Design of a bioactive mimetic of soluble klotho for the treatment of chronic kidney disease

项目摘要 在美国，超过3700万人患有慢性肾脏疾病（CKD），并处于高死亡风险之中。 心血管并发症虽然在改善CKD护理和透析途径方面取得了很大进展， 在延缓或逆转肾损伤和相关疾病的药物开发方面， 病理学目前，尚不存在预防CKD心血管损害的治疗选择， CKD的治疗方法是肾移植。血清磷酸盐和成纤维细胞生长因子水平升高 (FGF)23是CKD的标志，与心血管死亡风险增加相关。表达水平 klotho是肾脏磷酸盐代谢的调节剂，在CKD中减少。Klotho可以从 肾脏作为可溶性klotho（sKL）在血液中循环，并作为FGF受体的结合伴侣 （FGFR）在各种组织中的作用。血清sKL水平的降低已被证明有助于CKD相关的 病理学sKL似乎通过替代肾klotho来保护组织，从而促进FGF 23/FGFR 1- 诱导肾脏磷酸盐排泄，降低全身磷酸盐水平，以及阻断直接病理性磷酸盐释放。 FGF 23和旁分泌FGF的作用。虽然提高klotho表达已经显示出治疗潜力， 尽管在CKD动物模型中，sKL的半衰期短和技术困难阻碍了进一步的进展， 生产足够量的重组sKL蛋白，沿着缺乏测量sKL活性的工具。 Alpha Young LLC已经开发了一种新的方法来生产重组sKL蛋白以及一种新的 使用基于sKL结合FGF 23和FGFR 1的能力测定sKL的生物活性的测定法。我们已经生成 一种早期的模拟蛋白，在这里我们将引入额外的点突变来增加sKL的稳定性 和生物活性。在第一阶段，我们将修饰sKL的糖基化位点和肝素结合结构域， 筛选对FGF 23和FGFR 1具有增加的结合亲和力的突变变体以改善生物活性，和 降低肝素结合亲和力以增加半衰期。在第2阶段，我们将优化我们鉴定的sKL变体 通过利用基于噬菌体展示的方法将突变引入sKL的FGFR结合结构域， 目的是增加FGFR 1结合亲和力。候选人与所需的变化，结合性能将进行测试 使用可以确定sKL对FGF 23调节的细胞增殖的影响的细胞培养模型， 信号传导、肾磷酸盐摄取、心脏肥大和旁分泌FGF诱导的成纤维细胞活化。 最有希望的候选物的半衰期将通过大鼠注射研究进行测试。最后，最活跃的 将稳定的sKL变体注射到CKD小鼠模型中，然后分析肾磷酸盐 排泄和心血管损伤。我们建议，我们的sKL模拟物的管理可以作为一个 降低血清磷酸盐水平和保护免受损害作用的CKD新治疗方法 的FGF。我们的项目的成功完成将为我们提供一种有效的候选药物， 有机会寻求早期合作伙伴，以推进和验证其未来临床试验的潜力。

PROJECT SUMMARY Over 37 million individuals in the U.S. have chronic kidney disease (CKD) and are at high risk to die from cardiovascular complications. While great strides have been made to improve CKD care and dialysis access, minimal advances have been made in drug development to stall or reverse kidney damage and associated pathologies. Currently, therapeutic options to prevent cardiovascular damage in CKD do not exist, and the only cure for CKD is kidney transplantation. Elevations in serum levels of phosphate and fibroblast growth factor (FGF) 23 are a hallmark of CKD and associated with an increased risk of cardiovascular death. Expression levels of klotho, a regulator of phosphate metabolism in the kidney, are reduced in CKD. Klotho can be released from the kidney as soluble klotho (sKL) that circulates in the blood and acts as a binding partner for FGF receptors (FGFR) on various tissues. Reductions in serum sKL levels have been shown to contribute to CKD-associated pathologies. sKL seems to protect tissues by substituting for renal klotho thereby promoting FGF23/FGFR1- induced renal phosphate excretion and lowering systemic phosphate levels, as well blocking the direct pathologic actions of FGF23 and of paracrine FGFs. While elevating klotho expression has shown therapeutic potential in animal models of CKD, further advances have been stymied by sKL’s short half-life and technical difficulties to produce the recombinant sKL protein in sufficient amounts, along with a lack of tools to measure sKL activity. Alpha Young LLC has developed a novel method to produce the recombinant sKL protein as well as a novel assays to determine the bioactivity of sKL based on its ability to bind FGF23 and FGFR1. We have generated an early-stage mimetic protein, and here we will introduce additional point mutations to increase sKL’s stability and bioactivity. In Phase 1, we will modify sKL’s glycosylation sites and heparin binding domain, and we will screen for mutant variants with increased binding affinities for FGF23 and FGFR1 to improve bioactivity, and decreased heparin binding affinity to increase half-life. In Phase 2, we will optimize our identified sKL variants by utilizing a phage display-based approach to introduce mutations into sKL’s FGFR binding domain with the goal to increase FGFR1 binding affinity. Candidates with the desired changes in binding properties will be tested for their biological activity using cell culture models that can determine the effect of sKL on FGF23-regulated signaling, renal phosphate uptake, cardiac hypertrophy and on fibroblast activation induced by paracrine FGFs. The half-life of the most promising candidates will be tested by injection studies in in rats. Finally, the most active and stable sKL variant will be injected into mouse models of CKD, followed by the analysis of renal phosphate excretion and cardiovascular damage. We propose that the administration of our sKL mimetic can serve as a novel therapeutic approach in CKD to lower serum phosphate levels and to protect from the damaging actions of FGFs. A successful completion of our project would provide us with a potent drug candidate and the opportunity to pursue early-stage partners for advancing and validating its potential for future clinical trials.