Homogeneous Screening Assay for Cytokine Receptor Modulators

项目概要/摘要 “组织保护性”细胞因子受体的激活产生细胞保护性、抗炎性和抗肿瘤性。 在临床前研究中，在体外和体内研究了细胞凋亡作用，并在早期 罕见的神经肌肉疾病，弗里德赖希共济失调的临床研究。这种异聚体受体，由 EPOR（促红细胞生成素受体）和CSF 2 RB（集落刺激因子2受体β亚单位，也称为“β 共同受体”和CD 131）在非造血组织中广泛表达，在非造血组织中它被局部激活， 产生EPO，特别是在氧化应激和组织损伤部位附近。选择性EPOR/CSF 2 RB激活剂 包括重组蛋白，例如，EPO衍生物和促红细胞生成素活性小分子， 增加细胞中的EPOR和CSF 2 RB磷酸化并增加线粒体蛋白的表达， 共济失调蛋白，弗里德赖希共济失调的生物标志物。与重组蛋白相比，小分子的发展 提供了优化药物性质的独特机会， 选择性、药代动力学和耐受性/安全性，并产生了有前途的探针化合物。然而，在这方面， 小分子细胞因子受体调节剂的发现具有挑战性并且进展缓慢，尽管 为研究蛋白质-蛋白质相互作用调节剂的作用而开发的技术做出了巨大努力。 在第一阶段，我们开发了一种创新的基于邻近的方法来检测EPOR/CSF 2 RB的二聚体 使用与高通量完全相容的同质形式的受体胞外结构域 在筛选（“HTS”）中，证明了EPO和小分子的调节作用。在第二阶段，我们将重点关注三个方面 主要目的：（1）继续开发同质EPOR/CSF 2 RB受体寡聚化试验， HTS和筛选一系列化学上不同的化合物，我们将用这些化合物来识别新的小分子先导化合物 并建立受体结合SAR;（2）扩大三种密切相关的细胞因子的均相测定 受体作为EPOR/CSF 2 RB的反筛选以建立靶选择性;和（3）证明活性 体外细胞模型中EPOR/CSF 2 RB受体活化和共济失调蛋白的选择性小分子 增加通过这些研究，我们将开发一种前所未有的小分子SAR用于受体结合， 药理活性，支持鉴定弗里德赖希共济失调的新候选者。

PROJECT SUMMARY/ABSTRACT Activation of the “tissue-protective” cytokine receptor produces cytoprotective, anti-inflammatory, and anti- apoptotic effects in vitro and in vivo in preclinical studies, and has demonstrated therapeutic utility in early clinical studies of the rare neuromuscular disease, Friedreich’s ataxia. This heteromeric receptor, composed of EPOR (erythropoietin receptor) and CSF2RB (colony-stimulating factor 2 receptor beta subunit, also “beta common receptor,” and CD131), is expressed widely in non-hematopoietic tissues where it is activated by locally produced EPO, especially near sites of oxidative stress and tissue damage. Selective EPOR/CSF2RB activators include recombinant proteins, e.g., EPO derivatives, and pharmacologically active small molecules which increase EPOR and CSF2RB phosphorylation in cells and increase expression of the mitochondrial protein, frataxin, a biomarker for Friedreich’s ataxia. Development of small molecules compared to recombinant proteins provides distinct opportunities for optimization of pharmaceutical properties including receptor sub-type selectivity, pharmacokinetics, and tolerability/safety and has produced promising probe compounds. However, discovery of small molecule cytokine receptor modulators has been challenging and slow to advance, despite substantial efforts to develop technologies for studying the effects of modulators of protein-protein interactions. In Phase I, we developed an innovative proximity-based method for detecting dimerization of the EPOR/CSF2RB receptor extracellular domains using homogeneous formats which are fully compatible with high-throughput screening (“HTS”) and demonstrated modulation by EPO and small molecules. In Phase II, we will focus on three major Aims: (1) continue developing the homogeneous EPOR/CSF2RB receptor oligomerization assay to enable HTS and screen a collection of chemically diverse compounds with which we’ll identify new small molecule leads and establish a receptor-binding SAR; (2) expand homogeneous assays for three closely related cytokine receptors as counter-screens to EPOR/CSF2RB to establish target selectivity; and (3) demonstrate the activity of selective small molecules in cellular models in vitro for EPOR/CSF2RB receptor activation and frataxin increase. Through these studies, we will develop an unprecedented small molecule SAR for receptor binding and pharmacological activity, supporting identification of new candidates for Friedreich’s ataxia.