Enzymatic synthesis of UTPγS, a potent hydrolysis resistant agonist of P2U‐purinoceptors

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Abstract
1 The defective Cl− secretion characteristic of cystic fibrosis airway epithelial cells can be bypassed by an alternative Ca2+ dependent Cl− secretory pathway that is activated by extracellular nucleotides, e.g. uridine-5′ triphosphate (UTP), acting on P2U purinoceptors. Since UTP is susceptible to hydrolysis by nucleotidases and phosphatases present in the airways, the identification of stable P2U-purinoceptor agonists would be of therapeutic relevance. 2 Uridine-5′-0-(3-thiotriphosphate) (UTPγS) was synthesized by nucleoside diphosphate kinasecatalyzed transfer of the γ-phosphorothioate from guanosine-5′-0-(3-thiotriphosphate) (GTPγS) or adenosine-5′=O-(3-thiotriphosphate) (ATPγS) to UDP. Formation of UTPγS was illustrated by observation of transfer of 35S from [35S]-GTPγS and transfer of 3H from [3H]-UDP. The chemical identity of high performance liquid chromatography (h.p.l.c.)-purified UTPγS was confirmed by nuclear magnetic resonance analysis. 3 Human 1321N1 astrocytoma cells stably expressing the phospholipase C-coupled human P2U-purinoceptor were utilized to test the activity of UTPγS. UTPγS (EC50 = 240 nM) was essentially equipotent to UTP and ATP for stimulation of inositol phosphate formation. 4 Unlike [3H]-UTP, [3H]-UTPγS was not hydrolyzed by alkaline phosphatase, acid phosphatase, or apyrase. Moreover, no hydrolysis was detected during a 1 h incubation with human nasal epithelial cells. 5 UTPγS was equally potent and efficacious with UTP for stimulation of Cl− secretion by human nasal epithelium from both normal donors and cystic fibrosis patients. Based on its high potency and resistance to hydrolysis, UTPγS represents a promising compound for treatment of cystic fibrosis.

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