Engineered Substrate for Cyclooxygenase-2: A Pentapeptide

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Article Cite This: J. Med. Chem. 2019, 62, 6363−6376

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Engineered Substrate for Cyclooxygenase-2: A Pentapeptide Isoconformational to Arachidonic Acid for Managing Inflammation Baljit Kaur,ψ Manpreet Kaur,ψ Navjot Kaur,ψ Saweta Garg,Φ Rajbir Bhatti,Φ and Palwinder Singh*,ψ ψ

Department of Chemistry and Centre for Advanced Studies and ΦDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar 143005, India

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ABSTRACT: Beyond the conventional mode of working of anti-inflammatory agents through enzyme inhibition, herein, COX-2 was provided with an alternate substrate. A prolinecentered pentapeptide isoconformational to arachidonic acid, which exhibited appreciable selectivity for COX-2, overcoming acetic acid- and formalin-induced pain in rats to almost 80%, was treated as a substrate by the enzyme. Remarkably, COX-2 metabolized the pentapeptide into small fragments consisting mainly of di- and tripeptides that ensured the safe breakdown of the peptide under in vivo conditions. The kinetic parameter Kcat/Km for COX-2-mediated metabolism of the peptide (6.3 × 105 M−1 s−1) was quite similar to 9.5 × 105 M−1 s−1 for arachidonic acid. Evidenced by the molecular dynamic studies and the use of Y385F COX-2, it was observed that the breakage of the pentapeptide has probably been taken place through H-bond activation of the peptide bond by the side chains of Y385 and S530.

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this enzyme.31−35 Under these circumstances, we planned another strategy to minimize the formation of inducible COX2-mediated metabolites of AA by providing an alternative substrate to the enzyme. In fact, while keeping in mind the selectivity for COX-2 over its isozyme COX-1, we underpinned the COX-2 specificity by replacing AA with an isoconformational substrate, the rationally designed small peptide, expecting that, instead of formation of PGE2 from AA, alternative safe metabolites may form in case that these substrates are used as anti-inflammatory drugs. COX-2 is a highly conserved enzyme specific to arachidonic acid.36,37 The catalytic domain of the enzyme is constituted by Y385, R120, Y355, H206, H386, H388, and S530,38−40 wherein AA is present in a U-shaped geometry occupying the large hydrophobic pocket (Figure 1).41 Besides the hydrophobic interaction of the main chain of AA, it exhibits H-bond contacts through its carboxyl group with S530 and Y385 (Figure 1).42 Mechanistically, Y385 is responsible for the transfer of a radical from heme to AA for initiating the metabolism of AA (Scheme S1).43−46 In continuation to the previous report on peptide-based COX-2 inhibitors,47 it was hypothesized that a stereo-strained proline-centered tetra/ pentapeptide may acquire a geometry similar to that of AA and hence occupy the same space in the COX-2 active site where AA sits. Accordingly, peptides 1−3 (Chart 1) were designed. Remarkably, the crystal coordinates of COX-2 in a complex with peptide 2 showed that the peptide has occupied the same

INTRODUCTION In addition to the primary source of energy, fatty acids generate metabolites that play a regulatory role in controlling various biochemical processes.1−8 Arachidonic acid (AA) is one of those fatty acids that profoundly impinge the physiological state of the organism through its metabolites in maintaining body temperature, working of cardiovascular system, induction of labor pain, keeping up the immune system, and regulation of inflammation.9−14 These metabolites including prostaglandins, prostacyclins, prostanoids, and leukotrienes are being generated by the catalytic activity of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), lipoxygenase (LOX), and cytochrome P450 (CytP450)15−21 on AA. Irrespective of the 70% structural homology, COX-1 and COX-2 constitutively generate functionally diverse prostaglandins, prostacyclins, and prostanoids for regulating body temperature, smooth working of cardiovascular system, and maintenance of the immune system, respectively.22,23 However, under inflammatory conditions, the inducible enzyme COX-2 synthesizes PGE2, resulting in the emergence of inflammatory diseases.24,25 Therefore, the natural mode of working of COX-1 and COX-2 and the consequent generation of metabolites in the AA cascade imply that a strategic design of molecules for selectively controlling the inducible COX-2 must be made to avoid the side effects of the anti-inflammatory drugs. Overcoming the limitations of nonselective antiinflammatory drugs, the launch of selective COX-2 inhibitors (COXIBS) was the result of the above-mentioned tactical approach.26−30 But excessive suppression of COX-2 by the use of COXIBS relegated the normal immunological functions of © 2019 American Chemical Society

