Abstract
Background: In order to reduce toxicity and to enhance anticancer activity of nitrogen mustards, three hybrid steroidal esters were synthesized and tested in vitro against human pancreatic cancer cells expressing uridine phosphorylase (UPase). The inhibition potency against a target protein implicated in the chemotherapy of solid tumors, such as UPase, is of fundamental importance in the design and synthesis of new anticancer drugs. Materials and Methods: MTT colorimetric assay and molecular docking were employed for the in vitro and in silico drug evaluation, respectively. Results: A difference in cell sensitivity was found, which followed the known different UPase expression in the cell lines. Molecular docking studies on UPase protein, revealed the tested compounds to be bound to the binding cavity of the protein, with different affinity. Between the two D-modified compounds, the D-homo-aza (lactam)-hybrid compound (C2) was found to interact with the protein in a more efficient way. Conclusion: The molecular docking data were in accordance with the in vitro results, where the lactam steroid alkylator showed significantly higher cytostatic and cytotoxic activity than the non-D-modified compounds, which also correlated with the level of UPase expression in the pancreatic cancer cells.
- Uridine phosphorylase
- hybrid steroidal alkylators
- in silico molecular docking
- in vitro assay
- mitrogen mustards
- pancreatic cancer cells
Hybrid compounds, which combine two active molecules in one, such as steroid alkylators containing steroidal hormones as biological vectors for cytotoxic agents, have been introduced into anticancer therapy based on, the potential for increased intracellular concentration of these agents due to their lipophilic steroid carrier, low systemic toxicity and the possibility of targeting steroid receptors and increasing specificity particularly against hormone-dependent neoplasms (1, 2).
Although the majority of alkylating or similar agents are inactive in the treatment of tumours of the gastrointestinal system, the homo-aza-(lactam) steroids, which contain a –NH–CO– group inside the A or D steroid nucleus, as biological carriers for carboxylic derivatives of N,N-bis(2-chloroethyl)aniline (nitrogen mustards), demonstrated high activity against colon cancer both in vitro and in vivo, as well as presenting very good activity against pancreatic cancer (3-5). They are also effective against leukaemia (6-9), melanoma (8, 10) and non-small cell lung carcinoma (NSCLC) in vitro and in vivo (11) and a variety of cancer cell lines (12).
Several hypotheses of the structure–activity relationships that lead to the anticancer effect of the modified lactam steroid alkylators, have been proposed, but as yet, precise molecular mechanism of lactamic steroid alkylator action is still unclear. However, other cellular moieties and pathways, beside the typical alkylating impact, may be implicated. Our laboratory showed that these compounds highly interact with the enzymes that are involved in the metabolism of pyrimidines because of the structural similarity of the steroidal lactam ring with the common pyrimidine derivative uracil (3).
Uridine phosphorylases (UPases, EC 2.4.2.3) have been shown to be of medical and biological importance in the activation and catabolism of fluoropyrimidines, which are used in the chemotherapy of solid tumors (13). UPase can also catalyse the formation of nucleosides from uracil and ribose-1-phosphate and is the most important phosphorylase in the regulation of uridine homeostasis. UPase is a critical enzyme in the activation of 5-fluorouracil (5-FU) and its prodrug 5’-deoxy-5-fluorouridine (5’DFUR /capecitabine) via anabolism of 5-FU through the pyrimidine salvage pathway or the phosphorolysis of 5’DFUR into 5-FU. Additionally, UPase activity is elevated in various tumour tissues and is believed to contribute to drug selectivity and to confer 5-FU therapeutic advantage (14-16). However, the clinical effectiveness of these fluoropyrimidine antimetabolites is impeded by their toxicity to normal tissue (17-19). On the other hand, decreased levels of UPase provide a mechanism of acquired resistance to 5-FU in colorectal cancer (20). A high rate of UPase expression is also related to a higher incidence of tumour relapse, more advanced cancer stage, and worse survival in patients with head and neck or breast cancer (21, 22).
Pancreatic cancer is normally presented at an advanced stage and survival rates remain low despite the routine use of chemotherapy and radiotherapy and the development of targeted therapies. Novel more effective therapeutic agents and approaches are urgently needed (23). UPase is highly expressed in most pancreatic carcinomas and could potentially be useful in tumour targeting (24).
