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

Abstract

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 (100mg·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.