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Published as doi: 10.1096/fj.08-115121.
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(The FASEB Journal. 2009;23:565-574.)
© 2009 FASEB

Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation

Fang Wu1 and Heinz Gehring2

Department of Biochemistry, University of Zurich, Zurich, Switzerland

2Correspondence: Department 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
 
Creating transition-state mimics has proven to be a powerful strategy in developing inhibitors to treat malignant diseases in several cases. In the present study, structurally diverse coenzyme-substrate derivatives mimicking this type for pyridoxal 5'-phosphate-dependent human ornithine decarboxylase (hODC), a potential anticancer target, were designed, synthesized, and tested to elucidate the structural requirements for optimal inhibition of intracellular ODC as well as of tumor cell proliferation. Of 23 conjugates, phosphopyridoxyl- and pyridoxyl-L-tryptophan methyl ester (pPTME, PTME) proved significantly more potent in suppression proliferation (IC50 up to 25 µM) of glioma cells (LN229) than {alpha}-DL-difluoromethylornithine (DFMO), a medically used irreversible inhibitor of ODC. In agreement with molecular modeling predictions, the inhibitory action of pPTME and PTME toward intracellular ODC of LN229 cells exceeded that of the previous designed lead compound POB. The inhibitory active compounds feature hydrophobic side chain fragments and a kind of polyamine motif (-NH-(CHX)4-NH-). In addition, they induce, as polyamine analogs often do, the activity of the polyamine catabolic enzymes polyamine oxidase and spermine/spermidine N1-acetyltransferase up to 250 and 780%, respectively. The dual-action mode of these compounds in LN229 cells affects the intracellular polyamine metabolism and might underlie the more favorable cell proliferation inhibition in comparison with DFMO.—Wu, F., Gehring, H. Structural requirements for novel coenzyme-substrate derivatives to inhibit intracellular ornithine decarboxylase and cell proliferation.


Key Words: transition-state mimics • DFMO • polyamine metabolism • pyridoxyl-amino acid analogs • drug design • anticancer compounds


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
POLYAMINE (PA) METABOLISM is closely linked to cancer and other hyperproliferative diseases (1) . Ornithine decarboxylase (ODC) catalyzes the first and rate-limiting step in PA biosynthesis converting ornithine into putrescine (PUT). Odc is the first target of the oncogene c-myc (1) . ODC expression is rapidly induced after exposing cells with cell proliferation-stimulating agents (2 3 4) . High levels of ODC observed in cancer cells are closely related to tumor promotion (5 , 6) . Overexpression of ODC induces transformation of cells (7) , and inhibition of ODC abolishes transformation and is associated with tumor suppression (2 , 8) . Even modest reductions in ODC activity can lead to markedly reduced tumor development (2 , 8) . Thus, ODC is a target in the development of antiproliferative agents (2 , 6) . The most widely used inhibitor of ODC is {alpha}-DL-difluoromethylornithine (DFMO). Clinical studies proved that DFMO is a well-tolerated agent but turns out to be not so efficient as a treatment of cancer (1 , 9 , 10) . However, a combination chemotherapy for recurrent anaplastic gliomas, the most aggressive form of brain tumors with poor prognosis, looked promising (11) . A lot of attempts have been made to improve the efficiency of DFMO by modifying its skeleton structure. Although the binding affinity of modified compounds for ODC improved in vitro, they had no favorable effect in inhibiting cell proliferation (6) . Synthetic mimics or analogs of PA, namely PUT, spermidine (SPD) or spermine (SPM), as well as natural PA derivatives, decrease tumor cell growth (12) . In contrast to DFMO, these agents strongly induce the PA catabolic enzymes SPM/SPD N1-acetyltransferase (SSAT) and polyamine oxidase (PAO) and down-regulate ODC by a cellular feedback mechanism without directly inhibiting ODC activity (1) . Different derivatives of SPD and SPM, as a single agent or in combination with other drugs, are currently in preclinical or clinical studies (1 , 6) . However, they seem to exert, like SPM, unacceptable toxicities (1 , 6) . Therefore, novel types of inhibitors acting in the PA metabolism, which overcome or diminish these problems are in high demand.

