SMA is caused by loss of the SMN1 gene, which results in reduced cellular concentration of full-length SMN protein, expressed from the paralogous gene SMN2. One promising strategy for SMA therapy is to increase SMN protein abundance or function in SMA patients. A logical approach to achieve this goal is to improve the efficiency of splicing of exon 7 from SMN2 and thereby increase full-length SMN2 mRNA and protein concentration expressed from the gene. To this end, we performed a directed screening of small-molecule compounds for their ability to improve SMN2 exon 7 splicing in a cell-free splicing assay. We identified a novel small-molecule compound, PTK-SMA1, which increases SMN2 exon 7 splicing and SMN protein concentrations in vitro and in vivo. To our knowledge, this compound is the only molecule identified to date that has been demonstrated to alter splicing by directly targeting the splicing reaction to promote a specific splicing pathway.
The degree to which SMN2
mRNA and SMN protein concentrations must increase to achieve a therapeutically valuable effect is not clear. SMA carriers who only have one functional copy of SMN1
are asymptomatic. Individuals with homozygous deletions of SMN1
but multiple copies of SMN2
have complete protection from the disease or are less severely affected (8
). These studies suggest that increasing SMN2
expression by a factor of 2 could be clinically beneficial. In the cell-free assay, PTK-SMA1 improved splicing of exon 7 in SMN2
pre-mRNA to a concentration exceeding that seen with SMN1
pre-mRNA, suggesting that the molecule has the potential to completely rescue the splicing defect in SMN2
. PTK-SMA1 administration to type III SMA mice resulted in a 50% increase in SMN2
exon 7 inclusion in the liver, which translated into a nearly fivefold increase in SMN protein concentrations compared to untreated animals. The fact that a modest increase in SMN2
exon 7 splicing results in a dramatic increase in SMN protein concentrations suggests additional levels of regulation in SMN gene expression and highlights the value of targeting the splicing reaction for SMA therapy. Although these results indicate that PTK-SMA1 is a promising SMA therapeutic candidate, the activity of this molecule in motor neurons must still be determined.
PTK-SMA1 is a tetracycline derivative that has been modified at the C7 position of the tetracycline backbone. Tetracyclines are anthracycline-type antibiotics that inhibit binding of bacterial transfer RNA to ribosomes (38
). These molecules are safe, well-characterized, and commonly used antibacterial drugs in humans. PTK-SMA1 has reduced antibacterial activity and phototoxicity relative to other tetracycline-derived molecules, such as doxycycline and minocycline, and these characteristics may improve its tolerability in humans.
The fact that PTK-SMA1 promotes SMN2
exon 7 splicing in our cell-free splicing assay indicates that the compound is targeting the splicing reaction directly to improve exon 7 inclusion. Tetracyclines in general can bind RNA and affect RNA structure, synthesis, and stability (19
). Tetracyclines are also able to bind and activate proteins, such as the tetracycline repressor (39
). The mechanism by which PTK-SMA1 stimulates SMN2
exon 7 splicing could be by direct binding to the SMN2
pre-mRNA or an RNA component of the splicing machinery (such as a U snRNA) or it could bind to a protein involved in splicing. It is possible that PTK-SMA1 could alter structure or activity in either of these scenarios. It is also possible that the tetracycline backbone binds to RNA nonspecifically and the PTK-SMA1–specific side chain contributes binding specificity or a protein interaction that alters splicing. Support for this idea comes from studies that have shown that tetracycline and other antibiotics can inhibit splicing at very high (>100 μM) concentrations (40
). Indeed, PTK-SMA1 appears to function in such a way as to stimulate a particular splicing reaction in a specific manner at lower concentrations and to nonspecifically inhibit splicing at higher concentrations.
One speculative idea for the mechanism of action of PTK-SMA1 is that it is fortuitously acting as part of a riboswitch to control exon 7 splicing. Riboswitches are structured RNA sequences within a tran script that change structure after the binding of a regulatory molecule. This change in structure interferes with gene expression, typically by altering transcription, translation, or RNA processing. Natural riboswitches have not yet been identified in mammalian cells. However, they are a common mechanism of gene control in bacteria and have been identified in fungi and plants (41
). It is intriguing that the thiamin pyrophosphate–responsive riboswitch that has been identified in eukaryotes controls splicing and alternative 3′-end processing of mRNAs (43
). This finding indicates that natural riboswitches can regulate RNA splicing. Synthetic riboswitches that are recognized by tetracycline have been engineered in yeast to regulate pre-mRNA splicing (44
), demonstrating the ability of the tetracycline compound class to alter splicing through recognition of a specific RNA structure. Indeed, secondary structure is an important splicing-regulatory feature of the SMN1
RNA transcript (45
The structure of PTK-SMA1 is critical for splicing activity. Aclarubicin, the tetracycline derivative that has activity in cells and provided the impetus for investigating the tetracycline scaffold as a platform for screening SMN2
exon 7 activators, did not improve splicing of exon 7 in the cell-free splicing assay. Aclarubicin is most commonly used as a chemotherapeutic agent for cancer therapy and is extremely cytotoxic (46
). It is possible that the effect of aclarubicin on SMN2
splicing and SMN protein concentrations in cells (17
) is a secondary effect resulting from global changes in the cell. Nonetheless, when modified with particular side chains, such as that on PTK-SMA1, the tetracycline scaffold forms the basis of a potent activator of SMN2
exon 7 splicing.
