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 (BH₄) is an essential cofactor for nitric oxide biosynthesis. Substantial clinical evidence indicates that intravenous BH₄ restores vascular function in patients. Unfortunately, oral BH₄ has limited efficacy. Therefore, orally bioavailable pharmacological activators of endogenous BH₄ biosynthesis hold significant therapeutic potential. GTP-cyclohydrolase 1 (GCH1), the rate limiting enzyme in BH₄ 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 BH₄ 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 BH₄ 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 BH₄, 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 BH₄ 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 BH₄ biosynthesis for numerous cardiovascular disorders.