Identification of a Novel Allosteric Inhibitory Site on Tryptophan Hydroxylase 1 Enabling Unprecedented Selectivity Over all Related Hydroxylases

Figures

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  FIGURE 1. Identification of selectivity for TPH1 over TPH2. Compounds pCPA (A), LP533401 (B) and NVS-TPH146 (C) were pre-incubated with hTPH1 and hTPH2 enzymes for 15 min. Reactions were initiated with the addition of the substrates BH4 and Tryptophan. The reactions were quenched with the addition of 30% Sulfuric Acid at 30 min for hTPH1 and 60 min for hTPH2. The plates were read immediately with a PerkinElmer Envision reader (excitation = 280, emission = 535).

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  FIGURE 2. Surface Plasma Resonance (SPR) sensorgrams for the interaction of TPH1 with various ligands. Ligand (L-Tryp – blue) and compound (LP533401 or NVS-TPH146 – red) were injected singularly or in combination (green). Comparison of the resulting equilibrium responses between the individual or simultaneous injection allowed the classification of their binding mode for LP533401 (A) and NVS-TPH146 (B).

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  FIGURE 3. Location of the novel TPH1 allosteric inhibitory site. The TPH1 protein is shown in gray ribbon with the catalytic site Iron and cofactor shown. The allosteric site is indicated by the brown solvent accessible surface (A). The TPH1 protein residues that form close contacts with the allosteric ligands are shown in atom-type colored capped sticks with the site indicated by the transparent surface (B).

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  FIGURE 4. Mechanism of action of allosteric inhibitors. Double reciprocal plots of initial velocities at various concentration of the inhibitor NVS-TPH180 with varying (A) tryptophan concentration at a fixed non-saturating concentration of BH4 (10 μM) and (B), varying BH4 at fixed non-saturating concentrations of tryptophan (10 μM). The inhibitor concentrations (l) used are as indicated in the graph and are represented in μM concentrations. Data is a mean ± SD from a single experiment, with each point in triplicate. Data was analyzed using Sigmaplot and fitted to a global fit analysis. Best fit anaylsis indicates a mixed partial inhibition with tryptophan with an α-value of 0.477 indicating a preference for the tryptophan bound complex.

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  FIGURE 5. BON (human carcinoid cell) cell responses to TPH1 inhibitors. (A) Inhibition of D4-5-HT production. Compounds were incubated with BON cells for 15 min prior to addition of deuterated substrate. After 2 h intracellular D4-5-HT was extracted and quantified using LCMS/MS. (B) Table of compound efficacy vs. cell toxicity IC50 data. The data are expressed as means ± SEM; (n = 3).

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  FIGURE 6. In vivo Inhibition of de novo synthesis of 5HT-d4 by NVS-TPH120. The study was conducted as a 3-way crossover design. Male beagle dogs received orally vehicle or NVS-TPH120 2 h before and 3 h and 8 h after subcutaneous injection of TRP-d5 at 50 mg kg-1 (n = 6). Serially bled samples were analyzed by LC-MS/MS. (A) Metabolic pathway of TRP-d5 conversion to 5HT-d4. (B) Real values of blood 5-HT-d4 recovered from blood (baseline subtracted). (C) Blood 5HT-d4 levels % relative to the vehicle group. ∗P < 0.05; ∗∗P < 0.01. (D) Blood pharmacokinetic profiles of NVS-TPH120 (n = 6). AAAD: aromatic L-amino acid decarboxylase. The data are expressed as means ± SEM.