exon 7 splicing is controlled by a number of cis-acting sequence elements and trans-acting factors. The C residue at position 6 of SMN1
exon 7 is part of an ESE element that is recognized by the serine-arginine-rich (SR) protein SF2/ASF (21
). This ESE is critical for recognition and splicing of exon 7. In SMN2
pre-mRNA, position 6 of exon 7 is a U rather than aC. The U changes the ESE such that it is not effectively recognized by SF2/ASF and exon 7 is skipped most of the time. In the absence of the ESE, inhibitory interactions between splicing silencers and heterogeneous nuclear RNP A1 predominate and result in exon skipping (21
). Although the single-nucleotide difference between SMN1
is sufficient to prevent efficient SMN2
exon 7 splicing, other cis-acting motifs are involved in splicing of exon 7, including an ESE that is recognized by the Tra2 family of SR-like proteins (6
). Additional cis-acting elements within the flanking introns and in exon 7 are also important for exon 7 inclusion. Some of these elements are recognized by trans-acting splicing factors and others appear to be regulatory secondary structures (52
). Binding of splicing factors to the 3′ splice site is also impaired in SMN2
relative to SMN1
). Any of the splicing factors or sequence elements that control exon 7 splicing could be a potential target for PTK-SMA1.
A number of molecules have been identified that modulate alternative splicing in general (12
), and some have also been identified that increase the concentration of full-length SMN2
mRNA in cells (18
). However, there is no evidence that these act directly on splicing as opposed to, for example, mRNA stability. We have tested a number of these compounds in the cell-free splicing assay and found that none of them altered splicing of SMN2
exon 7 (). These molecules may influence splicing by modulating transcription, nuclear transport, stability, signaling pathways, or some other cellular process that can affect splicing indirectly. Although kinetin improves exon inclusion of a number of transcripts (25
), indicating that the compound may have a general effect on the splicing reaction, it did not significantly affect SMN2
exon 7 splicing in our assay (). Several small molecules that act specifically on components of the splicing machinery have been described. To date, these compounds have all been demonstrated to be inhibitors of splicing (57
). PTK-SMA1 appears to be unique in its ability to influence splicing directly and promote a specific splicing event.
PTK-SMA1 improves exon 7 splicing in two ways: by stimulating splicing of the intron upstream of exon 7 and by inhibiting splicing from exon 6 to exon 8 (exon 7 skipping) (). It is possible that both of these activities are mediated by a single protein or sequence element. ESEs, for example, which typically function to promote splicing of nearby splice sites, also block skipping of the exon in which they are located (62
Although the precise molecular target of PTK-SMA1 is not yet known, our screening assay has narrowed the target down to the splicing reaction itself. Discovering the mechanism of action of PTK-SMA1 in splicing is an important goal toward its development as a therapeutic and would also increase our understanding of how splicing can be targeted by small molecules. Because many diseases are caused by defects in RNA splicing (63
), the therapeutic impact of this drug class could be significant. Tetracycline may be a chemical scaffold that can be manipulated to generate agents for context-specific repair of aberrant splicing. Of more immediate impact, PTK-SMA1 and its derivatives are promising therapeutic candidates for the treatment of SMA.
A major goal for SMA therapy is the development of a drug that can penetrate the blood-brain barrier and increase SMN protein concentration in the motor neurons to stop or reverse disease progression. PTK-SMA1 does not cross the blood-brain barrier, and hence, we examined its activity in the liver of transgenic mice as a proof of principle for the compound's ability to promote SMN2 exon 7 splicing in vivo. Ongoing structure-activity relation studies are focused on modifying PTK-SMA1 to produce a molecule that efficiently crosses the blood-brain barrier and improves SMN protein expression in the central nervous system.