Rational Design of Small Peptides for Optimal Inhibition of Cyclooxygenase-2: Development of a Highly Effective Anti-Inflammatory Agent

Article abstract of DOI:10.1021/acs.jmedchem.6b00134

Among the small peptides 2-31, (H)Gly-Gly-Phe-Leu(OMe) (30) reduced prostaglandin production of COX-2 with an IC₅₀ of 60 nM relative to 6000 nM for COX-1. The 5 mg kg^-1 dose of compound 30 rescued albino mice by 80% from capsaicin-induced paw licking and recovered it by 60% from carrageenan-induced inflammation. The mode of action of compound 30 for targeting COX-2, iNOS, and VGSC was investigated by using substance P, l-arginine, and veratrine, respectively, as biomarkers. The interactions of 30 with COX-2 were supported by isothermal calorimetry experiments showing a K_a of 6.10 ± 1.10 × 10⁴ M^-1 and ΔG of -100.3 kJ mol^-1 in comparison to a K_a 0.41 × 10³ ± 0.09 M^-1 and ΔG of -19.2 ± 0.06 kJ mol^-1 for COX-1. Moreover, compound 30 did not show toxicity up to a 2000 mg kg^-1 dose. Hence, we suggest peptide 30 as a highly potent and promising candidate for further development into an anti-inflammatory drug.

Full text of DOI:10.1021/acs.jmedchem.6b00134

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Article
Rational Design of Small Peptides for Optimal Inhibition of Cyclooxygenase-2
(COX-2): Development of a Highly Effective Anti-inflammatory Agent
Palwinder Singh, Sukhmeet Kaur, Jagroop Kaur, Gurjit Singh, and Rajbir Bhatti
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00134 • Publication Date (Web): 28 Mar 2016
Downloaded from http://pubs.acs.org on March 29, 2016
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Rational Design of Small Peptides for Optimal Inhibition of Cyclooxygenase-2
(COX-2): Development of a Highly Effective Anti-inflammatory Agent
a,*
a
a
b
b
Palwinder Singh , Sukhmeet Kaur , Jagroop Kaur , Gurjit Singh , Rajbir Bhatti
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a
UGC Sponsored Centre for Advanced Studies - Department of Chemistry, Guru Nanak
Dev University, Amritsar- 143005, India
b
Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar-143005,
India
Abstract: Amongst the small peptides 2–31; (H)GlyꢀGlyꢀPheꢀLeu(OMe) (30) reduced
ꢀ1
prostaglandin production of COXꢀ2, IC 60 nM vs 6000 nM for COXꢀ1. The 5 mg Kg dose of
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0
compound 30 rescued albino mice by 80% from capsaicin induced paw licking and recovered it
by 60% from carageenan induced inflammation. The mode of action of compound 30 for
targeting COXꢀ2, iNOS and VGSC was investigated by using substance P,
Lꢀarginine and
veratrine, respectively as the biomarkers. The interactions of 30 with COXꢀ2 were supported by
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ꢀ1
ꢀ1
the isothermal calorimetry experiments showing K 6.10±1.10x10 mol and ꢁG ꢀ100.3 k J mol
a
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ꢀ1
ꢀ1
in comparison to K 0.41x10 ±0.09 mol and ꢁG ꢀ19.2±0.06 k J mol for COXꢀ1. Moreover,
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ꢀ1
compound 30 did not show toxicity up to 2000 mg Kg dose. Hence, we suggest peptide 30 as a
highly potent and promising candidate for its further development into antiꢀinflammatory drug.
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. Introduction
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Besides their key role as biocatalysts and hormones, small peptide/proteins are also
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identified with antiꢀcancer, antiꢀdiabetic, antiꢀmicrobial and antiꢀfungal activities. The
biological acceptability and the substrate specific properties of the proteins/enzymes have played
a guiding role in the design of target specific small peptides and consequently, they have taken
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the centreꢀstage in the development of new drugs.
Given the major role of cyclooxygenase
enzymes (housekeeping COXꢀ1 and inducible COXꢀ2) in regulating the arachidonic acid
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metabolic pathway
and the production of proꢀinflammatory prostaglandins
by the
inducible form of cyclooxygenase, COXꢀ2 is the selective target of antiꢀinflammatory drugs.
Starting with the use of aspirin, diclofenac, indomethacin as the nonꢀsteroidal antiꢀinflammatory
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drugs (NSAIDs),
the development of COXIBs
has provided some relief to the patients
suffering from wide spectrum of inflammatory diseases. However, the cardiovascular side effects
associated with the use of COXIBs is the limiting factor that has subdued the medicinal
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applications of this class of antiꢀinflammatory drugs
and hence still keeping the search for
new chemical entities open. Various small peptides have been recently described as an
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alternative class of antiꢀinflammatory drugs.
But a major issue in the development of COXꢀ2
inhibitors is to achieve optimal inhibition of the enzyme so that the side effect risks are kept at
the possible minimum.
Based on our recent success in identifying the antiꢀinflammatory potential of indole
appended peptides wherein the peptide component plays key role for the activity of the
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molecules and also taking into consideration the detailed analysis of arachidonic acid (AA)
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bound COXꢀ2 crystal coordinates;
here, we designed a series of small peptides. Specifically,
the molecular modelling studies helped us in: (i) the design of such peptides which can occupy
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the same place in the COXꢀ2 active site which otherwise is inhabited by AA, (ii) incorporation of
specific amino acids in the peptide so that the lipophilic – hydrophilic balance is maintained and
(iii) providing information about the selectivity of the peptide for COXꢀ2 over COXꢀ1 (Table
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S1). The flexibility of the peptides to enter the COXꢀ2 active site may help to selectively target
the inflammatory tissues due to the higher concentration of the enzyme therein (more expression
of COXꢀ2) than the normal tissues (less concentration of the enzyme) and advantageously the
side effects associated with COXꢀ2 inhibitors may come down. Probably, the COXꢀ2
concentration gradient in the inflammatory – nonꢀinflammatory tissues has worked in our
experiments (details will be given separately) for the identification of a highly promising antiꢀ
inflammatory agent. The synthesis of newly designed peptides was succeeded by their inꢀvitro
and inꢀvivo COXꢀ2 inhibition and antiꢀinflammatory properties.
