A Forward Chemical Screen in Zebrafish Identifies a Retinoic Acid Derivative with Receptor Specificity

Background: Retinoids regulate key developmental pathways throughout life, and have potential uses for differentiation therapy. It should be possible to identify novel retinoids by coupling new chemical reactions with screens using the zebrafish embryonic model. Principal Findings: We synthesized novel retinoid analogues and derivatives by amide coupling, obtaining 80–92% yields. A small library of these compounds was screened for bioactivity in living zebrafish embryos. We found that several structurally related compounds significantly affect development. Distinct phenotypes are generated depending on time of exposure, and we characterize one compound (BT10) that produces specific cardiovascular defects when added 1 day post fertilization. When compared to retinoic acid (ATRA), BT10 shows similar but not identical changes in the expression pattern of embryonic genes that are known targets of the retinoid pathway. Reporter assays determined that BT10 interacts with all three RAR receptor sub-types, but has no activity for RXR receptors, at all concentrations tested. Conclusions: Our screen has identified a novel retinoid with specificity for retinoid receptors. This lead compound may be useful for manipulating components of retinoid signaling networks, and may be further derivatized for enhanced activity

BT10 is selective for RAR receptors. Shown are the results of reporter assays in which specific activated receptors mediate luciferase reporter activity.

<p>Hek293T cells were transfected with the indicated RA receptor construct, in addition to the relevant reporter luciferase plasmid, and a non-retinoid regulated renilla reporter plasmid to control for transfection and lysate recovery. Twenty-four hours following transfection, cells were incubated with ATRA (blue diamonds) or BT10 (orange squares), at the doses indicated on the X axis (with the number (x) representing 10^x M final concentration). Control transfections lacked the receptor expression construct (yellow triangles or green marked boxes, for ATRA or BT10, respectively). Data is plotted relative to values obtained with transfected cells that were treated with DMSO, with that value set to 1.0, not shown on the graphs. ATRA activates robustly all 6 receptors, while BT10 shows specificity to RARs and does not activate RXRs at all concentrations tested.</p

Shown are qPCR results for the markers as indicated, using embryos that were taken at various developmental stages, also indicated.

<p>Transcript levels are calculated relative to those from vehicle (DMSO) treated embryos (light blue), for embryos treated with 0.1 µM ATRA (medium blue) or 5 µM BT10 (dark blue). The three markers in the left panel are repressed by both compounds, while <i>cyp26a</i> (right panel) is activated. The asterisks indicate statistically significant differences compared to control (p<0.05), based on a Student's T-test.</p

BT10, similar to RA, inhibits cardiomyocyte differentiation when added early during embryogenesis.

<p>(A) Embryos were cultured starting at 5 hpf in the presence of vehicle alone (DMSO, control), or “low” (0.1 µM, 5.0 µM) or “high” (0.3 µM, 15 µM) concentrations of ATRA or BT10, respectively. RNA was harvested at 24 hpf and used to measure by qPCR relative transcript levels, as indicated. All cardiac markers are significantly reduced in transcript levels by ATRA or BT10, and for both compounds, the ventricular marker is relatively more sensitive. (B) Embryos were treated in the same manner starting at 24 hfp, and RNA was harvested at 48 hpf. Under these conditions there was not a consistent significant inhibition of cardiomyocyte differentiation, based on levels of the same cardiac markers. These data are from one representative experiment, showing the mean relative transcript levels (normalized to the control) of triplicate qPCR assays, using 30 embryos per sample.</p

Compounds tested for activity altering normal zebrafish development.

<p>Note that compounds 10, 11, and 20 were found to generate similar phenotypes, while compound 10 (red) was chosen for further study, since it had highest activity based on a dose response study. For simplicity, highly related compounds are grouped on the right.</p

BT10 added at 24 hpf causes a defect in heart tube looping, similar to excess RA.

<p>Shown are representative embryos (n = 30) containing a <i>cmlc2:gfp</i> transgene were cultured in vehicle alone (control) or in the presence of ATRA or BT10 at the concentrations indicated, and as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010004#pone-0010004-g003" target="_blank">Fig. 3B</a>, beginning at 24 hpf. They were then photographed at 48 hpf in both brightfield and fluorescence, and the images overlayed to document the morphology of the heart tube (green). In control embryos the ventricle (V) is fully looped relative to the atrium (A), indicated by the dotted white line. In contrast, embryos cultured in the presence of ATRA or BT10 develop a relatively linear heart tube (at “low” or “high” concentrations), which is the major obvious morphological abnormality.</p

BT10 is selective for RAR receptors even at super-pharmacologic concentrations.

<p>Since BT10 has lower apparent affinity to all receptors compared to ATRA, the activity of BT10 was tested at 10 and 100 µM with each RXR isoform. Even at the highest concentration, there is no activation of RXR-dependent reporters, in contrast to activation of the control RARα receptor.</p

BT10 causes specific cardiovascular defects when added at 24 hpf.

<p>Shown are representative embryos (from 3 experiments, for each sample n = 20) that were cultured between 24–60 hpf in the presence of DMSO alone (A, control) or BT10 (B). Note the defined morphological structure of the ventral yolk sac (arrow). A normal looping heart tube fails to develop in these embryos. Panels C and D show higher magnification views of representative control (C) and BT10-treated (D) embryos, respectively. The red block arrow indicates blood flowing through the normal control heart, which is not evident in the BT10-treated embryos. Views are lateral, anterior to the right, dorsal at the top.</p