Effect of pharmacological chaperones on brain tyrosine hydroxylase and tryptophan hydroxylase 2

Abstract

J. Neurochem. (2010) 114, 853–863. Abstract Phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPH1 and TPH2) are structurally and functionally related enzymes that share a number of ligands, such as amino acid substrates, pterin cofactors and inhibitors. We have recently identified four compounds (I‐IV) with pharmacological chaperone effect for PAH and phenylketonuria mutants (Pey et al. (2008) J. Clin. Invest. 118, 2858–2867). We have now investigated the effect of these compounds on the brain enzymes TH and TPH2, comparative to hepatic PAH. As assayed by differential scanning fluorimetry each of the purified human PAH, TH and TPH2 was differently stabilized by the compounds and only 3‐amino‐2‐benzyl‐7‐nitro‐4‐(2‐quinolyl)‐1,2‐dihydroisoquinolin‐1‐one (compound III) stabilized the three enzymes. We also investigated the effect of compounds II‐IV in wild‐type mice upon oral loading with 5 mg/kg/day. Significant effects were obtained by treatment with compound III – which increased total TH activity in mouse brain extracts by 100% but had no measurable effects either on TPH activity nor on monoamine neurotransmitter metabolites dopamine, dihydroxyphenylacetic acid, homovanillic acid, serotonin and 5‐hydroxyindolacetic acid – and with 5,6‐dimethyl‐3‐(4‐methyl‐2‐pyridinyl)‐2‐thioxo‐2,3‐dihydrothieno[2,3‐d]pyrimidin‐4(1H)‐one (compound IV) – which led to a 10–30% decrease of these metabolites. Our results indicate that pharmacological chaperones aiming the stabilization of one of the aromatic amino acid hydroxylases should be tested on other members of the enzyme family. Moreover, compound III stabilizes in vitro the human TH mutant R202H, associated to autosomal recessive l‐DOPA‐responsive dystonia, revealing the potential of pharmacological chaperones for the treatment of disorders associated with TH misfolding.

Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms

“…S3). This effect of compound 5 resembles what we have previously found with compound IV (Table 2), which was previously tested on TH as a result of its pharmacological chaperone activity on the homologous enzyme PAH [17,28]. Actually, compound IV has a lower effect on the thermal stability of hTH1 than compound 5 (a tiny ΔT m ≈ 0.3°C is measured upon binding of compound IV by DSF) [28].…”

“…A complete characterization of its effects was therefore not possible. Also included for further characterization was compound IV from earlier studies [17,28] (see also Section 3.2) ( Table 2). The name and structure of compounds 1-5 and compound IV are shown in Table 2.…”

“…Whereas compound III behaved as a classical pharmacological chaperone that increased both the thermal stability of TH in vitro and the steady state TH protein level in mice brain, compound IV leads to higher TH activity in brain extracts without apparently affecting the conformational stability of the enzyme [28]. Despite the potential effect of compounds III and IV for the treatment of TH deficiencies, these compounds are not TH-specific and compound IV also inhibits isoform 2 of tryptophan hydroxylase (TPH2) [28], the BH 4 -dependent aromatic amino acid hydroxylase that catalyzes the ratelimiting step in the synthesis of serotonin. We thus aimed to obtain more effective stabilizing compounds with higher specificity for TH.…”

“…Furthermore, a number of studies have revealed several small molecular weight compounds with pharmacological chaperone potential for PKU [17,27]. Some of these compounds, such as compounds III (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) and IV (5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno [2,3-d]pyrimidin-4(1H)-one) from Pey et al [17], have also been tested on TH in vitro and supplemented to mice [28]. Whereas compound III behaved as a classical pharmacological chaperone that increased both the thermal stability of TH in vitro and the steady state TH protein level in mice brain, compound IV leads to higher TH activity in brain extracts without apparently affecting the conformational stability of the enzyme [28].…”

“…Some of these compounds, such as compounds III (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) and IV (5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno [2,3-d]pyrimidin-4(1H)-one) from Pey et al [17], have also been tested on TH in vitro and supplemented to mice [28]. Whereas compound III behaved as a classical pharmacological chaperone that increased both the thermal stability of TH in vitro and the steady state TH protein level in mice brain, compound IV leads to higher TH activity in brain extracts without apparently affecting the conformational stability of the enzyme [28]. Despite the potential effect of compounds III and IV for the treatment of TH deficiencies, these compounds are not TH-specific and compound IV also inhibits isoform 2 of tryptophan hydroxylase (TPH2) [28], the BH 4 -dependent aromatic amino acid hydroxylase that catalyzes the ratelimiting step in the synthesis of serotonin.…”