What is tryptophan hydroxylase

Two binding pockets were defined for docking to TPH1; 1 the cofactor pocket, defined by the center of the cofactor in the crystal structure, and 2 the postulated allosteric pocket, where the center of the docking grid box was defined as the center of residues that form close contacts with allosteric ligands as described by Petrassi et al. Omeprazole is a racemate of two stereoisomers which have been found to have a pKa-value of 7. The animals in the different treatment groups were matched for age.

Animals were housed and tested in compliance with the guidelines described in the Guide for the Care and Use of Laboratory Animals Clark et al. All efforts were made to minimize animal suffering and the number of animals used. While all treated animals were tested in the tail suspension test, brain tissue from eight of the vehicle-treated and nine of the omeprazole-treated mice was used for subsequent analysis of 5-HT content.

Serum samples for 5-HT analysis were drawn from another nine vehicle-treated and eight omeprazole-treated mice. Immobility time in the tail suspension test is known to be sensitive to manipulations that alter brain 5-HT content Garcia-Miralles et al.

Animals were subjected to the tail suspension test 20 h after the last administration of vehicle or omeprazole. By use of surgical tape, a cotton thread was affixed to the mouse tail, 1. The cotton thread was fastened to a horizontal metal bar, leaving the mouse suspended in the air, 20 cm above the floor of a cage containing sawdust. Each animal was suspended for 6 min and the time spent immobile during suspension was counted using a stopwatch. Immobility time was defined as the absence of movements of limbs or trunk.

Thus, movements of the head alone, such as sniffing, or swinging, pendulum-like motion of the animal resulting from previous bouts of activity, were not considered as mobility. The Mann-Whitney test was used for statistical comparison of immobility time between vehicle- and omeprazole-treated animals.

Mice were rapidly sacrificed by cervical dislocation and whole blood was obtained by cardiac puncture, collected in clean polypropylene tubes and placed on ice. Mice were rapidly sacrificed by cervical dislocation and one brain hemisphere from each animal dissected in 1 ml of ice-cold H 2 O and homogenized in a 1 ml Potter-Elvehjem glass tube using a homogenizer-stirrer HSE; Witeg Labortechnik GmbH, Wertheim, Germany with 10 strokes at — rotations per min.

Mobile phase was A Ultrapure water with 0. Sample-to-sample differences in recovery, liquid handling, and ionization efficiency were compensated for by normalizing to the amount of 5-HT- d 4 detected in the samples.

Analyst 1. A calibration curve was constructed with 5-HT standard solutions between 0. The following Eq. As TPH1 is responsible for the majority of 5-HT production in peripheral tissues, we were primarily interested in finding new TPH1 inhibitors, rather than TPH2 inhibitors, due to the potential usefulness of the former in treating dysregulation of peripheral 5-HT.

The T m of TPH1 was measured by monitoring the fluorescence intensity of a dye, SYPRO orange, the fluorescence of which increases upon interaction with hydrophobic parts of the denatured protein.

Primary hits identified using DSF. Following up on the discovery of omeprazole as a TPH1 inhibitor, enzymatic assays of both TPH1 and TPH2 activity were used to investigate the effect of several proton pump inhibitors; esomeprazole, ilaprazole, lansoprazole, R-lanzoprazole, pantoprazole, rabeprazole, and tenatoprazole, which are structural analogues of omeprazole.

Pantoprazole, lansoprazole, and R -lansoprazole were 6-fold less potent inhibitors of TPH1 than omeprazole, but their potencies were in the same range as omeprazole at TPH2. TABLE 1. In vitro enzyme activity assays were used to evaluate the effect of proton pump inhibitors to inhibit tryptophan hydroxylase 1 TPH1 ; tryptophan hydroxylase 2 TPH2. We chose to focus our further investigation on omeprazole, since it is the most widely used of the proton pump inhibitors. The enzyme assays were performed as described above.

The mechanism of action of omeprazole at TPH1 was investigated by measuring TPH1 inhibition at different concentrations of omeprazole, L-Trp substrate, and tetrahydrobiopterin BH 4 cofactor.

When enzyme activity was measured as a function of substrate L-Trp concentration 0. As shown in Table 2 and Figure 3A , there was a progressive decrease in V max whereas K M remained essentially unchanged with increasing concentrations of omeprazole.

Likewise, when enzyme activity was measured as a function of co-factor concentration 2. These results suggest that omeprazole is a non-competitive inhibitor, both with respect to L-Trp and to BH 4. TABLE 2. Enzyme kinetic parameters of tryptophan hydroxylase one in the absence and presence of omeprazole. Mechanism of TPH1 inhibition by omeprazole.

