Characterization of ENTPD1 as a drug target in PXE - This research will focus on pseudoxanthoma elasticum (PXE), the prototype of heritable ectopic mineralization disorders. Aberrant calcium phosphate deposition in PXE results in characteristic cutaneous findings which signify the potential for development of ocular complications leading to loss of visual acuity and blindness, as well as cardiovascular involvement, including nephrogenic hypertension, intermittent claudication, bleeding from gastrointestinal arteries, early myocardial infarct and stroke. There is no effective treatment for PXE and the disease therefore progresses after diagnosis. PXE is caused by mutations in the ABCC6 gene which encodes ABCC6, an efflux transporter expressed primarily in the liver. Our group elucidated the pathophysiology underlying PXE, by showing that ABCC6 in hepatocytes mediates release of ATP into the blood circulation. Released ATP is extracellularly converted by the ecto-nucleotidase ENPP1 into 1 molecule of AMP and 1 molecule of the potent mineralization inhibitor inorganic pyrophosphate (PPi). PXE patients bearing defective ABCC6 have low PPi plasma concentrations, explaining their progressive calcification of soft connective tissues. Plasma PPi homeostasis is maintained through a complex network of proteins involved in ATP release, and ecto-nucleotidases metabolizing the released nucleotides. ENPP1 is crucial in this respect, as its activity underlies all PPi present in plasma. The ~40% of residual PPi present in plasma of PXE patients is formed out of ATP released by cells independent of ABCC6. Extracellular ATP is also subject to metabolism by other ecto- nucleotidases and the most important competitor of ENPP1 in the circulation is ENTPD1, which converts ATP into 1 molecule of AMP and 2 molecules of inorganic phosphate. Importantly, ENPP1 and ENTPD1 have opposing effects on extracellular PPi homeostasis and, consequently, ectopic calcification. In this study we propose to test the hypothesis that inhibition of ENTPD1 enhances extracellular PPi formation and prevents ectopic calcification in PXE patients We have developed two Specific Aims to test our hypothesis: (1) Thoroughly characterize how competition between ENPP1 and ENTPD1 affects extracellular PPi homeostasis in vitro and (2) Determine the effect of ENTPD1 on plasma PPi concentrations and ectopic calcification in Abcc6-/- mice in vivo. These plans are based on solid preliminary data, and they take advantage of a well-characterized mouse model developed and characterized in our department – the Abcc6-/- mouse –, which recapitulates the clinical, histopathologic, ultrastructural and genetic features of PXE in humans. Collectively, our state-of-the-art studies utilizing both in vitro and in vivo model systems are expected to provide critical preclinical information of the therapeutic potential of ENTPD1 inhibition to restore plasma PPi levels in PXE, with subsequent inhibition of ectopic calcification. Our findings are expected to not only inform development of pharmacologic treatments for PXE, but also for other ectopic calcification disorders, for which no effective or specific therapy is currently available.