Received: May 20, 2019 Published: June 17, 2019 6363

DOI: 10.1021/acs.jmedchem.9b00823 J. Med. Chem. 2019, 62, 6363−6376

Journal of Medicinal Chemistry

Article

2 (Scheme 1). Similar to the sequence of reactions for the synthesis of peptide 2, another pentapeptide 20 was prepared by the coupling of tripeptide 16 and dipeptide 19 (Scheme 2). Peptides 21 and 22 were coupled to procure tetrapeptide 23 (Scheme 3), whereas coupling of dipeptides 24 and 25 gave tetrapeptide 26 (Scheme 4). The synthesis of hexapeptide 3 is depicted in Scheme 5. The percent purity of more than 98% of the compounds was ascertained with the q1H NMR spectrum. Due to the better solubility of compound 12 (in CDCl3 in comparison to its precursor 2 in DMSO and MeOH), its structure was established with the help of various 2D NMR experiments (Figures S31−S38). The observation of the nuclear Overhauser effect (nOe) between the terminal NH of one arm and ester CH3 of the second arm (Figure 2) indicated the closeness of the two arms acquiring a U-shaped geometry by molecule 12 (Figure 3) similar to the conformation attained by peptide 2 in the active site pocket of COX-2 (Figure 1). COX-1 and COX-2 Assays. The potency of the synthesized peptides against COX-1 and COX-2 was screened using enzyme immunoassay kits.48 Excitingly, in concurrence with the results of molecular docking studies, pentapeptide 2 exhibited superior enzyme inhibitory profile in comparison to the other peptides screened in the present investigation. Although it is difficult to draw a conclusive structure−activity relationship, a comparison of the IC50 and selectivity of peptide 2 for COX-2 was made with the other small peptides (Table 1). Peptide 12, the Cbz-protected 2, with IC50 of 0.4 μM for COX-2 was relatively less potent than 2. The presence of Phe residues in 20 increased its IC50 for COX-2 to 5 μM in comparison to that of 12. This may be attributed to the increase in the size of the peptide 20 than that of peptide 12. The hexapeptide 3 and tetrapeptides 1, 23, and 26 exhibited poor inhibition of COX-2. These results made us choose compounds 2, 12, and 20 for further investigations on animal models. Peptides 23 and 26 were included for the comparisons. Human Whole Blood Assay.48,49 The desirable selectivity of compound 2 for COX-2 over COX-1, as observed in the enzyme immunoassays, was further supported with the help of human whole blood assay in which LPS-induced expression of COX-2 was reversed (Figure 4B), but no effect was observed on calcium ionophore-induced expression of COX-1 (Figure 4A). In Vivo Studies on Rat Models. Effect of Compound Treatments in Acetic Acid-Induced Pain Model. Intra-

Figure 1. Crystal coordinates of COX-2 in association with AA (PDB ID: 1CVU).41 Molecular docking of peptide 2 (Chart 1) in the active site pocket of the enzyme was performed. The peptide has occupied the same space where AA binds.

hydrophobic pocket where AA was present (Figure 1). The molecular docking of peptides 1 and 3 in the active site pocket of the enzyme showed that 1 falls short of the size of the active site pocket and does not occupy the whole space as covered by AA (Figure S2), whereas the flexibility in peptide 3 made its one terminus displace away from the AA (Figure S3). Therefore, enthused by the results of molecular docking studies, compounds 1−3 were synthesized along with some other proline-containing peptides. These compounds were screened for anti-inflammatory and analgesic activity in addition to the investigations about their mode of action. The breakdown of the peptides under in vivo and in vitro conditions was also examined.

