Indoprofen Selectively Increases Luminescence from an SMN2-Minigene-Reporter Construct
We developed a high-throughput reporter assay designed to detect SMN production. We used C33a cells stably expressing an SMN
-minigene with luciferase reporter [13
] that consisted of exons 6 through 8 and intervening introns, for either SMN1
. Luciferase, the reporter gene’s product, is only produced when proper splicing and translation occurs.
We tested 20,000 compounds from a combinatorial library, 1040 compounds from a National Institute of Neurological Disorders and Stroke (NINDS) library, 2337 compounds from our Annotated Compound Library [14
], and 23,685 compounds from our TIC Library, which is a composite of compounds purchased from TimTec, IBS, and ChemBridge that were selected for specific properties, including stereochemical complexity [11
]. We discovered that indoprofen significantly increased reporter activity from SMN2
-luc cells relative to SMN1
-luc cells ( and ). The most effective concentration, 2.8 µg/ml (~10 µM, MW = 281.3), resulted in a 3-fold increase in luminescence. The raw data is available in the Supplemental Data
of our companion paper in this issue [11
] (available online at http://www.chembiol.com/cgi/content/full/11/11/1489/DC1/
Indoprofen and Related Compounds: Chemical Structures and Maximum Percent Luminescence Enhancement from SMN1and SMN2 Minigene-Reporter Constructs for Treated versus Untreated Cells
Indoprofen Increases Minigene-Reporter Luminescence, SMN Protein Level, and Number of Nuclear Gems
To find related active compounds, we tested those with structures similar to indoprofen, including NSAIDs, and found none that increased luminescence. Compounds with only the 2-phenylpropionic acid group or the isoindolinone group did not enhance luminescence. Methyl, ethyl, and isopropyl esters of indoprofen retained some degree of activity and selectivity, but this may have been due to simple hydrolysis in cells and generation of indoprofen ().
Despite the noteworthy increase in SMN2-minigene-reporter activity, real-time RT-PCR using SMA patient fibroblasts failed to show an increase in the ratio of full-length to truncated transcripts, or in the absolute level of transcripts following indoprofen treatment (data not shown). Considering indoprofen’s selective activity for SMN2-luc, it is unlikely that indoprofen is acting post-translationally, as both SMN1-luc and SMN2-luc encode the same protein. We propose that indoprofen has a pre- or cotranslational effect on protein production from SMN2, through a cyclooxygenase-independent mechanism. For example, it is possible that indoprofen, resembling a nucleotide, binds to the SMN2 pre-mRNA and displaces proteins that reduce the rate of translation. We sought evidence as to whether indoprofen also affects the level of endogenous SMN protein in human cells.
Indoprofen Increases SMN Protein Level in Human Type I SMA Patient Fibroblasts
To assess whether indoprofen treatment affected SMN protein production, we treated type I SMA patient fibroblasts (3813) with indoprofen and assessed protein level by Western blotting (). We treated 3813 cells with 5 and 20 µM indoprofen for 3 days with daily media and compound changes, even though liquid chromatography-mass spectroscopy (LC-MS) revealed that there was no significant degradation of indoprofen over 4 days in cell culture medium (data not shown). Both concentrations of indoprofen treatments yielded similar effects on SMN protein increase, although occasionally higher levels were observed from either treatment concentration (). Combining data from both treatments, indoprofen-treated cells resulted in a mean 13% increase in SMN protein production versus untreated cells (independent, one-tailed t test; n = 17 [9 treated samples, 8 control samples], ν = 15, p < 0.014).
Indoprofen Increases Number of Nuclear Gems in Human Type I SMA Patient Fibroblasts
The increase in SMN protein production led us to inquire about the effect of indoprofen on the overall number of gems—punctate structures in the nucleus. The number of gems (short for “Gemini of coiled [Cajal] bodies”) directly correlates with SMN protein production [5
], and they are found in many adult cell types (especially neurons) and in all fetal tissues. Fibroblasts from normal patients, SMA carriers, and type I SMA patients have ~80 gems, ~40 gems, and ~1 to 2 gems per 100 nuclei, respectively [5
We treated human type I SMA fibroblasts (2806) with indoprofen (5 and 15 µM) to observe changes in gem count. Both indoprofen treatments yielded a significant increase in gem count (5 µM: independent, one-tailed t test; n = 17 [8 treated, 9 untreated], ν = 15, p = 6.9 × 10−4
and 15 µM: independent, one-tailed t test; n = 16 [7 treated, 9 untreated], ν = 4, p = 1.7 × 10−4
) (). Pooling the two treatments also generated a significant result (independent, one-tailed t test; n = 24 [15 treated, 9 untreated], ν = 22, p = 3.2 × 10−7
]. Additionally, we tested one other type I SMA fibroblast cell line (3813) and one mouse fibroblast cell line (25). These cell lines yielded comparable results to 2806 (see Supplemental Data
available online at http://www.chembiol.com/cgi/content/full/11/11/1489/DC1/
Effect of Indoprofen in a Mouse Model of SMA
In an attempt to find a maximum effective dose, the pharmacokinetics of indoprofen in mice were studied. Pregnant mice were treated with a single dose of 20 mg/kg of indoprofen, using either intraperitoneal (IP) injection or oral gavage, to determine how much indoprofen enters the brain, blood, and embryo after treatment. Consistent with previous rodent studies using 14
C-labeled indoprofen [15
], liquid chromatography-mass spectrometry (LC-MS) revealed that no indoprofen was present in brain tissue 4 hr after treatment (data not shown). Indoprofen was found in the both the plasma of the pregnant mice and their embryos one hour after treatment. Both routes of administration resulted in ~5.3 µM indoprofen plasma concentration, while the IP method was shown to be more effective than gavage at delivering indoprofen to embryonic tissue. An average concentration of ~3.0 µM indoprofen was found in embryos of IP-treated mice at embryonic day 13 (E13).
We tested the effect of indoprofen on the viability of SMA model mice, which lack murine Smn
but contain a human SMN2
]. To generate litters that contained 25% Smn−/−
mice, we mated Smn+/−
mice with Smn+/−
mice. Our SMA model mice were derived from the published transgenic model created in C57BL/6 and crossed with FVB (Taconic Labs) [16
]. These mice were backcrossed once with a wild-type FVB mouse to generate our C57BL/6/FVB mice. We found that in this strain of Smn−/−
, embryos die at approximately embryonic day 11 (E11).
To determine whether indoprofen could increase the viability of these SMA model embryos, we treated such pregnant mice twice daily for the first 14 days of pregnancy by IP injection with 5 mg/kg indoprofen in phosphate buffered saline, the maximum dose that exhibited no toxicity within the 14 days. On embryonic day 14 (E14), we genotyped the embryos and ascertained the number of Smn−/−; TgSMN2+/− (SMA genotype) embryos (). At E14, none of the 7 untreated litters harbored any SMA genotype embryos, whereas 3 of 7 indoprofen-treated litters harbored SMA genotype embryos (Fisher Exact test, ν = 1, p = 0.096). Thus, there was a trend in which indoprofen increased the viability of SMA model mice. As expected, indoprofen treatment significantly increased the mean litter size from 5.7 embryos to 6.9 embryos (independent, one-tailed t test; n = 14, ν = 12, p = 0.040). There was also a trend in which indoprofen increased the number of SMA embryos (Fisher Exact test, ν = 1, p = 0.073).
Numbers of Non-SMA and SMA Litters and Embryos from Untreated and Indoprofen-Treated Mother Mice