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Published as doi: 10.1096/fj.07-9566com.
 (The FASEB Journal. 2008;22:890-897.)
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(The FASEB Journal. 2008;22:890-897.)
© 2008 FASEB

Inhibitory and structural studies of novel coenzyme-substrate analogs of human histidine decarboxylase

Fang Wu*, Jing Yu{dagger} and Heinz Gehring*,1

* Department of Biochemistry, University of Zurich, Zurich, Switzerland; and

{dagger} Institute of Molecular Pharmacy, University of Basel, Basel, Switzerland

1Correspondence: Dept. of Biochemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. E-mail: gehring{at}bioc.uzh.ch


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histamine, a biogenic amine with important biological functions, is produced from histidine by histidine decarboxylase (HDC), a pyridoxal 5'-phosphate-dependent enzyme. HDC is thus a potential target to attenuate histamine production in certain pathological states. Targeting mammalian HDC with novel inhibitors and elucidating the structural basis of their specificity for HDC are challenging tasks, because the three-dimensional structure of mammalian HDC is still unknown. In the present study, we designed, synthesized, and tested potentially membrane-permeable pyridoxyl-substrate conjugates as inhibitors for human (h) HDC and modeled an active site of hHDC, which is compatible with the experimental data. The most potent inhibitory compound among nine tested structural variants was the pyridoxyl-histidine methyl ester conjugate (PHME), indicating that the binding site of hHDC does not tolerate groups other than the imidazole side chain of histidine. PHME inhibited 60% of the fraction of 12-O-tetradecanoylphorbol-13-acetate-induced newly synthesized HDC in human HMC-1 cells at 200 µM and was also inhibitory in cell extracts. The proposed model of hHDC, containing phosphopyridoxyl-histidine in the active site, revealed the binding specificity of HDC toward its substrate and the structure-activity relationship of the designed and investigated compounds.—Wu, F., Yu, J., Gehring, H. Inhibitory and structural studies of novel coenzyme-substrate analogs of hHDC.


Key Words: mammalian histidine decarboxylase • pyridoxyl-substrate analogs • binding specificity of histidine decarboxylase • histamine


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THE BIOGENIC AMINE HISTAMINE PLAYS an important role in a number of physiological processes, including inflammatory as well as allergic reactions, control of gastric acid secretion, and neurotransmission (1 2 3 4 5) . Histidine decarboxylase (HDC), a pyridoxal 5'-phosphate (PLP)-dependent homodimeric enzyme catalyzes the decarboxylation of histidine to histamine (Fig. 1 ) and is regarded as a potential target to attenuate histamine production in certain pathological states (3) .


Figure 1
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  		Figure 1. Reaction mechanism of eukaryotic HDC with L-histidine. E-PLP, HDC-pyridoxal 5'-phosphate (internal aldimine); ES-PLP, enzyme-coenzyme substrate intermediate (external aldimine).

Mammalian HDC is initially translated as a 74-kDa form and then post-translationally processed to the 53- to 55-kDa form through a complex reaction (6 7 8 9) . In mouse mastocytoma cells, caspase-9 cleaves off the C-terminal part of HDC (1) . The 74-kDa form of HDC has very low enzymatic activity and is mainly recovered in the insoluble fraction, whereas the 53- to 55-kDa form is soluble and has a high activity if expressed in baculovirus-insect or mammalian cells (7 8 9) . Endogenous human (h) HDC is expressed only in histamine-releasing cells such as mast cells, basophils, enterochromaffin-like cells in the stomach, neurons in the brain, or macrophages (10) .

HDC shares high homology (51% identity) in primary sequence with dopa or aromatic L-amino acid decarboxylase (11) . To date, the crystal structure of mammalian HDC is unknown because of difficulties in preparation and its heterogeneity and instability (3 , 11) . A structure of dimeric rat (r) HDC with the cofactor PLP has been constructed by homology modeling using pig (p) dopa decarboxylase (DDC) with the inhibitor carbidopa (Fig. 2 A) as template (3 , 11 , 12) . The proposed model contains no ligand, and the interactions of binding-site residues of HDC with its ligands are still undefined.


