H. Alex Brown
Principle Investigator; Professor of Pharmacology, Chemistry, and Biochemistry; Associate Director, VICB;
Last active: 2/12/2015

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

Lazarowski ER, Watt WC, Stutts MJ, Brown HA, Boucher RC, Harden TK
Br J Pharmacol. 1996 117 (1): 203-9

PMID: 8825364 · PMCID: PMC1909382 · DOI:10.1111/j.1476-5381.1996.tb15175.x

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.

MeSH Terms (13)

Astrocytoma Chlorides Cystic Fibrosis Epithelium Humans Inositol Phosphates Magnetic Resonance Spectroscopy Membrane Potentials Nasal Cavity Purinergic Agonists Thionucleotides Tumor Cells, Cultured Uridine Triphosphate

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