Non-selective inhibition of cyclooxygenase enzymes by aminoacetylenic isoindoline 1,3-diones

Nidal A. Qinna1¶, Zuhair A. Muhi-eldeen2, Mohammad Ghattas3, Tawfiq M. Alhussainy1, Jenan Al-Qaisi1, Khalid Z. Matalka1

1Department of Pharmacology and Biomedical Sciences, Faculty of Pharmacy and Medical Sciences, Petra University, Amman, Jordan

2Department of Pharmaceutical Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy and Medical Sciences, Petra University, Amman, Jordan

3Faculty of Pharmacy, Alzaytoonah University, Amman, Jordan

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Abstract

The reported pharmacological activities of acetylenic and phthalimide groups promoted our interest to synthesize a novel series of N-[4-(t-amino-yl)-but-2-yn-1-yl] isoindoline-1,3-diones as an anti-inflammatory compounds. The aim of this research is to investigate the selectivity of two compounds, ZM4 and ZM5, on inhibiting cyclooxygenase (COX) in vitro and in silico as well as reducing carrageenan-induced edema in rats. Oral administration of 5-20 mg/kg ZM4 and ZM5 reduced significantly carrageenan-induced edema in dose-and time dependent manner. Furthermore, the IC50 induced by ZM4 and ZM5 were in the range of 3.0-3.6 µM for COX1 and COX 2 but were higher than those induced by Diclofenac and Celecoxib, respectively. Docking of ZM4 and ZM5 in both COX enzymes, on the other hand, exhibited the conventional binding modes that usually adopted by different non-steroidal anti-inflammatory drugs (NSAIDs). Furthermore, ZM4 and ZM5 bind to COX enzymes as strongly as Flurbiprofen and Celecoxib. In conclusion, aminoacetylenic isoindoline 1, 3-dione compounds have shown anti-inflammatory activity by inhibiting COX-1 and COX-2 enzymes. Interestingly, the best hits showed inhibition at low micromolar levels although they are not selective at this stage. Further research will be conducted to improve both selectivity and potency.

Keywords: COX inhibtors; aminoacetylenic isoindoline: docking

Introduction

The pathophysiological process of inflammation involves a coordinated response between the immune system and the local tissues in response to injury [1]. Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most commonly prescribed medications for controlling various forms of inflammation, despite their well-documented adverse effects especially on moderate to long term use [2].

In 1971, John Vane concluded that the pharmacological action of aspirin was due to the decreased production of prostaglandins (PGs) via irreversible inhibition of cyclooxygenase (COX) [3, 4]. Since the discovery of COX enzyme, at least three isoforms of this enzyme were reported: COX-1, COX-2 and COX-3 [5, 6]. The housekeeping isoform, COX-1, is expressed in most tissues and mediates most of the physiological functions attributed to PGs. However, inhibiting this latter isoform by NSAIDs drugs prevents the production of physiologic PGs which protect the gastric mucosa from hydrochloric acid-induced damage, maintain kidney and platelets function and therefore, explaining the shared side effects of the NSAIDs [7]. On the other hand, the constitutive isoform, COX-2, is the form expressed mainly in response to injurious stimuli and inflammation. Hence, COX-2 is the isoform involved in the production of prostanoids in the pathological processes [8]. Since conventional NSAIDs interfere with the enzymatic activity of both COX-1 and COX-2 at therapeutic doses, it is thought that the selective inhibition of COX-2 might have therapeutic actions similar to those of conventional NSAIDs, but without causing the undesirable side effects [9, 10]. Nevertheless, the cardiovascular problems associated with selective COX-2 inhibition still necessitate new approaches in the chemical development of new COX inhibitors [11, 12].

The high similarity between the two COX structures and the great difference in their functions in the human body make the discovery of new COX inhibitors extremely important. The first step, however, in any drug discovery is to develop inhibitors against the target protein regardless of their selectivity; afterwards attempts should be performed to optimize activity through enhancing selectivity of the obtained hits as well as their potency and pharmacokinetics properties. Recently, new COX inhibitors were tested and introduced as potential anti-inflammatory candidates [13, 14]. These COX inhibitors were designed based on the reported pharmacological activities of acetylenic and phthalimide groups, and promoted our interest to synthesize a novel series of N-[4-(t-amino-yl)-but-2-yn-1-yl] isoindoline-1,3-diones (Figure 1) [14]. The aim of this research is to investigate the selectivity of two compounds designed, ZM4 and ZM5, on inhibiting cyclooxygenase (COX) in vitro and in silico as well as reducing carrageenan-induced edema in rats.

Materials and Methods

Drugs and chemicals

ZM4 and ZM5 were synthesized and characterized as described earlier [14]. Carrageenan was obtained from Fluka, USA. Ibuprofen sodium and Diclofenac sodium were kindly provided by the Jordanian Pharmaceutical Manufacturing Co. “JPM” (Naor, Jordan). Celecoxib as Celebrex® of 200 mg capsule (Pfizer Inc, USA) was used in the study.