Figure 2
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  		Figure 2. Chemical structure and inhibitory activity of various compounds. A) Substrate analogs of HDC and DDC. B) Designed and synthesized pyridoxyl-substrate analogs. The compounds 1–9 were synthesized, purified, and verified as described in Materials and Methods. MSa = theoretical mass; MSb = measured mass [M+H]+. Inhibitiona: HMC-1 cells were pretreated with the indicated compounds (150 µM) for 3 h, then incubated with 100 nM TPA for additional 3 h before cells were collected and lysed. HDC activity was measured by trapping the released 14CO2 from L-[1-14C]histidine in assay buffer (see Materials and Methods). The inhibition values are expressed as a percentage of control (0%). Inhibitionb: HMC-1 cells were pretreated with 100 nM TPA for 3 h and then lysed and incubated with the indicated compounds (150 µM). HDC activity was measured as described (inhibition of control, 0%). The results are given as the average ± SD (n=3).

Numerous inhibitors of mammalian HDC have been developed (13 , 14) . {alpha}-Fluoromethylhistidine, an enzyme-activated irreversible inhibitor of mammalian as well as pyruvoyl-dependent bacterial HDC, covalently attaches to active site residues after being activated by HDC itself, an inhibitory mechanism similar to {alpha}-DL-difluoromethylornithine, a suicide inhibitor of ornithine decarboxylase (ODC) (15 , 16) . Most other inhibitors are substrate analogs and inhibit the enzyme competitively. They were developed in the 1970s, by adding substituents to histidine in the hope of gaining additional affinity (17) . However, the binding affinity did not improve much, with the methyl ester of histidine (HME) (Fig. 2A ) being an exception (IC50 of ~1.8 µM in vitro). Unfortunately, HME proved to be inactive in cells, because it is hydrolyzed to histidine, the substrate of HDC, after being taken up by cells (14) . Bacterial pyruvoyl-dependent HDC was also reported to be inhibited by HME in vitro (14) . Recently, (–)-epigallocatechin-3-gallate extracted from green tea was reported to inhibit rHDC in vitro and in rat RBL-2H3 cells (18) , but it also inhibited other PLP-dependent enzymes, such as ODC (19) .

PLP is the cofactor for numerous enzymes, which catalyze a wide variety of amino acid transformations (20 , 21) . External aldimine, a Schiff base of PLP with the {alpha}-amino group of the amino acid substrate (ES-PLP, Fig. 1 ), is the common intermediate of all transformation of amino acids catalyzed by PLP-dependent enzymes (20) . A stable analog of PLP-substrate adducts can be obtained by reduction of the Schiff base, producing phosphopyridoxyl-amino acids. Such analogs of coenzyme-substrate adducts are, for obvious reasons, high-affinity inhibitors for PLP-dependent apoenzymes (22 , 23) . Based on the knowledge that such analogs strongly interact with the corresponding apoenzymes, we recently developed a strategy for delivering a novel Schiff base analog as an inhibitor for intracellular PLP-dependent human ODC. The inhibitor, a phosphopyridoxyl-ornithine-based precursor, efficiently suppressed ornithine decarboxylase activity in cells and also cell proliferation (24) .

To develop novel bioavailable inhibitors for mammalian HDC and explore their interaction with the active site of mammalian HDC, we designed and synthesized several pyridoxyl-amino acid-based precursor inhibitors for hHDC and deduced a structural model composed of PLP as well as histidine analogs in the active site of hHDC. One of the designed compounds efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) -induced hHDC activity in cells and in cell extracts without affecting human ODC activity. The interaction model generated describes the structure-activity relationship of the tested compounds and of previously reported inhibitors. Moreover, it explains the binding specificity of mammalian HDC for histidine.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
His · HCl and histamine · 2HCl were bought from Sigma-Aldrich (St. Louis, MO, USA). His-OMe · 2HCl, His(1-Me)-OMe, Phe-OMe · HCl, β-(3-pyridyl)-D-Ala-OMe · 2HCl, Trp-OMe · HCl, and {alpha},{gamma}-diaminobutyric acid (Boc)-OMe · HCl were from Bachem (Bubendorf, Switzerland). Protease inhibitors were obtained from Roche (Basel, Switzerland). L-[1-14C]Histidine (55 mCi/mmol) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). Pyridoxal · HCl and TPA were from Fluka (Buchs, Switzerland).

