Virtual screening for novel COX-2 inhibitors using the ZINC database

Cyclooxygenase-2 (COX-2) enzyme binds to arachidonic acid and releases metabolites that are used to induce pain and inflammation. COX-2 selective inhibitors such as celecoxib, rofecoxib and valdecoxib are currently used to reduce inflammatory response. However, they lack anti-thrombotic activity and hence lead to cardiovascular and renal liabilities apart from gastrointestinal irritation. Therefore, there is still a need to develop more potent COX-2 inhibitors. In this paper, we report the screening of various compounds from the ZINC database (contains 4.6 million small molecule compounds) using the eHiTS (electronic High Throughput Screening) software tool against the COX-2 protein. The strategy employed can be conveniently split into two categories, viz. screening and docking, respectively. Screening was performed using molecular constraints tool to filter compounds with physico-chemical properties similar to the 6COX bound ligand SC-558. The analysis resulted in 1042 Lipinski compliant hits which are docked and scored to identify structurally novel ligands that make similar interactions to those of known ligands or may have different interactions with other parts of the binding site. Our screening approach identified two molecules ZINC00663976 (eHITS score of -7.135 kcal/mol) and ZINC02062094 (eHITS score of -7.242 kcal/mol) from the ZINC database. Their energy scores are better than the 6COX bound co-crystallized ligand SC-558 with an eHiTS score of -6.559 kcal/mol. Both the ligands were docked within the binding pocket forming interactions with Leu352, Phe518, Met522, Val523, Ala527 and Ser353. Visual inspection suggested similar orientation and binding mode for ZINC02062094 with SC-558 ligand. The NH group of the ligand formed hydrogen bond interactions with the backbone NH of Ala527.     

A double prodrug system for colon targeting of benzenesulfonamide COX-2 inhibitors

The design, synthesis and delivery potential of a new type of benzenesulfonamide cyclo-oxygenase-2 (COX-2) inhibitor prodrug is investigated using celecoxib. The approach involves a double prodrug that is activated first by azoreductases and then by cyclization triggering drug release. We studied the intramolecular aminolysis of the acylsulfonamide. The cyclization was surprisingly rapid at physiological pH and very fast at pH 5. The prodrug is activated specifically under conditions found in the colon but highly stable in the presence of human and rodent intestinal extracts. Finally, the prototype with celecoxib was transported much more slowly in the Caco-2 transepithelial model than the parent. The design therefore shows significant promise for the site specific delivery of benzenesulfonamide COX-2 inhibitors to the colon.     

A prodrug approach to COX-2 inhibitors with methylsulfone

2,2-dimethyl-4-phenyl-5-[4-(methylsulfinyl)phenyl]-3(2H)furanone derivatives, 3 and 6, were shown to be effectively transformed in vivo into the corresponding methylsulfone derivatives 1 and 4, when orally administered to rats. Pharmacological implications for use of sulfoxide analogues 3 and 6 are discussed as prodrugs to potent selective COX-2 inhibitors 1 and 4.     

Molecular docking analysis of COX-2 for potential inhibitors

Cyclooxygenase-2 (COX-2) is liked with breast cancer. Therefore, it is of interest to design and develop new yet effective compounds against COX-2 from medicinal plants such as the natural alkaloid compounds. We document the optimal binding features of aristolochicacid with COX-2 protein for further consideration.     

Mucosal damage induced by preferential COX-1 and COX-2 inhibitors: role of prostaglandins and inflammatory response

