Synthesis and Biological Evaluation of Novel 2,4-Diaminoquinazoline Derivatives as SMN2 Promoter Activators for the Potential Treatment of Spinal Muscular Atrophy

deCODE Chemistry Inc, Woodridge, IL 60517, USA.
Journal of Medicinal Chemistry (Impact Factor: 5.45). 03/2008; 51(3):449-69. DOI: 10.1021/jm061475p
Source: PubMed


Proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder characterized by death of motor neurons in the spinal cord that is caused by deletion and/or mutation of the survival motor neuron gene ( SMN1). Adjacent to SMN1 are a variable number of copies of the SMN2 gene. The two genes essentially differ by a single nucleotide, which causes the majority of the RNA transcripts from SMN2 to lack exon 7. Although both SMN1 and SMN2 encode the same Smn protein amino acid sequence, the loss of SMN1 and incorrect splicing of SMN2 have the consequence that Smn protein levels are insufficient for the survival of motor neurons. The therapeutic goal of our medicinal chemistry effort was to identify small-molecule activators of the SMN2 promoter that, by up-regulating gene transcription, would produce greater quantities of full-length Smn protein. Our initial medicinal chemistry effort explored a series of C5 substituted benzyl ether based 2,4-diaminoquinazoline derivatives that were found to be potent activators of the SMN2 promoter; however, inhibition of DHFR was shown to be an off-target activity that was linked to ATP depletion. We used a structure-guided approach to overcome DHFR inhibition while retaining SMN2 promoter activation. A lead compound 11a was identified as having high potency (EC50 = 4 nM) and 2.3-fold induction of the SMN2 promoter. Compound 11a possessed desirable pharmaceutical properties, including excellent brain exposure and long brain half-life following oral dosing to mice. The piperidine compound 11a up-regulated expression of the mouse SMN gene in NSC-34 cells, a mouse motor neuron hybrid cell line. In type 1 SMA patient fibroblasts, compound 11a induced Smn in a dose-dependent manner when analyzed by immunoblotting and increased the number of intranuclear particles called gems. The compound restored gems numbers in type I SMA patient fibroblasts to levels near unaffected genetic carriers of SMA.

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    • "For example, experiments using gene therapy approaches to restore SMN1 expression have yielded impressive amelioration in neuromuscular dysfunction and large increases in the lifespan of mice with SMA [11-14]. Other approaches aimed at increasing the amount of SMN protein produced by the SMN2 gene by promoter activation or reduction of alternative splicing of SMN2 exon 7 have also shown therapeutic benefit in animal models [15-17]. As a result, there is a growing desire to undertake clinical trials in human patient cohorts in order to evaluate the potential benefits of these therapeutic approaches. "
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    ABSTRACT: Spinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations in the survival motor neuron 1 (SMN1) gene. Recent breakthroughs in preclinical research have highlighted several potential novel therapies for SMA, increasing the need for robust and sensitive clinical trial platforms for evaluating their effectiveness in human patient cohorts. Given that most clinical trials for SMA are likely to involve young children, there is a need for validated molecular biomarkers to assist with monitoring disease progression and establishing the effectiveness of therapies being tested. Proteomics technologies have recently been highlighted as a potentially powerful tool for such biomarker discovery. We utilized label-free proteomics to identify individual proteins in pathologically-affected skeletal muscle from SMA mice that report directly on disease status. Quantitative fluorescent western blotting was then used to assess whether protein biomarkers were robustly changed in muscle, skin and blood from another mouse model of SMA, as well as in a small cohort of human SMA patient muscle biopsies. By comparing the protein composition of skeletal muscle in SMA mice at a pre-symptomatic time-point with the muscle proteome at a late-symptomatic time-point we identified increased expression of both Calreticulin and GRP75/mortalin as robust indicators of disease progression in SMA mice. We report that these protein biomarkers were consistently modified in different mouse models of SMA, as well as across multiple skeletal muscles, and were also measurable in skin biopsies. Furthermore, Calreticulin and GRP75/mortalin were measureable in muscle biopsy samples from human SMA patients. We conclude that label-free proteomics technology provides a powerful platform for biomarker identification in SMA, revealing Calreticulin and GRP75/mortalin as peripherally accessible protein biomarkers capable of reporting on disease progression in samples of muscle and skin.
    Genome Medicine 10/2013; 5(10):95. DOI:10.1186/gm498 · 5.34 Impact Factor
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    • "An ultrahigh-throughput screen of compounds that induce SMN2 promoter activity in motor neuron-like cells identified 2,4-diaminoquinazoline as a chemical scaffold on which derivatives with stronger potency and favorable pharmacological properties can be synthesized ( Jarecki et al., 2005). One such derivative, D156844, increases SMN protein expression in cultured SMA fibroblasts as well as in—albeit modestly—spinal cord extracts from SMND7 SMA mice (Thurmond et al., 2008; Butchbach et al., 2010). Oral administration of this compound significantly increases (by ~21–30%) the mean life span and motor function of SMND7 SMA mice when given before motor neuron loss (Butchbach et al., 2010). "

    Human gene therapy 02/2011; 22(2):121-5. DOI:10.1089/hum.2011.903 · 3.76 Impact Factor
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    ABSTRACT: Spinal muscular atrophy (SMA), the leading genetic cause of infant death results from loss of spinal motor neurons causing atrophy of skeletal muscle. SMA is caused by loss of the Survival Motor Neuron 1 (SMN1) gene, however, an identically coding gene called SMN2 is retained, but is alternatively spliced to produce approximately 90% truncated protein. Most SMA translational and preclinical drug development has relied on the use of SMA mice to determine changes in SMN protein levels. However, the SMA mouse models are relatively severe and analysis of SMN-inducing compounds is confounded by the early mortality of these animals. An antibody that could detect SMN protein on a Smn background could circumvent this limitation and allow unaffected, heterozygous animals to be examined. Here we describe the generation and characterization of a monoclonal anti-SMN antibody, 4F11, which specifically recognizes human SMN protein. 4F11 detects SMN (human) but not native Smn (mouse) protein in SMN2 transgenic mice and in SMA cell lines. We demonstrate the feasibility of using 4F11 to detect changes in SMN2-derived SMN protein in SMA patient fibroblasts and in healthy SMN2 transgenic mice. This antibody is, therefore, an excellent tool for examining SMN2-inducing therapeutics in patient cells as well as in transgenic mice.
    Journal of Neuroscience Methods 09/2008; 175(1):36-43. DOI:10.1016/j.jneumeth.2008.07.024 · 2.05 Impact Factor
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