General procedure for synthesis of the compounds
Compounds 1–6 (Fig. 2B ) were synthesized as described for the synthesis of pyridoxylhistamine (25) . Briefly, pyridoxal (1 mmol) and NaHCO3 (2 mmol) were dissolved in 5 ml of ethanol, and the substrate analogs (1.05 mmol) together with NaHCO3 (3 mmol) were dissolved in 10 ml of ethanol. Both solutions were mixed at 0°C and stirred at room temperature for 2 h. Then, NaBH4 (125 mg) was added in small portions to the solution on ice. Acetic acid (100%) was added 30 min later to stop the reaction until pH 5 was reached. The solvents were removed with a vacuum dryer, and the residues were dissolved in water and subjected to fast protein liquid chromatography (FPLC) or HPLC for purification and analysis. Compound 8 (Fig. 2B ) was synthesized as reported previously for compound 7 (24) . Compound 9 (cyclized form of compound 3) was obtained according to a reported procedure (26) . Briefly, histamine (1.02 mmol) and KOH (4.2 mmol) were dissolved in water (2.5 ml) and stirred at 0°C for 10 min. Then, solid pyridoxal (1 mmol) was added to the solution followed by ethanol (20 ml). The reaction mixture was stirred at 30°C for 7 h, and the solvent was evaporated, yielding a white powder.

Compounds 1, 2, 3, and 9 were analyzed and purified with FPLC using a chromatograph equipped with a mono S HR5/5 column (Amersham Pharmacia Biotech, Uppsala, Sweden) and a multiwavelength detector. The samples were loaded and separated with a flow rate of 0.5 ml/min using the following gradient: 0–5 min, 100% solvent A (50 mM acetic acid, pH 4.6) and 0% solvent B (50 mM acetic acid, pH 4.6, and 1M NaCl); 5–30 min, 0–100% B; and 30–40 min, 100% B. Detection was at 290 nm.

Compounds 4, 5, 6, and 8 were analyzed and purified with HPLC using a chromatograph equipped with a Kromasil C18 column (4.6x250 mm; Akzo Nobel, Bohus, Sweden). The samples were loaded and separated with a flow rate of 0.15 ml/min using the following gradient: 0–36 min, 100% solvent A [3% acetonitrile/0.1% trifluoroacetic acid (TFA)] and 0% solvent B (80% acetonitrile/0.1% TFA); 36–92 min, 0–80% B; and 92–120 min, 80–100% B, followed by 100% B. Elution was recorded at 290 nm. A preparative Kromasil C18 column (50.8x250 mm; Akzo Nobel) with a flow rate of 15 ml/min was used under the same conditions to prepare larger amounts of compounds. All compounds were vacuum-dried several times after addition of water and stored at a concentration of ~10 mM (water stock solution) at –20°C. The purified compounds obtained (purity >95%) were analyzed and verified by electrospray ionization-mass spectrometry.

Cell culture
HMC-1 (a human mastocytoma cell line) cells were a generous gift from Dr. J. Butterfield (Mayo Clinic, Rochester, MN, USA) and maintained in Iscove’s modified Dulbecco’s modified Eagle’s medium supplemented with 10% FBS in a humidified 5% CO2 atmosphere at 37°C.

HDC activity
HDC activity was measured by the release of 14CO2 from labeled histidine as described (2 , 27 , 28) . For measuring TPA-induced HDC activity, cells were cultured in six-well plates at a density of ~6 x 105 cells/well (2 ml) with 5% FBS for 1 day and then treated in the absence or in the presence of the indicated compound for 3 h. The cells were induced with 100 nM TPA for 3 h, collected, and washed twice with ice-cold PBS (pH 6.8). The collected cells were lysed by freezing (liquid nitrogen) and thawing twice (37°C, 2 min) in 310 µl of lysis buffer (10 µM EDTA, 10 µM PLP, 20 µM dithiothreitol, and protease inhibitor cocktail in 100 mM PBS, pH 6.8). The reaction was started by addition of 30 µl of L-[1-14C]histidine (final concentration, 130 µM; 0.09 µCi) to a 270-µl supernatant of lysed cells. After a 3-h incubation, the reaction was stopped by injecting 200 µl of 4 N H2SO4, and the mixture was kept for 1 h at room temperature to ensure complete absorption of released CO2 by the capture reagent (tissue solubilizer NCSII, Amersham Pharmacia Biotech). The captured 14CO2 was measured by scintillation counting in a Wallac 1450 MicroBeta liquid scintillation counter. The protein concentration in the supernatant of lysed cells was determined by a Bio-Rad protein assay kit.

To measure inhibition in cell extracts of TPA-induced untreated HMC-1 cells, supernatants of lysed cells were incubated with the respective compound. HDC activity was then determined as described above.

