Doupnik Craig A, Parra Katherine C, Guida Wayne C
Department of Molecular Pharmacology & Physiology, University of South Florida College of Medicine, 12901 Bruce B. Downs Boulevard, Tampa, FL 33612, United States.
Department of Chemistry, University of South Florida, 4202 E. Fowler Avenue, Tampa, FL 33620, United States.
Comput Struct Biotechnol J. 2014 Nov 7;13:85-94. doi: 10.1016/j.csbj.2014.11.004. eCollection 2015.
Ion channels represent a large family of membrane proteins with many being well established targets in pharmacotherapy. The 'druggability' of heteromeric channels comprised of different subunits remains obscure, due largely to a lack of channel-specific probes necessary to delineate their therapeutic potential in vivo. Our initial studies reported here, investigated the family of inwardly rectifying potassium (Kir) channels given the availability of high resolution crystal structures for the eukaryotic constitutively active Kir2.2 channel. We describe a 'limited' homology modeling approach that can yield chimeric Kir channels having an outer vestibule structure representing nearly any known vertebrate or invertebrate channel. These computationally-derived channel structures were tested ""in silico for 'docking' to NMR structures of tertiapin (TPN), a 21 amino acid peptide found in bee venom. TPN is a highly selective and potent blocker for the epithelial rat Kir1.1 channel, but does not block human or zebrafish Kir1.1 channel isoforms. Our Kir1.1 channel-TPN docking experiments recapitulated published in vitro ""findings for TPN-sensitive and TPN-insensitive channels. Additionally, in silico site-directed mutagenesis identified 'hot spots' within the channel outer vestibule that mediate energetically favorable docking scores and correlate with sites previously identified with in vitro thermodynamic mutant-cycle analysis. These 'proof-of-principle' results establish a framework for virtual screening of re-engineered peptide toxins for interactions with computationally derived Kir channels that currently lack channel-specific blockers. When coupled with electrophysiological validation, this virtual screening approach may accelerate the drug discovery process, and can be readily applied to other ion channels families where high resolution structures are available.
离子通道代表了一大类膜蛋白，其中许多是药物治疗中已确立的靶点。由不同亚基组成的异聚通道的“可药用性”仍然不清楚，这主要是由于缺乏在体内描述其治疗潜力所需的通道特异性探针。我们在此报告的初步研究，鉴于真核组成型活性Kir2.2通道有高分辨率晶体结构，对内向整流钾（Kir）通道家族进行了研究。我们描述了一种“有限”的同源建模方法，该方法可以产生具有代表几乎任何已知脊椎动物或无脊椎动物通道的外前庭结构的嵌合Kir通道。这些通过计算得出的通道结构在计算机上进行了测试，以“对接”蜂毒中发现的21个氨基酸肽tertiapin（TPN）的核磁共振结构。TPN是上皮大鼠Kir1.1通道的高度选择性和强效阻滞剂，但不阻断人或斑马鱼Kir1.1通道亚型。我们的Kir1.1通道 - TPN对接实验重现了已发表的关于TPN敏感和TPN不敏感通道的体外研究结果。此外，计算机定点诱变确定了通道外前庭内的“热点”，这些热点介导了能量有利的对接分数，并与先前通过体外热力学突变循环分析确定的位点相关。这些“原理验证”结果为虚拟筛选重新设计的肽毒素与目前缺乏通道特异性阻滞剂的通过计算得出的Kir通道的相互作用建立了框架。当与电生理验证相结合时，这种虚拟筛选方法可能会加速药物发现过程，并且可以很容易地应用于其他有高分辨率结构的离子通道家族。