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RESULTS AND DISCUSSION Syntheses. N-Cbz-Ala (4) was prepared by the reaction of L-Ala with benzyl chloroformate (Cbz), and it was coupled with glycine methyl ester by using ethyl chloroformate (ECF) as the coupling reagent to obtain N-Cbz-Ala-Gly(OMe) (5, Scheme 1). Hydrolysis of dipeptide 5 to 6 and further coupling with L-Pro(OMe) provided tripeptide 7, and it was hydrolyzed to get N-Cbz-Ala-Gly-Pro(OH) (8, Scheme 1). Treatment of 8 with alanine methyl ester hydrochloride followed by deprotection of the Cbz group gave tetrapeptide 1. Alagly(OMe) 11 was obtained by the coupling of Boc-Ala(OH) (9) with glycine methyl ester hydrochloride and then the removal of Boc from the resulting dipeptide 10. Coupling of tripeptide 8 and dipeptide 11 gave peptide 12. Treatment of 12 with Pd/C in methanol−acetic acid provided pentapeptide Chart 1. Designed Peptides as the Probable Substitutes of AA

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DOI: 10.1021/acs.jmedchem.9b00823 J. Med. Chem. 2019, 62, 6363−6376

Journal of Medicinal Chemistry

Article

Scheme 1. Reaction Conditions: (i) TEA in THF, ECF, −10−25 °C, 12 h; (ii) NaOH (1 M), Acetone−H2O (3:2), 45 min; (iii) Acetic Acid−MeOH, Pd/C, H2, 4 h

Scheme 2. Reaction Conditions: (i) TEA in THF, ECF, −10−25 °C, 12 h; (ii) NaOH (1 M), Acetone−H2O (3:2), 45 min; (iii) TFA (10 equiv), 12 h

Scheme 3. Reaction Conditions: (i) TEA in THF, ECF, −10−25 °C, 12 h

reduced the number of writhings as compared to the acetic

peritoneal administration of acetic acid induced abdominal contraction, trunk twisting, and extension of hind legs (writhing) in rats. Treatment with vehicle (control), indomethacin (10 mg kg−1), and compounds 2, 12, 20, 23, and 26 at a dose of 5 mg kg−1 and 10 mg kg−1 significantly

acid treated groups. Compounds 2 and 12 were found to be the most efficacious with almost 80% reduction in acetic acidinduced writhings (Figure 5A). 6365

DOI: 10.1021/acs.jmedchem.9b00823 J. Med. Chem. 2019, 62, 6363−6376

Journal of Medicinal Chemistry

Article

Scheme 4. Reaction Conditions: (i) TEA in THF, ECF, −10−25 °C, 12 h

Scheme 5. Reaction Conditions: (i) TEA in THF, ECF, −10−25 °C, 12 h; (ii) NaOH (1 M), Acetone−H2O (3:2), 45 min; (iii) Acetic Acid−MeOH, Pd/C, H2, 4 h

Figure 2. 1H−1H nOe spectrum of N-Cbz AGPAG-OMe (12).

Effect of Compound 2 Treatment on Formalin-Induced Pain. Formalin was injected into the intraplantar region of a rat and was found to produce biphasic hyperalgesia. An increase in the number of flinching in the neurogenic phase (0−5 min), that is, the first phase, and inflammatory hyperalgesia (20−30 min), that is, the second phase, was observed. A significant reduction in the number of flinchings in the inflammatory phase occurred after the treatment with the standard drug indomethacin, but no significant effect was observed in the

neurogenic phase. Compound 2 reduced the number of flinching by 56 and 80% in both neurogenic and inflammatory phases, respectively, as compared to the control groups (Figure 5B). Effect of Substance P Treatment on the Analgesic Effect of 2 in Neurogenic and Inflammatory Phases of FormalinInduced Pain. A reverse in the effect of compound 2 in both phases was found to be significant with pretreatment of substance P, a neurokinin receptor agonist and stimulator of 6366

DOI: 10.1021/acs.jmedchem.9b00823 J. Med. Chem. 2019, 62, 6363−6376

Journal of Medicinal Chemistry

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Figure 4. (A) TXB2 and (B) PGE2 inhibition in whole blood by indomethacin (1 μM) and compound 2 (1 μM). TXB2 and PGE2 formed in each experiment are mentioned over the bars. Calcium ionophore and LPS were used as stimulants of COX-1 and COX-2, respectively. Figure 3. Configuration of compound 12 as depicted from the nOe (black arrows) and COSY cross-peaks (brown arrows, Figure S35).

