['We report three novel inhibitors of the physiological pyrophosphatase activity of alkaline phosphatase and show that these compounds are capable of reducing calcification in two models of vascular calcification (i.e., they suppress in vitro calcification by cultured Enpp1(-/-) VSMCs and they inhibit the increased pyrophosphatase activity in a rat aortic model).', 'Genetic ablation of tissue-nonspecific alkaline phosphatase (TNALP) leads to accumulation of the calcification inhibitor inorganic pyrophosphate (PP(i)). TNALP deficiency ameliorates the hypermineralization phenotype in Enpp1(-/-) and ank/ank mice, two models of osteoarthritis and soft tissue calcification. We surmised that the pharmacological inhibition of TNALP pyrophosphatase activity could be used to prevent/suppress vascular calcification.', 'Comprehensive chemical libraries were screened to identify novel drug-like compounds that could inhibit TNALP pyrophosphatase function at physiological pH. We used these novel compounds to block calcification by cultured vascular smooth muscle cells (VSMCs) and to inhibit the upregulated pyrophosphatase activity in a rat aortic calcification model.', 'Using VSMC cultures, we determined that Enpp1(-/-) and ank/ank VSMCs express higher TNALP levels and enhanced in vitro calcification compared with wildtype cells. By high-throughput screening, three novel compounds, 5,361,418, 5,923,412, and 5,804,079, were identified that inhibit TNALP pyrophosphatase function through an uncompetitive mechanism, with high affinity and specificity when measured at both pH 9.8 and 7.5. These compounds were shown to reduce the calcification by Enpp1(-/-) VSMCs. Furthermore, using an ex vivo rat whole aorta PP(i) hydrolysis assay, we showed that pyrophosphatase activity was inhibited by all three lead compounds, with compound 5,804,079 being the most potent at pH 7.5.'] Do novel inhibitors of alkaline phosphatase suppress vascular smooth muscle cell calcification?