New Peptides for the Treatment of Multiple Sclerosis

描述（由申请人提供）：多发性硬化症（MS）是一种中枢神经系统慢性炎症性疾病，可导致严重残疾和死亡。在目前FDA批准的用于复发性MS的药物中，所有药物都有明显的副作用。我们建议使用多学科、基于结构的方法来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。该通道由终末分化效应记忆T（TEM）淋巴细胞高度表达，在MS发病机制中发挥重要作用。广泛的体外和体内疗效和安全性研究已经验证了Kv1.3作为免疫治疗的靶点，并表明最初从海葵中分离的肽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，它缺乏对Kv1.3通道的选择性，通过与其他钾通道的相互作用产生高毒性风险。我们已经开发了第一代合成类似物的ShK通过修改其N-末端与非蛋白加合物。这些类似物对Kv1.3的特异性比其他离子通道增强，同时保留皮摩尔效力，并且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，这些类似物之一（ShK-186）抑制炎症组织中的TEM细胞扩大和运动，抑制迟发型超敏反应，并有效治疗慢性复发性实验性自身免疫性脑脊髓炎（CR-EAE; MS模型）和降植烷诱导的关节炎。虽然ShK-186和相关类似物在大鼠中具有极好的安全性，并且不损害对病毒急性感染的保护性免疫应答，但它们在大鼠中的毒性是非常低的。（流感）或细菌（衣原体）病原体，它们遭受几个限制：（i）它们对pH和温度的变化敏感;（ii）它们具有非常短的体内半衰期;（iii）ShK-186上的磷酸化残基可以被去磷酸化;（iv）它们含有对氧化敏感的Met残基;和（v）它们的非蛋白加合物是免疫原性的。我们现在建议设计，生成和评估ShK的新类似物。在具体目标1下，我们将使用分子建模和高分辨率NMR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-和C-末端延伸的ShK类似物。在特定目标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，并将其聚乙二醇化以增加其循环半衰期。在特定目标3下，我们将评估最有效和选择性的ShK类似物的体内药代动力学、免疫原性、安全性和有效性。该项目将产生Kv1.3的新型肽阻断剂，我们相信这将是开发MS和其他慢性炎症性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.