Animals

Male Sprague Dawley rats (200-280g) were obtained from Yarmouk University Animal House Unit (Irbid, Jordan) and housed in Petra University Animal Care Unit (Amman, Jordan). Animals were accommodated in a 12-hr light/dark cycle and a temperature of 20±2 ºC. All animals were acclimatized for at least 5 days prior to any experiment with free access to standard diet and drinking water. All animal experiments were performed in compliance with FELASA guidelines (Federation of European Laboratory Animal Science Association) and study protocols were approved by the Ethical Committee of the Deanship of Scientific Research at Petra University, Amman, Jordan.

In vivo rat paw inflammation

All tested compounds were weighed and dissolved in 0.1N HCl followed by sonication at 40ºC for 5 min. Rats were randomized into groups (n=8/group), assigned and received ZM4, ZM5 (5, 10, and 20 mg/kg), Diclofenac sodium (5 and 10 mg/kg), Celecoxib (6 and 12 mg/kg), while the control group received 4 ml/kg of 0.1N HCL. Initially, the thickness of the left hind paw was measured for each rat using an electronic caliper (Mitutouo Corporation, Japan) then the rats were received the treatments by oral gavage using a stainless steel oral needle (Harvard Apparatus, USA). After 1 hr, 0.1 ml of 1% carrageenan solution prepared in 0.9% saline was injected subcutaneously into the plantar region of the left hind paw. The size of edema represented by paw thickness of each rat was determined at 1, 3 and 5 hours post carrageenan injection. The increase of paw edema post carrageenan injection was calculated as percentage compared with the paw size determined immediately before carrageenan injection (Zero time) for each rat.

In vitro COX inhibition assay

COX-1 and COX-2 inhibition assay was performed to determine the activity of the tested compounds using an immunoassay from Caynman, Chemical Co., MI, USA.

In silico docking of ZM4 and ZM5 into COX-1 and COX-2

Crystal structures of COX-1 and COX-2 were downloaded from the protein data bank [16] (PDB, ID: 3N8Z [17] and 3NT1 [18]. Next, all water molecules were removed and partial charges were assigned to all atoms using Kollman united atom model in the Autodock Tool program [19, 20]. Subsequently, the active site of COX-1 and COX-2 was identified by their own co-crystallized ligand then a grid box of a 50 x 50 x 50 Å size was created with a grid spacing of 0.375 Å using Autogrid (part of the Autodock software package [21, 22].

The ligand 3D structures were built using Maestro software [23], and were then minimized using OPLS force field [25]. Gasteiger-Marsili partial charges [25] were assigned for all prepared ligand in the Autodock program. Tertiary amines in ZM4 and ZM5 were assigned as protonated.

Following the preparation of the protein and ligands structures, the ligands were docked into the previously identified active site using Autodock [21, 22]. Lamarckian Genetic Algorithm was employed for the conformational sampling of the ligand structures while the protein was treated as rigid (default settings used) [21]. Docked conformations were rated by the Autodock scoring function that includes terms for van der Waals, hydrogen bond, electrostatic interactions, and the ligand internal energy.

Statistical analysis

The in vivo results are expressed as mean ±SEM for each group, whereas data for the COX inhibition assay are expressed as mean ± SD. Data were assessed by one way ANOVA, followed by one- tailed Dunnettۥs t-test (SPSS 17, USA). P value <0.05 was considered significant.

Results

Rat paw inflammation

Oral administration of ZM4 (20 mg/kg) showed a significant reduction of carrageenan-induced inflammation at 1 hr post oral administration and remained reduced for 5 hr (p<0.01). On the other hand, oral administration of ZM5 produced a varying degree of inhibition of inflammation at 1, 3 and 5 hr post carrageenan-induced inflammation with a maximum inhibition at 20 mg/kg after 3 and 5 hr (p<0.05), which was equipotent to Diclofenac and Celecoxib (Fig. 2).

COX inhibition

The IC50 values induced by ZM compounds against COX-1 and COX-2 activities are presented in Table 1. The maximum inhibitory activity observed by either ZM compounds, Diclofenac or Celecoxib were at 5 µM. However, the IC50 values induced by ZM4 and ZM5 were less against COX-2 than COX-1 and were higher than those induced by Diclofenac and Celecoxib as non-selective and COX-2 inhibitor, respectively (Table 1).

Docking of ZM4 and ZM5 into COX-1 and COX-2

Two groups of key amino acids are responsible for many NSAIDs binding in the COX-1 and COX-2 active site: Ser530 and Tyr385 in the top of the catalytic pocket (e.g. Diclofenac) or Arg120 and Tyr355 in the bottom of the pocket (e.g. Flurbiprofen) [18]. The above interactions are electrostatic mainly formed by the NSAIDs' carboxylate. In addition, the benzene rings and other aliphatic groups of NSAIDs usually form van der Waals interactions with the hydrophobic residues lining the active pocket.