Creating transition-state mimics is a powerful strategy in developing novel potent and specific inhibitors of enzymes to treat malignant diseases (13 , 14) . Designing inhibitors by mimicking well-defined reaction intermediates of pyridoxal 5'-phosphate (PLP) -dependent enzymes has been used in developing novel inhibitors for these enzymes (15 16 17 18 19) . In all PLP-dependent enzymes, the external aldimine, a Schiff base formed between the cofactor PLP and the amino group of the amino acid substrate (ES-PLP), is a common intermediate (19 , 20) . PLP binds with high affinity to PLP-dependent enzymes (21) . Reduction of the Schiff base produces phosphopyridoxyl-amino acid conjugates, analogs of the covalent intermediates. These compounds are a kind of transition-state analog that bind to the corresponding apoprotein with high affinity and specificity (22 23 24) . Because the compounds cannot be transformed by the enzymes, they inhibit the enzymes potently. We recently developed a strategy to deliver pyridoxyl-substrate analogs as bioavailable prodrug for intracellular PLP-dependent enzymes (18 , 19) . The precursor inhibitor POB, a mimic of ES-PLP intermediate of the ODC-catalyzed reaction (19) , can be taken up by cells and efficiently inhibits, after being phosphorylated by intracellular pyridoxal (PL) kinase (25 , 26) , newly synthesized ODC in competition with endogenous PLP (~20–50 µM) (27) and suppresses proliferation of tumor cells without affecting the proliferation of nontumorigenic smooth muscle cells (19) .

In the present study, structurally diverse enzyme-substrate mimics were created to study the structural requirements for optimal inhibition of intracellular human ODC as well as of tumor cell proliferation. On the basis of the previously developed structural model (19) , 23 structurally diverse conjugates of PL or PLP and substrate or PA analogs were designed, synthesized, and tested to explore the essential binding requirements to ODC and the inhibitory potential of the compounds as well as their mode of action.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Compounds 1 to 3 (tyrosine, alanine and lysine analogs) were kind gifts from Dr. P. Christen (Department of Biochemistry, University of Zurich) (refs. 28 , 29 ). The starting materials for synthesis of other compounds (Fig. 1 ), were bought from commercial sources: His-OMe·2HCl from Bachem (Bubendorf, Switzerland) for compounds 4 and 9; His·HCl (Sigma-Aldrich Corp., St. Louis, MO, USA), 5; His(1-Me)-OMe His·HCl (Bachem), 6; histamine·2HCl (Sigma-Aldrich), 7 and 8; Trp-OMe·HCl (Bachem), 10 to 13; Orn(BOC)-OMe·HCl (BOM; Bachem), 14 and 15; Phe-OMe·HCl (Bachem), 16; β-(3-pyridyl)-D-Ala-OMe·2 HCl (Bachem), 17; {alpha},{gamma}-diaminobutyric acid-(BOC)-OMe·HCl (Bachem), 18 and 19; PUT·2HCl (Fluka, Buchs, Switzerland), 20; SPD·3HCl (Fluka), 21 and 23; and SPM·4HCl (Fluka), 22. Protease inhibitors were obtained from Roche (Basel, Switzerland). L-[1-14C]Acetyl CoA (55 mCi/mmol) and L-[1-14C]ornithine (55 mCi/mmol) were from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA). Pyridoxal·HCl and PLP were purchased from Fluka, pyridoxamine 5'-phosphate and aminoguanidine dicarbonate from Sigma-Aldrich, and BOC-protected lysine methyl ester (BOL) from Bachem.


Figure 1
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  		Figure 1. Chemical structures of designed pyridoxyl-substrate derivatives. The compounds were synthesized, purified, and verified by ESI-MS methods, as described in Materials and Methods. MSa, theoretical mass; MSb, measured mass [M+H]+.

