Date Published: June 29, 2017
Publisher: Public Library of Science
Author(s): Cinsley Gentillon, Andrew J. Connell, Ryan W. Kirk, Matthew E. R. Butchbach, Ravindra N Singh.
C5-substituted 2,4-diaminoquinazolines (2,4-DAQs) ameliorate disease severity in SMA mice. It is uncertain, however, that these compounds increase SMN protein levels in vivo even though they were identified as activators of the SMN2 promoter. These compounds also regulate the expression of other transcripts in neuroblastoma cells. In this study, we investigate the mechanism by which the 2,4-DAQs regulate the expression of SMN2 as well as other targets. D156844, D158872, D157161 and D157495 (RG3039) increased SMN2 promoter-driven reporter gene activity by at least 3-fold in NSC-34 cells. These compounds, however, did not significantly increase SMN2 mRNA levels in type II SMA fibroblasts nor in NSC-34 cells, although there was a trend for these compounds increasing SMN protein in SMA fibroblasts. The number of SMN-containing gems was increased in SMA fibroblasts in response to 2,4-DAQ treatment in a dose-dependent manner. ATOH7 mRNA levels were significantly lower in type II SMA fibroblasts. 2,4-DAQs significantly increased ATOH7, DRNT1 and DRTN2 transcript levels in type II SMA fibroblasts and restored ATOH7 levels to those observed in healthy fibroblasts. These compounds also increase Atoh7 mRNA expression in NSC-34 cells. In conclusion, 2,4-DAQs regulate SMN2 by increasing protein levels and gem localization. They also increase ATOH7, DRNT1 and DRNT2 transcript levels. This study reveals that the protective effects of 2,4-DAQs in SMA may be independent of SMN2 gene regulation. These compounds could be used in concert with a proven SMN2 inducer to develop a multi-faceted approach to treating SMA.
Proximal spinal muscular atrophy (SMA) is an early-onset neurodegenerative disease characterized by the loss of α-motor neurons in the anterior horn of the spinal cord which leads to muscle weakness and atrophy [1;2]. SMA is an autosomal recessive disease that is a leading genetic cause of infant death worldwide with an incidence of 1 in ~10,000 births [3;4]. SMA can be classified into five clinical grades based on age of onset and the highest achieved motor milestone. Regardless of clinical grade, proximal SMA results from the loss or mutation of SMN1 (survival motor neuron 1) on chromosome 5q13 . SMN protein is involved the assembly of small nuclear ribonucleoprotein (snRNP) complexes required for splicing . SMN is also implicated in stabilizing messenger ribonucleoprotein (mRNP) complexes in axons . In addition to these well characterized functions, SMN is involved in many other intracellular processes .
SMN2 is an endogenous genetic modifier of SMA disease severity . Many SMA therapeutics discovery programs aim to increase the amount of SMN protein from SMN2 by exploring multiple mechanisms including increasing transcription from the SMN2 promoter, enhancing the inclusion of exon 7 in the SMN2 mRNA or stabilizing SMNΔ7 protein . Initially identified from an ultrahigh throughput drug screen , C5-substituted 2,4-DAQs activate SMN2 promoter activity and also increase SMN localization to subnuclear gems in SMA patient fibroblasts . D156844 and D157495 (RG3039) have been shown to improve motor neuron function and extend survival of SMA mice models [20–24]. In this study, we examined the effects of four 2,4-DAQs—D156844, D158872, D157161 and D157495—on different levels of SMN2 gene regulation. Our results show that these compounds increased reporter gene activity which is driven by a 3.4-kb fragment of the SMN2 promoter by least 3-fold in the motor neuron-like NSC-34 cell line, with D157495 being the most potent inducer. Surprisingly, these compounds had no detectable effects on SMN2 mRNA levels in type II SMA fibroblasts but they tended to increase SMN protein levels in these cells.