2. Results and Discussion
The structural and volume similarity between the peptides and arachidonic acid (natural
substrate of COXꢀ2) strengthened the design of the new molecules. Given in the preferred
primary structure, triꢀ and tetraꢀ peptides were expected as the most suitable candidates for
creating a competition with arachidonic acid for binding to the active site of the enzyme.
Therefore, by selecting the proper amino acids (keeping in mind the optimal LogP of the
molecule), the desired peptides were synthesized. The hydrophobic nature of the COXꢀ2 active
site made us to prefer the use of Gly, Ala, Val, Leu, Phe for making the peptide.
2.1. Chemistry
The desired peptides were procured by the coupling of NꢀCbz protected amino acid with
amino acid methyl ester hydrochloride by using ethyl chloroformate as the coupling reagent. For
the synthesis of Gly–Ala–Val peptide; solution of triethylamine, ethylchloroformate and Nꢀ
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CbzGly in THF was maintained at ꢀ10 C. Addition of alanine methyl ester hydrochloride to the
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above solution and stirring the reaction mixture at 0 – 10 C and then at room temperature (25 –
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0 C) for 12 h provided NꢀCbzGly–Ala(OMe) peptide. NꢀCbzGly–Ala(OMe) was hydrolyzed
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with 1N NaOH and the resulting NꢀCbzGly–Ala(OH) was further coupled with Val for procuring
NꢀCbzGly–Ala–Val peptide. Treatment of this peptide with PdꢀC/H removed the Cbz group and
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finally Gly–Ala–Val peptide was isolated. Other peptides were synthesized through the same
procedure (Table 1, Scheme S1ꢀS5). All the synthesized compounds were characterized with
NMR, mass and IR spectral techniques. The percentage purity was checked with qNMR
®
(
quantitative NMR) and optical purity was ascertained by using Chirobiotic T 10 µm reverse
1
phase chiral HPLC column. Reproducible H NMR spectra of the compounds after 2/5 days
ensured stability of the compounds in the solution (CHCl /DMSO) as well as in the solid form.
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2.2. Biological studies
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.2.1. In-vitro COX-1, COX-2 inhibitory activity
All the peptides were subjected to enzyme immunoassays. For making the comparison, the
dipeptides were also included in the enzyme inhibitory studies. Compounds were screened for
ꢀ5
ꢀ8
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COXꢀ1 and COXꢀ2 inhibition at four different molar concentrations (10 M – 10 M). All the
compounds were tested in triplicate and the average of three values with deviation <3% was
taken for calculation. The enzyme inhibition assay is based on COXꢀ1, COXꢀ2 catalyzed
production of prostaglandins during AA metabolism.
Enzyme inhibition = 1/conc of prostaglandin produced in each enzymatic reaction
The dipeptide 2 and 3 exhibited IC 1 ꢂM and 2 ꢂM, respectively for COXꢀ2 whereas these
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two dipeptides did not affect COXꢀ1 catalyzed reaction. Amongst compounds 6 – 9, the COXꢀ2
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Table 1. Peptides synthesized for the present experiments and their IC (µM) against COXꢀ1,
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COXꢀ2.
ψ
IC (µM)
Selectivity Index*
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Compd
Structure
% yield
COXꢀ1
COXꢀ2
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NꢀCbzꢀGlyꢀGlyꢀOMe
NꢀCbzꢀGlyꢀGlyꢀOH
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>10
>10
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>10
>10
80
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NꢀCbzꢀGlyꢀAlaꢀValꢀOMe
NꢀCbzꢀGlyꢀAlaꢀValꢀOH
>10
>3
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45
>10
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>1
H NꢀGlyꢀAlaꢀValꢀOMe
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45
25
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0.2
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H NꢀGlyꢀAlaꢀValꢀOH
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>10
NꢀCbzꢀGlyꢀAlaꢀValꢀLeuꢀOMe
NꢀCbzꢀGlyꢀAlaꢀValꢀLeuꢀOH
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>10
>10
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ꢀꢀ
ꢀꢀ
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NꢀCbzꢀGlyꢀValꢀAlaꢀLeuꢀOMe
NꢀCbzꢀGlyꢀValꢀAlaꢀLeuꢀOH
NꢀCbzꢀValꢀGlyꢀOMe
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18
19
18
65
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0.3
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ꢀꢀ
>10
>10
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NꢀCbzꢀValꢀGlyꢀOH
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>10
>10
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NꢀCbzꢀValꢀGlyꢀLeuꢀOMe
NꢀCbzꢀValꢀGlyꢀLeuꢀOH
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H NꢀValꢀGlyꢀLeuꢀOMe
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>10
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2
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2
H NꢀValꢀGlyꢀLeuꢀOH
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>10
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>10
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20
>125
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NꢀCbzꢀValꢀGlyꢀLeuꢀAlaꢀOMe
NꢀCbzꢀValꢀGlyꢀLeuꢀAlaꢀOH
NꢀCbzꢀGlyꢀGlyꢀPheꢀLeuꢀOMe
NꢀCbzꢀGlyꢀGlyꢀPheꢀLeuꢀOH
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H NꢀGlyꢀGlyꢀPheꢀLeuꢀOMe
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Average of the three values with deviation <3%. *IC (COXꢀ1)/IC (COXꢀ2)
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inhibitory activity of compound 6 and 9 was appreciable, showing respective IC 0.1 and 0.2
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ꢂM. The selectivity index 80 for compound 6 was also significant whereas the selectivity of
compound 9 for COXꢀ2 over COXꢀ1 was poor. Compounds 11, 17 and 24 provided an excellent
view of structure activity relationship. It was apparent that the difference in the sequence of
amino acids in these three isomeric peptides was responsible for the difference in the activity of
the compounds. Compound 24 with IC 0.08 ꢂM for COXꢀ2 displayed better enzyme inhibitory
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profile than that of compound 11 and 17. After hydrolyzing the ester group of 24, the IC of the
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resulting compound 25 for COXꢀ2 was improved. Amongst the last category of compounds, 30
displayed higher efficacy for the inhibition of COXꢀ2 enzyme with IC 0.06 µM and selectivity
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index 100 whereas its corresponding acid 31 exhibited poor inhibition of COXꢀ2 (Table 1).
