designed two Am580-biotin con-
jugates, namely Am580-biotin-1 (4) and Am580-biotin-2 (5), bearing
biotinylated side chains at the ortho-position of the benzoic acid moiety
(Fig. 1).
These conjugates were synthesized as shown in Scheme 1. Con-
densation of tetramethyltetrahydronaphthalene carboxylic acid (6) and
methyl 4-aminosalicylate afforded amide 7. An N-protected amino-
propyl moiety was introduced at the phenolic group of 7 to afford 8,
and removal of the Boc group under acidic conditions gave the inter-
mediate 9. The biotinylated side chain was connected to the amino-
propyl moiety of 9 to afford 10 using the commercially available bio-
tinylation reagent 14, and then hydrolysis of the ester group afforded
the target compound 4. The conjugate 5 was also synthesized via the
intermediate 9. Six-atom elongation of the side chain of 9 gave 11, and
removal of the Boc group under an acidic condition gave 12. The bio-
tinylated moiety was connected to the side chain of 12 under the same
conditions as used for 10, and hydrolysis of the ester group afforded the
target compound 5 (Scheme 1).
2

S. Fujii et al.
Bioorganic&MedicinalChemistryLettersxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of the designed Am580-biotin conjugates 4 and 5. Reagents and conditions: (a) i) SOCl2, DMF, reflux, ii) Methyl 4-aminosalycilate, pyridine,
CH2Cl2, rt, 63%; (b) N-Boc-3-aminopropyl bromide, K2CO3, 2-butanone, reflux, 62%; (c) TFA, CH2Cl2, rt, 99%; (d) 14, Et3N, CH3CN, rt, 67%; (e) NaOHaq, CH3OH, rt,
75%; (f) N-Boc-5-aminovaleric acid, EDC, DMAP, Et3N, rt, 63%; (g) TFA, CH2Cl2, rt; (h) 14, Et3N, CH3CN, rt, 82% for 2 steps; (i) NaOHaq, CH3OH, rt, 94%.
Fig. 2. Quantification of induction of mRNAs of retinoid-responsive genes determined by RT-qPCR, and the concentration dependence for 4 and 5. Fold induction
was calculated relative to the vehicle control. Gene expressions were standardized by 36B4. Data are presented as means
SD.
Fig. 3. Time courses of induction of mRNAs of retinoid-responsive genes by retinoids 1 and 2 (1 μM each) and the synthesized retinoid-biotin conjugates 4 and 5
(10 μM each). Fold induction was calculated relative to the vehicle control. Gene expressions were standardized by 36B4. Data are presented as means SD.
chemical pulldown experiments using the conjugate 4 to confirm its
Science (JSPS) Core-to-Core Program, A. Advanced Research Networks,
practical utility.
and Grants in-Aid for Scientific Research from JSPS (KAKENHI Grant
Nos. 17H03887 and 16K15138 (S.F.)). A part of this research is based
on the Cooperative Research Project of Research Center for Biomedical
Engineering. Work in the laboratory of H.G. was supported by funds
from the Plan Cancer, AVIESAN-ITMO Cancer, the Ligue National
Acknowledgments
This work was supported by the Japan Society for the Promotion of
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