Molecular modeling
The protein sequence of hHDC (Swiss-Prot access number P19113) was aligned with the sequence of pDDC obtained from the Protein Data Bank (PDB) structure [PDB code 1JS3 (12) ] and rat HDC (rHDC) (Swiss-Prot access number P16453) with ClustalIW (29) . On the basis of the sequence alignment of pDDC and hHDC, the structure of the dimeric hHDC was constructed by superposing two monomeric HDCs, obtained from Swiss-Model database (http://swissmodel.expasy.org/), into the crystal structure of dimeric (subunits A and B) pDDC by using InsightII (version 2000; Accelrys Corporate, San Diego, CA, USA). The structure of phosphopyridoxyl-histidine was constructed into hHDC on the basis of the known three-dimensional coordinates of the phosphopyridoxyl-carbidopa (PDB code 1JS3), followed by energy minimization performed with the InsightII/Built module [steepest descents until a maximum derivative of 20 kcal/mol, constant valence force field (CVFF)]. The assembled structures consisting of hHDC and the phosphopyridoxyl-histidine analog were further optimized by energy minimization using the InsightII/Discover module (conjugation gradient until a maximum derivative of 0.01 kcal/mol, CVFF).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Coenzyme-substrate analogs as inhibitors of hHDC
To target HDC in mammalian cells, we designed and synthesized potential coenzyme-histidine-based precursor inhibitors for hHDC (compounds 1, 2, 3, and 9; Fig. 2B ), which we assumed to be membrane permeable and phosphorylated by intracellular pyridoxal kinase (30 , 31) . Recently we showed that intracellular ornithine decarboxylase (ODC) could be targeted with this kind of transition state-based inhibitor (24) . ODC (half-life, 20–60 min) and HDC (half-life, 1–2 h) are inducible and short-lived enzymes (16 , 32) , and the freshly synthesized apoenzymes will be excellent targets for these inhibitors. As the knowledge of the structural prerequisite in the active site environment was marginal, we synthesized and tested compounds with variations in the substrate moiety (compounds 4–8; Fig. 2B ).

To identify effective inhibitors for newly synthesized apo-HDC, HMC-1 cells were induced with TPA, and HDC activities were measured after cells had been pretreated with the various compounds (Fig. 2B ). Among the nine tested structurally diverse compounds, pyridoxyl-histidine methyl ester (PHME) (compound 1; Fig. 2B ) was found to be the most potent inhibitor for HDC in TPA-induced HMC-1 cells and in their extracts if subjected to 150 µM pyridoxyl-substrate conjugate. The phosphorylated PHME was also found to be a potent inhibitor (IC50 ~50 µM) for hHDC when tested in the cell extracts of HMC-1 but was inactive in cells. None of the other analogs significantly inhibited HDC in HMC-1 cells, although some (compounds 2, 6, and 9) slightly inhibited HDC activity in the cell extracts (Fig. 2B ).

Noninduced (TPA) HMC-1 cells already exhibited basal activity, which was slightly (15%) affected by 200 µM PHME (Fig. 3 ). Under these conditions, the inhibitor has to compete with already bound PLP [intracellular concentration, ~20–50 µM (33) ] in the active site of HDC, which probably is a slow process. After induction (black columns, Fig. 3 ), freshly synthesized apoenzyme, indicated by a 2.5-fold increase in enzyme activity, is the main target of the inhibitor in the cells. The fraction of TPA-induced activity (0.97 nmol h–1 mg–1) is inhibited to ~60%, comparable to the inhibition caused by HME (Fig. 2A ) in vitro. Furthermore, PHME specifically inhibited hHDC in cells without affecting the activity of human ODC and cell viability (unpublished observation).


Figure 3
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  		Figure 3. PHME inhibition of TPA-induced HDC activity in HMC-1 cells. HMC-1 cells were treated with or without PHME for 3 h followed by a 3-h incubation with or without TPA (100 nM). Cells were collected and lysed, and HDC activities were measured as described in the legend to Fig. 2 . The results are given as the average ± SD (n=3).