Procedure for synthesis
Compounds 4 to 7, 9 to 13, and 20 to 23 (Fig. 1 ) were essentially synthesized as described previously (refs. 18 , 30 , 31 ). Briefly, PL or PLP (1 mmol) and NaHCO3 (2 mmol) were dissolved in 5 ml ethanol, and the substrate analogs (1.05 mmol) together with NaHCO3 (3 mmol), in 10 ml ethanol. Both solutions were mixed at 0°C and stirred at room temperature for 2 h. NaBH4 (125 mg) was then added in small portions to the solution on ice. After 30 min, acetic acid (100%) was added to pH 5 to stop the reaction. The solvents were removed with a vacuum dryer; the residue was dissolved in water and subjected to FPLC or HPLC for analysis and purification. Compounds 15 to 19 were synthesized as described for POB (compound 14) (ref. 19 ). Compound 8 was obtained according to procedures described (ref. 32 ). Briefly, histamine (1.02 mmol) and KOH (4.2 mmol) were dissolved in 2.5 ml water and stirred at 0°C for 10 min. Solid PL (1 mmol) was then added, 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 4 to 9 and 20 to 23 were analyzed and purified with FPLC equipped with a mono S HR5/5 column and compounds 10 to 19 with a Kromasil C18 column (ref. 18 ). Compounds 11, 13, 15 and 19 were hydrolyzed byproducts generated during synthesis and were eluted and collected as individual peaks from the C18 columns.

The purified compounds (purity >95%) were analyzed and verified by ESI-MS and ESI-MS-MS. All compounds were vacuum-dried several times after adding water and stored at a concentration of ~10 mM (aqueous stock solution) at –20°C.

Cell culture
Glioma cell line LN229 (from brain) was a generous gift from Dr. K. Frei (Department of Neurosurgery, University Hospital Zurich). LN229 cells were maintained in Dulbecco modified Eagle medium (DMEM) supplemented with 1 g/L glucose, 10% FBS (Life Technologies, Gaithersburg, MD, USA), 20 µg/ml gentamycine (Fluka) in a humidified 5% CO2 atmosphere at 37°C. HMC-1 (a human mastocytoma cell line) cells were a generous gift from Dr. J. Butterfield (Mayo Clinic, Rochester, MN, USA) and were maintained in Iscove’s modified DMEM supplemented with 10% FBS in a humidified 5% CO2 atmosphere at 37°C. For measuring cell growth, cells were seeded (2.5–5x104 cells/well) and incubated in 0.5 ml medium containing 5% FBS or newborn calf serum and 1% penicillin/streptomycin in a 24-well plate. After 1 day, cells were incubated for the indicated times in the absence or presence of the compounds at the indicated concentrations. Cells were counted with a Coulter counter (ZM Coulter Electronics, Hialeah, FL, USA) as described previously (33) .

Enzyme activities
ODC activity was measured by the released 14CO2 from L-[1-14C]ornithine as described previously (19) . PAO was assayed as described previously (34) . LN229 cells were seeded in 6-well plates at a density of ~1.6 x 105 cells/well with 5% FBS for 1 day and treated in the absence or presence of the indicated compound for 2 days. Cells were then lysed, and PAO activities measured. SSAT activity was determined essentially according to a published procedure (35) . LN229 cells were seeded in 6-well plates at a density of ~1.6 x 105 cells/well with 5% FBS for 1 day and treated in the absence or presence of the indicated compound for 2 days. Cells were then lysed in 50 mM Tris-HCl (pH 7.8) by 2 cycles of freezing (liquid nitrogen) and thawing (37°C, 2 min). After centrifugation, the supernatants (70 µl) were incubated with 0.4 µCi [14C]acetyl-CoA and unlabeled SPD (3 mM) for 15 min.

