New Peptides for the Treatment of Multiple Sclerosis

描述（由申请人提供）：多发性硬化症（MS）是一种慢性中枢神经系统炎症性疾病，可导致严重残疾和死亡。目前fda批准的治疗复发型多发性硬化症的药物都有明显的副作用。我们建议采用多学科、基于结构的方法，开发电压门控钾通道Kv1.3的新型多肽阻滞剂，作为MS的新干预措施。该通道在MS发病机制中起主要作用的终末分化效应记忆T （TEM）淋巴细胞中高度表达。大量的体外和体内有效性和安全性研究证实了Kv1.3作为免疫治疗的靶点，并表明最初从海葵中分离出来的肽ShK不仅是该通道的有效阻断剂，而且是有效的免疫调节剂。然而，它对Kv1.3通道缺乏选择性，通过与其他钾通道相互作用产生毒性的风险很高。我们通过用非蛋白加合物修饰其n端，开发了第一代ShK的合成类似物。与其他离子通道相比，这些类似物对Kv1.3的特异性增强，同时保留了皮摩尔的效力，它们选择性地抑制细胞因子的产生和人TEM细胞的增殖，而不影响其他T细胞亚群。在大鼠研究中，其中一种类似物（ShK-186）抑制炎症组织中TEM细胞的扩大和运动，抑制延迟型超敏反应，并有效治疗慢性复发性实验性自身免疫性脑脊髓炎（CR-EAE；一种多发性硬化症模型）和前列腺素诱导的关节炎。虽然ShK-186和相关类似物在大鼠中具有良好的安全性，并且不会损害对病毒（流感）或细菌（衣原体）病原体急性感染的保护性免疫反应，但它们存在以下几个局限性：(i)它们对pH和温度的变化敏感；（ii）它们在体内的半衰期非常短；（iii） ShK-186上的磷酸化残基可被去磷酸化；（iv）它们含有易氧化的Met残留物；(v)它们的非蛋白加合物具有免疫原性。我们现在建议设计，生成和评估新的ShK类似物。在Specific Aim 1下，我们将使用分子建模和高分辨率核磁共振来确定ShK类似物在Kv1.3上的对接构型，从而设计和合成更有效和选择性的N端和c端扩展的ShK类似物。在Specific Aim 2下，我们将评估ShK类似物的体外效力、选择性、稳定性和对T淋巴细胞活化的影响，并对其进行聚乙二醇化以增加其循环半衰期。在Specific Aim 3下，我们将评估体内最有效和选择性的ShK类似物的药代动力学、免疫原性、安全性和有效性。该项目将产生新的Kv1.3肽阻滞剂，我们相信这将是开发MS和其他慢性炎症疾病新疗法的有价值的线索。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system that leads to severe disability and death. Of the current FDA-approved medications for relapsing forms of MS, all have significant side effects. We propose to use a multi-disciplinary, structure-based approach to developing novel polypeptide blockers of the voltage-gated potassium channel Kv1.3 as new interventions for MS. This channel is highly expressed by terminally-differentiated effector memory T (TEM) lymphocytes that play a major role in MS pathogenesis. Extensive in vitro and in vivo efficacy and safety studies have validated Kv1.3 as a target for immunotherapy and shown that the peptide ShK, originally isolated from a sea anemone, is not only a potent blocker of this channel but also an effective immunomodulator. However, its lack of selectivity for Kv1.3 channels creates a high risk of toxicity through interactions with other potassium channels. We have developed a first generation of synthetic analogs of ShK by modifying its N-terminus with non-protein adducts. These analogs show enhanced specificity for Kv1.3 over other ion channels while retaining picomolar potency, and they selectively suppress cytokine production and proliferation of human TEM cells without affecting other T cell subsets. In rat studies, one of these analogs (ShK-186) suppresses TEM cell enlargement and motility in inflamed tissues, inhibits delayed-type hypersensitivity, and effectively treats chronic-relapsing experimental autoimmune encephalomyelitis (CR-EAE; a model of MS) and pristane-induced arthritis. While ShK-186 and related analogs have an excellent safety profile in rats and do not compromise the protective immune response to acute infection with viral (influenza) or bacterial (chlamydia) pathogens, they suffer from several limitations: (i) they are sensitive to changes in pH and temperature; (ii) they have very short in vivo half-lives; (iii) a phosphorylated residue on ShK-186 can be dephosphorylated; (iv) they contain a Met residue that is susceptible to oxidation; and (v) their non-protein adducts are immunogenic. We now propose to design, generate, and evaluate novel analogs of ShK. Under Specific Aim 1 we will use molecular modeling and high-resolution NMR to determine the docking configuration of ShK analogs on Kv1.3 and thereby to design and synthesize more potent and selective N- and C-terminally extended ShK analogs. Under Specific Aim 2 we will assess ShK analogs for their in vitro potency, selectivity, stability, and effects on T lymphocyte activation, and PEGylate them to increase their circulating half-life. Under Specific Aim 3 we will evaluate the most potent and selective ShK analogs in vivo for pharmacokinetics, immunogenicity, safety, and efficacy. This project will generate novel peptide blockers of Kv1.3, which we believe will be valuable leads in the development of new treatments for MS and other chronic inflammatory diseases.