Cherry Jonathan J, DiDonato Christine J, Androphy Elliot J, Calo Alessandro, Potter Kyle, Custer Sara K, Du Sarah, Foley Timothy L, Gopalsamy Ariamala, Reedich Emily J, Gordo Susana M, Gordon William, Hosea Natalie, Jones Lyn H, Krizay Daniel K, LaRosa Gregory, Li Hongxia, Mathur Sachin, Menard Carol A, Patel Paraj, Ramos-Zayas Rebeca, Rietz Anne, Rong Haojing, Zhang Baohong, Tones Michael A
Rare Disease Research Unit, Pfizer Worldwide Research and Development, Cambridge, Massachusetts, United States of America.
Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, United States of America.
PLoS One. 2017 Sep 25;12(9):e0185079. doi: 10.1371/journal.pone.0185079. eCollection 2017.
C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.
已开发出用于治疗脊髓性肌萎缩症（SMA）的C5取代的2,4 - 二氨基喹唑啉去帽清除酶DcpS抑制剂（DAQ - DcpSi），SMA由存活运动神经元（SMN）蛋白的基因缺陷引起。据称这些化合物可作为SMN2转录激活剂，但支持该说法的数据并不明确。此外，尚不清楚所声称的对SMN2的作用是DcpS抑制剂的直接结果，还是可能是溶酶体趋向性的结果，已知溶酶体趋向性具有神经保护作用。利用DcpS敲低和7 - 甲基类似物作为DcpS与非DcpS介导作用的探针，在体外细胞中对DAQ - DcpSi的作用进行了表征。我们还对Smn转录水平进行了分析，对转录组和SMN蛋白进行了RNA测序分析，以确定治疗效果背后受影响的途径，并研究了溶酶体趋向性和非溶酶体趋向性DAQ - DCpSi在2B / - SMA小鼠中的作用。用细胞处理导致SMN2 mRNA适度且短暂增加，而SMNΔ7无变化或减少，SMN1转录本或SMN蛋白无变化。对DAQ - DcpSi处理的N2a细胞进行RNA测序分析发现，广泛途径中约2000个基因的表达（上调和下调）有显著变化。用溶酶体趋向性和非溶酶体趋向性DAQ - DcpSi化合物处理2B / - SMA小鼠对疾病表型有相似影响，表明治疗作用机制不是溶酶体趋向性的结果。与体外研究结果形成鲜明对比的是，Smn转录本在组织中发生了显著变化，但脊髓中SMN蛋白水平没有增加。我们得出结论，DAQ - DcpSi在SMA小鼠中具有可重复的益处，在体外和体内具有广泛的生物学效应，但这些效应是复杂的、依赖于具体情况的，并非简单的SMN2转录激活的结果。