Compound 29 showed considerable inhibition of catalytic activities of both COXꢀ2 and COXꢀ1,
exhibiting negligible selectivity for COXꢀ2 over COXꢀ1. Amongst the other compounds tested
here, compound 17 with IC 3 µM and 0.3 µM for COXꢀ1 and COXꢀ2, respectively exhibited
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poor selectivity for COXꢀ2 over COXꢀ1. IC of compounds 8, 11 and 31 for COXꢀ2 was 8 µM,
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µM and 0.4 µM, respectively. Overall, the enzyme immunoassays identified compounds 24
and 30 with significant COXꢀ2 inhibitory activity which was comparable to that of diclofenac
and celecoxib. Desirably, the selectivity of compound 30 for COXꢀ2 over COXꢀ1 was higher
than that of diclofenac and lower than the COXꢀ2 selectivity of celecoxib.
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The selectivity of compound 30 for COXꢀ2 over COXꢀ1 was also ascertained using human
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whole blood assay.
Concentration of TxB (as a measure of TxA formation and hence COXꢀ
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activity) and PGE (as a measure of COXꢀ2 activity) was determined by ELISA. Compound 30
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did not alter the formation of TxB in comparison to the control whereas significant reduction in
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the PGE formation was observed (Table 2). These results support the enzyme immunoassay for
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COXꢀ2 selectivity of compound 30.
Therefore, the trend of enzyme inhibitory activity of the peptides is parallel to the results of
docking studies and explicitly justifies their design. Compound 6, 24 and 30 were further
screened for antiꢀinflammatory activity on mice and their physical interaction with COXꢀ2 was
also checked with the help of isothermal titration calorimetric (ITC) experiments.
Table 2. Inhibition of TxB in calcium ionophore stimulated whole blood and PGE in LPS
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stimulated whole blood by compound 30
TXB ng/mL
PGE ng/mL
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ꢀ
calcium Ionophore +calcium Ionophore
ꢀLPS
2.2
+LPS
15.5
2.1
control
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8.5
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Indomethacin (1 ꢂM)
Compound 30 (1 ꢂM)
0.08
2.2.2. In-vivo biological studies.
Swiss albino mice of either sex (25–35 g) were used in the present study. The animals were
maintained at 22 ± 2 °C under 12 h light/12 h dark cycle with free supply of food and water. The
study has been approved by the IAEC of Guru Nanak Dev University, Amritsar, Punjab, India.
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.2.2.1. Models for analgesic and anti-inflammatory activity. Capsaicin induced algesia as
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described in our previous studies was used. Briefly, pain response was quantified as the number
of paw lickings following capsaicin injection (1.6 µg in 20 µL) in the plantar surface of the hind
paw. Formalin induced algesia was used to assess neurogenic and inflammatory phases of pain
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according to the method described by Hunskaar and Hole. In brief, 20 µL of 2% formalin was
injected into the plantar surface of the right hind paw of mouse after 30 min of treatment with
standard/test compound and number of hind paw flinching were observed for first 5 min and at
Figure 1. Schematic representation of experimental protocol for analgesic and antiꢀinflammatory
studies.
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0 min. The first phase indicates the neurogenic pain whereas the second phase indicates the
inflammatory phase. To study the antiꢀinflammatory activity, carageenan induced paw edema
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model as previously described was used. The schematic representation of the experimental
protocol is given in figure 1.
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For studying the involvement of cyclooxygenase and lipooxygenase pathway in the mode of
action of the compound, the animals were pretreated with substance P. In order to check the
involvement of nitric oxide pathway during the inhibition of algesia, the animals were pretreated
with Lꢀarginine and LꢀNAME (Figure 2). For investigating the involvement of voltage gated
sodium channels (VGSC), calcium influx and potassium channels, the animals were pretreated
with veratrine, A 23187 and glipizide, respectively (Figure 2).
Figure 2. Schematic representation of experimental protocols for exploring the involvement of
cyclooxygenase, lipooxygenase pathway (substance P), nitric oxide pathway (Lꢀarginine, Lꢀ
NAME), VGSC (veratrine), Calcium channel (A 23187) and potassium channels (Glipizide) in
the observed analgesic effect of compound 30.
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.2.2.2. Analgesic activity and anti-inflammatory activity. The treatment of animals with
compound 30 (5 mg and 10 mg), compound 6 (5 mg and 10 mg) and compound 24 (5 mg and 10
mg) were found to produce a marked decrease in the number of paw lickings after capsaicin
injection. Compound 30 was the most potent showing its effect comparable to the standard drug
indomethacin (Figure 3) and it was subjected to further studies. It was observed that compound
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0
3
0 treated mice produce a marked decrease in inflammation in carageenan induced paw edema
and here the effect was similar to that of the standard drug diclofenac (Figure 4). In the formalin
induced pain, the neurogenic phase (0ꢀ5 min) was not significantly decreased, whereas the
inflammatory phase (15ꢀ30 min) of pain was attenuated by both the standard drug as well as
ꢀ1
compound 30 at a dose of 5 mg Kg (Figure 5). Therefore, the animal based experiments
substantiated the results of enzyme immunoassays and the potential of compound 30 as an antiꢀ
inflammatory drug.
