Tetracycline Derivatives to Correct SMN2 Splicing

The most up-to-date status about the program can be found here in our recent Drug Disovery Compass.

Paratek Pharmaceuticals’ interest in developing a small molecule drug discovery program in the Spinal Muscular Atrophy (SMA) therapeutic area started back in 2002 when a portion of Paratek’s library of over 2,000 modified Tetracycline (TC) derivatives were screened in a SMA relevant in-vitro assay.

The initial concept of screening TC derivatives for SMA came from their structural similarity with Aclarubicin A (see Figure 1).  This chemotherapeutic drug was reported in 2001 to be active in cellular assays relevant to SMA:

• it enhanced the inclusion of exon 7 in the splicing of SMN2 pre-messenger RNA (pre-mRNA); and
• it restored normal SMN protein levels in an SMA patient-derived cell line.

However, Aclarubicin is toxic and not suitable for clinical development.  Paratek surmised that nontoxic TC derivatives could potentially increase full-length mRNA production and SMN protein synthesis from the SMN2 gene. The discovery of a nontoxic TC derivative would be an important finding leading to a potential treatment for SMA.

 
Figure 1.  Structural Comparison of Aclarubicin to Paratek's Tetracycline Derivatives.

This is because the SMN2 gene is an ideal target for drug intervention to induce the synthesis of normal SMN protein in SMA patients.  Therefore, therapies that specifically increase inclusion of exon 7 into the SMN2 mRNA are likely to be effective treatments for even severe SMA type I patients.

SMN2 fails to compensate for the SMN1 mutation, and thus to protect from development of SMA, because its mRNA undergoes alternative splicing to encode for an unstable SMN protein, known as Δ7SMN.  After transcription of a gene into its corresponding premessenger RNA (pre-mRNA), the nuclear splicing machinery is responsible for processing that pre-mRNA into its corresponding messenger RNA (mRNA) by eliminating introns (bits of pre-mRNA unnecessary for protein synthesis) and joining together exons (bits of pre-mRNA coding for protein synthesis). The aberrant production of the truncated Δ7SMN is due to exon 7 being skipped during splicing of the SMN2 pre-mRNA, resulting in insufficient amounts of full length SMN protein and consequently in decreased motor functions in SMA patients. See the figure below for an illustration of this process. 

In a collaboration involving Paratek, Adrian Kranier's lab at Cold Spring Harbor Laboratory, and MIchelle Hastings's lab at Rosalind Franklin Unversity, several TC derivatives from Paratek’s compound library were tested in an in vitro assay for SMN2 splicing. Among several active “hit compounds”, PTK-SMA1 emerged as the most promising.   PTK-SMA1 showed an increase in the percentage of exon 7 inclusion during mRNA splicing of SMN2 by 2.6-fold.  The same compounds were evaluated in a whole cell “Gems” assay which looks at the concentration of full length SMN protein in nuclear studies called “Gems.” PTK-SMA1 showed promising results in that assay with an increase of SMN protein levels in a Type I SMA patient fibroblast cell line. In addition, PTK-SMA1 again increased SMN protein levels in the same fibroblast patient cell line when visualized by Western Blot analysis instead of gems count. Giving PTKSMA1 to mice carrying the human SMN2 gene increases SMN protein levels in multiple tissues in animals. The project focus is now on a medicinal chemsitry effort to build blood brain barrier penetration into these compound classes, a characteristic critical to an SMA drug. 

Please click here to view poster on the project presented at the 2008 American Academy of Neurology Meeting.

Please click here to read a recent journal publication in Science Translational Medicine describing the early lead compound.

Paratek Pharmaceuticals, armed with its expertise and intellectual property coverage in chemical modifications of the Tetracycline class of molecules, is perfectly positioned to implement a drug discovery project for the treatment of SMA.

Using funding from FSMA to generate the preliminary data for a NIH grant application, Paratek has been awarded a multi-million dollar coorporative agreement from the NINDS to continue the research that FSMA funded for the past 3 years.  The overall goal for this project is to develop a drug candidate for SMA resulting in an Investigational New Drug (IND) application with the Food and Drug Administration (FDA) within 4 to 5 years.

Please click here to read the press release from Paratek and FSMA announcing the award of a multi-million dollar cooperative agreement from NINDS.

Figure 2.  Splicing of the SMN Genes.

The major difference between the two SMN gene copies is the C (SMN1) or T (SMN2) nucleotide change in exon 7 of the DNA comprising the two genes. Because of this difference, SMN2 mostly makes mRNA that excludes exon 7 and produces a smaller, unstable SMN protein, while SMN1 makes mRNA that includes exon 7 and makes stable full-length SMN protein. This is due to a defect in mRNA splicing caused by the T nucleotide change in the SMN2 gene.  This process is explained below.

(a) The SMN1 and SMN2 gene organization on chromosome 5. 

(b) The SMN genes are turned on by their respective promoters (non-blueprint regions of genes that work to turn genes on and off) in a process call transcription.  Turning a gene on (transcription) results in a preliminary RNA message which contains an intermediate blueprint from which specific proteins can eventually be produced.

(c)  The preliminary RNA message must then be processed in an event called RNA  splicing in order to become a useful blue print for protein production. The process of RNA splicing removes chunks of RNA from the preliminary message, which are not part of the protein blueprint. The non-blueprint regions that must be removed are called introns.  The blueprint regions are called exons.  The splicing process results in the final mRNA message which is the contiguous protein blueprint.  The final mRNA message that results is used as the template for protein production in a process called translation.