Molecular modeling
Modeling of phosphopyridoxyl conjugates into the active site of hODC was based on the crystal structure of Trypanosoma brucei ODC (PDB code: 1F3T) (ref. 36 ), containing PLP and PUT as ligands, and was performed as described for POB (compound 14, Fig. 1 ) (ref. 19 ). Extensive sequence and structural identity exist between hODC and tODC. Superposition of C{alpha} of the crystal structures of hODC and tODC (residues 35–411 of hODC, sequence identity: 65%) yields an overall root mean square deviation value of 0.8 Å (ref. 37 ), and the active site of hODC and tODC shares a high similarity (Supplemental Fig. 1). A crystal structure composed of hODC, PLP, and 1-amino-oxy-3-amino-propane (APA), which is a well-known inhibitor of ODC, has recently also been reported (PDB code 2OO0) (ref. 38 ). However, it did not form the conjugate between PLP and APA (the aminooxy group of APA is expected to form an oxime with PLP under physiological pH) (refs. 38 , 39 ). One possible explanation for the absence of the oxime formation between PLP and APA could be the presence of cadaverine in close vicinity to the active site (ref. 38 ). Therefore, the conjugate of PLP and PUT of 1F3T remains the only physiologically relevant intermediate in the active site of a crystal structure of ODC.

In the present study, DOCK4 (University of California, San Francisco, CA, USA) was employed for predicting the geometry of the ligands bound to the ODC according to the standard procedures (ref. 40 ). The orientation of the ligand relative to the receptor was evaluated by a grid-based energy with a distance-dependent dielectric constant to model bulk solvent effects. Electrostatic and van der Waals energies were evaluated on a grid with a 0.3 Å spacing in the region spanning all residues within 6 Å of phosphopyridoxyl-PUT adduct (ref. 19 ). The structures of phosphopyridoxyl-substrate derivatives (Fig. 2 ) were drawn based on the known 3-dimensional coordinates of the phosphopyridoxyl-PUT conjugate, as described for phosphopyridoxyl-ornithine (ref. 41 ). The carboxyl group was oriented perpendicular to the pyrindine plane of PLP on the re face. The structures were energy minimized with the InsightII/Built module (steepest descents until a maximum derivative of 20 kcal/mol, constant valence force field). The optimized structures were docked into the modeled active site of ODC by using the program DOCK4, and the complexes of ODC and analogs were further optimized by energy minimization with the InsightII/Discover module (conjugation gradient until a maximum derivative of 0.01 kcal/mol, constant valence force field). The intramolecular binding energy of the phosphopyridoxyl-substrate derivatives and hODC, as shown in Fig. 2 , was then calculated by using the InsightII/Docking module.


Figure 2
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  		Figure 2. Stereo view of the binding model of the phosphopyridoxyl conjugates in the active site of hODC. A) Putative strong binding mode of phosphopyridoxyl (yellow) -tryptophan (white). The active site residues within 3 Å of the phosphopyridoxyl-tryptophan conjugate and the hydrogen bond between phosphopyridoxyl-tryptophan and hODC are displayed in white. B) Putative binding mode of phosphopyridoxyl-histidine (cyan), phosphopyridoxyl-phenylalanine (blue), phosphopyridoxyl-pyridine-D-Ala (red), and the BOC-protected phosphopyridoxyl-{alpha},{gamma}-diaminobutyric acid (brown). Hydrogen bonds between phosphopyridoxyl derivatives and hODC are white.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Design of potential inhibitors to inhibit the proliferation of tumor cells and cellular hODC
As demonstrated previously (ref. 19 ), a hydrophobic pocket is present in the active site of hODC, where the {epsilon}-amino group of ornithine is embedded between the 2 hODC subunits. The pocket is formed by the aromatic residues Tyr-A389, Tyr-A331, Phe-B397, and Tyr-B323. Only the dimer of ODC is catalytically active, as an active site consists of residues from both subunits A and B. This hydrophobic pocket is not fully occupied by the substrate ornithine (ref. 19 ) and can be utilized in designing hydrophobic derivatives of inhibitors with improved binding affinity. Indeed, this pocket favored the binding of the leading compound POB, which carried a BOC group at the {epsilon}-amino group. Here, we conjugated PL or PLP, the cellular active forms of vitamin B6, with different amino acid derivatives or PAs (compounds 1 to 23, Fig. 1 ). As shown in Fig. 3A , DFMO, the synthesized and purified (see Materials and Methods) vitamin B6 conjugates 1 to 23 (Fig. 1 ), the PAs (PUT, SPD or SPM), and the cellular active forms of vitamin B6 (PL, PMP, or PLP) as well as amino acid derivatives (BOM and BLM) were screened for their property to prevent proliferation of LN229 cells. Only the compounds 10, 12, 14, 16, and 17, which contain a hydrophobic amino acid side chain like an aromatic ring or a BOC group, together with a methyl ester at the {alpha}-carboxylate group, inhibited proliferation of LN229 cell at 100 µM concentration more or equally efficiently than DFMO at 500 µM concentration (Fig. 3A ). Cell inhibition was totally abolished, if the side chain of the conjugate was positively (compounds 4–9 and 20–23) and negatively charged (compound 3) (Fig. 3A ). The de-esterified forms (compounds 11, 13, and 15) of compounds 10, 12, and 14 also lost the inhibitory effect.


