ENZYMATIC-SYNTHESIS OF UTP-GAMMA-S, A POTENT HYDROLYSIS RESISTANT AGONIST OF P-2U-PURINOCEPTORS

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 P-2U purinoceptors. Since UT P is susceptible to hydrolysis by nucleotidases and phosphatases prese nt in the airways, the identification of stable P-2U-purinoceptor agon ists would be of therapeutic relevance. 2 Uridine-5'-O-(3-thiotriphosp hate) (UTP gamma S) was synthesized by nucleoside diphosphate kinase-c atalyzed transfer of the gamma-phosphorothioate from guanosine-5'-O-(3 -thiotriphosphate) (GTP gamma S) or adenosine-5'=O-(3-thiotriphosphate ) (ATP gamma S) to UDP. Formation of UTP gamma S was illustrated by ob servation of transfer of S-35 from [S-35]-GTP gamma S and transfer of H-3 from [H-3]-UDP. The chemical identity of high performance liquid c hromatography (h.p.l.c.)-purified UTP gamma S was confirmed by nuclear magnetic resonance analysis. 3 Human 1321N1 astrocytoma cells stably expressing the phospholipase C-coupled human P-2U-purinoceptor were ut ilized to test the activity of UTP gamma S. UTP gamma S (EC(50)=240 nM ) was essentially equipotent to UTP and ATP for stimulation of inosito l phosphate formation. 4 Unlike [H-3]-UTP, [H-3]-UTP gamma S was not h ydrolyzed by alkaline phosphatase, acid phosphatase, or apyrase. Moreo ver, no hydrolysis was detected during a 1 h incubation with human nas al epithelial cells. 5 UTP gamma S was equally potent and efficacious with UTP for stimulation of Cl- secretion by human nasal epithelium fr om both normal donors and cystic fibrosis patients. Based on its high potency and resistance to hydrolysis, UTP gamma S represents a promisi ng compound for treatment of cystic fibrosis.