We use cookies to improve your experience. By continuing to browse this site, you accept our cookie policy.×
Research ArticleOpen Accesscc iconby iconnc iconnd icon

Discovery and biological characterization of a novel scaffold for potent inhibitors of peripheral serotonin synthesis

    Nibal Betari

    *Author for correspondence:

    E-mail Address: nibal.betari@uib.no

    Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Postboks 7804, 5020 Bergen, Norway

    ,
    Kristoffer Sahlholm

    Department of Integrative Medical Biology, Wallenberg Centre for Molecular Medicine, Umeå University, Johan Bures väg 12, 901 87 Umeå, Sweden

    ,
    Yuta Ishizuka

    Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Postboks 7804, 5020 Bergen, Norway

    ,
    Knut Teigen

    Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Postboks 7804, 5020 Bergen, Norway

    &
    Jan Haavik

    Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Postboks 7804, 5020 Bergen, Norway

    Division of Psychiatry, Haukeland University Hospital, Jonas Lies vei 65, 5021 Bergen, Norway

    Published Online:https://doi.org/10.4155/fmc-2020-0127

    Aim: Tryptophan hydroxylase 1 (TPH1) catalyzes serotonin synthesis in peripheral tissues. Selective TPH1 inhibitors may be useful for treating disorders related to serotonin dysregulation. Results & methodology: Screening using a thermal shift assay for TPH1 binders yielded Compound 1 (2-(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one), which showed high potency (50% inhibition at 98 ± 30 nM) and selectivity for inhibiting TPH over related aromatic amino acid hydroxylases in enzyme activity assays. Structure–activity relationships studies revealed several analogs of 1 showing comparable potency. Kinetic studies suggested a noncompetitive mode of action of 1, with regards to tryptophan and tetrahydrobiopterin. Computational docking studies and live cell assays were also performed. Conclusion: This TPH1 inhibitor scaffold may be useful for developing new therapeutics for treating elevated peripheral serotonin.