Figure 3
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  		Figure 3. Effect of pyridoxyl-substrate derivatives on cells. A) Effect on cell proliferation. Glioma LN229 cells were treated for 3 days with the indicated compounds: DFMO, compounds 1 to 23 (Fig. 1 ), PUT, SPD, SPM, PL, PMP, PLP, BOM (BOC-protected ornithine methyl ester), and BLM (BOC-protected lysine methyl ester). The concentration used was 100 µM except for DFMO (500 µM), compound 12 (50 µM), and PLP, BOM, and BOL (200 µM each). Cells were counted as described in Materials and Methods. Error bars = SD; n = 3. To prevent exogenous oxidation of PUT, SPD, and SPM, 2 mM aminoguanidine was added to inhibit serum oxidases. B) Effect on serum-induced intracellular ODC activity. LN229 cells were treated with the indicated compounds for 3 days. Cells were collected and lysed, and ODC activity was measured as described in Materials and Methods. Error bars = SD; n = 3.

In agreement with their inhibitory effect on cell proliferation and molecular modeling prediction (Fig. 2 ), pyridoxyl- and phosphopyridoxyl-L-tryptophan methyl ester (PTME, compound 10, and pPTME, compound 12), the BOC-protected pyridoxyl-L-ornithine methyl ester (POB, compound 14), and the pyridoxyl-L-phenylalanine methyl ester (compound 16) also significantly affected serum-induced intracellular ODC activity. Whereas conjugates with an imidazole motif (compounds 4 and 6), pyridine D-alanine methyl ester (compound 17) or BOC-protected {alpha}, {gamma}-diaminobutyric acid methyl ester (compound 18) did not (Fig. 3B ). Phosphorylated PTME suppressed the induced intracellular ODC activity nearly completely if LN229 cells were treated at a concentration of 50 µM for 3 days, an inhibition comparable to that with PTME and POB at 100 µM (Fig. 3B ). The pPTME (compound 12) and phosphopyridoxyl-L-tryptophan (compound 13), which is the assumed cellular active form of pPTME and PTME (refs. 18 , 19 , 26 ), were found to inhibit the ODC activity when added to LN229 cell extracts with an IC50 of ~50 µM, whereas the unphosphorylated compounds PTME, POB, 16, 17, 18, and 20 did not. Furthermore, PTME and pPTME indeed showed dose-dependent inhibition on the proliferation of LN229 with an IC50 value ~25 and ~50 µM, respectively (Fig. 4 ).


Figure 4
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  		Figure 4. Dose-dependent cell growth inhibition by PTME (compound 10) and pPTME (compound 12). Glioma LN229 cells were incubated with different concentrations of PTME (•) or with pPTME ({blacksquare}) for 3 days, counted, and compared with the untreated controls (0%). Error bars = SD; n = 3.

