Glycolate Oxidase Is a Safe and Efficient Target for Substrate Reduction Therapy in a Mouse Model of Primary Hyperoxaluria Type I

Abstract

Primary hyperoxaluria type 1 (PH1) is caused by deficient alanine-glyoxylate aminotransferase, the human peroxisomal enzyme that detoxifies glyoxylate. Glycolate is one of the best-known substrates leading to glyoxylate production, via peroxisomal glycolate oxidase (GO). Using genetically modified mice, we herein report GO as a safe and efficient target for substrate reduction therapy (SRT) in PH1. We first generated a GO-deficient mouse (Hao1(-/-)) that presented high urine glycolate levels but no additional phenotype. Next, we produced double KO mice (Agxt1(-/-) Hao1(-/-)) that showed low levels of oxalate excretion compared with hyperoxaluric mice model (Agxt1(-/-)). Previous studies have identified some GO inhibitors, such as 4-carboxy-5-[(4-chlorophenyl)sulfanyl]-1,2,3-thiadiazole (CCPST). We herein report that CCPST inhibits GO in Agxt1(-/-) hepatocytes and significantly reduces their oxalate production, starting at 25 µM. We also tested the ability of orally administered CCPST to reduce oxalate excretion in Agxt1(-/-) mice, showing that 30-50% reduction in urine oxalate can be achieved. In summary, we present proof-of-concept evidence for SRT in PH1. These encouraging results should be followed by a medicinal chemistry programme that might yield more potent GO inhibitors and eventually could result in a pharmacological treatment for this rare and severe inborn error of metabolism.

An Investigational RNAi Therapeutic Targeting Glycolate Oxidase Reduces Oxalate Production in Models of Primary Hyperoxaluria

“…Recent results reported by Martin-Higueras et al 13 support the therapeutic potential for ALN-GO1 to profoundly lower oxalate levels in patients by silencing HAO1 mRNA. In these studies, GO-deficient mice (Hao1 2 /2 ) exhibit asymptomatic glycolate elevation, but no phenotypic differences in urine sediment, crystal deposition, histology, or breeding.…”

“…In fact, as recently described, crossing GO-deficient mice (Hao1 2/2 ) into the AGXT deficient background nearly resolved disease, with oxalate levels near normal, indicating the near total dependence of this pathway in mice on GO. 13 Rodents have evolved with AGTexpressed in both mitochondria and peroxisomes in contrast to only a peroxisomal localization in humans, suggesting that glyoxylate production in rodent mitochondria may be significant. As a result, mice lacking mitochondrial AGT will excrete greater amounts of glyoxylate produced in the mitochondria into the cytosol compared with wild-type mice.…”

“…Although they display hyperoxaluria and crystalluria, rodents can eliminate relatively high oxalate concentrations without renal damage. 13,30 However, it is known that PH1 mice fed ethylene glycol (to further enhance oxalate synthesis) do develop even more profound hyperoxaluria, severe nephrocalcinosis, and renal failure whereas healthy mice Figure 4. Silencing liver HAO1 mRNA leads to increased serum glycolate in cynomolgus monkeys.…”

“…Inhibiting glycolate oxidase (GO), a hepatic, peroxisomal enzyme upstream of AGT, is one possible mechanism for depleting diseased livers of substrate for oxalate synthesis, to potentially prevent the pathology that develops in PH1. [10][11][12][13] GO, encoded by the hydroxyacid oxidase (HAO1) gene, catalyzes the oxidation of glycolate to glyoxylate, the immediate precursor to oxalate synthesis in hepatocytes ( Figure 1). Suppression of GO activity should inhibit oxalate production while causing an accumulation of glycolate.…”

Hydroxyproline Metabolism and Oxalate Synthesis in Primary Hyperoxaluria

“…This cycling most likely plays an important role in the pathophysiology of PH1, due to the hydrogen peroxide produced with each cycle. Recent studies by our group and others have shown that reducing GO activity with siRNA in the Agxt-deficient mouse results in a significant decrease in urinary oxalate excretion, 19,31,32 further supporting that glycolate metabolism is a major source of oxalate in PH1, independent of Hyp catabolism. Subsequent work with rats and monkeys 33 further supported GO as a viable siRNA knock down target for oxalate reduction therapy, and clinical trials in patients with PH1 using GO siRNA are being conducted.…”