Peptide-based nanoparticle inhibition of CCR3-mediated inflammatory cell recruitm

描述（由申请人提供）：趋化因子信号通过激活嗜酸性粒细胞、Th2淋巴细胞、嗜碱性粒细胞和中性粒细胞向肺的迁移和募集，在哮喘的发病机制中起关键作用。最近的研究表明，在正确诊断的嗜酸性粒细胞哮喘患者中靶向嗜酸性粒细胞可以提供显着的健康益处。然而，非嗜酸性哮喘内型患者表现出更普遍的嗜中性粒细胞炎症，但目前尚无成功的治疗方法。越来越多的人认识到，要有效地干扰哮喘炎症细胞的趋化性，需要抑制不止一种趋化因子受体。目前，还没有药物能够从多方面干扰嗜酸性粒细胞和中性粒细胞的化学吸引。CCR3趋化因子受体结合其首选趋化因子eotaxins，并促进嗜酸性粒细胞向肺的募集。除了eotaxins， CCR3还结合了Th2淋巴细胞、嗜碱性细胞、肥大细胞和中性粒细胞的趋化因子。这种结合是由受体的细胞外环介导的。我们建议设计一种新的肽基纳米颗粒，具有CCR3细胞外环呈递，作为CCR3受体的诱饵或直接抑制受体的功能。这些纳米粒子将隔离针对嗜酸性粒细胞和潜在的其他炎症细胞（如中性粒细胞）的趋化因子。与通常用于药物递送的纳米级被动载体不同，我们的新型CCR3纳米颗粒将自己作为药物。我们使用这种方法开发了基于肽的纳米颗粒，可以拮抗CXCR4，一种类似于CCR3的趋化因子受体。X4-2-6是一种包含细胞外环1的跨膜螺旋肽类似物，靶向CXCR4受体抑制HIV进入和乳腺癌转移。基于x4-2-6组装成纳米颗粒和隔离SDF-1的能力，我们同样建议开发含有CCR3跨膜和细胞外环部分的肽。这些肽序列由具有高螺旋倾向的疏水氨基酸组成，包括N端和c端电荷。这些肽的性质是肽在水溶液中组装成球形纳米颗粒的原因。我们假设CCR3的肽类似物可以被设计成具有类似于CXCR4肽x4-2-6的特性，即它们将能够形成纳米颗粒并隔离参与嗜酸性粒细胞和可能的中性粒细胞迁移/募集到组织中的趋化因子（即eotaxins）或直接抑制CCR3的功能。为了解决这一假设，提出了两个具体目标：(1)确定CCR3跨膜肽组装成纳米颗粒的能力，并隔离参与嗜酸性粒细胞和中性粒细胞募集的趋化因子；(2)在小鼠三抗原过敏性哮喘模型中，验证CCR3肽纳米颗粒在体外和体内抑制趋化因子依赖性炎症反应的能力。拟议的项目极有可能对新型哮喘治疗药物的开发产生重大影响。

DESCRIPTION (provided by applicant): Chemokine signaling plays a critical role in the pathogenesis of asthma through the activation of eosinophil, Th2 lymphocyte, basophil, and neutrophil migration and recruitment into the lung. Recent studies indicate that targeting eosinophils in patients with properly diagnosed eosinophilic asthma can provide a significant health benefits. However, patients with the non-eosinophilic endotype of asthma exhibit more prevalent neutrophilic inflammation, but there are currently no successful treatments for this endotype. There is a growing realization that to efficiently interfere with the chemotaxis of inflammatory cells in asthma, more than one chemokine receptor needs to be inhibited. Currently, there are no drugs capable of multi-faceted interference with chemoattraction of eosinophils and neutrophils. The CCR3 chemokine receptor binds its preferred chemokines, the eotaxins, and facilitates recruitment of eosinophils to the lungs. In addition to eotaxins, CCR3 also binds chemokines responsible for the chemotaxis of Th2 lymphocytes, basophils, mast cells and neutrophils. This binding is mediated by the receptor's extracellular loops. We propose to design novel peptide- based nanoparticles with CCR3 extracellular loop presentation to act as decoy CCR3 receptors or to directly inhibit receptor function. These nanoparticles would sequester chemokines that target both eosinophils and potentially other inflammatory cells such as neutrophils. Unlike commonly available nanoscale passive carriers used for drug delivery, our novel CCR3 nanoparticles will act as drugs themselves. We used this approach to develop peptide-based nanoparticles that antagonize CXCR4, a chemokine receptor similar to CCR3. X4-2-6, a transmembrane helical peptide analog that includes the extracellular loop 1, targets CXCR4 receptors for inhibition of HIV entry and breast cancer metastasis. Based on the ability of x4-2-6 to assemble into nanoparticles, and to sequester SDF-1, we similarly propose to develop peptides containing transmembrane and extracellular loop portions of CCR3. These peptide sequences consist of hydrophobic amino acids with a high helical propensity and include N- and C-terminal charges. These peptide properties are responsible for the peptide's assembly into spherical nanoparticles in aqueous solution. We hypothesize that peptide analogs of CCR3 can be designed that will display properties similar to the CXCR4 peptide x4-2-6, namely, they will be able to form nanoparticles and sequester chemokines (i.e. eotaxins) that are involved in eosinophil and possibly neutrophil migration/recruitment into tissues or directly inhibit CCR3 function. To address this hypothesis, two specific aims are proposed: (1) Determine the ability of CCR3 transmembrane peptides to assemble into nanoparticles and to sequester chemokines involved in eosinophil and neutrophil recruitment; and (2) Demonstrate the ability of CCR3 peptide nanoparticles to inhibit chemokine-dependent inflammatory responses in vitro and in vivo in the mouse triple antigen allergic asthma model. The proposed project is highly likely to significantly impact the general approach to the development of novel asthma therapeutics.