Nonsteroidal anti-inflammatory drugs (NSAID) are well known to induce gastric mucosal damage including bleeding, ulceration and perforation in humans and animals too. These effects are related with the inhibition of the enzyme cyclooxygenase, which is the main established mechanism of action for these drugs. Fasted rats were given piroxicam, preferential COX-1 inhibitor (10-20 mg/kg) or meloxicam, preferential COX-2 inhibitor (7.5-15 mg/kg) orally. Six or nine hours (h) later, respectively, the stomach was excised, the severity of the damage assessed and myeloperoxidase (MPO) activity measured, as well as prostaglandin PGE(2) content. Furthermore, in order to assess the effects of these oxicams over previously damaged gastric mucosa, 1 ml of 0.6 N HCl was administered p.o. followed, 1 h after, of the correspondent dose of each NSAID, and the same parameters were determined. Oral administration of both drugs dose-dependently caused acute gastric haemorrhage erosions. Myeloperoxidase activity was significantly increased by piroxicam administration. In addition, PGE(2) content was significantly reduced. The association between the administration of the acid and NSAID caused a worsening of the damage and, while myeloperoxidase activity did not modify by both piroxicam and meloxicam, PGE(2) levels were reduced. These results suggest that the PG derived from both COX-1 and COX-2 pathway plays a beneficial role in the gastroprotection, and thus caution should be exercise in the clinical use of preferential COX-2 inhibitors.     

Practical approaches to minimizing gastrointestinal and cardiovascular safety concerns with COX-2 inhibitors and NSAIDs

Nonsteroidal anti-inflammatory drugs (NSAIDs) are highly effective in treating the pain and inflammation associated with osteoarthritis and rheumatoid arthritis, but it is well recognized that these agents are associated with substantial gastrointestinal toxicity. Treatment guidelines suggest that patients with one or more risk factors for NSAID associated ulcers should be prescribed preventive treatment. However, well over 80% of such patients may not receive an appropriate therapeutic intervention. Multiple strategies are available to reduce the risk for NSAID associated gastrointestinal complications. First, risk may be reduced by using non-NSAID analgesics. Second, use of the minimum effective dose of the NSAID may reduce risk. Third, co-therapy with a proton pump inhibitor or misoprostol may be desirable in at-risk patients. Use of cyclo-oxygenase-2 inhibitors may also reduce the risk for gastrointestinal events, although this benefit is eliminated in patients who receive aspirin, and cyclo-oxygenase-2 inhibitors may increase cardiovascular adverse events. The optimal management of NSAID related gastrointestinal complications must be based on the individual patient's risk factors for gastrointestinal and cardiovascular disease, as well as on the efficacy and tolerability of both the NSAID and accompanying gastroprotective agent.     

A novel genetic model of selective COX-2 inhibition: comparison with COX-2 null mice

Prostaglandin H Synthase (PGHS) is a bi-functional enzyme with a cyclooxygenase (COX) activity and a functionally linked peroxidase (POX) activity that exists in two isoforms (COX-1, COX-2). Non-steroidal anti-inflammatory drugs (NSAIDs), including the selective COX-2 inhibitors, block COX activity while leaving POX activity unscathed. Recently, some selective COX-2 inhibitors were withdrawn from the market due to elevated cardiovascular risk in placebo-controlled trials. Mice deficient for PGHS2 were developed in 1995 and through numerous subsequent studies have revealed significant roles in renal development, ductus arteriosus patency/closure, skin carcinogenesis and cardiovascular function. In this short review, we compare a novel genetic COX-2 selective inhibition mouse model with the originally described COX-2 null mice in these different physiological functions.     

Hybrid fluorescent conjugates of COX-2 inhibitors: Search for a COX-2 isozyme imaging cancer biomarker

The observation that the cyclooxygenase-2 (COX-2) isozyme is over-expressed in multiple types of cancer, relative to that in adjacent non-cancerous tissue, prompted this investigation to prepare a group of hybrid fluorescent conjugates wherein the COX inhibitors ibuprofen, (S)-naproxen, acetyl salicylic acid, a chlororofecoxib analog and celecoxib were coupled via a linker group to an acridone, dansyl or rhodamine B fluorophore. Within this group of compounds, the ibuprofen-acridone conjugate (10) showed potent and selective COX-2 inhibition (COX-2 IC₅₀ = 0.67 μM; SI = 110.6), but its fluorescence emission (λ_em = 417, 440 nm) was not suitable for fluorescent imaging of cancer cells that over-express the COX-2 isozyme. In comparison, the celecoxib-dansyl conjugate (25) showed a slightly lower COX-2 potency and selectivity (COX-2 IC₅₀ = 1.1 μM; SI > 90) than the conjugate 10, and it possesses a better fluorescence emission (λ_em = 500 nm). Ultimately, a celecoxib-rhodamine B conjugate (28) that exhibited moderate COX-2 potency and selectivity (COX-2 IC₅₀ = 3.9 μM; SI > 25) having the best fluorescence emission (λ_em = 580 nm) emerged as the most promising biomarker for fluorescence imaging using a colon cancer cell line that over-expresses the COX-2 isozyme.     

