Facilitated forward chemical genetics using a tagged triazine library and zebrafish embryo screening

Article abstract of DOI:10.1021/ja035334d

An improved forward chemical genetics approach was successfully demonstrated using a tagged library concept. A small-molecule triazine library with linkers was used to screen for brain/eye developmental phenotypes in a zebrafish embryo system. This approa

Full text of DOI:10.1021/ja035334d

Published on Web 09/09/2003
Facilitated Forward Chemical Genetics Using a Tagged Triazine Library and
Zebrafish Embryo Screening
Sonya M. Khersonsky,† Da-Woon Jung,† Tae-Wook Kang,† Daniel P. Walsh,† Ho-Sang Moon,†
Hakryul Jo,‡ Eric M. Jacobson,§ Vivekananda Shetty,| Thomas A. Neubert,| and Young-Tae Chang*,†
Department of Chemistry, New York UniVersity, New York, New York 10003, Whitehead Institute,
Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, DeVelopmental Genetics Program,
Department of Cell Biology, and Department of Pharmacology, Skirball Institute of Biomolecular Medicine,
New York UniVersity School of Medicine, New York, New York 10016
Received March 26, 2003; E-mail: yt.chang@nyu.edu
Forward chemical genetics is an emerging field that offers
powerful tools to search for novel drug candidates and their targets.1
It differs from classical genetics by substituting small molecules
for mutation-inducing agents or X-ray irradiation. Using combi-
natorial techniques,2 one is able to rapidly screen a large number
of small molecules and identify those that induce a novel phenotype
in a cellular or embryonic system. Once a phenotype effect is found,
the next step is to identify the biological target using an affinity
Figure 1. Tagged library approach for forward chemical genetics. (a)
Synthesis of the linker library, (b) screening for a novel phenotype, (c)
affinity matrix step facilitated by linker, and (d) protein analysis and
identification.
matrix made of the immobilized hit compound. However, the
synthesis of an efficient affinity matrix without the hit compound’s
activity loss has been shown to be challenging, or sometimes totally
impossible, due to the difficulties of adequate linker attachment.
In this paper, we demonstrate a novel tagged library approach to
accelerate the conversion of a hit compound to an efficient affinity
matrix, thus making forward chemical genetics a more systematic
strategy (Figure 1).
The design of our tagged library was based on a triazine scaffold
due to its ease of manipulation and structural similarity to purine
and pyrimidine, which have already been demonstrated to be active
in various biological systems.3 In addition, the triazine scaffold has
three-fold symmetry, and the positional modification is much more
flexible than that in the purines or pyrimidines. In our previous
report, we described an orthogonal solid-phase method to synthesize
a triazine-based combinatorial library4,5 and demonstrated anti-
microtubule activities among the library entities.4 A similar
Figure 2. Zebrafish brain and eye morphogenesis upon treatment with
derivatives of 1. (a) Wild-type embryo; (b) 50 µM of 1; (c) 10 µM of 1-B;
and (d) 25 µM of 1-B treatment in 8-cell-stage embryos. (e) Structures of
derivatives of 1. The concentration in parentheses indicates MIC.
chemistry has been applied to construct a novel tagged triazine
library, where three building blocks were prepared separately and
assembled orthogonally to yield 1536 highly pure compounds (see
Supporting Information). Each library compound contains one of
a variety of triethyleneglycol (TG) linkers at one of the diversity
sites of the triazine scaffold. Traditionally, selected and modified
active molecules, after biological screening, are fitted with a linker,
to provide an attachment point to the affinity bead. In many cases,
this can lead to activity loss, and thus a time-consuming and
laborious structure-activity relationships (SAR) study is required.
The incorporation of the linkers, before biological screening,
provides for a straightforward method of isolation of the target
protein without compromising the lead compound’s activity or
performing further SAR experiments.
play an important role in the biological activity. However, this can
be easily probed by detaching or modifying the linker. It should
be noted that removing an already existing linker is much easier
than adding a new one into the molecule via SAR.
The library compounds were screened for brain/eye morphologi-
cal changes in a zebrafish (Danio rerio) embryo assay using the
96-well plate format. Zebrafish has proven to be a powerful
screening system for forward genetic research because of its small
size and high fecundity.4,6 Among the 1536 triazines we have tested,
initially we found one library compound, 1, to generate significant
phenotype changes on zebrafish brain and eyes at 50 µM (Figure
2b). To see if the TG tag and amino end functionality are important
for the activity, two derivatives 1-A and 1-B were synthesized and
tested (Figure 2e). 1-B, a Boc attached derivative of 1, was found
to be more effective than the mother compound, which shows that
the amino end is not critical for the activity. The shape of the head
region was flatter and smaller than that of wild-type, and also the
eye development was retarded by 10 µM of 1-B treatment (Figure
2c). At a higher concentration (25 µM) of 1-B, eyes completely
Two possible problems may be envisioned. One is that the linker
may interfere with the biological activity and we may miss the