Figure 3. Effect of various compounds on capsaicin induced hyperalgesia. All values are
expressed as mean ± SEM. * p<0.05 vs. control.
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Figure 4. Effect of compound 30 on carageenan induced inflammation in mice. All values are
expressed as mean ± SEM. * p<0.05 vs. control.
Figure 5. Effect of compound 30 on formalin induced hyperalgesia. All values are expressed as
mean ± SEM. * p< 0.05 vs. formalin treated control.
2.2.2.3. Studies supporting the probable mode of action of the compounds. The excellent
inhibition of COXꢀ2 by compound 30 and its probable mode of action was checked by using
substance P– a stimulator of COXꢀ2, 5ꢀLOX and promoter of prostaglandin/leukotriene
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production.
effect of compound 30 (Figure 6) which indicated that COXꢀ2 may be the probable target of 30
as it exhibited poor inhibition of 5ꢀLOX (IC 6 ꢂM). Nitric oxide formation by inducible nitric
Pretreatment of the animal with substance P was found to reverse the analgesic
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4
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oxide synthase (iNOS) is well documented in pain mechanisms. Therefore the modulation of
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nitric oxide pathway was explored in the analgesic effect of compound 30. Pretreatment with Lꢀ
arginine, a nitric oxide precursor was found to reverse the analgesic effect of compound 30
whereas pretreatment with LꢀNAME, a nitric oxide synthase inhibitor did not alter the analgesic
effect of compound 30 (Figure 6). These results indicate that a combination of modulation of
COXꢀ2 and nitric oxide formation might be involved in the analgesic effect of compound 30.
Remarkably, the pretreatment with A23187 (TxA2 stimulator) did not alter the analgesic effect
of compound 30 and hence supporting the results of human whole blood assay, this compound
does not affect COXꢀ1.
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Capsaicin
+
ꢀ
ꢀ
ꢀ
ꢀ
+
+
ꢀ
ꢀ
ꢀ
+
+
+
ꢀ
+
+
ꢀ
+
ꢀ
+
+
ꢀ
ꢀ
+
+
+
ꢀ
ꢀ
ꢀ
Compound 30
Substance P
LꢀArginine
LꢀNAME
ꢀ
Aꢀ23187
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
Figure 6. Effect of Substance P, Lꢀarginine and LꢀNAME on the analgesic effect of compound
30. All values are expressed as mean ± SEM. * p<0.05 vs. control; # p< 0.05 vs. compound 30.
In addition to the COXꢀ2 and nitric oxide pathways, voltage gated sodium channels (VGSC)
and increased calcium influx is also known to be involved in pain and inflammatory
4
9,50
processes.
Furthermore, an increased influx of potassium culminating in hyperpolarization of
5
1
free nerve endings is proposed to mediate analgesia. Therefore, the involvement of these ion
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channel pathways was investigated in the observed analgesic effect of compound 30.
Pretreatment with veratrine, a VGSC opener was found to ameliorate the analgesic effect of
compound 30 partially although the reversal was not complete whereas pretreatment with
glipizide, an ATP sensitive potassium channel (KATP) blocker, did not alter the analgesic effect
of compound 30. A comparison of the results of these experiments (Figure 6 and Figure 7)
indicates that the inhibition of COXꢀ2 is probably the major contributor to the analgesic effect of
compound 30.
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Capsaicin
Compound 30
Veratrine
+
ꢀ
ꢀ
+
+
ꢀ
+
+
+
ꢀ
+
+
ꢀ
Glipizide
ꢀ
ꢀ
+
Figure 7. Modulation of VGSC, calcium and KATP channels in the analgesic effect of compound
30. All values are expressed as mean ± SEM. * p<0.05 vs. control; # p< 0.05 vs. compound 30.
52
2
.2.2.4. Acute toxicity studies. OECD guidelines (OECD, 2001) were followed for acute
toxicity studies of compound 30. Four groups of animals with three animals per group were
taken. The first group was administered the vehicle and served as the control group; the second,
ꢀ1
ꢀ1
third and the fourth groups were treated with compound 30 at doses of 50 mg Kg , 300 mg Kg
ꢀ1
and 2000 mg Kg respectively. All the treatments were administered after 4 h of fasting.
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Thereafter, the animals were observed continuously for the first four hours and periodically for
ꢀ1
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4 h. After 14 days, one animal each in control and highest dose (2000 mg Kg ) was sacrificed
and histological studies were conducted by using H and E staining. No gross behavioral
abnormality was observed in any of the four groups of animals. No significant changes were
evident in the myocardium or liver of the animals treated with the highest dose of compound 30
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(
2000 mg Kg ) as compared to the control group (Figure 8).
A
B
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D
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F
Figure 8. Histology myocardium of control (A, 20x) and compound 30 treated (B, 20x); liver of
control (C, 20x) and compound 30 treated (D, 20x); kidneys of control (E, 20x) and compound
30 treated (F, 20x).
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.2.3. Isothermal calorimetric (ITC) experiments
ITC is one of the most precise techniques to measure the affinity between the
macromolecule and the small molecule. Since ITC measures heat released or absorbed during
binding, it is the only technique which allows simultaneous determination of all binding
parameters viz. the binding constant (K), enthalpy (ꢁH) and entropy (ꢁS) in a single experiment.
The solution of peptide 30 (in the syringe) was injected into the enzyme solution in HEPES
buffer (pH 7.4) taken in the sample cell. In one experiment, 19 consecutive injections of 2 ꢂL of
100 ꢂM compound were given to the sample cell after regular time intervals of 120 s so that
equilibrium is ensured in each titration point. Upon each titration, the amount of heat released or
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absorbed was measured and used to determine the association constant (K ), binding enthalpy
a
(ꢁH) and entropy (ꢁS). The total heat Q produced/absorbed in the active cell, determined at
th
fractional saturation Θ after the i injection, is given by equation 1
Q = n ΘM ꢁHV
(1)
t
o
where M is the total concentration of the enzyme, V is the cell volume, n is the total
t
0
number of binding sites in the enzyme, and ꢁH is the molar heat of ligand binding.
th
The enthalpy change for the i injection ꢁH(i) for an injection volume dVi is defined by
equation 2
ꢁ
H(i) = Q(i) + dV /V [Q(i)ꢀQ(iꢀ1)/2] – Q(iꢀ1)
(2)
i
o
The various parameters determined from ITC experiment and the binding isotherm for
the titration of compound 30 against COXꢀ2 and COXꢀ1 are given in Table 2.