Binding mode of designed compounds
The presumably active form of PTME (compound 10) and pPTME (compound 12) in the cell is phosphopyridoxyl-tryptophan (compound 13). Molecular modeling showed it is tightly interacting with the active site of hODC (Fig. 2A ). The phosphate group forms hydrogen bonds with Arg-A277 and Tyr-A389, also observed in the crystal structures of tODC and hODC with PLP (refs. 36 , 37 , 42 ). The side chain of tryptophan is embedded in a hydrophobic pocket composed of residues Tyr-A331, Tyr-A389, Tyr-B323, and Phe-B397, and binding is supported by stacking effects between the aromatic rings of the indole and Tyr-A389. The N atom of the indole ring is able to form a hydrogen bond with Asp-A332, which was described to interact with the {epsilon}-amino group of DFMO or APA (refs. 36 , 38 ), known inhibitors of ornithine decarboxylase. The active form of POB (compound 14), phosphopyridoxyl-ornithine (BOC), also bound favorably into the active form of hODC, as reported previously (ref. 19 ). In addition to the hydrogen-bond network observed between the phosphate group and Arg-A277 and Tyr-A389, the carbonyl group of the side chain of POB (compound 12) formed a hydrogen bond with Arg-A277. Phosphopyridoxyl-histidine and phosphopyridoxyl-phenylalanine, the assumed active forms of compounds 4 (cyan, Fig. 2B ) and 16 (blue), shared a similar active-site occupancy with phosphopyridoxyl-tryptophan (compound 13) but formed no hydrogen bond with Asp-A332 or other surrounding residues (Fig. 2B ). The presumed intracellular forms of the inactive compounds 17 and 18 cannot properly occupy the cofactor or substrate binding site and miss the favorable interactions as well (Fig. 2B , red and brown). The calculated intramolecular binding energies of –179 and –167 kcal/mol for phosphopyridoxyl-tryptophan and phosporylated de-esterified POB, respectively, support the favorable interactions of these compounds over the others.

Mode of action of the designed inhibitory compounds
Cell inhibition caused by DFMO can be reversed by adding PAs to the cells (ref. 43 ). This effect was considered one reason for ineffectiveness of DFMO (ref. 6 ). PTME and pPTME inhibited proliferation of LN229 and HMC-1 cells more efficiently than DFMO, but their inhibition could not be reversed by the PAs PUT, SPD, or SPM (Fig. 5 ), as observed and discussed previously with POB (Supplemental Fig. 2) (ref. 19 ).


Figure 5
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  		Figure 5. Effect of exogenous polyamines on cell growth inhibition. LN229 cells were treated or not (control, 100%) as indicated during 3 days in the presence of 2 mM aminoguanidine, which inhibits the oxidation of polyamines by serum oxidases. Open columns, LN229 cells; solid columns, HMC-1 cells. Error bars = SD; n = 3.

PA analogs strongly induce the catabolic enzymes SSAT and PAO of PA metabolism by acting in a complex PA regulatory system (ref. 44 ) and stop cell proliferation with or without depleting the PA pools (ref. 12 ). The designed cellular active pyridoxyl-amino acid derivatives seem not to reduce the PA pools below that of the control as measured and reported with POB, the representative proliferation inhibitor of this type (Supplemental Fig. 3) (ref. 19 ). PTME showed a similar behavior; the PA concentrations were nearly the same as with POB when measured for comparison after 3 days of incubation. The reason that the intracellular PA concentration was not depleted as drastically as with DFMO (ref. 19 ) could be that despite the inhibition of intracellular ODC, intracellular conversion of PAs was accelerated by PTME, pPTME, and POB through induction of polyamine metabolic enzymes in a complex interplay whose regulation is not yet understood (refs. 2 , 34 , 45 , 46 ). Indeed, intracellular PAO activity was up-regulated to 195, 270, and 206%, and SSAT activity was also induced to 620, 790, and 194%, when LN229 cells were incubated with PTME, pPTME, and POB, respectively, whereas DFMO and inactive conjugates (compounds 4, 16, 17 and 18) had no effect (Fig. 6 ).