In order to reduce toxicity and to enhance the anticancer activity of nitrogen mustards, we have synthesized three new androstan D-modified steroidal alkylators, 3β-hydroxy-5α-androstan-[p-[N,N-bis(2-chloroethyl)amino]phenyl]acetate (compound C1), 3β-hydroxy-13α-amino-13,17-seco-5α-androstan-17-oic-13,17-lactam-[p-[N,N-bis(2-chloroethyl) amino]phenyl]acetate (compound C2) and 3β-hydroxy-17-oxo-17α-oxa-5α-androstan-[p-[N,N-bis(2-chloroethyl)amino] phenyl]acetate (compound C3) (Figure 1a) (25, 26). The anticancer activity of these three compounds against three human pancreatic cancer cell lines that differentially express the UPase enzyme is here described.
This study also aimed at the elucidation of the underlying mechanism of action of the three modified steroidal esters using molecular docking studies (based on previous crystallographic and molecular modeling studies) to identify the binding interactions of the compounds with amino acid residues of the UPase protein within the active site of the protein target.
Materials and Methods
Drug preparations. The three esters of p-[N,N-bis(2-chloroethyl)aminophenyl]acetic acid with, androstan (C1), D-homo-aza (lactam)-androstan (C2) and D-lactone androstan (C3) were synthesized as previously described (26, 27). Stock solutions of the tested compounds, were made immediately before use. C1, C2 and C3 were initially dissolved in a small volume of 10% dimethyl sulfoxide (DMSO).
Cell cultures. Human pancreatic adenocarcinoma cell lines Panc1, MiaPaCa2 and Hs766T (obtained from the American Type Culture Collection (ATCC); Manassas, VA, USA), which differentially express UPase (Hs766T>Panc1>MiaPaCa2) (10), were grown in a monolayer in RPMI-1640 medium supplemented with 10% fetal calf serum, penicillin, streptomycin and 1 mM sodium pyruvate and maintained at 37°C in air containing 5% CO2.
In vitro testing. The cells were seeded out at a density of 50,000 cells/ml and after 24 h culture were treated with 10–100 μM of the tested compounds C1, C2 and C3 for 48 h. The viability of the cultured cells was estimated by MTT assay (28). MTT (Sigma, St. Louis, MO, USA) was dissolved in PBS at a concentration of 5 mg/ml, filter sterilized and stored at 4°C. MTT (0.2 ml of stock solution per ml) was added to each culture and incubated for 3 h at 37°C to allow metabolization. The formazan crystals produced were solubilized by acidic isopropanol (0.04 N HCl in absolute isopropanol in a ratio 1:3 v/v). Absorbance of the converted dye was measured at a wavelength of 540 nm on an Anthos Labtec AR 8001 microplate reader (Diagnostics Pasteur, Sanofi). The mean concentrations of each drug that generated 50% or total (100%) growth inhibition (GI50 and TGI, respectively) as well as the drug concentrations that produced cytotoxicity against 50% of the cultured cells (IC50) were calculated by the linear regression method (29, 30). Using seven absorbance measurements (time 24 h [Ct24], control growth 72 h [Ct72], and test growth in the presence of drug at five concentration levels [Tt72×]), the percentage of growth was calculated at each drug concentration. The percentage growth inhibition was calculated according to National Cancer Institute (NCI) as: ([Tt72×][Ct24]/[Ct72][Ct24])×100 for concentrations for which Tt72× ≥Ct24, and ([Tt72×][Ct24]/[Ct24])×100 for concentrations for which Tt72×<Ct24. GI50 was calculated from ([Tt72×][Ct24]/[Ct72] [Ct24])×100=50, TGI from ([Tt72×][Ct24]/[Ct72][Ct24])×100=0 and IC50 from [(Tt72×)Ct24)/Ct24]×100=50. All the experiments were conducted in triplicate.