The Michaelis-Menten equation was fitted to data using nonlinear regression. The fits are consistent with a decrease in V max with increasing concentrations of compound, while K M remained virtually unchanged. The selectivity of omeprazole to inhibit TPH1 over the other aromatic amino acid hydroxylases was also investigated. Finally, the inhibitory potency of omeprazole at full-length TPH1 was studied and found to be virtually identical to that observed in experiments with doubly truncated TPH1 Supplementary Figure S2.

Selectivity of omeprazole to inhibit TPH1 over other aromatic amino acid hydroxylases. The effects of omeprazole, esomeprazole, ilaprazole, lansoprazole, R-lanzoprazole, pantoprazole, rabeprazole, and tenatoprazole on MAO-A enzymatic activity were tested using a fluorescence-based MAO-A activity assay measuring the formation of H 2 O 2 as a byproduct of the enzymatic activity Figure 5. Inhibitory activities of omeprazole and its analogues at MAO-A.

In vitro enzyme activity assays were used to evaluate the potencies of omeprazole and its analogues to inhibit monoamine oxidase-A MAO-A. Omeprazole overlaps with the binding site of the known inhibitor, harmine, present in the original crystal structure Son et al.

Docking of omeprazole to TPH1. A Docking of omeprazole to the active site of TPH1. Omeprazole is shown in ball and stick representation with carbons colored gray while the biopterin cofactor is shown in sticks with green carbons. The active site iron is shown as an orange sphere. Superimposed is also the inhibitor from Lexicon Pharmaceuticals LP in sticks with yellow carbons. Omeprazole partially overlaps with the binding pockets of both the cofactor and LP when docked to the active site of TPH1.

B Docking of omeprazole to the postulated allosteric site of TPH1. Omeprazole shown as ball and sticks docks close to the surface of the postulated allosteric pocket of TPH1, near the four phenylalanines defining the entrance of the site F, F, F and F , shown as green sticks in the figure.

The docking pose of omeprazole with the highest docking score is shown with in ball and stick with gray carbons. Also shown is the binding pose of harmine in sticks with green carbons. The docking pose of omeprazole overlaps entirely with harmine in the binding pocket of MAO-A. TABLE 3. Omeprazole has commonly been administered i. We were interested in finding out how the seemingly opposing actions of omeprazole, as a TPH inhibitor on the one hand and an MAO-A inhibitor on the other, might affect its in vivo profile.

Twenty hours after the last dose, animals were subjected to the tail suspension test; a behavioral readout which is sensitive to CNS 5-HT levels and commonly used to assess antidepressant-like activity of experimental compounds. For analysis of brain and serum 5-HT content, the animals were sacrificed by cervical dislocation immediately after behavioral testing and brain tissue and serum were harvested.

Omeprazole-treated animals displayed significantly higher 5-HT content in both brain Figure 8A and serum Figure 8B compared to vehicle controls. In the present investigation, we showed that the proton pump inhibitor, omeprazole, as well as several analogues, inhibit TPH1 and TPH2 with low micromolar potency. Omeprazole was the first proton pump inhibitor when approved for clinical use in Lindberg and Carlsson, It has been designated an essential medicine by the World Health Organization, was the most commonly prescribed drug in the United States in and can be obtained without a prescription in many countries WHO, Although, for example, ilaprazole showed higher potency to inhibit TPH1, we chose to focus our further investigative efforts on omeprazole, given its widespread use.

Kinetic measurements of omeprazole inhibition of TPH1 suggest that omeprazole binds in a noncompetitive fashion with respect to both the cofactor and the substrate. This may suggest that omeprazole does not bind to the tryptophan or cofactor binding sites, but rather to an allosteric site in the protein. Our computational docking results are also compatible with an allosteric mode of action see below.

Allosteric TPH1 inhibitors are of potential interest for further development, as such ligands can be expected to bind outside the conserved active site and thus, potentially, show greater selectivity between the different aromatic amino acid hydroxylases. Selective TPH1 inhibitors are of interest from a medicinal chemistry perspective, since elevated peripheral 5-HT is an important component of several disease conditions, such as carcinoid syndrome; a malignancy-related hyperserotonergic state characterized by severe diarrhea, as well as irritable bowel syndrome and infectious and autoimmune states associated with gut inflammation, including inflammatory bowel disease Manocha and Khan, ; Margolis et al.

TPH1 inhibitors with potencies in the nanomolar range have been reported by Lexicon Pharmaceuticals, out of which one compound, telotristat, was recently approved for treatment of carcinoid syndrome Jin et al.

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