Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy

Abstract

Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of survival motor neuron (SMN) protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN lacking exon 7 (SMNDelta7) SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMNDelta7 SMA mice by approximately 21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMNDelta7 SMA mice.

Figures

Figure 1.

The chemical structures of the three 2,4-diaminoquinazoline derivatives (D152344, D153249 and D156844) that were used in these experiments. These compounds are labeled as 5b, (S)-7a and 11a, respectively, in Ref. (42).

Figure 2.

Drug bioavailability and mSmn promoter activities in the CNS of neonatal SMNΔ7 carrier mice treated with 2,4-diaminoquinazoline derivatives. (A–C) Dose-dependent serum (dashed line with triangles) and brain (solid line with circles) levels of D152344 (A), D153249 (B) and D156844 (C) in neonatal SMNΔ7 carrier mice. (D–F) mSmn promoter-dependent β-galactosidase activity in the forebrains and spinal cords of SMNΔ7 carrier mice receiving differing doses of D152344 (D), D153249 (E) and D156844 (F). The β-galactosidase activity at each dose was normalized to the activity of vehicle-treated (0 mg/kg/day) samples. The doses administered for each compound are listed under each bar (n = 4/treatment dose). Key: *P ≤ 0.05 when compared with 0 mg/kg/day-treated mice.

Figure 3.

Oral administration of D156844 alters SMN gene expression in the spinal cord of SMNΔ7 SMA mice. (A) Changes in human-specific, full-length SMN (FL-SMN) mRNA levels in the spinal cord of mice treated with D156844 or vehicle for 5 days (n = 3/treatment group). (B) Changes in human-specific, SMNΔ7 mRNA levels in the spinal cord of mice treated with D156844 or vehicle for 5 days (n = 3/treatment group). (C) Representative immunoblot showing SMN and β-actin protein expression in spinal cord extracts from SMNΔ7 SMA mice treated with either D156844 or vehicle for 5 days. (D) Quantitation of SMN protein levels relative to β-actin protein in D156844- or vehicle-treated spinal cord extracts (n = 3/treatment group). SMN protein levels are expressed relative to those observed in age-matched carrier mice. (E) snRNP assembly activity in the spinal cord of SMNΔ7 SMA mice treated with either D156844 or vehicle for 5 days (n = 3/treatment group). snRNP assembly activity is measured by the amount of 32P-labeled U1 snRNA precipitated by antibodies against snRNP proteins (Sm proteins) as a consequence of SMN-dependent Sm core formation taking place in vitro using spinal cord extracts (50,69). snRNP assembly activity is expressed relative to that observed in age-matched carrier mice.

Figure 4.

Oral administration of D156844 improves the survival of and delays the onset of loss of body mass in neonatal SMNΔ7 SMA mice. (A) Kaplan–Meier survival plot for SMNΔ7 SMA mice receiving either vehicle (light grey solid line) or D156844 (3 mg/kg/day) beginning at either PND04 (black dashed line) or PND09 (grey dotted line). (B) Kaplan–Meier onset of body mass loss plot for SMNΔ7 SMA mice receiving either vehicle (light grey solid line) or D156844 (3 mg/kg/day) beginning at either PND04 (black dashed line) or PND09 (grey dotted line). (C) Body mass curves for SMNΔ7 SMA mice receiving either vehicle (triangles) or D156844 (3 mg/kg/day; circles) beginning at PND04.

Figure 5.

Oral administration of D156844 improves the motor phenotype of SMNΔ7 SMA mice and reduces motor neuron loss in the lumbar spinal cord. (A) Righting reflex latencies at PND07 and PND11 for SMNΔ7 SMA mice treated with D156844 (3 mg/kg/day; dark grey bar) or vehicle (light grey bar) as compared with carrier (black bar) mice. (B) Vectorial movement latencies at PND07, PND11 and PND14 for SMNΔ7 SMA mice treated with D156844 or vehicle. (C) Spontaneous locomotor activity measured as the number of grids crossed in 1 min at PND07, PND11 and PND14 for SMNΔ7 SMA mice treated with D156844 or vehicle. (D) The number of pivots (90° turns) made in 1 min at PND07, PND11 and PND14 for SMNΔ7 SMA mice treated with D156844 or vehicle. For the phenotype data shown in (A–D), the sample sizes (n) for vehicle-, D156844-treated SMA mice and carrier mice at PND07 were 7, 5 and 5, respectively, at PND11, 5, 5 and 3, respectively and at PND14, 3, 5 and 3, respectively. Key for (A–D): *P < 0.05 when comparing vehicle-treated SMNΔ7 SMA mice to D156844-treated SMNΔ7 SMA mice. (E–G) Representative images of Cresyl violet-stained lumbar spinal cord sections from carrier mice (E) and SMNΔ7 SMA mice treated with vehicle (F) or D156844 (G) from PND04 until PND11. The dashed ovals represent the regions of interest in the ventral horn and arrow in (E) points to a motor neuron. Scale, 100 µm. (H) The number of motor neurons in the lumbar (L4-5) spinal cord of SMNΔ7 SMA mice (PND11) treated with D156844 or vehicle (n = 3/group). Key for (H): * P < 0.001 when comparing vehicle-treated SMNΔ7 SMA mice to D156844-treated SMNΔ7 SMA mice.

Figure 6.

CNS bioavailability and mSmn promoter activity in prenatal mice receiving D156844. (A) D156844 levels in prenatal mouse brains whose dams were dosed with differing amounts of D156844 (0–60 mg/kg/day; n = 3/group) beginning at ED11.5. (B) β-Galactosidase activity—a marker for mSmn promoter activity—in the brains of mice whose dams received different doses of D156844 (0–60 mg/kg/day; n = 3/group) beginning at ED11.5. The β-galactosidase activity at each dose was normalized to the activity of vehicle-treated (0 mg/kg/day) samples. Key: *P ≤ 0.05 when compared with 0 mg/kg/day-treated mice.

Figure 7.

Prenatal administration of D156844 improves the survival and phenotype of SMNΔ7 SMA mice. (A) Kaplan–Meier survival plot for SMNΔ7 SMA mice receiving either vehicle (light grey solid line) or D156844 (3 mg/kg/day) beginning ED11.5 and continuing after birth (black dashed line) or ending at birth (grey dotted line). (B) Kaplan–Meier onset of body mass loss plot for SMNΔ7 SMA mice receiving either vehicle (light grey solid line) or D156844 [beginning ED11.5 and continuing after birth (black dashed line) or ending at birth (grey dotted line)]. (C) Body mass curves for SMNΔ7 SMA mice receiving either vehicle (triangles) or D156844 (3 mg/kg/day; circles) beginning at ED11.5.

Figure 8.

Therapeutic window of opportunity for protective effects of SMN2 induction by D156844.