Synthesis and evaluation of 1,5-diaryl-substituted tetrazoles as novel selective cyclooxygenase-2 (COX-2) inhibitors

A series of 1,5-diaryl-substituted tetrazole derivatives was synthesized via conversion of readily available diaryl amides into corresponding imidoylchlorides followed by reaction with sodium azide. All compounds were evaluated by cyclooxygenase (COX) assays in vitro to determine COX-1 and COX-2 inhibitory potency and selectivity. Tetrazoles 3a-e showed IC₅₀ values ranging from 0.42 to 8.1 mM for COX-1 and 2.0 to 200 μM for COX-2. Most potent compound 3c (IC₅₀ (COX-2) = 2.0 μM) was further used in molecular modeling docking studies.     

A novel mechanism of action of chemically modified tetracyclines: inhibition of COX-2-mediated prostaglandin E2 production.

Tetracyclines (doxycycline and minocycline) inhibit inducible NO synthase expression and augment cyclooxygenase (COX)-2 expression and PGE2 production. In contrast, chemically modified tetracyclines (CMTs), such as CMT-3 and -8 (but not CMT-1, -2, and -5), that lack antimicrobial activity, inhibit both NO and PGE2 production in LPS-stimulated murine macrophages, bovine chondrocytes, and human osteoarthritis-affected cartilage, which spontaneously produces NO and PGE2 in ex vivo conditions. Furthermore, CMT-3 augments COX-2 protein expression but inhibits net PGE2 accumulation. This coincides with the ability of CMT-3 and -8 to inhibit COX-2 enzyme activity in vitro. The action of CMTs is distinct from that observed with tetracyclines because 1) CMT-3-mediated inhibition of PGE2 production coincides with modification of COX-2 protein, which is distinct from the nonglycosylated COX-2 protein generated in the presence of tunicamycin, as observed by Western blot analysis and 2) CMT-3 and -8 have no significant effect on COX-2 mRNA accumulation. In contrast, CMT-3 and -8 do not inhibit COX-1 expression in A549 human epithelial cells at the level of protein and mRNA accumulation or modification of COX-1 protein. CMT-3 and -8 inhibit the sp. act. of COX-2 (but not COX-1) in cell-free extracts. These results demonstrate differential action of CMT-3 (Metastat) on COX-1 and -2 expression, which is distinct from other tetracyclines.     

Cardiovascular and gastrointestinal effects of COX-2 inhibitors and NSAIDs: achieving a balance

Conventional 'nonselective' nonsteroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of pain and inflammation; however, the potential gastrointestinal risks associated with their use can be a cause for concern. In response to the adverse effects that can accompany nonselective NSAID use, selective cyclo-oxygenase (COX)-2 inhibitors were developed to target the COX-2 isoenzyme, thus providing anti-inflammatory and analgesic benefits while theoretically sparing the gastroprotective activity of the COX-1 isoenzyme. Data from large-scale clinical trials have confirmed that the COX-2 inhibitors are associated with substantial reductions in gastrointestinal risk in the majority of patients who do not receive aspirin. However, some or all of the gastrointestinal benefit of COX-2 inhibitors may be lost in patients who receive low, cardioprotective doses of aspirin, and recent evidence suggests that some of these agents, at some doses, may be associated with an increased risk for cardiovascular adverse events compared with no therapy. The risks and benefits of conventional NSAIDs and of COX-2 inhibitors must be weighed carefully; in clinical practice many patients who might benefit from NSAID or COX-2 therapy are likely to be elderly and at relatively high risk for gastrointestinal and cardiovascular adverse events. These patients are also more likely to be taking low-dose aspirin for cardiovascular prophylaxis and over-the-counter NSAIDs for pain. Identifying therapies that provide relief from arthritis related symptoms, confer optimum cardioprotection, and preserve the gastrointestinal mucosa is complex. Factors to consider include the interference of certain NSAIDs with the antiplatelet effects of aspirin, differences in the adverse gastrointestinal event rates among nonselective NSAIDs and selective COX-2 inhibitors, emerging data regarding the relative risks for cardiovascular events associated with these drugs, and the feasibility and cost of co-therapy with proton pump inhibitors.     