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Table 2. Isothermal calorimetric data and binding isotherm of compound 30 for COXꢀ2 and
COXꢀ1.
Physical parameters obtained from ITC data*
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COXꢀ2
COXꢀ1
ꢀ1
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K (M )
(6.10±1.10)x10
(0.41±0.09) x 10
a
ꢁ
H (kJ/mol)
ꢀ107.0±1
ꢀ6.7±0.03
ꢀ100.3±1
ꢀ99.2±1
ꢀ80.0±1
TꢁS (kJ/mol)
ꢁG (kJ/mol)
ꢀ19.20±0.06
*average of the three experiments
Binding isotherm for COX-1
Binding isotherm for COX-2
2.2.4. Molecular docking studies
In order to support the experimental results and to have an insight into the nature of
interactions between the test compounds and the active site amino acid residues of the enzyme,
53
molecular docking studies were carried out. Crystal coꢀordinates of COXꢀ2 (PDB ID 1CVU)
were downloaded from the protein data bank (www.rcsb.org). The molecular docking was
performed using Schrodinger (Schrödinger Release 2014ꢀ4: Maestro, version 10.0, Schrödinger,
54ꢀ58
LLC, New York, NY, 2014)
software package. Docking of arachidonic acid in the active site
of COXꢀ2 and root mean square deviation (RMSD) 1.18 Å to the Xꢀray structure validated the
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docking procedure (Figure S81, supporting information). The most active compound 30,
identified in the present investigations, showed Hꢀbond interactions through its carbonyl group
with Y355 (1.89 Å) and R120 (1.68 Å, 2.20 Å). Hence, the two most crucial amino acids of
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COXꢀ2 catalytic site (Y355 and R120)
were captured by compound 30. The NH group of
2
compound 30 was also Hꢀbonded to Y115 (2.14 Å) and S119 (2.17 Å) (Figure 9). It was
apparent from the comparison between the docking pose of compound 30 and AA that the
hydrophobic part of compound 30 (benzyl and isoꢀbutyl groups) occupies the same place where
AA was present (as seen in the COXꢀ2 crystal coordinates with AA, 1CVU) (Figure 10).
Additionally, the polar part of compound 30 constituted by two amide groups and one amino
group extends into the polar subꢀpocket of the active site (Figure S85). The positioning of
compound 30 in the interacting pocket of COXꢀ2 is clearly visible in figure 11.
Figure 9. Compound 30 docked in active site of COXꢀ2 (PDB ID 1CVU). Red lines represent Hꢀ
bond with bond length in Å.
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Figure 10. Compound 30 (green) and arachidonic acid (purple) in the active site of COXꢀ2 (pdb
ID 1CVU).
Figure 11. NꢀCbzꢀGlyꢀGlyꢀPheꢀLeuꢀOMe (30) was positioned in the interacting pocket of
1CVU with hydrogen bonds (dotted lines in Magenta).
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Figure 12. NꢀCbzꢀGlyꢀGlyꢀPheꢀLeuꢀOMe (30) (green) docked in the active site of COXꢀ1 (PDB
ID 1EQG).
Further to support the selectivity of compound 30 for COXꢀ2 over COXꢀ1, as observed during
the enzyme immunoassays, compound 30 was also docked in the active site of COXꢀ1. As it is
apparent from figure 12, compound 30 entered into the active site of COXꢀ1 but in contrast to its
contacts in the active site of COXꢀ2, it does not interact with Y355 and R120. However, the Hꢀ
bond interactions of compound 30 with R83 and P86 were visible. Moreover, the docking score
of compound 30 for COXꢀ2 and COXꢀ1 was quite different whereas expectedly, arachidonic acid
exhibited similar docking score for COXꢀ2 and COXꢀ1 (Table S1). Evidently, a nice correlation
between the experimental results and the results of molecular docking studies was observed.
For getting more support to the docking results of compound 30, the molecular docking of
compound 6 in the active site of COXꢀ2 and COXꢀ1 was also investigated. Compound 6 showed
Hꢀbond interactions of 1.94 Å, 2.44 Å and 2.44 Å between its carbonyl group and Y355, R120
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residues of COXꢀ2 active site (Figure 13) whereas no such type of interactions were visible when
it was docked in the active site of COXꢀ1 (Figure S84).
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Figure 13. Compound 6 (green) docked in active site of COXꢀ2 (PDB ID 1CVU). Red lines
represent HꢀBond with bond length in Å.
Since compound 30 was found to inhibit the NOS pathway also, its interaction in the active
site of iNOS was checked through molecular docking. NOS is present in three different
isoformsꢀ neuronal NOS or nNOS (Type I or NOSꢀ1), inducible NOS or iNOS (Type II or NOSꢀ
2) and endothelial NOS or eNOS (Type III or NOSꢀ3). In contrast to nNOS and eNOS, which are
constitutively active, expression of iNOS is induced by cytokines and pathogens. Docking of the
peptides in the active site of iNOS was performed. Crystal coꢀordinates of iNOS (PDB ID
6
4
3
E7G) were downloaded from the protein data bank (www.rcsb.org). Compound 30 showed
interactions with the active site residues though its docking score (ꢀ5.97 Kcal/mol) was less than
that for COXꢀ2. Compound 30 showed Hꢀbond interactions through its carbonyl group with
amino group of Q263 (2.10 Å) and hydroxyl group of Y347 (2.14 Å). It displayed three Hꢀbond
interactions with propionate group of heme of iNOS protein (Figure 14, Figure 15).