Figure 6
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  		Figure 6. Effect of pyridoxyl-substrate derivatives on polyamine catabolic enzymes. LN229 cells were treated with the indicated compounds or not (control, 100%) for 2 days. Cells were collected and lysed, and the activity of PAO (A) or of SSAT (B) was measured as described in Materials and Methods. Error bars = SD; n = 3 (A), 2 (B).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Essential features of the active coenzyme-substrate derivatives
Studying the interaction between analogs of reaction intermediates and the active site of enzymes and exploring the 3-dimensional geometry of the binding site by using structurally diverse mimics is a reasonable strategy for discovering improved inhibitors. To find more potent cell-permeable inhibitors for intracellular hODC, different molecular mimics of reaction intermediates were designed and synthesized through conjugating PL or PLP with substrate derivatives. The achieved inhibition of cell proliferation and ODC activity by these newly designed inhibitors (compounds 10 and 12) as well as compound 14 (POB, ref. 19 ) bearing an ornithine-like side chain with a -NH-(CHx)4-NH- motif and a bulky hydrophobic group (Fig. 7 ) was more pronounced than that of compounds, which either lack a hydrophobic group (compounds 4–9) or the distal N atom in the side chain (compound 16) or have it in the wrong position (compounds 18 and 19). Compound 18, a mimic of POB, was synthesized as a control compound. It has a hydrophobic BOC group but 1 methylene group less and thus a -NH-(CH2)3-NH- instead of the -NH-(CHx)4-NH- PA motif. The missing inhibition of hODC and cell proliferation with the various compounds (Fig. 1 ) strongly indicate that the inhibitory effect of POB, pPTME, and PTME is the result of specific interactions. The conjugate of PL with pyridyl-D-alanine methyl ester (compound 17), which has a -NH-(CHx)4-NH- motif and a hydrophobic side chain but a D chiral center, showed no inhibition of hODC activity in cells and in cell extracts. These results demonstrate that maintaining the -NH-(CHx)4-NH- motif of the L-ornithine backbone is essential for binding to ODC and that additional hydrophobic modifications at the {epsilon}-amino group of ornithine or appropriate bulky hydrophobic group in the side chain favors the binding to ODC.


Figure 7
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  		Figure 7. Essential features of the active inhibitors. Overlap of the structural skeleton of the active pyridoxyl-ligand derivatives, which inhibited intracellular ODC and cell proliferation. Bold solid line, required -N-(CHx)4-N- motif; bold dashed line, overlap of the hydrophobic fragments supporting binding; R1, moiety required for cell permeability.

The de-esterified forms (compounds 11, 13 and 15) of the active compounds 10, 12, and 14 lost the inhibitory effect (Fig. 3 ), indicating that successful exposure of this class of compounds to intracellular ODC requires the carboxyl group to be esterified (Fig. 7 ). Esterification seems to be crucial for uptake into cells (ref. 19 ).

The phosphorylated PTME (compound 12) exceeded the potency of PTME (compound 10) and POB (compound 14) (ref. 19 ) as a result of better binding properties, as suggested by molecular modeling (Fig. 2A ) and pharmacokinetics—indicated by the absence of the initial lag phase of pPTME found with PTME (Fig. 4 ). Apparently, its hydrophobicity is such that it can pass the cell membrane. Its overall calculated logP of 1.1, which is in the range of cell-permeable compounds (ref. 47 ) and the strong retardation of pPTME on a C18 column (see Materials and Methods) supports this conclusion. Because it is already phosphorylated, it will be more prone to bind to freshly synthesized apoODC than unphosphorylated conjugates, which have to be phosphorylated in cells by PL kinase (ref. 26 ).

Binding modes of coenzyme-substrate derivatives in the active site of hODC
The binding mode of the active compounds 10 and 12, having a -NH-(CHx)4-NH- motif, showed that their hydrophobic side chains are located in the hydrophobic pocket, which is formed in the interface of the 2 subunits of hODC (Fig. 2A ). The indole side chain stacks with the hydrophobic side chain of Tyr-A389. This hydrophobic interaction is also observed in the crystal structure of APA-bound hODC (ref. 38 ). Alternatively to the observed hydrogen bond formed with the functional binding residue Asp-A332 in this model, the N atom of -NH-(CHx)4-NH- motif could also form other hydrogen bonds via a water molecule to other functional binding residues, such as Asp-B361, the backbone carbonyl of Tyr-A331, and the hydroxyl group of Tyr-B323, as was observed and described in the APA-complex (ref. 38 ).

