6R l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for several enzymes including phenylalanine hydroxylase and the nitric oxide synthases (NOS). Oral supplementation of BH4 has been successfully employed to treat subsets of patients with hyperphenylalaninaemia. More recently, research efforts have focussed on understanding whether BH4 supplementation may also be efficacious in cardiovascular disorders that are underpinned by reduced nitric oxide bioavailability. Whilst numerous preclinical and clinical studies have demonstrated a positive association between enhanced BH4 and vascular function, the efficacy of orally administered BH4 in human cardiovascular disease remains unclear. Furthermore, interventions that limit BH4 bioavailability may provide benefit in diseases where nitric oxide over production contributes to pathology. This review describes the pathways involved in BH4 bio-regulation and discusses other endogenous mechanisms that could be harnessed therapeutically to manipulate vascular BH4 levels.

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GTP-cyclohydrolase-1 (GTPCH1) catalyses the rate-limiting step in the biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor for enzymes including aromatic amino acid hydroxylases and nitric oxide synthases. Strategies that increase vascular BH4 biosynthesis represent a promising therapeutic approach for the treatment of endothelial dysfunction. GTPCH1 is subject to feedback and feed-forward regulation by BH4 and L-phenylalanine (L-phe) respectively, via an allosteric protein interaction with GTPCH1 feedback regulatory protein (GFRP). The aim of this thesis was to investigate the GTPCH1-GFRP interaction using recombinant proteins, and to validate the functional significance of the interaction in the vasculature using animal models. Human GTPCH1 and GFRP proteins were recombinantly expressed. Studies compared the activity and protein interactions of native GTPCH1 with a truncated mutant. A kinetic GTPCH1 activity assay was modified to enable a high-throughput screen of a fragment library. GTPCH1-GFRP interactions were assessed using surface plasmon resonance (SPR). Finally, in-vivo and ex-vivo functional studies in rodents investigated the effects of L-phe on vascular function and BH4 levels. Studies using recombinant proteins revealed the activity of truncated GTPCH1 exceeds that of native GTPCH1. A high-throughput screen successfully identified four compounds that modulate GTPCH1 activity. Biophysical analysis (SPR) demonstrated that both native and truncated GTPCH1 bind to GFRP in the absence of natural ligands. The kinetics and binding rate constants have been reported for the first time. In functional studies, oral L-phe supplementation in rodents led to a rise of BH4 levels within aortic tissue, and reversed vascular dysfunction observed in vessels obtained from spontaneously hypertensive rats. This thesis demonstrates that modulation of the GTPCH1-GFRP interactions represents a novel therapeutic target to regulate endogenous BH4 levels. SPR data suggests that GTPCH1 and 2 GFRP are constitutively bound in-vivo and indicate that the N-terminal region of GTPCH1 may directly interact with GFRP and modulate basal enzyme activity. The functional effects of L-phe on vascular BH4 levels and function, validate the potential of the GTPCH1-GFRP pathway as a therapeutic target for cardiovascular disease. 3

Background and Purpose6R‐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 BH4 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 BH4 levels in vivo.Experimental ApproachDetailed 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 BH 4, an effect absent in GCH1(fl/fl)‐Tie2Cre mice.Conclusions and ImplicationsBiophysical 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 BH4 biosynthesis for numerous cardiovascular disorders.

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