Department of Internal Medicine and Clinical Pharmacology, Faculty of Medical Science in Katowice, Medical University of Silesia, Medykow 18, 40-752 Katowice, Poland.
Laboratory of Chemistry of Biologically Active Compounds, Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.
Int J Mol Sci. 2024 Jan 25;25(3):1463. doi: 10.3390/ijms25031463.
Proprotein convertase subtilisin/kexin 9 (PCSK9) is a protein that plays a key role in the metabolism of low-density lipoprotein (LDL) cholesterol. The gain-of-function mutations of the gene lead to a reduced number of surface LDL receptors by binding to them, eventually leading to endosomal degradation. This, in turn, is the culprit of hypercholesterolemia, resulting in accelerated atherogenesis. The modern treatment for hypercholesterolemia encompasses the use of biological drugs against PCSK9, like monoclonal antibodies and gene expression modulators such as inclisiran-a short, interfering RNA (siRNA). Peptide nucleic acid (PNA) is a synthetic analog of nucleic acid that possesses a synthetic peptide skeleton instead of a phosphate-sugar one. This different structure determines the unique properties of PNA (e.g., neutral charge, enzymatic resistance, and an enormously high affinity with complementary DNA and RNA). Therefore, it might be possible to use PNA against PCSK9 in the treatment of hypercholesterolemia. We sought to explore the impact of three selected PNA oligomers on gene expression. Using a cell-free transcription/translation system, we showed that one of the tested PNA strands was able to reduce the gene expression down to 74%, 64%, and 68%, as measured by RT-real-time PCR, Western blot, and HPLC, respectively. This preliminary study shows the high applicability of a cell-free enzymatic environment as an efficient tool in the initial evaluation of biologically active PNA molecules in the field of hypercholesterolemia research. This cell-free approach allows for the omission of the hurdles associated with transmembrane PNA transportation at the early stage of PNA selection.
前蛋白转化酶枯草溶菌素 9（PCSK9）是一种在低密度脂蛋白（LDL）胆固醇代谢中起关键作用的蛋白质。基因的功能获得性突变通过与它们结合导致 LDL 受体表面数量减少，最终导致内体降解。反过来，这就是高胆固醇血症的罪魁祸首，导致动脉粥样硬化加速形成。高胆固醇血症的现代治疗方法包括使用针对 PCSK9 的生物药物，如单克隆抗体和基因表达调节剂，如inclisiran——一种短干扰 RNA（siRNA）。肽核酸（PNA）是一种核酸的合成类似物，它具有合成的肽骨架而不是磷酸-糖骨架。这种不同的结构决定了 PNA 的独特性质（例如，中性电荷、酶抗性和与互补 DNA 和 RNA 的极高亲和力）。因此，有可能在高胆固醇血症的治疗中使用 PNA 针对 PCSK9。我们试图探讨三种选定的 PNA 寡聚物对基因表达的影响。使用无细胞转录/翻译系统，我们表明，在测试的 PNA 链中，有一种能够将基因表达降低到 74%、64%和 68%，分别通过 RT-实时 PCR、Western blot 和 HPLC 测量。这项初步研究表明，无细胞酶环境作为一种高效工具，在高胆固醇血症研究领域中对具有生物活性的 PNA 分子进行初步评估具有很高的适用性。这种无细胞方法允许在 PNA 选择的早期阶段省略与跨膜 PNA 转运相关的障碍。
