#1723 A positive functional modulator of ABCC6 decreases vascular calcification and improves kidney function in a rat adenine diet model of chronic kidney disease

Abstract Background and Aims Vascular calcification (VC) is one consequence of the ionic and metabolic imbalance observed in chronic kidney disease (CKD). Greater VC content is demonstrated in advanced CKD stages and leads to significantly higher rates of cardiovascular events and mortality. Multiple factors influence the progression of VC, including the levels of inorganic pyrophosphate (PPi), which inhibits VC. Several proteins maintain the homeostatic balance of anti-calcific PPi with pro-calcific inorganic phosphate (Pi). Among these, ABCC6, which is expressed on the basolateral membrane of hepatocytes, facilitates ATP release into circulation where it can be broken down into AMP and PPi by enzymes such as ENPP1. Rectify has developed an efficient platform capable of delivering small-molecule, positive functional modulators (PFMs) which enhance the function of proteins. We hypothesized that an ABCC6 PFM may increase ATP release from hepatocytes, leading to increased PPi levels and subsequent slowing or prevention of VC progression in the context of CKD. Method Rectify's proprietary small molecule library was screened to identify PFMs that increased ABCC6 expression. These PFMs were assessed in primary human hepatocytes (PHH) for their potential to impact ABCC6 protein levels using a novel AlphaLISA assay. In parallel, secreted PPi levels were measured from the media using an assay adapted from the literature. Additional SAR optimization provided ABCC6 PFM analogs with improved in vivo pharmacokinetic properties. One such potent and selective PFM, RTY-822, was dosed orally at 30 and 100 mg/kg once daily for 6 weeks in a rat adenine diet VC model with modified diet composition and supplemented with calcitriol dosing 3x/week. Calcium levels were assessed in the aorta and femoral artery using mass spectrometry and calcification was visualized via von Kossa histological staining. PPi levels were measured in terminal plasma samples and liver ABCC6 expression was measured by western blot. Kidney function was assessed using serum creatinine, urea, and estimated glomerular filtration rate (eGFR). KIM-1 levels were determined in plasma and in kidney tissue lysates. Results RTY-822, a potent and selective ABCC6 PFM, was identified using the Rectify platform. RTY-822 demonstrated significant and correlated increases in ABCC6 protein and PPi levels in PHHs. Rats on the modified adenine diet for 6 weeks showed decreased kidney function and significant VC in both the aorta and femoral artery. 100 mg/kg RTY-822 significantly reduced VC in both vascular beds as determined by both types of calcium measurement. RTY-822 also dose-dependently decreased serum creatinine and urea levels and increased eGFR, indicating improved kidney function. RTY-822 dosing led to reduced KIM-1 levels in the plasma and increased KIM-1 in the kidney, consistent with less kidney damage and corroborating the improved kidney function measurements. Conclusion Rectify's platform enabled rapid identification of compounds with the potential to increase ABCC6 protein and PPi levels in vitro, including in translationally relevant PHHs. Compound dosing in vivo led to a significant reduction in VC and significant improvements in kidney function in a modified CKD model, thereby demonstrating the importance of ABCC6 in the progression of VC-associated with CKD.