Therapeutic Na+ channel blockers: Receptor & Drug Design

描述（由申请人提供）：本项目的长期目标是开发与疼痛管理相关的长效Na+通道阻滞剂，并更好地了解这些阻滞剂的作用机制。传统的局部麻醉药（LA）由于神经阻滞持续时间不足，通常不适用于慢性或顽固性癌痛。我们的具体目标是（1）鉴定和合成有效阻断开放和/或失活Na+通道的化合物，（2）评估有效Na+通道阻断剂的使用和状态依赖性阻断，（3）测试其作为长效LA的体内效力，以及（4）绘制其在Na+通道α亚基内的受体位点。最近的筛选已经确定了几个新的铅结构，阻止开放的Na+通道具有高亲和力。早期的筛选表明，口服治疗神经性疼痛的药物，如阿米替林、氟卡尼和美西律，在其治疗血浆浓度下也能有效阻断开放的Na+通道。我们推测，这些药物减轻异位高频放电损伤神经，由于其高亲和力的开放Na+通道的阻滞。阿米替林，这太有力地阻止失活的Na+通道，确实作为一个长效LA。我们计划识别和合成新的开放和/或失活通道阻滞剂的基础上，这些铅结构。将在人HEK 293细胞中表达的野生型和/或失活缺陷型突变Na+通道中测定其在重复脉冲期间对Na+电流的使用依赖性阻断以及静息、开放和失活通道阻断的50%抑制浓度（IC 50）。在通过各种途径注射强效Na +通道阻滞剂之前和之后，将在大鼠或绵羊中评价神经阻滞的感觉和运动功能。最后，我们计划通过定点诱变和配体-受体复合物的计算机模拟来确定Na+通道α亚基内所选阻断剂的受体。这些信息将反过来促进基于受体的药物设计。总之，这些研究应该为我们提供新的铅结构的选择性靶向开放和/或失活的Na+通道的长效LA的发展。这些药物，无论是口服或局部注射，可能是有益的慢性和顽固性癌症疼痛的患者。

DESCRIPTION (provided by applicant): The long-term objectives of this project are to develop long-acting Na+ channel blockers pertinent for pain management and to gain a better understanding of how these blockers work mechanistically. Traditional local anesthetics (LAs) are often inept for chronic or intractable cancer pain due to their insufficient duration of nerve block. Our specific aims are (1) to identify and synthesize compounds that potently block the open and/or inactivated Na+ channels, (2) to assess the use- and state-dependent block of potent Na+ channel blockers, (3) to test their in vivo potency as long-acting LAs, and (4) to map their receptor site within the Na+ channel alpha-subunit. Recent screening has identified several new lead structures that block open Na+ channels with high affinities. Earlier screening demonstrated that drugs taken orally for neuropathic pain, such as amitriptyline, flecainide, and mexiletine, also block open Na+ channels effectively at their therapeutic plasma concentrations. We hypothesize that these drugs alleviate ectopic high-frequency discharges found in injured nerves due to their high-affinity block of open Na+ channels. Amitriptyline, which too potently blocks the inactivated Na+ channels, indeed acts as a long-acting LA. We plan to identify and synthesize novel open and/or inactivated-channel blockers based on these lead structures. Their use-dependent block of Na+ currents during repetitive pulses and the 50% inhibitory concentration (IC50) of resting-, open-, and inactivated-channel block will be determined in wild-type and/or in inactivation-deficient mutant Na+ channels expressed in human HEK293 cells. Sensory and motor functions of nerve block will be evaluated in rats or sheep before and after injection of potent Na + channel blockers via various routes. Finally, we plan to delimit the receptor for selected blockers within the Na+ channel alpha-subunit by site-directed mutagenesis and by computer simulation of the ligand-receptor complex. This information will in turn facilitate receptor-based drug design. Together, these studies should provide us new lead structures for the development of long-acting LAs that selectively target open and/or inactivated Na+ channels. Such drugs, either taken orally or injected locally, may be beneficial for patients with chronic and intractable cancer pain.