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Figure 14. NꢀCbzꢀGlyꢀGlyꢀPheꢀLeuꢀOMe (30, green) docked in the active site of iNOS (PDB
ID 3E7G). Magenta lines represent HꢀBond with bond length in Å.
Figure 15. 2D diagram of binding pose of compound 30 in the active site of iNOS.
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.2.5. 3D-QSAR (Quantitative Structure Activity Relationship Studies) model
Being a new class of COXꢀ2 inhibitors and showing appreciable results, it was interesting to
perform QSAR studies with the series of peptides under present investigation. By using PLS
with three factors, the Gaussianꢀbased QSAR model was generated by correlating with five
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fields: steric, electrostatic, hydrophobic, HBD, and HBA (Figure 16, Figure S88). An R CV
value of 0.68 were derived from the LOO crossꢀvalidation method and nonꢀcrossꢀvalidation
2
analysis yielded an R value of 0.96 with a standard error of estimate of 0.21 and an F ratio of
9
6.3 (Table 3, Table S3). The contributions of the steric, electrostatic, hydrophobic, HBD, and
HBA fields were 0.39, 0.08, 0.27, 0.21 and 0.03, respectively. The field contributions of steric
0.39) and hydrophobic (0.27) intensities were higher when compared to the electrostatic, HBA,
(
and HBD which indicate their significant role in the protein–ligand interactions. Figure 16a
explains steric contours map with green region on the ligand molecules represent favorable effect
of bulky substituents and grey region shows negative effect of bulky substituents and substitution
in this region may reduce the activity. The positive effect of bulky substituent was clearly
observed in the case of tetrapeptide 30. Replacement of alanine residues of tetrapeptide 16 with
phenyl alanine (bulky substituent) resulted in higher activity of tetrapeptide 30. Similar effect
was observed in case of tetrapeptide 24 where the bulky leucine residue was present in the green
region.
Figure 16b illustrates electrostatic contours map. Blue contours in the molecule represent that
electropositive groups may increase the activity while red regions represent that electronegative
group may enhance the activity. The electrostatic contribution to the activity is 0.08 which is
quite less. In Figure 16c, white contour in the hydrophobic plot indicates that hydrophobic
groups at those regions are disfavored. A yellow contour indicates that hydrophobic groups are
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tolerated at that position. The yellow contour closer to the leucine residue in compound 11, 17
and 24 resulted in better activity. The effect of hydrophobicity on the activity of the compound
was quite evident from the difference in the LogP of compound 30 and 31 and the corresponding
change in their IC for COXꢀ2 (Table S2). Figure 16d shows the HBA contour maps. The red
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contour in the HBA indicates that HBA groups lead to the improved activity whereas magenta
contour indicates that presence of HBA groups lead to reduced activity. In general, in all the
molecules the carbonyl group of amide bond is very much responsible for establishing hydrogen
bonds with the Arg 120 and Y355 (these two amino acids of COXꢀ2 are required for the
inhibition of NSAID’s). Figure 16e shows the HBD contours where areas favored by donors are
shown in blue violet, and unfavorable areas are shown in cyan color. The contribution of HBD is
negligible (0.03). The QSAR model also showed significant fitness of observed activity with the
predicted activity for the training and test set of compounds (Table S3) (Figure 17). A common
pharmacophores model was generated by the alignment of all ligands (active and inactive) to the
pharmacophore (Figure 18).
Table 3: PLS statistical parameters of the selected 3D-QSAR model
Pearson
2
2
2
2
PLS Factors
SD
R
R CV
R Scramble
Stability
F
P
RMSE
Q
-R
1
2
3
0.8715
0.4211
0.2116
0.6291 0.2862
0.8193 0.4638
0.9602 0.6811
0.4916
0.7289
0.8436
0.576
34.2
52.3
96.3
0.0256
1.19
0.8610
0.5211
0.7532
0.2917
0.432
0.694
3.91ꢀ007 0.98
0.2210
0.5102
1.21eꢀ008 0.73
2
SD, standard deviation of the regression; R, squared value of R for the regression; F, variance
ratio. Large value of F indicate a more statistically regression; P, significance level of variance
ratio. Smaller value indicate a greater degree of confidence; RMSE, root mean square error; Q,
2
squared value of Q for the predicted activities; PearsonꢀR, PearsonꢀR value for the correlation
between the predicted and observed activity for the test set.
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Figure 16. QSAR visualization of various subsituents effects: (a) steric contour map (grey,
negative saturation and green is positive saturation); (b) Electrosatatic contour map (blue positive
saturation and red negative saturation; (c) Hydrophobic contour map (white, negative saturation
and yellow positive saturation), (d) HꢀBond contour map (magenta negative saturation and
maroon positive saturation); (e) HꢀBond Donor contour map (cyan represents negative
saturation and purple is positive saturation). Active molecules are shown in tube and inactive
molecules in wire.
Figure 17. Fitness graph between observed activity versus predicted activity for training and test
set compounds.
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Figure 18. Common pharmacophore generation (peptide backbone): alignment of all ligands
(active and inactive) to the pharmacophore.
3.
Conclusions
We have identified tetrapeptide 30 which exhibited inhibition of COXꢀ2 with IC 60 nM and
5
0
appreciable selectivity over COXꢀ1. Testing of this compound on albino mice showed reversal of
algesia and inflammation with as much efficacy but better selectivity for COXꢀ2 as shown by the
standard drug indomethacin and diclofenac. The mechanistic studies indicated that compound 30
was acting through the inhibition of COXꢀ2 channel of arachidonic acid pathway and inhibition
of inducible nitric oxide synthase whereas inhibition of VGSC and calcium influx was not
significant. Appreciable binding constant of compound 30 with COXꢀ2, calculated from the ITC
data, and its considerable interactions in the active site of COXꢀ2 and iNOS, as depicted from the
molecular modeling studies, supported the inꢀvitro and inꢀvivo biological results. Overall, we
came out with a peptide based lead molecule for antiꢀinflammatory drug.