In silico computational methods (molecular modeling and docking calculations). All the compounds were screened virtually against a large Protein Drug Target Database comprising almost 1,000 target proteins. The search for protein targets of known crystal structure and biological significance was restricted to three major therapeutic categories: neoplastic, inflammation and bacterial infection diseases. All the molecules were built in 3D coordinates and their best, most stable (lower energy) conformation was detected by geometrical optimization of their structure in the gas phase, as implemented in the Spartan ‘08 Molecular Modeling program suite (Spartan ’08 v.1.2.0, Wavefunction Inc., Irvine, CA, USA; www.wavefun.com). The structure of the molecules were initially optimised (via energy minimization) by conformational search using the Monte Carlo method with the MMFF94 molecular mechanics model, included in the Spartan ’08 program suite. Geometry optimization (leading to the most stable conformer with the lowest energy) was accomplished via quantum-chemical calculations by utilizing the ab initio Hartree–Fock method with a 6-31G* basis set (Figure 1b). The chemical structures of 5-phenylthioacyclouridine (PTAU or 1-[(2-hydroxyethoxy)methyl]-5-phenylthiouracil), a highly specific and potent inhibitor of UPase (31, 32), and the substrate used in the docking studies are shown in Figure 1c.
The molecular docking studies were carried out on two crystal structures of the UPase target protein (Protein Data Bank, PDB entry codes 1U1D and 1TGV), to investigate the effect of the synthesized compounds on this protein. Both X-ray structures of the UPase protein were obtained from the Brookhaven Protein Data Bank (operated by the Research Collaboratory for Structural Bioinformatics, RCSB) (33-35). The molecular docking simulations were performed by BioMedCAChe 6.1 computer-aided chemistry software package, which is part of the CAChe package (CAChe WorkSystem Pro version 7.5.0.85, Fujitsu Co. Ltd., Tokyo, Japan) (36). The compound–protein complexes produced were ranked by the energy score, including their binding conformations. Best docked poses, with both lower binding energies and stronger interaction pattern, were derived from a number of solutions (docking results), usually with the higher ranking. Three-dimensional (3D) models of the above protein crystal structures were developed, after the deletion of the co-crystallized bound inhibitor. The docking procedure enables the flexibility of the ligand within the protein binding site by means of a 4-point chiral pharmacophoric comparison between the ligand and the site. The final output of the docking procedure is a set of solutions (docking results) ranked according to the corresponding scoring function values, each defined by the 3D coordinates of its atoms and expressed as a PDB file. In this study, the docking procedure with the aid of BioMedCAChe, was shown to accurately reproduce experimentally observed binding modes of the studied UPase inhibitor PTAU and its substrate 5FU, in terms of RMSD (root-mean squared deviation). The BioMedCAChe provided excellent results as low values of RMSD (best docked solutions of docking results 0.23-0.45 Å) were observed between the experimental and the best-scoring docked structures derived by superimposition of these structures (the accuracy of the docking results are good when the RMSD is mostly below 1.0 Å). The ability to accurately predict the binding conformation of the enzyme inhibitor and substrate, gave confidence that the BioMedCAChe would also exhibit a similar accuracy with the investigated molecules in the study. The PyMol molecular graphics system (DeLano Scientific, San Carlos, CA, USA, version 0.99), was used to visualize the molecules and the results of the docking and to construct the molecular models (37).
Statistical analysis. Statistical analysis was performed with SigmaPlot software v. 10.0 (SigmaPlot Software, Inc., Germany) using two-tailed Student's t-test. In all tests, a criterion of p≤0.05 was considered necessary for statistical significance. For all values derived from the corresponding dose–effect curves and drawn from triplicate determinations, coefficient of variation (CV) calculated to be lower than 5%.
Results
In vitro testing. The D-lactam steroid alkylator (C2) presented significant activity (compared to compounds C1 and C3) against the 3 human pancreatic adenocarcinoma cell lines (Panc1, MiaPaCa2 and Hs766T) tested (p<0.0001, t-test). C2, in comparison to C1 and C3, was the most active (p<0.001, paired t-test) whereas the respective D-lactone steroid ester (C3) showed modest activity and the unmodified androsterone alkylator (C1) was inactive (Table I). Furthermore, the anticancer effects of C2 correlated with the expression of UPase in the pancreatic cancer cells. Thus, Hs776T cells that express the higher level of UPase were the most sensitive, Panc1 cells were less sensitive and MiaPaCa2 cells that present the lowest UPase expression demonstrated the least sensitivity to C2 treatment (Figure 2).