histological studies. Tissue histology did not show any lesion, confirming no toxicity of the compound even at 2000 mg kg−1 dose (Figure S164). Study of Peptide 2 Metabolism. As per the hypothesis made during the design of the peptides, it was worth checking how peptide 2 undergoes breakdown after exhibiting the antiinflammatory and analgesic effects. The blood serum samples of compound 2-treated animals were collected at different time intervals. The proteins were removed by centrifugation, and the mass spectra of the supernatants were recorded. Intriguingly, liquid chromatography−mass spectrometry (LC−MS) (Figure 6A and Figure S150) corresponding to various fragments of the peptide, as depicted in Figure 6D (Figures S151−S156 and Scheme S2), was observed. To confirm that, in in vivo conditions, peptide 2 was broken under the influence of COX-2 (although the anti-inflammatory effect of 2 and its targeting and selectivity for COX-2 are established above), we incubated a solution of peptide 2 and COX-2 in HEPES buffer at pH 7.0. LC−MS of this solution (Figure 6B and Figure S158) also shows the similar fragmentations, as observed for the in vivo samples. The kinetic constants of COX-2-mediated breakage of peptide 2, calculated from the LC−MS data (monitoring the disappearance of peptide 2 under in vitro conditions), were Km = 2.1 × 10−5 M and Kcat = 13.2 s−1, with Kcat/Km = 6.3 × 105 M−1 s−1. Km and Kcat for COX-2-mediated metabolism of AA (disappearance of AA) were 2.1 × 10−4 M and 2.0 × 102 s−1, respectively, making Kcat/Km = 9.5 × 105 M−1 s−1 (Figure S159). Apparently, the peptide was metabolized by COX-2. To have an insight into

COX-2. Thus, the results are indicative of the involvement of cyclooxygenase pathway in reversing inflammation by compound 2 (Figure 5C). Effect of L-NAME and L-Arginine on the Analgesic Effect of 2 in Neurogenic and Inflammatory Phases of FormalinInduced Pain. Pretreatment with L-arginine, a NO precursor, reversed the analgesic effect of compound 2, as evidenced by a significant increase in the number of flinching in both neurogenic and inflammatory phases as compared with the formalin-treated group. However, pretreatment with L-NAME, a nonselective nitric oxide synthase inhibitor, did not alter the effect of 2 in either phase in formalin-induced hyperalgesia (Figure 5D). The anti-inflammatory effect of compound 2 against carrageenan-induced paw inflammation in mice was similar to that of indomethacin (Figure 5E). Therefore, the screening of compound 2 over the animal models clearly supported its appreciable analgesic and anti-inflammatory effects that were mainly emanating by targeting COX-2. Acute Toxicity Studies. Toxicity of compound 2 at doses of 50, 300, and 2000 mg kg−1 was checked according to OECD guidelines.50 Four groups (three in each group) of rats were taken. After 4 h of fasting, the first group was given vehicle; the second, third, and fourth groups were treated with 50, 300, and 2000 mg of compound 2, respectively. All animals were observed for the next 14 days, and then one animal from the group with a higher dose and control was sacrificed for

Table 1. COX-1 and COX-2 Inhibitory Activities and Selectivity Index (SI) of the Compounds IC50 (μM)a compound

structure

COX-1

COX-2

SIb

1 2 3 7 8 12 20 23 24 26 celecoxib indomethacin

Ala-Gly-Pro-Ala(OMe) Ala-Gly-Pro-Ala-Gly(OMe) Ala-Gly-Gly-Pro-Ala-Gly(OMe) N-Cbz-Ala-Gly-Pro(OMe) N-Cbz-Ala-Gly-Pro(OH) N-Cbz-Ala-Gly-Pro-Ala-Gly(OMe) N-Cbz-Ala-Phe-Pro-Phe-Gly(OMe) N-Cbz-Met-Gly-Pro-Phe(OMe) N-Boc-Pro-Phe(OH) N-Boc-Pro-Phe-Met-Gly(OMe)

0.8 40 10

0.6 0.1 2 0.3 0.02 0.4 5 0.1 0.08 0.4 0.04 0.96

1.3 400 5

0.05 50 600 0.5 50 15 0.08

25 125 120 5 125 375 0.08

a

Average of the three values with deviation of