Cyclooxygenase (COX) is a key enzyme in the biosynthetic pathway leading to the formation of prostaglandins, which are mediators of inflammation. It exists mainly in two isoforms COX-1 and COX-2. The conventional nonsteroidal anti-inflammatory drugs (NSAIDs) have gastrointestinal side effects because they inhibit both isoforms. Recent data demonstrate that the overexpression of these enzymes, and in particular of cyclooxygenases-2, promotes multiple events involved in tumorigenesis; in addition, numerous studies show that the inhibition of cyclooxygenases-2 can delay or prevent certain forms of cancer. Agents that inhibit COX-2 while sparing COX-1 represent a new attractive therapeutic development and offer a new perspective for a further use of COX-2 inhibitors. The present study extends the evaluation of the COX activity to all 203 possible natural tripeptide sequences following a rational approach consisting in molecular modeling, synthesis, and biological tests. Based on data obtained from virtual screening, only those peptides with better profile of affinity have been selected and classified into two groups called S and E. Our results suggest that these novel compounds may have potential as structural templates for the design and subsequent development of the new selective COX-2 inhibitors drugs.
The main cause of the inflammation is the prostaglandins overproduction, which are synthesized by cyclooxygenase enzymes [
Prostaglandin-endoperoxide synthase, commonly called cyclooxygenase (COX), is an intracellular enzyme required for the conversion of arachidonic acid to prostaglandins. The two best-known COX isoforms are referred to as COX-1 and COX-2 for the order in which they were discovered [
Chemical structures of selective COX-2 inhibitors.
A common structural feature of these selective COX-2 inhibitors is the presence of two vicinal aryl rings attached to a central five- or six-membered heterocyclic or carbocyclic motif. Typical examples of selective COX-2 inhibitors like celecoxib, rofecoxib, valdecoxib, etoricoxib, and SC57666 demonstrate that a broad variety of five- or six-membered carbo- and heterocycles are acceptable for binding to the cyclooxygenase active site.
Recent reviews on the current status of COX-2 inhibitors further confirm the flexibility of the carbocyclic/heterocyclic core motif for COX-2 binding [
The PDB entries 1Q4G [
The 3D tripeptides library was built by an
The docking binding site, for both targets, has been defined by means of a regular box centered onto the protein chain A Tyr 385. Its volume was about 390,000 Å3 widely covering COX-1 and -2 binding sites. Glide grid maps have been computed using the standard precision algorithm. Our optimized library has been submitted to flexible Glide docking simulation with respect to both COX receptor models reducing the ligands van der Waals atom radius by a 0.8 factor. Default Glide interaction energies (ECvdW) have been adopted for scoring the tripeptide docking poses.
The synthesis of tripeptides (S1–E10) was performed according to the solid phase approach using standard Fmoc methodology in a manual reaction vessel [
The following protected amino acids were then added stepwise: N
In addition, after each step of deprotection and after each coupling step, Kaiser test was performed to confirm the completeremoval of the Fmoc protecting group, respectively, and to verify that complete coupling has occurred on all the free amines on the resin.
The N-terminal Fmoc group was removed as described above, and the peptide was released from the resin with TFA/iPr3SiH/H2O (90 : 5 : 5) for 3 h. The resin was removed by filtration, and the crude peptide was recovered by precipitation with cold anhydrous ethyl ether to give a white powder and then lyophilized.
All crude peptides were purified by RP-HPLC on a semipreparative C18-bonded silica column (Phenomenex, Jupiter, 250 × 10 mm) using a Shimadzu SPD 10A UV/VIS detector, with detection at 215 and 254 nm.
The column was perfused at a flow rate of 3 mL/min with solvent A (10%, v/v, water in 0.1% aqueous TFA), and a linear gradient from 10 to 90% of solvent B (80%, v/v, acetonitrile in 0.1% aqueous TFA) over 40 min was adopted for peptide elution. Analytical purity and retention time (tR) of each peptide were determined using HPLC conditions in the above solvent system (solvents A and B) programmed at a flow rate of 1 mL/min using a linear gradient from 10 to 90% B over 25 min, fitted with C-18 column Phenomenex, Jupiter C-18 column (
All analogues showed >97% purity when monitored at 215 nm. Homogeneous fractions, as established using analytical HPLC, were pooled and lyophilized.
