172 Discovery of small molecule compounds with readthrough potency at premature termination codon

Abstract

Nagashima-type palmoplantar keratosis (NPPK) is the most common palmoplantar keratoderma in the East Asian populations. NPPK is caused by biallelic loss-of-function mutations in SERPINB7. Notably, almost all NPPK patients carry the TGAA nonsense mutation c.796C>T (p.Arg266Ter) in SERPINB7, which creates a premature termination codon (PTC) and leads to highly reduced production of full-length, functional SERPINB7 polypeptides. The suppression of a PTC, also known as ‘readthrough’, is able to restore the full-length protein. As the currently available drugs, including topical vitamin D3 and topical keratolytic, yield very limited improvement of NPPK skin phenotypes, we sought to tackle the disease by the readthrough therapy. We first performed a high- throughput screening of 19,992 small molecule compounds for readthrough activity using firefly luciferase assay. A total of 64 compounds survived retest screening and were shortlisted as potential readthrough candidates. Next, we generated a FLAG-tagged mutant SERPINB7 cDNA vector which contains the nonsense mutation c.796C>T and perform single and multiple concentrations screening using this vector for the selected 64 compounds. 2 hit compounds were identified with readthrough efficiency at their optimal concentration. Additional small molecule compounds with similar chemical structures of the 2 hit compounds were purchased for hit expansion; the process in which derivatives with similar chemical structure are selected from databases. Along with the hit expansion compounds, they have shown to induce a higher readthrough potency than the currently available drugs such as gentamicin and ataluren (Translarna®). As approximately 10% of all described gene lesions in genetic diseases are caused by nonsense mutations, the discovery of potent hit compounds can benefit not only NPPK patients but also patients who suffer from diseases caused by nonsense mutations.