    References

    • 1. Matthes S, Bader M. Peripheral serotonin synthesis as a new drug target. Trends Pharmacol. Sci. 39(6), 560–572 (2018).
    • 2. Yadav VK, Balaji S, Suresh PS et al. Pharmacological inhibition of gut-derived serotonin synthesis is a potential bone anabolic treatment for osteoporosis. Nat. Med. 16(3), 308–312 (2010).
    • 3. Inose H, Zhou B, Yadav VK, Guo XE, Karsenty G, Ducy P. Efficacy of serotonin inhibition in mouse models of bone loss. J. Bone Miner. Res. 26(9), 2002–2011 (2011).
    • 4. Hallen A. Fibrosis in the carcinoid syndrome. Lancet 1(7336), 746–747 (1964).
    • 5. MacDonald RA, Robbins SL, Mallory GK. Dermal fibrosis following subcutaneous injections of serotonin creatinine sulphate. Proc. Soc. Exp. Biol. Med. 97(2), 334–337 (1958).
    • 6. Crane JD, Palanivel R, Mottillo EP et al. Inhibiting peripheral serotonin synthesis reduces obesity and metabolic dysfunction by promoting brown adipose tissue thermogenesis. Nat. Med. 21(2), 166–172 (2015).
    • 7. Oh CM, Namkung J, Go Y et al. Regulation of systemic energy homeostasis by serotonin in adipose tissues. Nat. Commun. 6, 6794 (2015).
    • 8. Margolis KG, Stevanovic K, Li Z et al. Pharmacological reduction of mucosal but not neuronal serotonin opposes inflammation in mouse intestine. Gut 63(6), 928–937 (2014).
    • 9. Dempsie Y, Morecroft I, Welsh DJ et al. Converging evidence in support of the serotonin hypothesis of dexfenfluramine-induced pulmonary hypertension with novel transgenic mice. Circulation 117(22), 2928–2937 (2008).
    • 10. Morecroft I, Dempsie Y, Bader M et al. Effect of tryptophan hydroxylase 1 deficiency on the development of hypoxia-induced pulmonary hypertension. Hypertension 49(1), 232–236 (2007).
    • 11. Jacobsen KK, Kleppe R, Johansson S, Zayats T, Haavik J. Epistatic and gene wide effects in YWHA and aromatic amino hydroxylase genes across ADHD and other common neuropsychiatric disorders: association with YWHAE. Am. J. Med. Genet. B Neuropsychiatr. Genet. 168(6), 423–432 (2015).
    • 12. Zhang X, Gainetdinov RR, Beaulieu JM et al. Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45(1), 11–16 (2005).
    • 13. Matthes S, Mosienko V, Bashammakh S, Alenina N, Bader M. Tryptophan hydroxylase as novel target for the treatment of depressive disorders. Pharmacology 85(2), 95–109 (2010).
    • 14. Patrick RP, Ames BN. Vitamin D hormone regulates serotonin synthesis. Part 1: relevance for autism. FASEB J. 28(6), 2398–2413 (2014).
    • 15. Eissa N, Al-Houqani M, Sadeq A, Ojha SK, Sasse A, Sadek B. Current enlightenment about etiology and pharmacological treatment of autism spectrum disorder. Front. Neurosci. 12, 304 (2018).
    • 16. Fitzpatrick PF, Ralph EC, Ellis HR, Willmon OJ, Daubner SC. Characterization of metal ligand mutants of tyrosine hydroxylase: insights into the plasticity of a 2-histidine-1-carboxylate triad. Biochemistry 42(7), 2081–2088 (2003).
    • 17. Abu-Omar MM, Loaiza A, Hontzeas N. Reaction mechanisms of mononuclear non-heme iron oxygenases. Chem. Rev. 105(6), 2227–2252 (2005).
    • 18. Walther DJ, Bader M. A unique central tryptophan hydroxylase isoform. Biochem. Pharmacol. 66(9), 1673–1680 (2003).
    • 19. Walther DJ, Peter JU, Bashammakh S et al. Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299(5603), 76 (2003).
    • 20. McKinney J, Knappskog PM, Haavik J. Different properties of the central and peripheral forms of human tryptophan hydroxylase. J. Neurochem. 92(2), 311–320 (2005).
    • 21. Amireault P, Sibon D, Cote F. Life without peripheral serotonin: insights from tryptophan hydroxylase 1 knockout mice reveal the existence of paracrine/autocrine serotonergic networks. ACS Chem. Neurosci. 4(1), 64–71 (2013).
    • 22. Cote F, Thevenot E, Fligny C et al. Disruption of the nonneuronal TPH1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc. Natl Acad. Sci. USA 100(23), 13525–13530 (2003).
    • 23. Malek ZS, Dardente H, Pevet P, Raison S. Tissue-specific expression of tryptophan hydroxylase mRNAs in the rat midbrain: anatomical evidence and daily profiles. Eur. J. Neurosci. 22(4), 895–901 (2005).
    • 24. Gutknecht L, Waider J, Kraft S et al. Deficiency of brain 5-HT synthesis but serotonergic neuron formation in TPH2 knockout mice. J. Neural Transm. (Vienna) 115(8), 1127–1132 (2008).
    • 25. Waloen K, Kleppe R, Martinez A, Haavik J. Tyrosine and tryptophan hydroxylases as therapeutic targets in human disease. Expert Opin. Ther. Targets 21(2), 167–180 (2017).
    • 26. Bader M. Inhibition of serotonin synthesis: a novel therapeutic paradigm. Pharmacol. Ther. 205, 107423 (2020).
    • 27. Engelman K, Lovenberg W, Sjoerdsma A. Inhibition of serotonin synthesis by para-chlorophenylalanine in patients with the carcinoid syndrome. N. Engl. J. Med. 277(21), 1103–1108 (1967).
    • 28. Zimmer L, Luxen A, Giacomelli F, Pujol JF. Short- and long-term effects of p-ethynylphenylalanine on brain serotonin levels. Neurochem. Res. 27(4), 269–275 (2002).
    • 29. Cianchetta G, Stouch T, Yu W et al. Mechanism of inhibition of novel tryptophan hydroxylase inhibitors revealed by co-crystal structures and kinetic analysis. Curr. Chem. Genomics 4, 19–26 (2010).