Peptides molecular weights were determined by ESI mass spectrometry. ESI-MS analysis in positive ion mode, were made using a Finnigan LCQ ion trap instrument, manufactured by Thermo Finnigan (San Jose, CA, USA), equipped with the Excalibur software for processing the data acquired. The sample was dissolved in a mixture of water and methanol (50/50) and injected directly into the electrospray source, using a syringe pump, which maintains constant flow at 5
Structures and analytical data of tripeptides synthesized.
Peptide | Structure | HPLC | ESI-MS | Yield | |
---|---|---|---|---|---|
tR | found | calc | |||
S1 | GMD | 10.50 | 322.08 | 321.10 | 75% |
S2 | ERA | 9.99 | 375.3 | 374.19 | 80 % |
S3 | GHE | 8.08 | 342.14 | 341.13 | 67% |
S4 | GER | 10.00 | 361.17 | 360.18 | 80% |
S5 | DRC | 9.89 | 393.20 | 392.15 | 62% |
S6 | ARA | 10.46 | 317.24 | 316.19 | 68% |
S7 | PER | 8.87 | 401.40 | 400.21 | 81% |
S8 | KHI | 10.98 | 397.11 | 396.25 | 80% |
S9 | AER | 11.00 | 375.32 | 374.19 | 75% |
S10 | AGR | 9.03 | 303.34 | 302.17 | 79% |
E1 | SRH | 8.95 | 399.30 | 398.20 | 68% |
E2 | SWE | 8.04 | 421.0 | 420.16 | 69% |
E3 | IRT | 8.03 | 389.3 | 388.24 | 76% |
E4 | SMD | 8.00 | 342.16 | 351.11 | 78% |
E5 | GRN | 8.43 | 346.2 | 345.18 | 65% |
E6 | SHE | 8.68 | 372.17 | 371.14 | 76% |
E7 | SQE | 8.45 | 363.17 | 362.14 | 67% |
E8 | SMH | 9.46 | 374.26 | 373.14 | 75% |
E9 | ARM | 8.03 | 377.6 | 376.19 | 77% |
E10 | AQE | 9.97 | 347.0 | 346.15 | 76% |
tR: peptide retention time.
Washed human platelets were prepared from blood anticoagulated with citrate-phosphate-dextrose, which was obtained from Centro de Transfusion de Galicia (Santiago de Compostela, Spain).
Bags containing buffy coat from individual donors were diluted with the same volume of washing buffer (NaCl, 120 mM; KCl, 5 mM; trisodium citrate, 12 mM; glucose, 10 mM; sucrose, 12.5 mM; pH 6) and centrifuged at 400 g for 9 min. The upper layer containing platelets (platelet-rich plasma) was removed and centrifuged at 1000 g for 18 min. The resulting platelet pellet was recovered, resuspended with washing buffer, and centrifuged again at 1000 g for 15 min. Finally, the platelet pellet from this step was resuspended in a modified Tyrode-HEPES buffer (HEPES 10 mM; NaCl 140 mM; KCl 3 mM; MgCl2 0.5 mM; NaHCO3 5 mM; glucose 10 mM; pH 7.4) to afford a cell density of 3–3.5
The potential effects of the test drugs on total hCOX activity (bis-dioxygenase and peroxidase reactions) were investigated by measuring their effects on the oxidation of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) to N,N,N′,N′-tetramethyl-p-phenylenedaimine, using AA as common substrate for both hCOX-1 and hCOX-2, microsomal COX-2 prepared from insect cells (Sf21 cells) infected with recombinant baculovirus containing cDNA inserts for hCOX-2 (Sigma Aldrich Química S.A., Alcobendas, Spain), and COX-1 from human platelet microsomes (obtained as described in the above paragraph since, unlike hCOX-2, hCOX-1 is not available commercially) [
The purpose of this work consists in the identification of new peptide ligands for COX that show, compared to the current state of the art, a greater power, a lower toxicity, and a high average degree of selectivity.
In particular, the attention has been focused on cyclooxygenase 2 (COX-2) as it has been recently shown to promote multiple events in the tumorigenesis process [
For several types of cancer, the real risk factor seems to be chronic inflammation that maintains a high level of COX-2 and increases events that promote tumor formation. A tragic example of this mechanism is malignant mesothelioma (MM), a rare tumor of the mesothelial surface of the pleural and peritoneal cavities [
The aim of this study is to demonstrate the possibility of modulation of the activity of COX through peptides that may be found in sites in which the inflammatory process is in place.