The substantial loss of affinity of compound 4 with an imidazole ring (cyan; Fig. 4B ) or of compound 16 with a bulky hydrophobic benzene ring (blue), could be the result of the loss of this hydrogen bond and less pronounced hydrophobic interactions and ring stacking effects. Compound 17, with a -NH-(CHx)4-NH- motif but having a D chiral center instead of L, showed a mismatching binding mode of the pyridine ring in the substrate binding site of hODC. The pyridoxyl ring of the compound 18, which has a -NH-(CHx)3-NH- motif rather than a -NH-(CHx)4-NH-, cannot be docked properly into the binding site. All of these indicate that the -NH-(CHx)4-NH- motif of L-ornithine is needed for binding to ODC.

In agreement with the inhibitory results discussed, the binding modes of coenzyme-substrate derivatives, as suggested by molecular modeling, also prove that the bulky hydrophobic group at the side chain and the -NH-(CHx)4-NH- motif is essential for efficient binding to ODC and responsible for the inhibitory effect of these compounds.

Mode of action of coenzyme-substrate derivatives in cells
In addition to the inhibition of intracellular ODC activity, PTME (compound 10), pPTME (compound 12), and POB (compound 14) induce, as polyamine analogs often do, the activity of the enzymes of the polyamine catabolism, namely that of PAO and SSAT. Probably, the PA motif (-NH-(CHx)4-NH-) present in PTME, pPTME, and POB seems to be recognized by the PA regulatory system. The up-regulation of PA catabolic enzymes regenerates PUT and SPD from SPD and SPM, respectively (ref. 48 ) and might, together with an inhibited export or up-regulated import system (ref. 1 ), explain why the PAs were not completely depleted even if ODC was inhibited. H2O2 is produced in the PAO-catalyzed reaction and is regarded as the main cause of PA analog-induced cell death (ref. 1 ) and thus also contributes to the more pronounced inhibition that the new kind of inhibitors exert. The dual mode of the 3 active compounds in LN229 cells (i.e., inhibition of ODC and induction of polyamine catabolic enzymes) interferes in the intracellular polyamine pools and might underlie their inhibitory effect on cell proliferation, which compares favorably with that of DFMO. The designed compounds bearing a PA motif might affect DNA conformation and aggregation as PAs seem to do and might even compete with or disrupt the normal interaction between PA and DNA, which has been proposed as a mechanism for the cytotoxic activity of certain PA analogs (ref. 1 ).

In conclusion, the application of structurally diverse compounds of reaction intermediates to map the binding requirements of such enzymes as ODC proved an efficient tool to develop new inhibitors of it. The structural requirements for inhibition of intracellular hODC as well as the proliferation of glioma LN229 cells is summarized and defined in Fig. 7 and are verified not only with inhibitory experiments but also by molecular modeling. The present investigation will serve in developing even better inhibitors for ODC for the treatment of tumors like gliomas.


   ACKNOWLEDGMENTS
 
We thank the COST Switzerland (Action 922, grant C02.0017) for financial support. We thank Dr. S. Bienz and collaborators (Institute of Organic Chemistry, University of Zurich) for the assistance in the synthesis of the designed analogs. We thank Dr. L. Persson and coworkers (Department of Experimental Medical Research, Lund University, Lund, Sweden) for measuring the polyamines, Dr. K. Frei (Department of Neurosurgery, University Hospital, University of Zurich) for kindly providing glioma cell lines, Dr. P. Christen (Department of Biochemistry, University of Zurich) for providing us with some of the compounds and for critical reading of the manuscript, Dr. J. Butterfield and coworkers (Mayo Clinic, Rochester, MN, USA) for providing HMC-1 cells, and Dr. R. D. Walter and Dr. I. B. Müller for helpful discussions.


   FOOTNOTES
 
1 Current address: Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne EPFL, CH-1015 Lausanne, Switzerland.

Received for publication June 14, 2008. Accepted for publication September 25, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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