Over 37 million individuals in the U.S. have chronic kidney disease (CKD) and are at high risk to die fromcardiovascular 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 associatedpathologies. Currently, therapeutic options to prevent cardiovascular damage in CKD do not exist, and the onlycure 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 levelsof klotho, a regulator of phosphate metabolism in the kidney, are reduced in CKD. Klotho can be released fromthe 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-associatedpathologies. 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 pathologicactions of FGF23 and of paracrine FGFs. While elevating klotho expression has shown therapeutic potential inanimal models of CKD, further advances have been stymied by sKL's short half-life and technical difficulties toproduce 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 novelassays to determine the bioactivity of sKL based on its ability to bind FGF23 and FGFR1. We have generatedan early-stage mimetic protein, and here we will introduce additional point mutations to increase sKL's stabilityand bioactivity. In Phase 1, we will modify sKL's glycosylation sites and heparin binding domain, and we willscreen for mutant variants with increased binding affinities for FGF23 and FGFR1 to improve bioactivity, anddecreased heparin binding affinity to increase half-life. In Phase 2, we will optimize our identified sKL variantsby utilizing a phage display-based approach to introduce mutations into sKL's FGFR binding domain with thegoal to increase FGFR1 binding affinity. Candidates with the desired changes in binding properties will be testedfor their biological activity using cell culture models that can determine the effect of sKL on FGF23-regulatedsignaling, 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 activeand stable sKL variant will be injected into mouse models of CKD, followed by the analysis of renal phosphateexcretion and cardiovascular damage. We propose that the administration of our sKL mimetic can serve as anovel therapeutic approach in CKD to lower serum phosphate levels and to protect from the damaging actionsof FGFs. A successful completion of our project would provide us with a potent drug candidate and theopportunity to pursue early-stage partners for advancing and validating its potential for future clinical trials.
Public Health Relevance Statement: PROJECT NARRATIVE
Patients with chronic kidney disease (CKD) are at high risk for developing cardiovascular disease, but as diseas
progression leads to costly dialysis or high-risk transplantation, therapies to protect the heart and vasculature
and to increase lifespan do currently not exits. Soluble klotho (sKL) is a protein in the blood with protective effects
on various organs, and sKL levels are reduced in CKD patients and are thought to contribute to CKD-associated
pathologies, such as cardiovascular damage. Alpha Young LLC has developed an early-stage mimetic protein
of sKL, and here we will introduce and test additional mutations to increase sKL's stability and bioactivity, in
order to develop a novel drug with widespread protective effects that can be injected into patients with CKD and
prolong their survival.
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