4
. Experimental
.1. General
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Melting points were determined in capillaries and are uncorrected. H and C NMR spectra
were recorded on Bruker 500 MHz and 125 MHz NMR spectrometer, respectively using CDCl3
and/or DMSOꢀd as solvent. Chemical shifts are given in ppm with TMS as an internal
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reference. J values are given in Hertz. Signals are abbreviated as singlet, s; doublet, d; doubleꢀ
doublet, dd; triplet, t; multiplet, m. Mass spectra were recorded on Bruker micrOTOF Q II Mass
spectrometer. The purity of compound was determined using qHNMR method (Absolute
6
5
qHNMR with Internal Calibration). The purity of >95% of the target analyte was determined
using the following equation:
[
P%] = n . Int . MW . m / n . Int . MW . m * P
IC
IC
t
t
IC
t
IC
IC
S
MW = molecular weights
P = purity of internal calibrant
m = amount of internal calibrant, m = amount of sample (compound)
IC
s
Int = integral, n = no of protons giving rise to a given NMR signal
IC = internal calibrant, t = target analyte or compound
General Procedure for peptide synthesis
Procedure A.
L
ꢀAA (amino acid) (0.01 mol) was dissolved in 10 ml (0.01 mol) of 4M sodium
o
hydroxide solution, cooled to 0 C and 0.01 mol of benzyl chloroformate and 0.01 mol of 4M
sodium hydroxide solution was added alternatively and portion wise with shaking and cooling
during 30 min. The mixture was allowed to stir for another 1 h. Then the reaction mixture was
extracted with 2x15 ml portions of ether and aqueous part was acidified with dil HCl. The
liberated oil was extracted with ether and dried over anhydrous sodium sulphate. The ether was
evaporated to obtain the desired NCbzꢀAA (90%).
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Procedure B. NꢀCbzꢀAA (1 mmol) was dissolved in the THF (50 ml). After cooling the solution
o
to ꢀ10 C, triethylamine (0.021 mol) was added followed by the addition of ethylchloroformate
o
(
0.014 mol). The mixture was stirred at ꢀ10 C for 15 min. A cooled solution of the neutralized
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AA methyl ester hydrochloride (1.2 mmol) in 5 ml THF was added to the reaction mixture. The
o
o
reaction mixture was stirred for 1 h at 0 C to ꢀ10 C at and then at room temperature for 12 h.
After completion of the reaction (TLC), 5% Na CO solution was added to the reaction mixture
2
3
and extracted with ethyl acetate. The organic layer was dried over anhydrous Na SO and
2
4
evaporated under vacuum and purified through column chromatography using ethyl acetateꢀ
hexane as eluent to get pure NꢀCbzꢀAAꢀAA methyl ester (70%). Acidification of aqueous part
with 10% HCl gave unreacted NꢀCbzꢀAA. Treatment of NꢀCbzꢀAA–AA methyl ester with 1N
NaOH in acetoneꢀwater (3:2) followed by acidification of the reaction mixture and extraction
with ethyl acetate provided pure NꢀCbzꢀAA–AA dipeptide.
Procedure C. To a solution of NꢀCbzꢀAAꢀAA in MeOH, 10% Pd/C (0.1%) was added. H gas
2
was passed through reaction mixture for 3ꢀ4 h. The completion of reaction mixture was
monitored by TLC. After completion of reaction, the catalyst was removed by filtration and the
filtrate was concentrated in vacuum to obtain the desired peptide.
4.1.1. N-Cbz-Glycine
NꢀCbzꢀGly was synthesized according to general procedure A as white solid, yield 95%, mp 117
o
o
C (lit mp 120 C).
.1.2. N-Cbz Gly-Gly-OMe (2)
Compound 2 was synthesized according to general procedure B as white solid; yield 55%, mp 65
4
o
1
C; H NMR (500 MHz, CDCl ) δ 3.76 (s, 3H, OCH ), 3.93 (d, J = 5.0 Hz, 2H, CH ), 4.05 (d, J
3
3
2
=
5.0 Hz, 2H, CH ), 5.14 (s, 2H, CH ), 5.58 (t, J=5.1 Hz, 1H, NHGly1), 6.69 (br, 1H, NHGly2),
2
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7
.32 – 7.37 (m, 5H, ArH), C NMR (125 MHz, normal/DEPTꢀ135) δ 41.1 (ꢀve, CH ), 44.4 (ꢀve,
2
CH ), 52.4 (+ve, OCH ), 67.3 (ꢀve, CH ), 128.1 (+ve, ArCH), 128.2 (+ve, ArCH), 128.5 (+ve,
2
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2
ArCH), 136.0 (ab, ArC), 156.6 (C=O), 169.3 (C=O), 170.1 (C=O); HRMS (microTOFꢀQII, MS,
0
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+
ESI): Calcd for C H N O ([M+Na] ) 303.0951, found 303.0959.
1
3
16
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4.1.3. N-Cbz Gly-Gly-OH (3)
Compound 3 was synthesized according to general procedure B as white solid; yield 50%, mp
o
1
1
10 C; H NMR (500 MHz, DMSOꢀd ) δ3.66 (d, J = 6.2 Hz, 2H, CH ), 3.77 (d, J = 5.8 Hz, 2H,
6
2
CH ), 5.04 (s, 2H, CH ),7.33 – 7.48 (m, 5H, ArH), 7.49 (t, J = 5.9 Hz, 1H, NHGly1), 8.15 (t, J =
2
2
13
5
.3 Hz, 1H, NHGly2),12.58 (br, 1H, OH); C NMR (125 MHz, normal/DEPTꢀ135) δ 41.0 (CH ),
2
4
3.8 (CH ), 65.9 (CH ), 128.1 (+ve, ArCH), 128.2 (+ve, ArCH), 128.8 (+ve, ArCH), 137.4 (ab,
2
2
ArC), 156.9 (C=O), 169.9 (C=O), 171.6 (C=O); HRMS (microTOFꢀQII, MS, ESI): Calcd for
+
C H N O ([M+H] ) 267.0978 found 267.0975.