active hits. However, this negative selection will be favorable for
a researcher, as it will reduce unfruitful efforts to modify the hit
compounds later. The other possibility is that the linker itself may
† New York University.
‡ Massachusetts Institute of Technology.
§ Department of Cell Biology, New York University School of Medicine.
| Department of Pharmacology, New York University School of Medicine.
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10.1021/ja035334d CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
S5 mutant has a “small head/eye” and S18 mutation causes CNS
necrosis phenotypes including inflated hindbrain or reduced fore-
brain.8 Therefore, we suggest that 1 may interfere with the function
of a protein complex which includes S5, S13, S18, and L28 and
plays an important role for brain/eye development in the early
zebrafish embryo.
In conclusion, we have successfully demonstrated the power of
a tagged library approach for efficient forward chemical genetics,
especially by expediting the connection of a hit compound to the
affinity matrix by incorporating a linker directly to the library
compounds prior to phenotypic screening. The current study
elucidated the first novel small-molecule inhibitors for several
ribosomal accessory proteins or their complex as the target, which
are important for the early brain/eye development before gastrula-
tion. A further study will be carried out to elucidate the exact
binding mode of the small molecules and target proteins. The same
tagged library approach will be applied to other biological systems
to facilitate the general paradigm of forward chemical genetics.
Figure 3. Isolation of 1-specific proteins by competitive affinity chroma-
tography. (a) Structure of 1-immobilized agarose bead (1-Affi). (b) The
proteins bound to 1-Affi. Lane P: whole protein extract. Lane 1: 1-Affi
alone. Lane 2: 1-Affi + 1-A (50 µM). Lane 3: 1-Affi + 2-A (50 µM).
Protein bands indicated by arrowheads were collected for further analysis.
disappeared (Figure 2d). It was also found that 1-A, 1 with removed
linker (Figure 2e), generated similar phenotype changes with even
higher activity (MIC-minimum inhibitory concentration: 2.5 µM).
Again, this clearly demonstrates that the TG linker moiety of 1 is
not important for the activity. The specific effects of these
compounds were confirmed by the inactivity of 2, which is a
regioisomer of 1 with a different methoxy group position (meta vs
para). The corresponding derivatives 2-A and 2-B were also
synthesized and shown to be inactive even at higher concentration
(100 µM) and were further used as negative control compounds
(Figure 2e).
We also tested 1-B in zebrafish embryos at different time
points: 1 cell (0.2 hpf: hour post fertilization), 8 cell (1.25 hpf),
1K (1000) cell (3 hpf), 50% epiboly (5.3 hpf), and 10 somite (10
hpf, postgastrulation) stages. Almost the same morphological
changes were observed at 1, 8, and 1K cell stages, which are all
pregastrulation, but not at the 50% epiboly and 10 somite stages
(Figure 4, Supporting Information). This demonstrates that the target
proteins of 1 play an important role in brain/eye development only
before the gastrulation.
To identify target proteins, we immobilized 1 on activated
agarose beads (Affi gel 10) to afford 1-Affi (Figure 3a). After
loading of 1, the remaining active sites of the agarose bead were
blocked by ethanolamine. Ethanolamine-only treated agarose beads
were employed as a negative control matrix. Freshly prepared
protein extracts, from 128 to 1000 cell stages, were loaded on the
beads for binding to affinity matrices and gently rotated at 4 °C
overnight. After extensive washing with bead buffer, the bound
proteins were resolved by 14% SDS-PAGE and detected by silver
staining.
Two strong bands (23 and 18 kDa) were identified from the
1-Affi matrix, which were absent in ethanolamine-only resin. To
confirm the specificities of the proteins selectively bound to the
1-Affi, competition assays were performed using 1-A, the strongest
inhibitor, and 2-A as a negative control. An addition of 50 µM of
1-A to 1-Affi beads caused a dramatic decrease of the two strong
protein bands, indicated by arrowheads, but not by 2-A (Figure
3b). Those two bands were excised from the gel, digested with
trypsin, and the peptides were analyzed by LC-MS/MS using a
nanoflow HPLC coupled directly to a Q-TOF mass spectrometer.
The 23 kDa protein matched the 40S ribosomal subunit protein S5
(Danio rerio). Three different proteins were retrieved from the 18
kDa protein bands: 40S ribosomal subunit protein S18 (Danio
rerio), Danio rerio EST (expressed sequence tags) sequence similar
to human 40S ribosomal subunit protein S13, and mouse 60S
ribosomal subunit protein L28 (Figure 5 in Supporting Information).
40S ribosomal subunit proteins S5, S13, and S18 have proven to
have extraribosomal functions related to malignant transformation
and development regulation in eukaryotics.7 Furthermore, an early
zebrafish development by insertional mutagenesis demonstrated that
Acknowledgment. We thank Dr. Victor Livengood and Dr.
Yeounjin Kim at the Laboratory of Bioorganic Chemistry, NIDDK,
NIH, for the high resolution mass spectrometry measurements.
Funding support from Luminogene (www.luminogene.com) is
acknowledged.
Supporting Information Available: Full experimental procedures
and characterization data (PDF). This material is available free of charge
via the Internet at http://pubs.acs.org.
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