In silico docking study of protein–ligand interactions. The computed binding energies (kcal/mol) of the tested compounds C1, C2 and C3 on the UPase protein are shown in Table II, and indicated that C2 established better attachment than C1 and C3, while C1 bound to protein better than C3.
The docking results of the C1, C2 and C3 molecules in complex with the crystal structure of the 1U1D UPase showed that in both subunits, in four out of the six chains of the hexameric structure of the UPase, the C1, C2 and C3 molecules were docked at the same place as PTAU was attached (Figure 3a). However, in addition to binding in the active site, in two docking poses for the compounds C1 and C2 and one for C3, the interaction of the C1, C2 and C3 molecules with separate binding pockets located near the center of the hexameric structure was identified (Figure 3a). The C1, C2 and C3 molecules were inserted in the binding pocket of the protein by their steroidal moieties, while the alkylating moieties were positioned at the opening of the binding pocket. Figure 3b illustrates the ligand-binding pocket of the protein in one chain of the hexameric structure with the docked conformations of the C1, C2, C3 and PTAU molecules. The stabilization of the compounds in the pocket was attributed to the formation of hydrogen bond, polar and hydrophobic contacts (the latter interactions for clarity are not shown in the figure).
The X-ray crystallography study revealed (Figure 3c, d) that the PTAU inhibitor anchored the UPase target protein via a network of hydrogen bond and hydrophobic binding interactions with the amino acid residues of the ligand-binding cavity of the protein. The C2 carbonyl O of the pyrimidine ring of PTAU interacted with the amino acid residue Gln166/NE2 (2.76 Å) and hydrogen bonded with water67/OH (3.48 Å). In addition, the C4 carbonyl O of the pyrimidine ring of PTAU contributed to the protein binding via polar and hydrogen bond interactions with the following amino acid residue and water molecules: Gln166/OE1 (3.45 Å), Arg168/NH1 (3.44 Å), Arg168/NH2 (2.81 Å) and water263/OH (2.74 Å). Additionally, the ethoxy oxygen atom of the (2-hydroxyethoxy)methyl group and the carbonyl oxygens of the pyrimidine ring of PTAU accepted hydrogen bonds from the amino acid residues His11 (2.79 Å), Thr97 (3.12 Å) and Gln169 (2.68 Å) and water molecule water299/OH (3.00 Å). Furthermore, in the UPase/PTAU complex, it was established that hydrophobic interactions involved the residues Ile714 (3.76 Å), Val715 (3.86 and 3.79 Å), Thr589 (3.88 Å), Gln660 (3.85 Å), Phe656 (3.86, 3.77, 3.65, 3.68, 3.69 and 3.79 Å) and Gly590 (3.59, 3.80, 3.89 and 3.74 Å).
C1 compound was stabilized in the complex with the 1U1D UPase protein through a number of hydrogen bond and hydrophobic contacts as shown in Table III.
The C2 molecule was found to be bound to the protein by many more interactions (polar, hydrogen bond or hydrophobic) compared to C1 and C3, with both its steroidal and alkylating moiety. The incorporation of the nitrogen atom in the D-ring of C2 (N18) improved its binding capacity with the involvement of the amino acid residues, water molecules and phosphate group as shown in Table IV.
The C3 compound was stabilized in the UPase/C3 complex more weakly than C2 and again through both the alkylating and steroidal moieties of the molecule. The stabilization of the molecule in the alkylating pocket was attributed to a hydrogen bond network involving: O1’ atom, C2’ carbonyl oxygen and nitrogen atom of the N-chloroethyl group (Table V). The steroidal moiety of the C3 molecule contributed to the binding through hydrogen bond and polar contacts of the D-ring C17 carbonyl oxygen (O21) atom and O18.
The stabilization of the compounds in the pocket was additionally attributed to hydrophobic contacts formed by the hydrophobic residues Ile220, Ile228, Leu121, Phe7, and Phe162. Hydrophobic interactions were also observed with the incorporation of Pro229, Thr95, Glu227, Glu196, Glu127, His8, Arg48, Lys3, Ser4 and His179 (Tables III, IV and V).
