Validating the GTP-cyclohydrolase 1-feedback regulatory complex as a therapeutic target using biophysical and in vivo approaches

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 (GCH1), the rate limiting enzyme in BH(4) synthesis, forms a protein complex with GCH1 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 GCH1 and GFRP and its potential to alter BH(4) levels in vivo. EXPERIMENTAL APPROACH: Detailed characterization of GCH1–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: GCH1 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 GCH1(fl/fl)-Tie2Cre mice. CONCLUSIONS AND IMPLICATIONS: Biophysical data indicate that GCH1 and GFRP are constitutively bound. In vivo, data demonstrated that L-phe elevated vascular BH(4) in an endothelial GCH1 dependent manner. Pharmacological agents which mimic the allosteric effects of L-phe on the GCH1–GFRP complex have the potential to elevate endothelial BH(4) biosynthesis for numerous cardiovascular disorders.