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

用于治疗慢性肾病的可溶性 klotho 生物活性模拟物的设计

• 批准号: 10716007
• 负责人: Christopher Yanucil
• 金额: $ 60.65万
• 依托单位: ALPHA YOUNG LLC
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10716007
• 关键词: Design; bioactive; mimetic; soluble; klotho

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.

项目摘要 美国超过3700万人患有慢性肾脏疾病（CKD），死于死亡的风险很高 心血管并发症。尽管已经取得了长足的进步来改善CKD护理和透析访问，但 在药物开发方面取得了最小的进步，以使肾脏损害或逆转肾脏损害和相关性 病理。目前，不存在预防CKD心血管损伤的治疗选择，而唯一 CKD治疗是肾脏移植。血清磷酸盐和成纤维细胞生长因子的升高 （FGF）23是CKD的标志，与心血管死亡的风险增加有关。表达水平 肾脏中磷酸盐代谢的调节剂克洛托（Klotho）在CKD中降低。克洛托可以从 肾脏作为固体klotho（SKL），它在血液中循环并充当FGF受体的结合伴侣 （FGFR）在各种组织上。血清SKL水平的降低已证明有助于CKD相关 病理。 SKL似乎通过替代肾脏Klotho来保护组织，从而促进FGF23/FGFR1- 诱导的肾磷酸排泄并降低全身磷酸盐水平，并阻止直接病理 FGF23和旁分泌FGF的动作。同时提高klotho表达已显示出治疗潜力 CKD的动物模型，SKL的短期半衰期和技术困难使进一步的进步困扰 产生足够量的重组SKL蛋白，以及缺乏测量SKL活性的工具。 Alpha Young LLC开发了一种新型方法来生产重组SKL蛋白以及一种新颖的方法 根据SKL结合FGF23和FGFR1的能力来确定其生物活性的测定。我们已经生成了 早期模拟蛋白，在这里我们将引入其他点突变以提高SKL的稳定性 和生物活性。在第1阶段，我们将修改SKL的糖基化位点和肝素结合域，我们将 筛选FGF23和FGFR1的结合亲和力增加的突变体变体以提高生物活性，并且 肝素结合亲和力降低以增加半衰期。在第2阶段，我们将优化已确定的SKL变体 通过使用基于噬菌体显示的方法将突变引入SKL的FGFR绑定域中 增加FGFR1结合亲和力的目标。将测试具有所需变化的候选人 使用可以确定SKL对FGF23调节的影响的细胞培养模型的生物学活性 信号传导，肾磷酸盐摄取，心脏肥大和由旁分泌FGF诱导的成纤维细胞激活。 最有希望的候选人的半衰期将通过大鼠注射研究来测试。最后，最活跃 稳定的SKL变体将注入CKD的小鼠模型，然后分析肾磷酸盐 排泄和心血管损伤。我们建议我们的Skl Mimetic的管理可以用作 CKD中的新型热方法至降低血清磷酸盐水平，并保护侵害动作 fgfs。我们项目的成功完成将为我们提供有力的毒品候选者和 追求早期合作伙伴的机会，以促进并验证其未来临床试验的潜力。