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

<div><p>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 <i>SMN2</i> transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on <i>SMN2</i> 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 <i>in vitro</i> utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of <i>Smn</i> 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 <i>SMN2</i> mRNA increases with either no change or a decrease in <i>SMNΔ7</i> and no change in <i>SMN1</i> 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 <i>in vitro</i>, <i>Smn</i> 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 <i>in vitro</i> and <i>in vivo</i>, but these are complex, context specific, and not the result of simple <i>SMN2</i> transcriptional activation.</p></div

Smn protein levels in spinal cord and muscle tissue from 2B/- SMA mice or 2B/+ healthy littermates treated with either vehicle, RG3039, or PF-06738066.

<p>Tissues were collected as described in Materials and Methods. Data is expressed as mean s.e.m. and statistical significance was evaluated using One-way ANOVA and Dunnett's multiple comparisons test. Tukey post-hoc tests were utilized to determine significance among the groups. <i>P</i><0.05 (*), <i>P</i><0.01(**), <i>P</i><0.001(***), <i>P</i><0.0001(****).</p

Effect of active DAQ-DcpSi and their 7-methyl analogues on <i>PAQR8</i> and <i>DPM3</i> transcripts.

<p>SMA-derived lymphoblasts were treated with active DAQ-DcpSi (RG3039, PF-06738066) or their 7-methyl analogues (PF-06802336 and PF-06832344 respectively) and the levels of <i>PAQR8</i> and <i>DPM3</i> transcripts measured using ddPCR. All data shown as mean ± s.e.m. and where not shown error bars are within the size of the symbols. Significant P-values were denoted as <i>P</i><0.05 (*), <i>P</i><0.01(**), <i>P</i><0.001(***), <i>P</i><0.0001(****).</p

Effect of DAQ-DcpSi treatment on <i>Smn</i> transcript levels in tissues from 2B/- SMA mice and healthy 2B/+ littermate controls.

<p>Animals were dosed with either vehicle, RG3039 (6 mg/kg) or PF-06738066 (10 mg/kg) BID via intraperitoneal injection from P4-P16 and were sacrificed 12 hours following the last dose for collection of tissues. RNA was prepared and analyzed using ddPCR as described in materials and methods. All gene expression was normalized to <i>PSMD14</i> expression and expressed relative to that in vehicle-treated 2B/- mice. All data shown as mean ± s.e.m. Numbers of animals in each data set were: 2B/- Vehicle (22); 2B/- RG3039(9); 2B/- PF-06738066 (13); 2B/+ Vehicle (7); 2B/+ RG3039(9); 2B/+ PF-06738066 (10). Significance using Student’s <i>t</i>-test: <i>P</i><0.05 (*), <i>P</i><0.01(**), <i>P</i><0.001(***), <i>P</i><0.0001(****).</p

Effect of DAQ-DcpSi on the performance of 2B/- SMA mice in a combined 55° negative geotaxis/ climb test.

<p>Data presented represent combined sexes for RG3039 (<i>n</i> = 20); vehicle (<i>n</i> = 21), PF-06738066 (<i>n</i> = 21) and vehicle treated healthy 2B/+ littermates (<i>n</i> = 24). The percent of mice able to pass the 55° negative geotaxis and climb tests simultaneously is shown. At P16, while a predominant number of vehicle treated SMA were alive, PF-06738066 or RG3039 showed improvement over vehicle SMA mice that did not reach statistical significance (Fisher’s exact test, <i>P</i>-value = 0.0708; 43% power to detect a benefit amongst SMA groups).</p

Effect of RG3039 on cellular SMN protein levels determined by Western blot.

<p>A. Neural progenitor cells treated for 48 hours (example) and B. quantification of data from 4 independent experiments; C. Fibroblasts treated for 72 hours (example) and D. quantification of data from 3 independent experiments; E. Fibroblasts treated for 6 days (example); F. SMA lymphoblasts treated for 72 hours (example). DMSO final concentration for lymphoblast cells was 0.1% and 0.2% for neural progenitors and fibroblast cell lines. All aggregate data is expressed as mean ± s.e.m. and statistical significance vs. SMA DMSO (B.) or 3813 DMSO controls (D) was assessed using Student’s <i>t</i>-test: <i>P</i><0.05 (*), <i>P</i><0.01(**) or otherwise non-significant where not indicated.</p

Effect of DcpS knockdown on putative DcpS-sensitive genes in stable clones of lentivirus-infected HEK-293 cells.

<p>Western blot (A) and transcript (B) analysis of DcpS lentiviral HEK293T knockdown clones. C. and D., Transcripts of putative DcpS-sensitive genes. All numerical data are shown as mean ± s.e.m. and where not shown error bars are within the size of the symbols. Levels of statistical significance were noted as <i>P</i><0.05 (*), <i>P</i><0.01(**), <i>P</i><0.001(***), <i>P</i><0.0001(****).</p

Volcano plots of differentially regulated genes detected by RNA-Seq analysis of spinal cord tissues from 2B/- SMA mice or 2B/+ healthy littermates treated with RG3039 or vehicle.

<p>The table (E.) summarizes the number of significantly (<i>P</i><0.05) differentially-regulated genes irrespective of the fold change for the various comparisons and as a percentage of the total numbers of genes detectable. Upregulated is defined such that the first condition in the comparison caused upregulation relative to the second e.g. 3,041 genes were upregulated by RG3039 treatment of SMA mice in comparison to vehicle-treated SMA mice. For all the comparisons the total number of detectable genes was between 20,514 and 20,517.</p

Effect of DAQ-DcpSi on luciferase activity driven by either the SMN2 promoter and SMN splicing cassette (filled triangles) or CMV promoter (open triangles).

<p>All data shown as mean ± range of duplicate determinations for a representative experiment of two performed which gave very similar results. Where not shown, error bars are within the size of the symbols.</p