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

项目总结 在美国，超过3700万人患有慢性肾脏疾病(CKD)，死亡风险很高 心血管并发症。虽然在改善慢性肾脏病护理和透析途径方面取得了很大进展， 在延缓或逆转肾损害和相关疾病的药物开发方面取得的进展甚微 病理学。目前，预防慢性肾脏病心血管损害的治疗方案还不存在，而且唯一的 慢性肾脏病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 (成纤维细胞生长因子)23是慢性肾脏病的标志，与心血管死亡风险增加有关。表达水平 在慢性肾脏病中，肾脏磷酸盐代谢调节剂Klotho的含量减少。Klotho可以从 肾脏作为可溶性Klotho(SKL)，在血液中循环，并充当成纤维细胞生长因子受体的结合伙伴 (FGFR)在各种组织上。血清SKL水平的降低已被证明与慢性肾脏病相关 病理学。SKL似乎通过替代肾Klotho而保护组织，从而促进FGF23/FGFR1- 诱导肾脏磷酸盐排泄和降低全身磷酸盐水平，以及阻断直接病理 FGF23和旁分泌FGFs的作用。虽然提高Klotho的表达显示出治疗潜力 CKD的动物模型，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，同时缺乏测量SKL活性的工具。 Alpha Young LLC开发了一种生产重组SKL蛋白的新方法以及一种新的 根据SKL与FGF23和FGFR1结合的能力，测定SKL的生物活性。我们已经产生了 一个早期的模拟蛋白，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，我们将修改SKL的糖基化位点和肝素结合结构域，我们将 筛选与FGF23和FGFR1结合亲和力增加的突变体以提高生物活性，以及 降低肝素结合亲和力以增加半衰期。在第二阶段，我们将优化我们确定的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结合域 目标是增加FGFR1结合亲和力。将对具有所需绑定属性更改的候选进行测试 利用细胞培养模型确定SKL对FGF23调节的影响 信号转导、肾脏磷酸盐摄取、心肌肥大和旁分泌成纤维细胞激活。 最有希望的候选者的半衰期将通过在老鼠身上进行注射研究来测试。最后，最活跃的 并将稳定的SKL变异体注射到CKD小鼠模型中，然后分析肾脏磷酸盐 排泄和心血管损害。我们建议我们的SKL模拟物的管理可以作为一个 降低血磷水平保护慢性肾功能不全的新治疗方法 关于FGFs的。我们项目的成功完成将为我们提供一个强有力的候选药物和 有机会寻找早期合作伙伴，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.