Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN, called SMN1 and SMN2. In the SMA disease state, various mutations in the SMN1 locus render that protein nonfunctional and induce the disease state. A therapy to either increase the amount of SMN2 product made, or to increase the inclusion of exon 7, has been proposed for the treatment of SMA. Previous probes have worked through non-specific mechanisms of activation (HDAC inhibition, RNA decapping), and effect an increase in protein abundance in both SMN2 and SMN1 reporter cell lines. The present probe ML104 (CID-6404603) appears to operate through a unique mechanism of action, showing an increase in SMN2 protein production in fibroblast line derived from an SMA patient, without having an effect on SMN1 protein production in the analogous cell line. The compound may alter splicing of SMN2 protein, but it has not been evaluated for use in vivo, and mode of action studies are ongoing.

Probe Structure & Characteristics

Recommendations for the scientific use of this probe

This probe should be useful for modulating the expression of SMN2 protein in cell-based models of SMN protein expression. This probe has not been evaluated for use in vivo, though it has been shown to increase SMN2 protein production in fibroblasts derived from SMA patients. Compound may alter splicing of SMN2 protein, but mode of action studies are ongoing. The goal of the project was to identify a compound 10-fold selective for SMN2 versus SMN1 expression in reporter-based assays which increase SMN2 protein abundance in primary SMA derived fibroblasts, or immortalized SMA derived fibroblasts. CID 6404603 satisfies these criteria, increasing protein expression in fibroblasts derived from SMA patients at a compound concentration of 1 μM, and was the most potent member of the series in the primary screening assay.

1. Scientific Rationale for Project

Spinal muscular atrophy (SMA) is caused by insufficient levels of the survival motor neuron protein SMN. The SMN locus on chromosome 5q13 contains two inverted copies of SMN, called SMN1 and SMN2, which are 99% identical at the amino acid level. At the splicing level, SMN1 mainly produces one splice variant (90%) containing exons 1–8; this variant is identified as SMN protein, which is the fully functional protein. Protein from SMN2 expression excludes exon 7 90% of the time. Skipping of exon 7 produces non-functional SMN protein product. In the SMA disease state, various mutations in the SMN1 locus render that protein nonfunctional and induce the disease state. The 10% of SMN2 translation that yields SMN protein is not sufficient to overcome the deficiency produced by the loss of the SMN1 expression product. A therapy to either increase the amount of SMN2 product made, or to increase the inclusion of exon 7, has been proposed for the treatment of SMA.

We have designed a luciferase reporter gene assay by combining the promoter and splicing-based cassettes in tandem with the major portion of the native SMN2 cDNA. A screen of this assay with a compound library can identify compounds that increase SMN2 levels by three mechanisms: modulating alternative splicing of SMN exon 7, increasing transcription from the SMN2 promoter, or stabilizing the SMN protein. Earlier screening assays for SMA only targeted compounds that either stimulate the SMN2 promoter or decrease exon 7 skipping.

2. Project Description

208,516 compounds were screened in the primary assay during the course of this screen. Some active compounds were chosen for confirmation according to the scheme shown in figure 1.

Figure 1

Overview of screening process.

a. Information for each Assay Implemented and Screening Run

i. qHTS Assay for modulators of SMN2 splicing [AID-1458; Primary DR]

Assay Description

Cells stably expressing the luciferase reporter gene fused to exon 8 were used as the primary screening cell line (figure 2). When exon 7 is included in the mRNA transcript, full length protein (fused to luciferase) is generated. Compounds that either increase the inclusion of exon 7 or increase the overall level of transcript produced will generate increased levels of luciferase. The assay measures the luminescent signal produced by luciferase, and compounds with increases in luminescent signaling will be flagged as potential actives. The luciferase enzyme requires ATP, luciferin, and oxygen as substrates for enzymatic activity. All of the required components are present in Promega’s One Glo Luicferase detection kit. The luminescent signal is read on the Viewlux CCD camera-based plate reader, which has a very sensitive, cooled CCD camera that captures data from the entire plate in one read. The reagents required for the cell propagation and the assay are listed in table 2.

