Towards the drug: tetracycline

Tetracycline derivatives to correct SMN2 splicing

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. 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 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.

The defective splicing

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. Therefore the SMN2 gene is an ideal target for drug intervention to induce the synthesis of normal SMN protein in SMA patients.
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.

The development of the drug

With the support of Families of SMA and in collaboration with Professor Adrian Krainer’s team at Cold Spring Harbor Laboratory in NY for in-vitro screening, several TC derivatives from Paratek’s compound library were screened. Among several “hit compounds”, PTK-SMA1 emerged as the most promising. When incubated in nuclear extracts from HeLa cells, PTK-SMA1 showed an increase in the percentage of exon 7 inclusion during mRNA splicing of SMN2 by 2.6-fold.
The same screening compounds were evaluated in collaboration with Professor Arthur Burghes at Ohio State University 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 of 8.3-fold increase. 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.
While further in vivo evaluation of PTKSMA1 is currently underway in an animal model of SMA with adult heterozygote transgenic mice expressing the human SMN2 gene, Paratek’s medicinal chemists were able to synthesize more than 30 derivatives, 13 of which showed activities similar to PTK-SMA1’s in the exon 7 splicing assay.
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. In addition to demonstrating sufficient increase of SMN protein levels in an in vivo model of SMA, Paratek’s objectives also include the optimization of the pharmacokinetic profile and of all other drug-like properties of the selected lead compound in order to develop a small molecule candidate for the treatment of SMA within the next few years..

(source: FSMA website)