Nonhydrolyzable ATP analogues as selective inhibitors of human NPP1: a combined computational/experimental study.

Elevated nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is implicated in health disorders including pathological calcification. Specific NPP1 inhibitors would therefore be valuable for studying this enzyme and as potential therapeutic agents. Here we present a combined computational/experimental study characterizing 13 nonhydrolyzable ATP analogues as selective human NPP1 inhibitors. All analogues at 100 µM inhibited (66-99%) the hydrolysis of pnp-TMP by both recombinant NPP1 and cell surface NPP1 activity of osteocarcinoma (HTB-85) cells. These analogues only slightly altered the activity of other ectonucleotidases, NPP3 and NTPDases. The Ki,app values of the seven most potent and selective inhibitors were in the range of 0.5-56 µM, all with mixed type inhibition, predominantly competitive. Those molecules were docked into a newly developed homology model of human NPP1. All adopted ATP-like binding modes, suggesting competitive inhibition with the endogenous ligand. NPP1 selectivity versus NPP3 could be explained in terms of the electrostatic potential of the two proteins that of NPP1 favoring negatively charged ligands. Inhibitor 2 that had the lowest Ki,app (0.5 µM) was also inactive toward P2Y receptors. Overall, analogue 2 is the most potent and selective NPP1 inhibitor described so far.

Diadenosine 5',5''-(boranated)polyphosphonate analogues as selective nucleotide pyrophosphatase/phosphodiesterase inhibitors.

Nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyze extracellular nucleotides and dinucleotides and thus control purinergic signaling. Enhanced NPP activity is implicated in health disorders such as osteoarthritis and cancer. We designed novel diadenosine polyphosphonate derivatives as potential NPP inhibitors. Analogues 1-4 bear a phosphonate and/or boranophosphate group and/or a 2'-H atom instead of a 2'-OH group. In comparison to ATP, analogues 1-4 were barely hydrolyzed by human NTPDase1, -2, -3, and -8 (<5% hydrolysis) and NPP1 and -3 (≤ 13%) and were not hydrolyzed by ecto-5'-nucleotidase, unlike AMP. These derivatives did not affect NTPDase activity, and analogues 1 and 2 did not inhibit ecto-5'-nucleotidase. All analogues blocked ∼80% of the NPP2-dependent hydrolysis of pnp-TMP, a specific NPP substrate, and inhibited the catabolism of pnp-TMP (K(i) and IC50 both found to be between 10 and 60 µM), Ap5A, and ATP by NPP1. The activity of NPP3 was inhibited to a lesser extent by the new analogues, with compounds 1 and 4 being the most effective in that respect. The analogues dramatically reduced the level of hydrolysis of pnp-TMP at the cell surface of both osteocarcinoma and colon cancer cells. Importantly, analogues 1-4 exhibited significantly reduced agonistic activity toward human P2Y1,11) receptors (except for analogue 1) and no activity with human P2Y2 receptor. Our data provide strong evidence that analogue 2 is the first specific NPP inhibitor to be described.

2-MeS-beta,gamma-CCl2-ATP is a potent agent for reducing intraocular pressure.

Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydrolyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37 degrees C. In human blood serum, analogues 8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t(1/2) 1 h-30 days). Analogues 8-14 were agonists of the P2Y(1) receptor (EC(50) 0.57-9.54 muM). Ocular administration of most analogues into rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC(50) 95.5 nM). Analogue 9 was more effective at reducing IOP than several common glaucoma drugs and represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.

A novel insulin secretagogue based on a dinucleoside polyphosphate scaffold.

Dinucleoside polyphosphates exert their physiological effects via P2 receptors (P2Rs). They are attractive drug candidates, as they offer better stability and specificity compared to nucleotides, the most common P2 receptor ligands. The activation of pancreatic P2Y receptors by nucleotides increases insulin secretion. Therefore, in the current study, dinucleoside polyphosphate analogues (di-(2-MeS)-adenosine-5',5''-P(1),P(4),alpha,beta-methylene-tetraphosphate), 8, (di-(2-MeS)-adenosine-5',5''-P(1),P(4),beta,gamma-methylene-tetraphosphate), 9, and di-(2-MeS)-adenosine-5',5''-P(1),P(3),alpha,beta-methylene triphosphate, 10, were developed as potential insulin secretagogues. Analogues 8 and 9 were found to be agonists of the P2Y(1)R with EC(50) values of 0.42 and 0.46 microM, respectively, whereas analogue 10 had no activity. Analogues 8-10 were found to be completely resistant to hydrolysis by alkaline phosphatase over 3 h at 37 degrees C. Analogue 8 also was found to be 2.5-fold more stable in human blood serum than ATP, with a half-life of 12.1 h. Analogue 8 administration in rats caused a decrease in a blood glucose load from 155 mg/dL to ca. 100 mg/dL and increased blood insulin levels 4-fold as compared to basal levels. In addition, analogue 8 reduced a blood glucose load to normal values (80-110 mg/dL), unlike the commonly prescribed glibenclamide, which reduced glucose levels below normal values (60 mg/dL). These findings suggest that analogue 8 may prove to be an effective and safe treatment for type 2 diabetes.

Identification of hydrolytically stable and selective P2Y(1) receptor agonists.

P2Y nucleotide receptors (P2YRs) are attractive pharmaceutical targets. Most P2YR agonists proposed as drugs consist of a nucleotide scaffold, but their use is limited due to their chemical and enzymatic instabilities. To identify drug candidates, we developed non-hydrolyzable P2YR agonists. We synthesized ATP-beta,gamma-CH(2) analogues 2-4, and evaluated their chemical and metabolic stabilities and activities at P2Y(1,2,4,6) receptors. Analogues 2-4 exhibited t(1/2) values of 14.5-65 h in gastric juice pH. They were completely resistant to alkaline phosphatase for 30 min at 37 degrees C and slowly hydrolyzed in human blood serum (t(1/2) 12.7-71.9 h). In comparison to ATP, analogues 2-4 were barely hydrolyzed by nucleoside triphosphate diphosphohydrolases, NTPDase1,2,3,8 (< 8% hydrolysis), and nucleotide pyrophosphatases, NPP1,3 (< or = 10% hydrolysis). Analogues 2 and 4B were selective agonists of the P2Y(1)R with EC(50)s of 0.08 and 17.2 microM, respectively. These features make analogues 2 and 4B potential therapeutic agents for health disorders involving the P2Y(1)R.