12
14
2
5
4
.1.4. N-Cbz Gly-Ala-Val-OMe (6)
Compound 6 was synthesized according to general procedure B as white solid; yield 50%, mp
o
25
o
1
o
1
08 C; [α]D = ꢀ50 (C=1, CHCl ); H NMR (500 MHz, CDCl , 25 C, TMS): δ 0.87ꢀ0.90 (m,
3 3
6
H, 2xCH ), 1.35 (d, J = 6. 4 Hz, 3H, CH ), 2.12ꢀ2.16 (m, 1H, CH), 3.71 (s, 3H, OCH ), 3.90
3
3
3
(
br, 2H, CH ),4.48ꢀ4.50 (m, 1H, CH ), 4.65ꢀ4.68 (m, 1H, CH ), 5.10 (s, 2H, CH ), 5.80 (d, J=
2 Val Ala 2
13
2
.60 Hz, 1H, NH ), 7.05 (br, 2H, NH +NH ) 7.26ꢀ7.34 (m, 5H, ArH); C NMR ( 125 MHz,
Gly Val Ala
normal/DEPTꢀ135) δ 17.7 (+ve, CH ), 18.4 (+ve, CH ), 18.9 (+ve, CH ), 31.1 (+ve, CH), 44.3 (ꢀ
3
3
3
ve, CH ), 48.8 (+ve, CH), 52.1 (+ve, OCH ), 57.3 (+ve, CH), 67.1 (ꢀve, CH ), 128.1 (+ve,
2
3
2
ArCH), 128.2 (+ve, ArCH), 128.3 (+ve, ArCH), 128.7 (+ve, ArCH), 128.8 (+ve, ArCH), 136.2
(ab, ArC), 156.6 (C=O), 168.9 (C=O), 172.1 (C=O), 172.2 (C=O); HRMS (microTOFꢀQII, MS,
+
ESI): Calcd for C H N O ([M+Na] ) 416.1792, found 416.1817.
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.1.5. N-Cbz Gly-Ala-Val-OH (7)
Compound 7 was synthesized according to general procedure B as white solid; yield 38%, mp
o
25
o
1
o
1
60 C, [α] = ꢀ50 (C=1, MeOH); H NMR (500 MHz, DMSOꢀd , 25 C, TMS): δ 0.88 (d, J =
D 6
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6
.6 Hz, 6H, 2xCH ), 1.20 (d, J=6.6 Hz, 3H, CH ), 2.04ꢀ2.08 (m, 1H, CH), 3.58ꢀ3.68 (m, 2H,
3
3
CH ),4.11ꢀ4.14 (m, 1H, CH ), 4.42ꢀ4.46 (m, 1H, CH ), 5.03 (s, 2H, CH ), 7.31ꢀ7.38 (m, 5H;
2
Ala
Val
2
1
3
ArH), 7.45 (t, J = 6.5 Hz, 1H, NH ), 7.95ꢀ7.98 (m, 2H, NH +NH ), 12.58 (br, 1H, OH); C
Gly
Ala
Val
NMR (125 MHz, normal/DEPTꢀ135) δ 18.4 (+ve, CH ), 18.7 (+ve, CH ), 19.5 (+ve, CH ), 30.2
3
3
3
(+ve, CH), 43.8 (ꢀve, CH ), 48.2 (+ve, CH), 57.6 (+ve, CH), 65.9 (ꢀve, CH ), 128.1 (+ve, ArCH),
2
2
1
28.2 (+ve, ArCH), 128.8 (+ve, ArCH), 137.5 (ab, ArC), 156.9 (C=O), 169.0 (C=O), 172.8
(C=O), 173.2 (C=O); IR (CHCl ): ν= 3329 (OH), 2966, 2927 (NH), 1712 (C=O), 1651 (C=O),
3
ꢀ1
+
1219 (CꢀN) cm ; HRMS (microTOFꢀQII, MS, ESI): Calcd for C H N O ([M+Na] )
1
8
25
3
6
4
02.1635, found 402.1626.
4
.1.6. NH Gly-Ala-Val-OMe (8)
2
Compound 8 was synthesized according to general procedure B and C as a semiꢀ solid; yield
25
o
1
o
4
5%; [α]D = ꢀ30 (C=1, MeOH); H NMR (500 MHz, DMSOꢀd , 25 C, TMS): δ 0.86ꢀ0.89 (m,
6
6H, 2xCH ), 1.20 (d, J = 6.5 Hz, 3H, CH ), 2.02ꢀ2.06 (m, 1H, CH), 3.13 (br, 2H, CH ), 3.63 (s,
3
3
2
3
H, OCH ), 4.14ꢀ4.17 (m, 1H, CH ), 4.45 (br, 1H, CH ), 8.09 (br, 2H, NH ), 8.22ꢀ8.23 (br,
3 Val Ala 2
1
3
2
H, NH +NH ); C NMR ( 125 MHz, normal/DEPTꢀ135) δ 18.6 (+ve, CH ), 19.2 (+ve,
Val
Ala
3
CH ), 19.3 (+ve, CH ), 30.2 (+ve, CH), 44.5 (ꢀve, CH ), 47.8 (+ve, CH), 52.1 (+ve, OCH ),
3
3
2
3
5
7.9 (+ve, CH), 172.2 (C=O), 172.3 (C=O), 173.0 (C=O); HRMS (microTOFꢀQII, MS, ESI):
+
Calcd for C H N O ([M+H] ) 260.1614, found 260.1605.
11 21
3
4
4
.1.7. NH Gly-Ala-Val-OH (9)
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