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. 1996 Jan;117(1):203–209. doi: 10.1111/j.1476-5381.1996.tb15175.x

Enzymatic synthesis of UTP gamma S, a potent hydrolysis resistant agonist of P2U-purinoceptors.

E R Lazarowski 1, W C Watt 1, M J Stutts 1, H A Brown 1, R C Boucher 1, T K Harden 1
PMCID: PMC1909382  PMID: 8825364

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'-O-(3-thiotriphosphate) (UTP gamma S) was synthesized by nucleoside diphosphate kinase-catalyzed 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 observation of transfer of 35S from [35S]-GTP gamma S and transfer of 3H from [3H]-UDP. The chemical identity of high performance liquid chromatography (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 P2U-purinoceptor were utilized to test the activity of UTP gamma S. UTP gamma S (EC50 = 240 nM) was essentially equipotent to UTP and ATP for stimulation of inositol phosphate formation. 4. Unlike [3H]-UTP, [3H]-UTP gamma S 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. UTP gamma S 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, UTP gamma S represents a promising compound for treatment of cystic fibrosis.

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Selected References

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