Enzymatic synthesis of UTP gamma S, a potent hydrolysis resistant agonist of P2U-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 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.
Lazarowski
E R
ER
Department of Pharmacology, University of North Carolina, Chapel Hill 27599, USA.
Watt
W C
WC
Stutts
M J
MJ
Brown
H A
HA
Boucher
R C
RC
Harden
T K
TK
D001254:Astrocytoma; D002712:Chlorides / Q000378:metabolism; D003550:Cystic Fibrosis / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D009682:Magnetic Resonance Spectroscopy; D008564:Membrane Potentials; D009296:Nasal Cavity / Q000187:drug effects / Q000378:metabolism; D058913:Purinergic Agonists; D013873:Thionucleotides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology
D002712:Chlorides; D007295:Inositol Phosphates; D058913:Purinergic Agonists; D013873:Thionucleotides; C099105:uridine-5'-O-(3-thiotriphosphate); D014544:Uridine Triphosphate