Design, synthesis and biological evaluation of pyridine acyl sulfonamide derivatives as novel COX-2 inhibitors

A series of pyridine acyl sulfonamide derivatives (1-24) have been designed and synthesized and their biological activities were also evaluated as potential cyclooxygenase-2 (COX-2) inhibitors. Among all the compounds, compound 23 displayed the most potent COX-2 inhibitory activity with an IC(50) of 0.8 μM. Antitumor and anti-inflammatory assays indicated that compound 23 owned high antiproliferative activity against B16-F10, HepG2 and MCF-7 cancer cell lines as well as COX-2-derived prostaglandin E(2) (PGE(2)) inhibitory activity of murine macrophage RAW 264.7 cell line with IC(50) values of 2.8, 1.2, 1.8 and 0.15 μM, respectively. Docking simulation was performed to position compound 23 into the COX-2 active site to determine the probable binding model.     

ACE2: A novel therapeutic target for cardiovascular diseases

Hypertension afflicts over 65 million Americans and poses an increased risk for cardiovascular morbidity such as stroke, myocardial infarction and end-stage renal disease resulting in significant mortality. Overactivity of the renin-angiotensin system (RAS) has been identified as an important determinant that is implicated in the etiology of these diseases and therefore represents a major target for therapy. In spite of the successes of drugs inhibiting various elements of the RAS, the incidence of hypertension and cardiovascular diseases remain steadily on the rise. This has lead many investigators to seek novel and innovative approaches, taking advantage of new pathways and technologies, for the control and possibly the cure of hypertension and related pathologies. The main objective of this review is to forward the concept that gene therapy and the genetic targeting of the RAS is the future avenue for the successful control and treatment of hypertension and cardiovascular diseases. We will present argument that genetic targeting of angiotensin-converting enzyme 2 (ACE2), a newly discovered member of the RAS, is ideally poised for this purpose. This will be accomplished by discussion of the following: (i) summary of our current understanding of the RAS with a focus on the systemic versus tissue counterparts as they relate to hypertension and other cardiovascular pathologies; (ii) the newly discovered ACE2 enzyme with its physiological and pathophysiological implications; (iii) summary of the current antihypertensive pharmacotherapy and its limitations; (iv) the discovery and design of ACE inhibitors; (v) the emerging concepts for ACE2 drug design; (vi) the current status of genetic targeting of the RAS; (vii) the potential of ACE2 as a therapeutic target for hypertension and cardiovascular disease treatment; and (viii) future perspectives for the treatment of cardiovascular diseases.     

The cardiovascular effects of the carotid sinus mechanism

The feedback mechanism by means of which the carotid sinus affects arterial blood pressure is represented with the use of the single chamber model of the cardiovascular system. The average pressure in the large arteries is the controlled variable. The resultant firstorder differential equation is easily solved. The theoretical results indicate that the feed-back mechanism of the carotid sinus serves to decrease the deviation of the average arterial pressure from an internal standard of pressure in the steady state and to hasten the stabilization of the pressure following environmental or pharmacological changes. This approach also suggests an experimentally feasible method for the determination of the internal standard of pressure.     

A novel antiinflammatory maintains glucocorticoid efficacy with reduced side effects

Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor gamma coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.     