    • 30. Goldberg DR, De Lombaert S, Aiello R et al. Optimization of spirocyclic proline tryptophan hydroxylase-1 inhibitors. Bioorg. Med. Chem. Lett. 27(3), 413–419 (2017).
    • 31. Markham A. Telotristat ethyl: first global approval. Drugs 77(7), 793–798 (2017).
    • 32. Masab M, Saif MW. Telotristat ethyl: proof of principle and the first oral agent in the management of well-differentiated metastatic neuroendocrine tumor and carcinoid syndrome diarrhea. Cancer Chemother. Pharmacol. 80(6), 1055–1062 (2017).
    • 33. Liu Q, Yang Q, Sun W et al. Discovery and characterization of novel tryptophan hydroxylase inhibitors that selectively inhibit serotonin synthesis in the gastrointestinal tract. J. Pharmacol. Exp. Ther. 325(1), 47–55 (2008).
    • 34. Shi H, Cui Y, Qin Y. Discovery and characterization of a novel tryptophan hydroxylase 1 inhibitor as a prodrug. Chem. Biol. Drug Des. 91(1), 202–212 (2018).
    • 35. Petrassi M, Barber R, Be C et al. Identification of a novel allosteric inhibitory site on tryptophan hydroxylase 1 enabling unprecedented selectivity over all related hydroxylases. Front. Pharmacol. 8, 240 (2017).
    • 36. Teigen K, McKinney JA, Haavik J, Martinez A. Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases. Curr. Med. Chem. 14(4), 455–467 (2007).
    • 37. Lo MC, Aulabaugh A, Jin G et al. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal. Biochem. 332(1), 153–159 (2004).
    • 38. Niesen FH, Berglund H, Vedadi M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2(9), 2212–2221 (2007).
    • 39. Wang L, Erlandsen H, Haavik J, Knappskog PM, Stevens RC. Three-dimensional structure of human tryptophan hydroxylase and its implications for the biosynthesis of the neurotransmitters serotonin and melatonin. Biochemistry 41(42), 12569–12574 (2002).
    • 40. McKinney J, Knappskog PM, Pereira J et al. Expression and purification of human tryptophan hydroxylase from Escherichia coli and Pichia pastoris. Protein Expr. Purif. 33(2), 185–194 (2004).
    • 41. Winge I, McKinney JA, Knappskog PM, Haavik J. Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2. J. Neurochem. 100(6), 1648–1657 (2007).
    • 42. Flydal MI, Chatfield CH, Zheng H et al. Phenylalanine hydroxylase from Legionella pneumophila is a thermostable enzyme with a major functional role in pyomelanin synthesis. PLoS ONE 7(9), e46209 (2012).
    • 43. Aubi O, Flydal MI, Zheng H et al. Discovery of a specific inhibitor of pyomelanin synthesis in Legionella pneumophila. J. Med. Chem. 58(21), 8402–8412 (2015).
    • 44. Haavik J, Flatmark T. Rapid and sensitive assay of tyrosine 3-monooxygenase activity by high-performance liquid chromatography using the native fluorescence of DOPA. J. Chromatogr. 198(4), 511–515 (1980).
    • 45. Szigetvari PD, Muruganandam G, Kallio JP et al. The quaternary structure of human tyrosine hydroxylase: effects of dystonia-associated missense variants on oligomeric state and enzyme activity. J. Neurochem. 148(2), 291–306 (2019).
    • 46. Friesner RA, Murphy RB, Repasky MP et al. Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J. Med. Chem. 49(21), 6177–6196 (2006).
    • 47. Dai R, Geders TW, Liu F et al. Fragment-based exploration of binding site flexibility in Mycobacterium tuberculosis BioA. J. Med. Chem. 58(13), 5208–5217 (2015).
    • 48. Morreale FE, Bortoluzzi A, Chaugule VK, Arkinson C, Walden H, Ciulli A. Allosteric targeting of the Fanconi anemia ubiquitin-conjugating enzyme Ube2T by fragment screening. J. Med. Chem. 60(9), 4093–4098 (2017).
    • 49. Sayegh J, Cao J, Zou MR et al. Identification of small molecule inhibitors of Jumonji AT-rich interactive domain 1B (JARID1B) histone demethylase by a sensitive high throughput screen. J. Biol. Chem. 288(13), 9408–9417 (2013).
    • 50. Calvo AC, Scherer T, Pey AL et al. Effect of pharmacological chaperones on brain tyrosine hydroxylase and tryptophan hydroxylase 2. J. Neurochem. 114(3), 853–863 (2010).
    • 51. Thony B, Calvo AC, Scherer T et al. Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase. J. Neurochem. 106(2), 672–681 (2008).
    • 52. Frantom PA, Seravalli J, Ragsdale SW, Fitzpatrick PF. Reduction and oxidation of the active site iron in tyrosine hydroxylase: kinetics and specificity. Biochemistry 45(7), 2372–2379 (2006).
    • 53. Renson J, Weissbach H, Udenfriend S. Hydroxylation of tryptophan by phenylalanine hydroxylase. J. Biol. Chem. 237, 2261–2264 (1962).
    • 54. Jin H, Cianchetta G, Devasagayaraj A et al. Substituted 3-(4-(1,3,5-triazin-2-yl)-phenyl)-2-aminopropanoic acids as novel tryptophan hydroxylase inhibitors. Bioorg. Med. Chem. Lett. 19(17), 5229–5232 (2009).
    • 55. Shi ZC, Devasagayaraj A, Gu K et al. Modulation of peripheral serotonin levels by novel tryptophan hydroxylase inhibitors for the potential treatment of functional gastrointestinal disorders. J. Med. Chem. 51(13), 3684–3687 (2008).
    • 56. Ouyang L, He G, Huang W, Song X, Wu F, Xiang M. Combined structure-based pharmacophore and 3D-QSAR studies on phenylalanine series compounds as TPH1 inhibitors. Int. J. Mol. Sci. 13(5), 5348–5363 (2012).