To achieve this goal, we relied on the data reported in the literature, focused the attention on the structure of COX-2. One of the known potent inhibitors of COX-2 is SC-558 (a dyaril heterocyclic inhibitor) which contains a bromophenyl ring, a pyrazole group, and a phenylsulphonamide moiety. Most of the sulphur containing NSAIDs are selective COX-2 inhibitors, and this sulphur atom reduces the toxicity of the compound.
Using this information, in a recent work, Somvanshi et al. have designed and synthesized several tripeptide sequences containing a hydrophobic amino acid with aromatic ring, a cysteine residue which contains sulphur atom, and a charged residue at the C-terminal end [
Though the chemical nature of sulphur atom of sulphonamide group in SC-558 and in cysteine residue is different, still similarities in binding constant of peptide with known NSAIDs SC-558 were observed. Thus, preventing the reaction of substrate arachidonic acid with the enzyme supports the possibility of peptide WCS as potent and competitive inhibitor of COX-2. As the phenyl ring of SC-558 interacts with residues in hydrophobic cavity of COX-2 formed by Phe 381, Leu 384, Tyr 385, Trp 387, Phe 518, and Ser 530, it can be assumed that the aromatic ring of tryptophan residue of peptide will also interact with residues in hydrophobic cavity. The free carboxylate group of the peptide can electrostatically interact to Arg 120. WCS can be considered as a potential
Extending the study of Somvanshi et al. [
COXs theoretical binding energies (be) in kcal/mol, number of van der Waals interacting enzyme residues (ir), and intermolecular hydrogen bonds (hb).
Peptide | COX-1 | COX-2 | ||||
---|---|---|---|---|---|---|
be | ir | hb | be | ir | hb | |
GMD | −1.20 | 23 | 1 | −39.87 | 21 | 3 |
GHE | −0.56 | 25 | 0 | −42.15 | 26 | 5 |
YYV | — | — | — | — | — | — |
The graphical inspection of the GMD and GHE COXs complexes revealed, for both peptides, remarkable different recognition of the two enzymes (Figure
COX-1 (a) and (b) and COX-2 (c) and (d) recognition of GMD and GHE. Tripeptides are depicted in polytube CPK colored notation, interacting enzyme residues in green carbons wireframes, HEME cofactor in green carbons spacefill, and the rest of the enzyme is showed in transparent green cartoon. Yellow dotted lines indicated hydrogen bond interaction.
In all cases our compounds have occupied the known NSAID binding pocket. Interestingly, the number of enzyme interacting residues, comprise between 21 and 26, is quite similar, but the COX-2 hydrogen bond network is much better than COX-1 and could explain the selectivity of our ligands. COX-2 Ser 530 side chain is involved in hydrogen bond to GMD backbone and, through a water molecule bridge, to the carboxylate groups of both our active peptides. Tripeptide carboxylate moieties are, also, involved in hydrogen bonds to COX-2 catalytic Tyr 385. GHE still reports such an interaction between its protonated N-terminal and Leu 352 backbone. Even if GMD, through its C-terminal, shows one hydrogen bond to Tyr 355 and favorable electrostatic interaction to COX-1 Arg 120, the steric hindrance of COX-1 Ile 523, bulkier than COX-2 Val, limits the enzyme cleft recognition preventing our compounds, in particular GHE, from establishing hydrogen bonds highlighted in COX-2. The remaining part of contribution to the COXs recognition of our peptides could be addressed an unspecific van der Waals interaction.
At the same time, based on data obtained from virtual screening, only those peptides with better profile of affinity have been selected and classified into two groups called S and E (Table
All peptides S1-E10 were synthesized by standard 9-fluorenylmethoxycarbonyl (Fmoc) chemistry using an appropriate orthogonal protection strategy. Peptides were released from the solid support using a cleavage cocktail of 90% TFA, 5% water, and 5% Et3SiH. All analogues showed >97% purity at HPLC analysis.
The biological assays were carried out to evaluate the inhibitory activity against COX-2 by the group of Professor Dr. Francisco Orallo, Department of Pharmacology, Faculty of Pharmacy, University of Santiago de Compostela, Spain.
Results shown in Table
Inhibitory activity of compounds synthesized and selectivity against COX-2 over COX-1.
