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Validating the GTP ‐cyclohydrolase 1‐feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches
Author(s) -
Hussein D,
Starr A,
Heikal L,
McNeill E,
Chan K M,
Brown P R,
Sutton B J,
McDonnell J M,
Nandi M
Publication year - 2015
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.13202
Subject(s) - gtp cyclohydrolase i , in vivo , tetrahydrobiopterin , allosteric regulation , gtp' , biochemistry , chemistry , phenylalanine , biosynthesis , biophysics , enzyme , pharmacology , biology , nitric oxide synthase , amino acid , microbiology and biotechnology
Background and Purpose 6R‐L‐erythro‐5,6,7,8‐tetrahydrobiopterin ( BH 4 ) is an essential cofactor for nitric oxide biosynthesis. Substantial clinical evidence indicates that intravenous BH 4 restores vascular function in patients. Unfortunately, oral BH 4 has limited efficacy. Therefore, orally bioavailable pharmacological activators of endogenous BH 4 biosynthesis hold significant therapeutic potential. GTP ‐cyclohydrolase 1 ( GCH 1), the rate limiting enzyme in BH 4 synthesis, forms a protein complex with GCH 1 feedback regulatory protein ( GFRP ). This complex is subject to allosteric feed‐forward activation by L ‐phenylalanine ( L ‐phe). We investigated the effects of L ‐phe on the biophysical interactions of GCH 1 and GFRP and its potential to alter BH 4 levels in vivo . Experimental Approach Detailed characterization of GCH 1– GFRP protein–protein interactions were performed using surface plasmon resonance ( SPR ) with or without L ‐phe. Effects on systemic and vascular BH 4 biosynthesis in vivo were investigated following L‐phe treatment (100 mg·kg −1 , p.o.). Key Results GCH 1 and GFRP proteins interacted in the absence of known ligands or substrate but the presence of L ‐phe doubled maximal binding and enhanced binding affinity eightfold. Furthermore, the complex displayed very slow association and dissociation rates. In vivo , L‐phe challenge induced a sustained elevation of aortic BH 4 , an effect absent in GCH 1(fl/fl)‐ T ie2Cre mice. Conclusions and Implications Biophysical data indicate that GCH 1 and GFRP are constitutively bound. In vivo, data demonstrated that L ‐phe elevated vascular BH 4 in an endothelial GCH 1 dependent manner. Pharmacological agents which mimic the allosteric effects of L‐phe on the GCH 1– GFRP complex have the potential to elevate endothelial BH 4 biosynthesis for numerous cardiovascular disorders.