Tetrahydrobiopterin: Beyond Its Traditional Role as a Cofactor

Abstract

Tetrahydrobiopterin (BH4) is an endogenous cofactor for some enzymatic conversions of essential biomolecules, including nitric oxide, and monoamine neurotransmitters, and for the metabolism of phenylalanine and lipid esters. Over the last decade, BH4 metabolism has emerged as a promising metabolic target for negatively modulating toxic pathways that may result in cell death. Strong preclinical evidence has shown that BH4 metabolism has multiple biological roles beyond its traditional cofactor activity. We have shown that BH4 supports essential pathways, e.g., to generate energy, to enhance the antioxidant resistance of cells against stressful conditions, and to protect from sustained inflammation, among others. Therefore, BH4 should not be understood solely as an enzyme cofactor, but should instead be depicted as a cytoprotective pathway that is finely regulated by the interaction of three different metabolic pathways, thus assuring specific intracellular concentrations. Here, we bring state-of-the-art information about the dependency of mitochondrial activity upon the availability of BH4, as well as the cytoprotective pathways that are enhanced after BH4 exposure. We also bring evidence about the potential use of BH4 as a new pharmacological option for diseases in which mitochondrial disfunction has been implicated, including chronic metabolic disorders, neurodegenerative diseases, and primary mitochondriopathies.

Keywords: antioxidant; inflammation; memory; mitochondrial enhancer; neopterin; oxidative stress; sepiapterin.

Figure 2

(a) Phenylalanine (PAH), tyrosine (TH), and tryptophan (TPH) hydroxylases are dependent on tetrahydrobiopterin (BH4) for their biological activities. Due to its cofactor function, BH4 is required to synthesize tyrosine from phenylalanine, dopamine from tyrosine, and serotonin from tryptophan. After functioning as a cofactor, BH4 is regenerated via the BH4 recycling pathway. Dihydrobiopterin quinoid (qBH2) is then formed by pterin 4α-carbinolamine dehydratase (PCD) and dihydropteridine reductase (DHPR). Protein levels are highest in kidney and liver for PAH; in the adrenal gland and brain for TH; and in the gut and brain for TPH1 and TPH2, respectively. (b) Tetrahydrobiopterin (BH4) availability is compromised by the uncoupling of nitric oxide synthase III (NOSIII). Coupled NOSIII: L-arginine and molecular oxygen (O₂) are the substrates for NOS III to produce _L-citrulline and nitric oxide (NO). The reaction requires as cofactors, the mandatory presence of O₂ and appropriate levels of BH4, as well as reduced nicotinamide adenine dinucleotide phosphate (NADPH). Coupled NOSIII is presented as a heme-containing dimer stabilized by zinc (Zn). Zn is responsible for connecting two NOSIII monomers at the heme group site. BH4 exerts structural and biochemical functions, helping to stabilize the dimer and controlling the coupling of O₂ to _L-arginine oxidation. When BH4 levels become deficient and the levels of its oxidized product dihydrobiopterin (BH2) increase (reduced BH4/BH2 ratio), the NOSIII-catalyzed reaction results in the formation of _L-citrulline, NO, and superoxide anion (O₂⁻). O₂⁻ is a very reactive radical species that produces peroxynitrite (ONOO⁻), a highly reactive oxidant that favors BH4 oxidation. Abbreviations: Fe²⁺: iron; NADP⁺: nicotinamide adenine dinucleotide phosphate. Adapted from Kim and Han, 2020 [53]. The symbol “^•” is used to indicate the presence of an unpaired electron in ^•NO (nitric oxide), O₂^• (superoxide anion), and ONOO⁻ (peroxynitrite).

