Mechanism of Inhibition of Novel Tryptophan Hydroxylase Inhibitors Revealed by Co-crystal Structures and Kinetic Analysis.

Current Chemical Genomics, 2010, 4, 19-26 19 Open Access Mechanism of Inhibition of Novel Tryptophan Hydroxylase Inhibitors Revealed by Co-crystal Structures and Kinetic Analysis Giovanni Cianchetta*,1, Terry Stouch1, Wangsheng Yu2, Zhi-Cai Shi1, Leslie W. Tari3, Ronald V. Swanson3, Michael J Hunter3, Isaac D. Hoffman3 and Qingyun Liu*,2 1 Department of Medicinal Chemistry, Lexicon Pharmaceuticals, Inc., 350 Carter Rd., Princeton, New Jersey, USA; Department of Pharmaceutical Discovery, Lexicon Pharmaceuticals, Inc., 8800 Technology Forest Pl., The Woodlands, Texas, USA; 3Activesight, Inc., San Diego, California, USA 2 Abstract: Trytophan Hydroxylase Type I (TPH1), most abundantly expressed in the gastrointestinal tract, initiates the synthesis of serotonin by catalyzing hydroxylation of tryptophan in the presence of biopterin and oxygen. We have previously described three series of novel, periphery-specific TPH1 inhibitors that selectively deplete serotonin in the gastrointestinal tract. We have now determined co-crystal structures of TPH1 with three of these inhibitors at high resolution. Analysis of the structural data showed that each of the three inhibitors fills the tryptophan binding pocket of TPH1 without reaching into the binding site of the cofactor pterin, and induces major conformational changes of the enzyme. The enzyme-inhibitor complexes assume a compact conformation that is similar to the one in tryptophan complex. Kinetic analysis showed that all three inhibitors are competitive versus the substrate tryptophan, consistent with the structural data that the compounds occupy the tryptophan binding site. On the other hand, all three inhibitors appear to be uncompetitive versus the cofactor 6-methyltetrahydropterin, which is not only consistent with the structural data but also indicate that the hydroxylation reaction follows an ordered binding mechanism in which a productive complex is formed only if tryptophan binds only after pterin, similar to the kinetic mechanisms of tyrosine and phenylalanine hydroxylase. Keywords: Serotonin, structure, kinetics, gastrointestinal disorder, carcinoid, monooxygenase. INTRODUCTION Serotonin (5-hydroxytryptamine, 5-HT) has a plethora of functions in both the central nervous system and the periphery. It is synthesized from tryptophan by the sequential action of two enzymes, tryptophan hydroxylase (TPH) and aromatic amino acid decarboxylase with TPH catalyzing the rate-limiting step. Two forms of TPH have been identified in mammals: TPH1, expressed highly in the gastrointestinal tract and the pineal gland, is responsible for >90% of 5-HT synthesis in the periphery, while TPH2, expressed in neuronal cells located in the dorsal raphe nucleus of the brain and the myenteric plexus of the gut, is responsible for the vast majority of 5-HT synthesis in the central nervous system [1-4]. The two enzymes, sharing an overall identity of ~80% at the amino acid level, are homologous to the other two aromatic amino acid hydroxylases, phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH), with approximately 50% amino acid sequence identity [3, 5]. The four enzymes use the same cofactors, tetrahydrobiopterin, iron, and oxygen to catalyze the hydroxylation of their respective substrates [6]. *Address correspondence to these authors at the Department of Medicinal Chemistry, Lexicon Pharmaceuticals, Inc., 350 Carter Rd., Princeton, NJ, USA; Tel: (609)466-6055; Fax: (609)466-3562; E-mail: Brown Foundation Institute of Molecular Medicine, 1825 Pressler St., Suite 330H, Houston, TX, USA; Tel: (713)500-3808; Fax: (713)500-0319; E-mail: 1875-3973/10 X-ray crystal structures have been determined for various forms of TPH1, PAH and TH [7-13]. The catalytic domains of all three enzymes have a very similar fold with a mixture f
and  structures. The active site is formed by two long channels, one occupied by the amino acid substrate, the other one by pterin, with iron sitting at the intersection of the two channels. For PAH, binding of 5,6,7,8-tetrahydro-Lbiopterin (BH4) caused small structural changes to the catalytic domain [8]. In contrast, binding of a phenylalanine analogue, such as L-norleucine or 3-(2-thienyl)-L-alanine (THA) in the presence of BH4 and iron ion led to large structural changes in the catalytic domain [9, 14]. Specifically, the loop containing residues 133-141 of PAH moved much closer to the catalytic site. For TPH1, a structure of the catalytic domain of the human enzyme with 7,8-dihydrobiopterin (BH2) and ferric iron ion bound was solved first, which showed remarkable similarity to the structure of the catalytic domain of PAH with BH4 bound [11]. More recently, a structure of the catalytic domain of chicken TPH1 with tryptophan bound was solved [12]. Compared with the BH2bound structure of human TPH1, binding of tryptophan caused major structural changes with two loops (Leu124Asp139 and Ile367-Thr369) brought closer to the catalytic site, as in the corresponding regions of PAH after substrate analogue binding [12]. The kinetic mechanism of mammalian TH and bacterial PAH has been determined. For both enzymes, all three substrates, i.e., amino acid, pterin, and oxygen, have to be bound before catalysis can happen [6]. For rat TH, the order of substrate binding is pterin first, followed by oxygen, and then 2010 Bentham Open 20 Current Chemical Genomics, 2010, Volume 4 tyrosine [15]. For bacterial PAH, one study reported oxygen binding first, followed by pterin and amino acid in random order [16]. A more recent study, however, conclusively showed that pterin binds first, followed by phenylalanine and then oxygen [17]. Such an ordered binding mechanism with pterin binding first is consistent with the observations that both enzymes are inhibited by their amino acid substrates at high concentrations [6]. TPH1 is also inhibited by high concentrations of Trp [18], implicating a similar kinetic mechanism , even though no detailed mechanism studies have been reported. Cianchetta et al. tions were carried using PHASER in the CCP4 suite2. The structures were refined using REFMAC in the CCP4 suite2. All electron density map visualization and manual model rebuilding was carried out using the XtalView/Xfit package [27]. Enzyme Kinetic Studies Compounds LP-521834, LP-533401, and LP-534193 were synthesized in-house as described before [23, 24]. All other reagents were purchased from Sigma-Aldrich. Full-length human TPH1 was expressed and purified as described before [4], to a specific activity of approximately 60 nmole/min/(mg of protein). Enzyme assays were carried out at room temperature with atmosphere oxygen in a volume of 0.1 ml containing 50 mM 3-(N-morpholino)propanesulfonate (MOPS), pH 7.2, 100 mM (NH4)2SO4, 0.05 mg/ml of catalase, 1 mg/ml of bovine serum albumin, 0.05 mM (NH4)2Fe(SO4)2 , various concentrations of tryptophan and 6-methyltetrahydropterin, and 0.5 μg of TPH1. The reactions were started with the (...truncated)