Keynote
A Quest for a Disease Modifying Drug for the Treatment of Osteoarthritis

Ecto-nucleotide pyro­phosphatase-1 (NPP1) hydrolyzes phospho­diester bonds of ATP resulting mainly in the formation of AMP and pyrophosphate (PP_i). NPP1’s activity plays a deleterious function in osteoarthritis (OA)/calcium pyrophosphate deposition (CPPD) disease. Hence, inhibitors of NPP1 represent a medical need. We synthesized ATP-α-thio-β,γ-CH₂ (1), ATP-α-thio-β,γ-CCl₂ (2), ATP-α-CH₂-γ-thio (3) and established their resistance to hydrolysis by NPP1,-3 and NTPDase1,-2,-3 and -8 (<5% hydrolysis). Analogues 1-3 at 100 mM inhibited NPP1 and NPP3 by > 90% and 23-43%, respectively, and only slightly affected (0-40%) hydrolysis of ATP by NTPDase1,-2,-3,-8. Analogue 3 is a potent NPP1 inhibitor, K_i 20 nM. Analogue 2a is a selective NPP1 inhibitor, K_i 685 nM. Analogues 1-3 were mostly non-agonists of P2Y₁/P2Y₂/P2Y₁₁ receptors. We next developed NPP1 inhibitors based on uridine 5’-P_α,α-di-thiophosphate analogues, 4-7, to maintain the favorable pharmacological properties of 1 and 2, while avoiding a chiral center at P_α-phosphate, and the waste of half of the nucleotide. Analogues 4-7 proved to be stable to air-oxidation and to acidic and basic pH. All analogues potently inhibited hNPP1 (80-100% inhibition) at 100 µM, with no inhibition of the related NPP3 and ectonucleotidases (NTPDase1,2,3,8). Likewise, these compounds showed nearly no activity at P2Y_2,4,6-receptors. A promising analogue was diuridine-5`,5”-O-P_α,α–tetrathiotetraphosphate, 7, Ki 27 nM. Docking simulations have indicated that the origin of enhanced NPP1 inhibitory activity and selectivity of 7 is the simultaneous occupancy of both binding-sites of the NPP1 by 7. NPP1 inhibitor, 2, was not toxic to chondrocytes and was stable in culture medium and human plasma. 2 effectively inhibited NPP1 activity in whole human cartilage and in chondrocytes and reduced extracellular PP_i accumulation. Therefore, we suggest potent and selective inhibitors of NPP1 for lowering extracellular PP_i levels in cartilage for preventing and treating OA/CPPD.