ABSTRACT Oxalate is an anion with no known biological function in humans. Oxalate is ingested through diet and also generated by liver as a metabolic waste product. The majority of oxalate (~90%) is excreted by the kidney with some excretion in stool. In the kidney, oxalate forms poorly soluble calcium oxalate crystals which can lead to nephrolithiasis, nephrocalcinosis and even chronic kidney disease (CKD). Hyperoxaluria is a major risk factor for calcium oxalate kidney stones (the most common type constituting 2/3 of all stones), and recently recognized as a risk factor for CKD progression. Importantly, certain gastrointestinal diseases (bariatric surgery, inflammatory bowel disease, pancreatic insufficiency) are associated with hyperabsorption of dietary oxalate in colon, significant hyperoxaluria and urinary stone burden (i.e. enteric hyperoxaluria). Here, we propose a novel strategy for treatment of hyperoxaluria by blocking oxalate uptake in colon and promoting stool excretion, which is predicted to reduce urinary oxalate burden and protect kidneys from the detrimental effects of hyperoxaluria. The target is SLC26A3, an anion (oxalate, Cl-, HCO3-) exchanger highly expressed in colon facilitating oxalate uptake. SLC26A3 inhibition is a compelling approach for treatment of hyperoxaluria as suggested by 50-70% lower urine oxalate excretion in knock-out mice and humans with rare SLC26A3 mutations. We recently discovered first-in-class SLC26A3 inhibitors with nanomolar potency and demonstrated proof-of-concept efficacy of a candidate in mouse models of hyperoxaluria and oxalate nephropathy. SLC26A3 inhibitors will be advanced as first-in-class drugs for hyperoxaluria and calcium oxalate kidney stones. Recognizing the importance of having back-up candidates, in Aim 1 additional high-throughput screening and medicinal chemistry will be done to identify novel scaffolds with nanomolar potency and good solubility with distinct sites of action (intracellular vs. extracellular), metabolic stability and good pharmacokinetics. The compounds identified and optimized in Aim 1 will be tested in established models of hyperoxaluria and oxalate nephropathy in mice, as well as in other clinically relevant models of hyperoxaluria including obesity, cystic fibrosis and bariatric surgery-associated hyperoxaluria, and primary hyperoxaluria. Candidates with good efficacy in these models will be tested in Aim 3 for in vitro and in vivo toxicity. The goal of these proposed experiments is to select one or two lead candidate SLC26A3 inhibitors with good animal efficacy and excellent safety profile for further pre-clinical development.