Modeling the active site of hHDC
To understand the structure-activity relationship of the synthesized compounds (Fig. 2B ) and the active site of HDC, we built up by molecular modeling the active site of HDC together with the presumed intracellularly active form of PHME (phosphorylated and deesterified compound 1; Fig. 2B ). The crystal structure of pDDC (51% sequence identity with mammalian HDC) (Fig. 4 ) served as a template for constructing dimeric hHDC. Residues of hHDC and pDDC as well as rat HDC surrounding the coenzyme-substrate analog within a distance of 4–5 Å are listed in Table 1 , aligned as reported previously (11) , and shown in Fig. 4 . Most residues in the active sites of pDDC and hHDC are identical (71%), except the seven conservative, semiconservative, or nonconservative substitutions [Ile-101 (pDDC) -> Leu-102 (hHDC), Gly-354 (pDDC) -> Ser-354 (hHDC), Phe-80 (pDDC) -> Tyr-81 (hHDC), Thr-82 (pDDC) -> Ala-83 (hHDC), Ser-147 (pDDC) -> Thr-149 (hHDC), Ala-148 (pDDC) -> Val-150 (hHDC), and His-302 (pDDC) -> Ser-304 (hHDC)] shown in Fig. 5 C and Table 1 . Among them, Ala-83 and Ser-304 have been shown to be important residues for maintaining the activity of mouse HDC (11) .


Figure 4
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  		Figure 4. Sequence alignment of hHDC, rHDC, and pDDC. The protein sequence of hHDC (Swiss-Prot access number P19113) was aligned with the sequence of pDDC obtained from the PDB structure [PDB code 1JS3 (12) ], and rHDC (Swiss-Prot access number P16453) with ClustalIW (29) . * = identical residues in all sequences in the alignment; : = conserved substitutions; · = semiconserved substitutions. Arrows indicate the nonidentical residues of active sites (see Table 1 and Fig. 5C ).


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  		Table 1. Comparison of residues in the active site of pDDC, hHDC, and rHDC


Figure 5
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  		Figure 5. Active site model of hHDC and pDDC, containing a coenzyme-substrate analog. A) The modeled active site of hHDC (green) and the phosphopyridoxyl-histidine conjugate (pink-yellow). B) The active site of pDDC together with the phosphopyridoxyl-carbidopa (pink-brown) (PDB code 1JS3). C) Superposed stereo view of the nonidentical residues of the active site of hHDC (red, the side chains are labeled with white fonts) and pDDC (blue, labels in cyan) together with phosphopyridoxyl (pink)-histidine (yellow) and phosphopyridoxyl (pink)-carbidopa (brown) conjugates. Residues surrounding the coenzyme-substrate analog within a distance of 4–5 Å are indicated.

In the active site of hHDC, the imidazole ring was located in a pocket composed of residues Tyr-81B, Asn-302B, Ser-304B, Lys-305B, Leu-102A, Phe-104A, and Ser-354A (Fig. 5A ), whereas in pDDC, the aromatic ring is surrounded by residues Phe-B80, Asn-B300, His-B302, Lys-B303, Ile-A101, Phe-A103, and Gly-A354 (Fig. 5B ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Exploring the steric constraints in the binding site by attaching additional groups to mimic a reaction intermediate is a robust methodology in understanding the structural requirements of the active site and in developing inhibitors (24 , 34) . The present study describes the successful action of PHME, a newly developed inhibitor of this kind of intracellular hHDC. The development strategy was based on external aldimine, an obligatory intermediate in the mechanism of action of hHDC. In the nine pyridoxyl derivatives tested, the 5'-phosphate group is eliminated and the carboxyl group is esterified with methanol. The rationale behind the design was that these compounds have no negative net charge and very likely cross the cell membrane as was reported for several pyridoxylamines (31 , 35) , a pyridoxyl-methionine analog (36) , and compound 7 (24) . The methyl ester group in the compounds will be hydrolyzed by endogenous esterase (14) , and the resulting inhibitors, after being phosphorylated by intracellular pyridoxal kinase (30 , 35) , will bind the cellular newly synthesized apo-HDC with high affinity.

Here, we tested several structural diverse compounds that mimic a reaction intermediate of HDC (ES-PLP) (Fig. 1) with variations in the substrate motif (Fig. 2) . Only PHME showed remarkable inhibition both in cell extract and in cells, which indicated that the binding site of hHDC is rather specific and seems not to accept variations in the substrate moiety.