Figure 2

Schematic of SMN2 reporter cell line.

Table 2

Reagents and resources for the SMN2 Luciferase screen.

Assay Protocol

The protocol is presented in table 3. Freshly trypsinized cells were resuspended in phenol red free DMEM containing 10% fetal bovine serum, 1x penicillin/streptomycin, and 1x sodium pyruvate at a density of 2000 cells/well in 5ul of media. The assay plates used were white solid bottom TC-treated 1536 well plates. Cells were incubated at 37 C for 10 to 12 hours to allow recovery. After incubation, cells were treated with control (sodium butyrate) or library compounds. Cells were then incubated at 37 C for 30 to 36 hours prior to being assayed. Luciferase activity was detected by adding 3 ul of OneGlo detection reagent, incubating for 5 minutes at room temperature and measuring luminescent signal in the Viewlux (using a 60 second integration time, high speed capture with 2x pixel binning).

Table 3

Assay protocol for the SMN2 luciferase cell line in 1536 well format.

Summary of Results

NCGC tested 1229 1536-well plates in the primary screen. The average Z’ for the screen was 0.47 +/− 0.12, excluding 8 plates which failed visual QC. Results are reported for 210,836 samples. 6,128 compounds gave significant concentration-responses in the primary screen. Compounds were deprioritized for confirmation if they were suspected to interfere with the readout of the assay platform, or cellular viability using internal NCGC SAR data from other related assays.

ii. Confirmation Concentration-Response Assay for SMN2 activators [AID-1740; Confirmatory DR]

Assay Description

To confirm activity in the original assay, select samples active in the primary screen were obtained in DMSO solution from the MLSMR and/or as powders from compound vendors.

Assay Protocol

The assay protocol and reagents are identical to: qHTS Assay for Enhancers of SMN2 Splice Variant Expression [AID-1458; Primary]

Summary of Results

Prioritized compounds had a high confirmation rate, but the most potent compounds from the MLSMR were only in the micromolar range using the HTRF assay format.

iii. Counterscreen in SMN1 cell line for SMN1 activators. [AID-1739; Confirmatory DR]

Assay Description

The SMN1 splicing pattern typically includes exon 7. A cell line harboring the SMN1 gene fused to luciferase was used as a counterscreen. Follow-up compounds exhibiting increased signal in the SMN1 cell line were flagged as non specific activators (i.e. transcription or protein stability).

Assay Protocol

The assay protocol and reagents are identical to: qHTS Assay for Enhancers of SMN2 Splice Variant Expression [AID-1458; Confirmatory DR]

Summary of Results

About half of the compounds from the MLSMR inhibited SMN1 protein production in the micromolar range using the HTRF assay format.

iv. Counterscreen of active compounds against purified luciferase enzyme [AID-1733; Confirmatory DR]

Assay Description

Luciferase inhibitors have been shown to stabilize the structure (and also the half life) of the enzyme. Compounds which act as luciferase inhibitors could show up in the SMN2 assay as false positives due to this phenomenon. Purified luciferase enzyme was used in a counter-screen of the active compounds.

Assay Protocol

The assay protocol and reagents are listed in table 4 and 5, respectively. A white solid bottom 1536 well plate was used in the assay. The enzyme mixture consisted of 50 mM Tris-acetate pH 7.6, 10 mM Mg acetate 0.05% BSA, 0.01% Tween and 5 nM firefly luciferase. Each well received 2.5 ul of enzyme and 23 nl of follow-up compound, and was incubated for 5 minutes at room temperature. After incubation, 5 ul of detection reagent (OneGlo) was added to each well and luminescent enzyme activity was recorded on the ViewLux (using medium sensitivity, 1x pixel binning and 1 second of integration). The control compound used was AMP at 100 nM and dose responses thereof.

Table 4

Purified Luciferase Enzyme Assay Protocol.

