Simvastatin Sodium Salt and Fluvastatin Interact with Human Gap Junction Gamma-3 Protein

Finding pleiomorphic targets for drugs allows new indications or warnings for treatment to be identified. As test of concept, we applied a new chemical genomics approach to uncover additional targets for the widely prescribed lipid-lowering pro-drug simvastatin. We used mRNA extracted from internal mammary artery from patients undergoing coronary artery surgery to prepare a viral cardiovascular protein library, using T7 bacteriophage. We then studied interactions of clones of the bacteriophage, each expressing a different cardiovascular polypeptide, with surface-bound simvastatin in 96-well plates. To maximise likelihood of identifying meaningful interactions between simvastatin and vascular peptides, we used a validated photo-immobilisation method to apply a series of different chemical linkers to bind simvastatin so as to present multiple orientations of its constituent components to potential targets. Three rounds of biopanning identified consistent interaction with the clone expressing part of the gene GJC3, which maps to Homo sapiens chromosome 7, and codes for gap junction gamma-3 protein, also known as connexin 30.2/31.3 (mouse connexin Cx29). Further analysis indicated the binding site to be for the N-terminal domain putatively ‘regulating’ connexin hemichannel and gap junction pores. Using immunohistochemistry we found connexin 30.2/31.3 to be present in samples of artery similar to those used to prepare the bacteriophage library. Surface plasmon resonance revealed that a 25 amino acid synthetic peptide representing the discovered N-terminus did not interact with simvastatin lactone, but did bind to the hydrolysed HMG CoA inhibitor, simvastatin acid. This interaction was also seen for fluvastatin. The gap junction blockers carbenoxolone and flufenamic acid also interacted with the same peptide providing insight into potential site of binding. These findings raise key questions about the functional significance of GJC3 transcripts in the vasculature and other tissues, and this connexin’s role in therapeutic and adverse effects of statins in a range of disease states.

发掘药物的多态性靶点，有助于识别新的治疗适应证或用药警示信息。作为概念验证，我们采用一种新型化学基因组学方法，为临床广泛应用的调脂前体药物辛伐他汀发掘额外靶点。我们从接受冠状动脉手术患者的乳内动脉中提取mRNA，利用T7噬菌体（T7 bacteriophage）构建了心血管病毒蛋白文库。随后我们在96孔板中，针对表面固定的辛伐他汀，研究了各表达不同心血管多肽的噬菌体克隆与其的相互作用。为最大程度提升识别辛伐他汀与血管多肽间功能性相互作用的概率，我们采用经验证的光固定化方法，使用一系列不同的化学连接臂结合辛伐他汀，使其组成成分可通过多种取向呈递给潜在靶点。经过三轮生物淘选（biopanning），我们发现与表达GJC3基因片段的克隆存在稳定相互作用；该基因定位于智人（Homo sapiens）7号染色体，编码间隙连接γ3蛋白，又名连接蛋白30.2/31.3（小鼠连接蛋白Cx29）。进一步分析显示，其结合位点位于N端结构域，该结构域被认为可调控连接蛋白半通道与间隙连接通道孔。我们通过免疫组化实验发现，连接蛋白30.2/31.3存在于与构建噬菌体文库所用动脉样本相似的动脉组织中。表面等离子体共振（Surface plasmon resonance）实验结果显示，一段代表上述发现的N端结构域的25氨基酸合成肽，无法与辛伐他汀内酯结合，但可与水解型HMG-CoA还原酶抑制剂辛伐他汀酸结合。氟伐他汀（fluvastatin）也可与该肽段产生此类相互作用。间隙连接阻断剂甘珀酸与氟芬那酸也可与该肽段结合，这为潜在结合位点的研究提供了线索。上述研究结果引出了若干关键问题：包括GJC3转录本在血管系统与其他组织中的功能意义，以及该连接蛋白在他汀类药物在多种疾病状态下的治疗作用与不良反应中所扮演的角色。