Drug repositioning strategy for the identification of novel telomere-damaging agents: A role for NAMPT inhibitors

Drug repositioning strategy represents a valid tool to accelerate the pharmacological development through the identification of new applications for already existing compounds. In this view, we aimed at discovering molecules able to trigger telomere-localized DNA damage and tumor cell death. By applying an automated high-content spinning-disk microscopy, we performed a screening aimed at identifying, on a library of 527 drugs, molecules able to negatively affect the expression of TRF2, a key protein in telomere maintenance. FK866, resulting from the screening as the best candidate hit, was then validated at biochemical and molecular levels and the mechanism underlying its activity in telomere deprotection was elucidated both in vitro and in vivo. The results of this study allow us to discover a novel role of FK866 in promoting, through the production of reactive oxygen species, telomere loss and deprotection, two events leading to an accumulation of DNA damage and tumor cell death. The ability of FK866 to induce telomere damage and apoptosis was also demonstrated in advanced preclinical models evidencing the antitumoral activity of FK866 in triple-negative breast cancer—a particularly aggressive breast cancer subtype still orphan of targeted therapies and characterized by high expression levels of both NAMPT and TRF2. Overall, our findings pave the way to the development of novel anticancer strategies to counteract triple-negative breast cancer, based on the use of telomere deprotecting agents, including NAMPT inhibitors, that would rapidly progress from bench to bedside.     

Synthesis of novel hybrids of pyrazole and coumarin as dual inhibitors of COX-2 and 5-LOX

In our previous study, we designed a series of pyrazole derivatives as novel COX-2 inhibitors. In order to obtain novel dual inhibitors of COX-2 and 5-LOX, herein we designed and synthesized 20 compounds by hybridizing pyrazole with substituted coumarin who was reported to exhibit 5-LOX inhibition to select potent compounds using adequate biological trials sequentially including selective inhibition of COX-2 and 5-LOX, anti-proliferation in vitro, cells apoptosis and cell cycle. Among them, the most potent compound 11g (IC₅₀ = 0.23 ± 0.16 μM for COX-2, IC₅₀ = 0.87 ± 0.07 μM for 5-LOX, IC₅₀ = 4.48 ± 0.57 μM against A549) showed preliminary superiority compared with the positive controls Celecoxib (IC₅₀ = 0.41 ± 0.28 μM for COX-2, IC₅₀ = 7.68 ± 0.55 μM against A549) and Zileuton (IC₅₀ = 1.35 ± 0.24 μM for 5-LOX). Further investigation confirmed that 11g could induce human non-small cell lung cancer A549 cells apoptosis and arrest the cell cycle at G2 phase in a dose-dependent manner. Our study might contribute to COX-2, 5-LOX dual inhibitors thus exploit promising novel cancer prevention agents.     

Paraoxonase-2 deficiency aggravates atherosclerosis in mice despite lower apolipoprotein-B-containing lipoproteins: anti-atherogenic role for paraoxonase-2

Paraoxonases (PONs) are a family of proteins that may play a significant role in providing relief from both toxic environmental chemicals as well as physiological oxidative stress. Although the physiological roles of the PON family of proteins, PON1, PON2, and PON3, remain unknown, epidemiological, biochemical, and mouse genetic studies of PON1 suggest an anti-atherogenic function for paraoxonases. To determine whether PON2 plays a role in the development of atherosclerosis in vivo, we generated PON2-deficient mice. When challenged with a high fat, high cholesterol diet for 15 weeks, serum levels of high density lipoprotein cholesterol, triglycerides, and glucose were not significantly different between wild-type and PON2-deficient mice. In contrast, serum levels of very low density lipoprotein (VLDL)/low density lipoprotein (LDL) cholesterol were significantly lower (-32%) in PON2-deficient mice compared with wild-type mice. However, despite lower levels of VLDL/LDL cholesterol, mice deficient in PON2 developed significantly larger (2.7-fold) atherosclerotic lesions compared with their wild-type counterparts. Enhanced inflammatory properties of LDL, attenuated anti-atherogenic capacity of high density lipoprotein, and a heightened state of oxidative stress coupled with an exacerbated inflammatory response from PON2-deficient macrophages appear to be the main mechanisms behind the larger atherosclerotic lesions in PON2-deficient mice. These results demonstrate that PON2 plays a protective role in atherosclerosis.