Table 5

Reagents for purified luciferase counterscreen.

v. SMN2 protein expression in fibroblasts derived from an SMA patient

Assay Description

Ideally, compounds modulating SMN2 protein production in the luciferase reporter line would also modulate SMN2 protein production in fibroblasts derived from patients. Protein production is quantified via a Western blot.

Assay Protocol

Human fibroblast cell lines from a 3-year-old type I SMA patient (GM03813; Coriell Cell Repository) with 2 copies of the SMN2 gene were maintained as previously described (1). For drug treatments, cells were either plated in media alone or with TSA (BIOMOL International), which was dissolved in DMSO immediately before each use. The cells were then harvested for either RNA or protein isolation and quantification as previously described (1).

Summary of Results

The probe compound enhanced SMN2 protein production in fibroblast line derived from an SMA patient.

3. Probe

b. Probe chemical structure

g. Detailed synthetic pathway for making probe

There several published methods for the synthesis of 3,6-disubtitued-1,2,4-triazines and analogues (2–4).

Scheme 1 shows a general method for the synthesis of this probe. Thus, commercially available benzimidohydrazide and 2-oxo-2-phenylacetic acid were reacted in the presence of phosphorus pentachloride using toluene as solvent. The intermediate 5-chloro-3,6-diphenyl-1,2,4-triazine reacted with methanol in the present of a suitable base such us Butyl Lithium to yield 5-methoxy-3,6-diphenyl-1,2,4-triazine.

Scheme 1

Synthesis of 5-methoxy-3,6-diphenyl-1,2,4-triazine.

4. Appendices

a. Activity of probe and structurally-related compounds

MLS-000763654 is uniquely active within the set of compounds tested. See Table 6

Table 6

Activity of MLS-000763654 and related compounds in reporter assays of SMN2 and SMN1 expression.

b. Comparative data showing probe specificity for target

Previous probes have worked through non-specific mechanisms of activation (HDAC inhibition, RNA decapping), and effect an increase in protein abundance in both SMN2 and SMN1 reporter cell lines. The present probe appears to operate through a unique mechanism of action, showing an increase in SMN2 protein production without having an effect on SMN1 protein production in the analogous cell line.

Activity of the probe molecule in the luciferase assay was initially of concern (figure 3), but the probe molecule exhibits similar activity in a luciferase-free assay – the patient fibroblasts shown below (figure 4). In addition, it is worth noting the luciferase inhibition SAR also does not track with SMN2 activity in the reporter cell line.

Figure 3

Activity of the probe molecule in SMN2, SMN1 reporter cell lines and in an in vitro assay against purified luciferase enzyme.

Figure 4

SMN protein abundance in patient fibroblasts after incubation with probe molecule. Sodium butyrate was used as a positive control; it has been observed to increase SMN protein roughly 10-fold. The probe molecule effected an approximately 2-fold increase (more...)

Data for this project is available from PubChem as shown in Table 7.

Table 7

Summary of data in PubChem.

5. Bibliography

1.

Lunn MR, et al. Indoprofen upregulates the survival motor neuron protein through a cyclooxygenase-independent mechanism. Chem Biol. 2004;11(11):1489–1493. [PMC free article: PMC3160629] [PubMed: 15555999]

2.

Kozhevnikov DN, et al. Transformations of 1,2,4-Triazines in Reactions with Nucleophiles: V. SNH and ipso-Substitution in the Synthesis and Transformations of 5-Cyano-1,2,4-triazines. Russian Journal of Organic Chemistry (Translation of Zhurnal Organicheskoi Khimii). 2002;38(5):744–750.

3.

Neunhoeffer Hans, et al. The chemistry of 1,2,4-triazines. XIV. Synthesis and reactions of 5-chloro-1,2,4-triazines. Liebigs Annalen der Chemie. 1990;(7):631–40.

4.

Konno Shoetsu, et al. as-Triazine derivatives. VIII. Synthesis of 5-substituted 1,2,4-triazines. Heterocycles. 1987;26(12):3259–64.