Figure 1

Metabolic pathways involved in the biosynthesis of tetrahydrobiopterin (BH4). De novo pathway: guanosine triphosphate cyclohydrolase (GTPCH), 6-pyruvolyl tetrahydropterin synthase (PTPS), and sepiapterin reductase (SPR) transform guanosine triphosphate into BH4. The last enzymatic step catalyzed by SPR can be overcome by the unspecific reductases aldose reductase (AR) and carbonyl reductase (CR). This is possible due to an active interaction between the de novo and the salvage pathways, where AR, CR, and/or SPR utilize intermediates of the de novo pathway to generate the key intermediate of the salvage pathway, sepiapterin. Salvage pathway: sepiapterin is transformed into dihydrobiopterin (BH2), then reduced to BH4 by dihydrofolate reductase (DHFR). Recycling pathway: Pterin-4-alpha-carbinolamine dehydratase (PCD) transforms BH2 in dihydrobiopterin quinoid (qBH2), which is reduced back to BH4 by dihydropteridine reductase (DHPR). BH4 is an obligatory cofactor for the activity of the aromatic amino acid hydroxylases, phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TPH), for all isoforms of nitric oxide synthase (NOS), and for alkylglycerol monooxygenase (AGMO). Abbreviations: IL-1β: interleukin-1 beta; TNF-α: tumor necrosis factor-alpha; INF-γ: interferon-gamma; IL-6: interleukin-6; LPS: lipopolysaccharide.

Figure 3

Tetrahydrobiopterin (BH4) pathway in the brain of naïve mice and the effect of its metabolites on mitochondrial parameters in sensory neurons. (a,b) Increased mitochondrial number in dorsal root ganglia neurons (sensory neurons) exposed to 50 nM neopterin for 24 h. (b) Arrows denote increased organelle number. Bars indicate 500 nm. Electron microscopy micrographs: 10,000× magnification. (c,d) Increased Nrf-2 and Tfam expression in different brain regions after 24 h of a single intracerebroventricular injection of 4 pmol BH4 (1 μL) in C57Bl6 mice. St: striatum; Hip: hippocampus; PFC: prefrontal cortex; aCSF: artificial cerebrospinal fluid. (e) Expression of the genes coding dihydropteridine reductase (DHPR; BH4 recycling pathway), dihydrofolate reductase (DHFR; BH4 salvage pathway), and sepiapterin reductase (SPR; BH4 de novo and salvage pathways) in the hippocampus of naïve C57Bl6 mice. 1, 3, 5, and 7 m: 1, 3, 5 or 7-month-old mice. * p < 0.05.

Figure 4

Food and Drug Administration (FDA)-approved clinical trials for the off-label use of tetrahydrobiopterin (BH4) for a variety of disorders not primarily affecting phenylalanine (Phe) metabolism. (a) Completed clinical trials where BH4 was administered to individuals affected by heart and vascular diseases, kidney, lung, liver, central nervous system (CNS), and genetic disorders. Other miscellaneous conditions were also included in the clinical trials, such as rheumatologic diseases, menopause, and aging. (b) Clinical trials involving the administration of BH4 in children affected by genetic disorders. Circled numbers correspond to the number of clinical trials based on BH4 administration. * The study was withdrawn before enrolling its first participant, due to contractual issues.

Figure 5

Tetrahydrobiopterin (BH4) metabolism as a central hub regulating physiological and toxic pathways. Normal BH4 levels (green circle): Physiological levels of BH4 sustain the traditional coenzyme activity of the pathway, favoring the correct metabolism of aromatic amino acids and ether lipids, and the biosynthesis of nitric oxide. Under these conditions, appropriate BH4 levels activate energy metabolism, enhance cellular resistance to oxidative stress, modulate the inflammatory response, facilitate learning and memory, regulate immune system activity, increase vascular activity, and exert neuroprotective effects. Reduced BH4 levels (orange circle): When BH4 levels are perturbed, ATP synthesis and brain lipid signaling are impaired, an oxidant status is induced, neurotransmission is compromised, and inflammation is favored. Pathologically augmented BH4 levels (brown circle): Excessive intracellular BH4 levels induce mitochondrial dysfunction, compromise memory and learning, increase the aggressivity of the immune system, promote the progression of inflammatory and autoimmune diseases, and elicit chronic pain. Thus, BH4 metabolism can be considered a double-edged sword: too little or too much results in cytotoxicity.