To understand the structural basis of the binding specificity of hHDC, a model consisting of dimeric hHDC and phosphopyridoxyl-histidine, the cellular active from of PHME, was constructed by homologous modeling methods. With this model, the substrate specificity of these two proteins can be explained by the substitutions Gly-354 (pDDC) -> Ser-354 (hHDC), Phe-80 (pDDC) -> Tyr-81 (hHDC), and His-302 (pDDC) -> Ser-304 (hHDC) (Fig. 5A-C and Table 1 ). The imidazole side chain of the substrate is in a favorite position to form hydrogen bonds with the hydroxyl groups of Tyr-81, Ser-304, or Ser-354 of hHDC, whereas the hydroxyl groups of carbidopa favorably interact with the imidazole ring of His-302 of pDDC as reported (11 , 37) . Mutation of Ser-304 of hHDC to Gly decreased the enzymatic activity but not completely (11) indicating that Ser-304 participates in substrate binding rather than in the catalytic conversion as also suggested by the present model. This binding mode of the coenzyme-substrate conjugate in the active site of hHDC demonstrates that the imidazole binding moiety of substrate consists of a tightly packed and specific pocket in contrast to that of pDDC (Fig. 5A-C ) (38) . Tyr-81B in hHDC adopted a "closed conformation" in contrast to the "open conformation" of the corresponding residue Phe-80B in pDDC and can form a stacking interaction with the imidazole ring and an additional hydrogen bond with the N1 atom of the imidazole ring (Fig. 5C ). This different conformation forms a small but specific binding pocket for the imidazole ring of the substrate histidine, which cannot accept bigger ligands as pDDC does (Fig. 5C ). The model also displays more tight packing in hHDC than in pDDC of active site residues surrounding the phosphate group (Fig. 5A-C ).

The small but specific binding site of hHDC as analyzed above explains why the conjugates with a larger amino acid moiety [His(1-Me), 2; Phe, displays 4; β-(3-pyridyl)-D-Ala, 5; Trp, 6; tert-butoxycarbonyl (Boc) -protected ornithine, 7; and Boc-protected {alpha},{gamma}-diaminobutyric acid, 8] (Fig. 2B ) do not significantly inhibit hHDC in cells. Compound 2, which has an additional methyl group at the imidazole ring, already showed much less inhibition than compound 1 (Fig. 2) . The model indicates that the methyl group at N1 of the imidazole ring disturbs the formation of a hydrogen bond to Tyr-81B, Ser-304B, or Ser-354A in the active site of hHDC and exerts steric constraints (Figs. 2B , 5 A, C). The failure of inhibition by compounds 3 and 9 (cyclized form of 3) is most likely due to the missing carboxylate group, which could interact with the conservative positively charged Arg-447B during catalysis (Figs. 2 and 5A) .

Besides the structure-activity relationship gained from the designed compounds, the proposed model reasonably explains previous experimental data. The hHDC model points out that hHDC contains a narrow side chain-binding pocket and a relatively large binding site for the carboxylate group. Additional modifications on histidine, such as {alpha}- and β-substitutions (13) as well as substitutions at the imidazole ring (14) , will decrease the binding affinity due to steric hindrance in this region. The small binding site does not accept a bulky fragment such as {alpha}-methyldopa either (Fig. 5C ), which is a potent inhibitor for DDC but not for rHDC (39) . The relatively large free binding region around the carboxylate group in hHDC (Fig. 5A ) could accept additional moieties, whereas in DDC this is not the case (Fig. 5B ). Indeed, PHME, phosphopyridoxyl-histidine methyl ester (see above), HME (14) , the dipeptide His-Phe (17 , 40) , and others (41) inhibited mammalian HDC in vitro. This fact could be used to develop more potent inhibitors for hHDC by modifications at the carboxylate group.

In conclusion, the present investigation with pyridoxyl-histidine analogs as possible precursor ligands for hHDC revealed a novel intracellular inhibitor of hHDC and a structural model of its active site containing not only the coenzyme but also substrate analogs. These findings can serve as the structural basis for functional studies of HDC and for the development of more potent inhibitors for this enzyme of pharmaceutical interest.


   ACKNOWLEDGMENTS
 
We acknowledge the financial support by COST Switzerland (Action 922, grant C02.0017). We thank Dr. S. Bienz and collaborators (Institute of Organic Chemistry, University of Zurich) for providing excellent facilities for organic synthesis, Dr. J. Butterfield and coworkers for providing HMC-1 cells, and Dr. P. Christen (Department of Biochemistry, University of Zurich) for his valuable advice and critical reading of the manuscript.

Received for publication August 16, 2007. Accepted for publication September 20, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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F. Wu and H. Gehring
Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation
FASEB J, February 1, 2009; 23(2): 565 - 574.
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