COX-1 (IC50) | COX-2 (IC50) | Ratio | |
---|---|---|---|
Indometacina | 12.16 ± 1.16 |
35.20 ± 1.41 |
2.9 |
Diclofenac | 18.23 ± 1.73 |
23.62 ± 1.97 |
1.3 |
FR122047 | 93.80 ± 6.55 |
*** | >1.066a |
Nimesulide | *** | 231.40 ± 19.84 |
<0.46a |
DuP697 | 22.61 ± 1.56 |
126.32 ± 7.41 |
0.0056 |
S1 | 150.33 ± 2.34 |
94.04 ± 2.59 |
0.6255 |
S2 | 143.21 ± 2.57 |
120.92 ± 2.33 |
0.8443 |
S3 | 152.44 ± 5.18 |
94.89 ± 2.12 |
0.6225 |
S4 | 99.32 ± 1.14 |
80.56 ± 2.14 |
0.8111 |
S5 | 161.43 ± 2.57 |
100.01 ± 2.33 |
0.6195 |
S6 | 102.31 ± 1.14 |
91.20 ± 2.41 |
0.8914 |
S7 | 100.33 ± 2.19 |
88.21 ± 3.01 |
0.8792 |
S8 | 122.48 ± 3.78 |
91.66 ± 2.98 |
0.7484 |
S9 | 221.57 ± 1.04 |
68.34 ± 5.43 |
0.308 |
S10 | 99.11 ± 1.55 |
79.20 ± 2.15 |
0.7991 |
E1–E10** | — | — | — |
Significant differences between the two means (
***No active at 500
aValue obtained whereas the corresponding IC50 to COX-1 or COX-2 is the highest concentration tested.
**Data not shown.
This regression was performed using the data obtained with 4–6 different concentrations of each compound assayed, which inhibited the enzymatic activity of COX control isoform between 20 and 80%.
Finally, they were calculated the corresponding indices of selectivity (SI) of COX-1. SI = [IC50(COX-2)]/[IC50(COX-1)].
As demonstrated by the virtual screening, all twenty tripeptides show a greater selectivity against COX-2 over COX-1.
In particular, peptide S9 shows a very interesting profile of both selectivity and inhibitory potency towards COX-2; in fact, the selectivity index between COX-2 and COX-1 is about 0.308, more selective than the nimesulide that has an index of about 0.46; moreover, this peptide also shows an increase in activity compared to the same drug (
Analyzing biological data, depending both on the chemical structure that the values of the energies of binding, the peptides S9, S10, S7 and S4 show an analogous biological profile (selectivity and affinity).
However, a complete analysis of the structure-activity relationship of these peptides cannot be performed because of the small number of peptides that limit the goodness of this report. It is possible to highlight two important aspects: the guanidine group of Arg at C-terminal and the carboxyl group in the side chain of the second amino acid are requirements for the interaction with the target, while all peptides that have a carboxyl group in the side chain on the first amino acid show a loss of selectivity that of affinity; the aromatic group present in WCS, peptide lead, is not essential to interact with the target.
In conclusion, previously reported peptides seem to reflect too high potency and selectivity; instead, peptides of series E do not result selective for COX-2 (data not shown). Further studies for the peptides E1–E10 are in progress.
There is an increasing interest in the development of new treatments based on cyclooxygenases-2 inhibitors, to prolong survival and even potentially cure various forms of cancer, as malignant mesothelioma.
The present study describes hit identification, synthesis, and biological evaluation of a series of linear tripeptides, most of them are able to selectively inhibit COX-2. Further, other experiments aimed to verify the potentiality of these peptides as anticarcinogenic drugs; as well as the preparation of novel; more potent and selective peptidomimetic derivatives are in progress.
Abbreviations used for amino acids follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972, 247, 977-983. Amino acid symbols denote L-configuration.
4-dimethylamino-pyridine
Trifluoroacetic acid
Dichloromethane
N,N-diisopropylethylamine
N,N-dimethylformamide
Triethylsilane
9-fluorenyl-methoxycarbonyl
N-hydroxy-benzotriazole
2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate
Reversed-phase high performance liquid chromatography
Electrospray ionization
Liquid chromatography-mass spectrometry
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.
The authors of the paper do not have a direct financial relation with the commercial identity mentioned in thier submitted paper that might lead to a conflict of interests for any of the authors.
The assistance of the staff is gratefully appreciated. Dr. Francisco Orallo died unexpectedly on December 9, 2009; The authors dedicate this work to his memory.