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.

项目概要 美国有超过 3700 万人患有慢性肾病 (CKD)，并且面临很高的死亡风险 尽管在改善 CKD 护理和透析服务方面取得了巨大进步， 在阻止或逆转肾脏损伤及相关疾病的药物开发方面取得的进展微乎其微 目前，尚不存在预防 CKD 心血管损害的治疗方案，也是唯一的治疗方案。 慢性肾病的治疗方法是肾移植，提高血清磷酸盐和成纤维细胞生长因子的水平。 (FGF) 23 是 CKD 的标志，与心血管死亡风险增加相关。 Klotho 是肾脏中磷酸盐代谢的调节剂，在 CKD 中可减少 Klotho 的释放。 肾脏作为可溶性 klotho (sKL) 在血液中进行检修并充当 FGF 受体的结合伴侣 (FGFR) 对各种组织的影响已被证明有助于 CKD 相关的血清 sKL 水平的降低。 sKL 似乎通过替代肾 klotho 来保护组织，从而促进 FGF23/FGFR1- 肾脏诱导磷酸盐排泄并降低全身磷酸盐水平，并阻断直接病理性磷酸盐 FGF23 和旁分泌 FGF 的作用同时提高 klotho 表达已显示出治疗潜力。 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，但缺乏测量 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 我们已经开发了基于 sKL 结合 FGF23 和 FGFR1 的能力来确定 sKL 生物活性的测定方法。 一种早期模拟蛋白，在这里我们将引入额外的点突变来提高 sKL 的稳定性 在第一阶段，我们将修改 sKL 的糖基化位点和肝素结合域。 筛选对 FGF23 和 FGFR1 的结合亲和力增加的突变体变体，以提高生物活性，以及 在第 2 阶段，我们将优化已识别的 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，将测试具有所需结合特性变化的候选者。 使用细胞培养模型来确定其生物活性，该模型可以确定 sKL 对 FGF23 调节的影响 信号传导、肾磷酸盐摄取、心脏肥大以及旁分泌 FGF 诱导的成纤维细胞活化。 最有希望的候选药物的半衰期将通过大鼠注射研究进行测试。 将稳定的 sKL 变体注射到 CKD 小鼠模型中，然后进行肾磷酸盐分析 我们建议使用我们的 sKL 模拟物可以作为一种治疗方法。 CKD 的新治疗方法，可降低血清磷酸盐水平并防止损害作用 FGF 的成功完成将为我们提供一种有效的候选药物和 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。