In vitro and in vivo effects of 2,4 diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS: Context-specific modulation of SMN transcript levels

Abstract

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.

Hyperexcitability precedes motoneuron loss in the Smn2B/− mouse model of spinal muscular atrophy

“…1). We found 2B/Ϫ FVB/N mice used in this study showed no drift [median survival: 20 days, this study; 21 days, historical data; log-rank (Mantel-Cox), 2 ϭ 0.58, P ϭ 0.45] (Cherry et al 2017;Gogliotti et al 2013). Importantly, mice from the 2B/Ϫ line had a slower course of disease progression and a longer lifespan than other severe SMA mouse models (Fig.…”

“…A single nucleotide transition in SMN2 alters its splicing pattern such that only a small amount of full-length survival motor neuron (SMN) protein is produced (Lorson et al 1999). Thus SMA is caused by low levels, not the complete absence, of the essential SMN protein (Coovert et al 1997;Lefebvre et al 1997;Schrank et al 1997).…”

“…The exact cellular and molecular mechanisms of motoneuron degeneration mediated by SMN deficiency are still unclear (Ahmad et al 2016;Burghes and Beattie 2009). SMN is a ubiquitously expressed essential protein that is involved in assembly of spliceosomal machinery (Coovert et al 1997;Liu et al 1997;Meister et al 2002;Paushkin et al 2002;Schrank et al 1997). Accordingly, SMN deficiency is associated with extensive changes in pre-mRNA splicing patterns and the differential expression of transcripts involved in a variety of cellular processes (Bäumer et al 2009;Doktor et al 2017;Huo et al 2014;Jangi et al 2017;Maeda et al 2014;Murray et al 2010Murray et al , 2015Ng et al 2015;O'Hern et al 2017;Saal et al 2014;Sareen et al 2012;Sison et al 2017;Staropoli et al 2015;Wertz et al 2016;Zhang et al 2008Zhang et al , 2013.…”

Design of Potent mRNA Decapping Scavenger Enzyme (DcpS) Inhibitors with Improved Physicochemical Properties To Investigate the Mechanism of Therapeutic Benefit in Spinal Muscular Atrophy (SMA)

“…These data and a comprehensive study of the biological effects of DcpS inhibitors in cells in vitro and in vivo are the subject of another report. 28 Design of Matched Inactive Analogs of DcpS Inhibition. Toward our ultimate goal of understanding if the efficacy observed in SMA mouse models by administering 1 is due to DcpS inhibition or due to the physicochemical or general structural characteristics of the chemotype, we were in need of structurally similar but DcpS inactive 2,4-diaminoquinazolines as tool compounds.…”

“…s., 1H), 1.76 (d, J = 12.05 Hz, 2H), 1.28−1.44 (m, 2H); 13 C NMR (101 MHz, DMSO-d 6 ) δ 162.5 (d, 1 J CF = 247.51 Hz), 162.1, 161.2, 156.9, 155.6, 143.0, 132.0 (d, 3 J CF = 5.03 Hz), 129.4 (d, 3 J CF = 8.32 Hz), 125.2 (d, 2 J CF = 14.77 Hz), 124.6 (d, 4 J CF = 3.5 Hz),, 117.2, 115.5 (d, 2 J CF = 23.96 Hz), 103.8, 99.7, 73.5, 55. 3 3-((2,4-Diamino-7-methylquinazolin-5-yl)methoxy)-5-fluorobenzonitrile (28). To a solution of 55 (1.47 g, 5.171 mmol) in DMA (16 mL) was added guanidine carbonate (1.86 g, 10.3 mmol) at 10 °C.…”