Similarly, docking of ZM4 and ZM5 in both COX enzymes exhibited the conventional binding modes that usually adopted by different NSAIDs. Regarding COX-1, both inhibitors demonstrated multiple binding modes with a polar head directed either to the roof or to the bottom of the active pocket (Figure 3A and 3B). However, in COX-2 docking, both compounds have a single binding mode where the polar head is electrostatically bound to the bottom of the active site; in particular with Tyr355 or Arg120, as shown in Figure 3C and 3D. Furthermore, Autodock scores listed in Table 2 indicates that ZM4 and ZM5 were able to bind to COX as strongly as Flurbiprofen and Celecoxib, which were used as a docking reference in our experiments. Indeed, such strong binding was not solely caused by the hydrogen bonding made by the cyclic carbonyl groups but also due to the good shape complementarily shown by these ligands with COX catalytic pocket. As shown in the Figure 3, ZM4 and ZM5 have many close contacts with the surrounding hydrophobic residues (e.g. Val349, Leu352, Leu357, Leu359, Trp389 and Phe518) which further stabilized these ligands in the COX-1 and COX-2 catalytic pocket.

The best docked pose of ZM5 has an unusual binding mode compared with the conventional binding mode normally adopted by many NSAIDs. As shown in Figure 3B, although the imide carbonyl is hydrogen bound to key amino acids, Tyr385 and Ser530, which are commonly involved in NSAIDs binding, the rest of the ligand sits in an adjacent pocket that involved many close contacts between the hexamethyleneimine ring and the surrounding hydrophobic residues (i.e. Phe205, Phe209, Val228 and Phe318). Furthermore, a hydrogen bonding between positively charged amino group of the hexamethyleneimine ring and the Ser530 carboxyl group is evident.

Discussion

In the present study, the anti-inflammatory effect of two aminoacetylenic compounds, ZM4 and ZM5, was investigated utilizing in vivo, in vitro and in silico methods. The carrageenan-induced rat paw edema test is usually considered the preferred in vivo method for testing acute phase inflammation due to its sensitivity [26]. This acute inflammatory response is generally characterized by an increase in vascular permeability and cellular infiltration that leads to edema, as a result of fluid and proteins and accumulation of leukocytes at the inflammatory site. However, a biphasic response for paw-injected carrageenan in mice but not in rats has been confirmed namely, the acute phase that develops in the first 6 hr followed by a second phase that starts at 24 hr post carrageenan injection [27]. Here, the effect of the tested compounds was determined only in the acute phase of carrageenan-induced rat paw inflammation. Both orally administered ZM compounds suppressed the edematous response induced by carrageenan injection in rat paws (Figure 2). However, only the high tested dose of ZM4 (20 mg/kg) showed intense reduction of paw edema starting from the first hour post carrageenan injection in comparison to Diclofenac, the non selective COX inhibitor and Celecoxib, the selective COX-2 inhibitor. On the other hand, all tested doses of ZM5 were effective in reducing edema formation. These results may indicate the rate of absorption of ZM4 is dose-dependent but both ZM4 and ZM5 are absorbed rapidly and have therapeutic bioavailability. More studies regarding plasma bioavailability, however, are warranted.

In vitro, ZM4 and ZM5 showed to inhibit both COX-1 and COX-2 but according to the IC50 they were 5 and 3 times less effective than Diclofenac and Celecoxib, respectively. Since, the rat paw edema inhibition were equivocal (with respect to the dose given) to Diclofenac and Celecoxib, other mechanisms of action induced by aminoacetylenic isoindoline 1,3-diones such as enhancing anti-inflammatory cytokines and/or suppressing proinflammatory cytokines might be evident.

In order to understand the molecular basis of the COX-1 and COX2 inhibition an in silco investigation was carried out using protein-ligand docking technique. Interestingly, ZM4 and ZM5 were able to score as low energy as Flurbiprofen and Celecoxib (Table 2) and adopted the typical binding mode in the COX catalytic pocket [18]. ZM4 and ZM5 possessed high shape complementarily with the catalytic pocket of both COX enzymes and were able to make hydrogen bonds with at least one of their key amino acids (e.g. Arg120, Tyr355 and Ser530) (Figure 3A and 3B). Probably, the high rigidity of ZM4 and ZM5 gained by the aminoacetylenic group, located in the connection segment of the two peripheral rings, was one of the strong reasons of why these inhibitors have such remarkable binding affinities and COX inhibition activities. Such rigidity gives these ligands a distinctive linear shape that makes them fit in the COX active site.

It is worth noting that the best docked posed of ZM5 has a different binding mode than the other obtained poses (Figure 3B). In this pose, the hexamethyleneimine ring is located in an adjacent pocket, making many hydrophobic interactions with the surrounding residues. Additionally, the protonated amino group has unusually bound to the catalytic residue, Ser530. Such an interaction is thought to be responsible for the high score that was given for this pose and may indicate for the importance of the cyclic amine ring in these inhibitors.