Figure 4 illustrates the docked compounds C1, C2 and C3 superimposed with 5-FU within the 1TGV UPase binding site. As illustrated from the ligand-binding site architecture, the ligands occupied the same place as 5-FU in the crystal structure. All the compounds were shown to be anchored inside the ligand-binding pocket via polar, H-bond and van der Waals hydrophobic interactions with residues Ile220, Val221, Asn222, Ile228, Pro229, Phe7, His8, Thr9, Arg91, Phe162, Arg168, Met197 and Glu198. The polar, H-bond and hydrophobic interactions between compounds C1, C2, C3 and 5-FU and the amino acid residues of the ligand binding cavity of UPase (1TGV) are shown in Figure 4c. Binding interactions between the atoms of the steroidal and alkylating moieties of compounds C1, C2 and C3 and the atoms of the residues of the active site of UPase are shown in Table VI.
Beyond the UPase target protein, for each compound, after extended virtual screening with the aid of molecular modelling and docking techniques, more than 20 chief target proteins were also found to be inhibited in a specific order of binding capacity. The produced compound–protein complexes were ranked by the energy score, including their binding conformations (Table VII).
Discussion
This preclinical research indicated that compound C2 – a promising D-homo-aza-steroid alkylating ester – is a very active compound with a relatively low toxicity rate and a high therapeutic ratio against pancreatic cancer cells. Previously, the compound C2 (NSC 290205) has shown in vivo cytostatic activity against murine pancreatic adenocarcinoma PAN02 in combination with adriamycin (38) was active in treating lung and breast xenografts, as well as a number of rodent tumors (25, 39) and leukaemia (40), and induced cytogenetic damage in human lymphocytes (41).
Prediction of the binding mode of a ligand within the active site of a protein of known structure is of paramount importance in rational drug design. The differences in activity of the studied compounds towards the tested cell lines, as shown in the in vitro experiments, could be partially explained by the molecular docking calculations which indicated better stabilization with C2 than C1 or C3 inside the binding cavity of the protein. The lower predicted binding energy of compound C2 (which included the lactamic group) compared to that of compounds C1 and C3 was in accordance with its extended contact network. The docking simulation studies showed that compounds C1, C2 and C3 entered into the binding pocket of UPase protein with their steroidal part and were positioned in the same place as the specific and potent co-crystallized inhibitor PTAU and substrate 5-FU of the protein, mimicking their action. The enzyme–inhibitor complexes were stabilized mostly by van der Waals hydrophobic interactions in the hydrophobic steroidal region and by hydrogen bonds and polar interactions in the hydrophilic alkylating region.
The significance of the insertion of a lactamic group in homo-aza-steroidal esters, to increase antitumor activity has been pointed out in many studies (1-3, 6-9). In a series of hybrid steroidal esters of nitrosourea with an A-lactamic ring and nitrosourea esterified compounds at position 17, we have shown anticancer activity against PANO2 murine pancreatic adenocarcinoma (5), which was probably due to the lactam group (–NHCO–) modification. The presence of the characteristic group –NHCO– of the homo-aza-steroid molecule C2 has proved important in binding with the target proteins, in order to lower acute toxicity and improve antitumor activity in cancer research (7, 8). Possibly the antineoplastic effects of these steroidal esters may be due to the multiple interactions of the –NHCO– group with similar groups or with specific structural domains which exist in DNA and proteins. It was suggested that the –NHCO– lactam group maybe transformed by a metabolic process or at least by an enzymatically catalyzed reaction to active species which strongly interact with similar groups existing in the DNA and proteins (–NH–CO– → –N(−)H + –C(+)=O) (7, 8). Molecular docking studies incorporating such active species active on DNA and various proteins are underway.
In conclusion, the in silico approach (molecular docking calculations) may provide a structural rationale of the receptor antagonism of the tested compounds, providing a molecular basis for understanding their inhibitory effect and suggesting that it could be attributed, at least in part, to the inhibition of the pathways involving UPase. On the other hand, taking into consideration the fact that cellular death and apoptosis are multifactor processes, a number of other pathways and target proteins could play a role in the observed in vitro inhibitory effect. More than 20 cancer-related target proteins were also inhibited by the studied compounds. The chief targets were proteins whose blockade might provide a mechanism to counteract cancer, inflammation and/or bacterial infection diseases.
- Received September 28, 2010.
- Revision received February 28, 2011.
- Accepted February 28, 2011.
- Copyright© 2011 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved