Summary /

ML200 and selected analogs (with potency below 150 nM in the reporter assay) were examined to evaluate the effect on the human SMN protein expression using fibroblasts from SMA patients. A fibroblast cell line from a type I SMA patient (3813 cell line) was incubated with different doses of  the probe or its analogs and assessed the SMN protein level by quantitative western blotting (Figure 1). Resutls showed that the probe (8m) at concentration of 37 nanomolar increased the SMN protein level by 2-3 fold. The SMN protein level was decreased with the increasing of the drug concentration. This result matched with of bell-shaped curve observed in the luciferase reporter assay. Analog 9a (CID 46907666, NCGC00187872)  showed a dose-dependent trend between 37 nM to 1 uM concentration and a decrease of protein level at 3 uM. Other selected agents 9c (CID 46907686, NCGC00187875), 8c (CID 46907733, NCGC00187892) and 8l  (CID 46907708, NCGC00187894) all showed an up-regulation of SMN protein at concentrations ranging between 37 nM and 333 nM.

Figure 1. Quantification of western blot of SMN levels after treatment with drug compounds as indicated with different doses.

Summary /

Increase in SMN protein production led us to explore whether ML200 and its analogs had effect of on the overall number of SMN positive foci or gems in the nucleus. This would corroborate that the increased protein amounts represent indeed functional SMN protein. SMN protein is predominately a cytoplasmic protein in the nucleous SMN that localized in distinct punctuate bodies often referred to as gems (gemini of coiled Cajal bodies). There is a direct correlation between the number of gems and total SMN protein production in the cell. Gem counts are commonly used as another metric to score the amount total SMN protein expressed on a cell to cell basis. The number of nuclei with gems and the number of gems per cell were both significantly reduced in type I SMA cells (Coovert, 1997). Only 3.3% of nuclei have gems in fibroblast cells from SMA type I patients (cell line 3813), while 24.8% of nuclei have gems in fibroblast from a carrier parent (3814 cell line). We treated human type I SMA fibroblasts with increasing doses (37 nM – 3000 nM) of arylpiperidine analogs 8a, ML200 (8m), and 9a for 3 days and the number of gems per 100 nuclei were examined (Figure 2). The probe compound treatment yielded more than 2 fold of increase of the number of the gems at low 37 nM doses. With the concentration increased, the numbers of gems was reduced which agreed with the previous findings. Analog 9a also showed an 80% increase of the gem numbers at 37 nM to 1000 nM concentrations and slightly decreased at 3000 nM. However, analog 8a didn’t show any activity in this assay.

Figure 2. Number of gems per 100 nuclei after treatment with drug compounds as indicated with different doses.

Summary /

As initial studies to have a better understanding of the mode of action, we decided to use RT-PCR to measure RNA expression levels and exon 7 inclusion within our reporter cell line. Some analogs showed a small increase in total SMN-luciferase transcripts but there was almost no increase in exon 7 mRNA. Although the mechanism of SMN protein induction by this class of arylpiperidine analogs was still unclear, this result indicated that SMN activity for this series might be post-transcriptional, potentially stabilizing SMN protein and somehow reducing its degradation.