Cyclooxygenase Inhibition Safety and Efficacy in Inflammation-Based Psychiatric Disorders

According to the World Health Organization, the major psychiatric and neurodevelopmental disorders include major depression, bipolar disorder, schizophrenia, and autism spectrum disorder. The potential role of inflammation in the onset and progression of these disorders is increasingly being studied. The use of non-steroidal anti-inflammatory drugs (NSAIDs), well-known cyclooxygenase (COX) inhibitors, combined with first-choice specific drugs have been long investigated. The adjunctive administration of COX inhibitors to classic clinical treatments seems to improve the prognosis of people who suffer from psychiatric disorders. In this review, a broad overview of the use of COX inhibitors in the treatment of inflammation-based psychiatric disorders is provided. For this purpose, a critical analysis of the use of COX inhibitors in the last ten years of clinical trials of the major psychiatric disorders was carried out

Three-dimensional structure of human cyclooxygenase (hCOX)-1

The beneficial effects of Cyclooxygenases (COX) inhibitors on human health have been known for thousands of years. Nevertheless, COXs, particularly COX-1, have been linked to a plethora of human diseases such as cancer, heart failure, neurological and neurodegenerative diseases only recently. COXs catalyze the first step in the biosynthesis of prostaglandins (PGs) and are among the most important mediators of inflammation. All published structural work on COX-1 deals with the ovine isoenzyme, which is easier to produce in milligram-quantities than the human enzyme and crystallizes readily. Here, we report the long-sought structure of the human cyclooxygenase-1 (hCOX-1) that we refined to an R/Rfree of 20.82/26.37, at 3.36 Å resolution. hCOX-1 structure provides a detailed picture of the enzyme active site and the residues crucial for inhibitor/substrate binding and catalytic activity. We compared hCOX-1 crystal structure with the ovine COX-1 and human COX-2 structures by using metrics based on Cartesian coordinates, backbone dihedral angles, and solvent accessibility coupled with multivariate methods. Differences and similarities among structures are discussed, with emphasis on the motifs responsible for the diversification of the various enzymes (primary structure, stability, catalytic activity, and specificity). The structure of hCOX-1 represents an essential step towards the development of new and more selective COX-1 inhibitors of enhanced therapeutic potential

Fluorochrome Selection for Imaging Intraoperative Ovarian Cancer Probes

The identification and removal of all gross and microscopic tumor to render the patient disease free represents a huge challenge in ovarian cancer treatment. The presence of residual disease is an independent negative prognostic factor. Herein, we describe the synthesis and the “in vitro” evaluation of compounds as cyclooxygenase (COX)-1 inhibitors, the COX-1 isoform being an ovarian cancer biomarker, each bearing fluorochromes with different fluorescence features. Two of these compounds N-[4-(9-dimethylimino-9H-benzo[a]phenoxazin-5-ylamino) butyl]-2-(3,4-bis(4-methoxyphenyl)isoxazol-5-yl)acetamide chloride (RR11) and 3-(6-(4-(2-(3,4-bis(4-methoxyphenyl)isoxazole-5-yl)acetamido)butyl)amino-6-oxohexyl)-2-[7-(1,3-dihydro-1,1-dimethyl-3-ethyl 2H-benz[e]indolin-2-yl-idene)-1,3,5-heptatrienyl]-1,1-dimethyl-3-(6-carboxilato-hexyl)-1H-benz[e]indolium chloride, 23 (MSA14) were found to be potent and selective inhibitors of cyclooxygenase (COX)-1 “in vitro”, and thus were further investigated “in vivo”. The IC50 values were 0.032 and 0.087 µM for RR11 and 23 (MSA 14), respectively, whereas the COX-2 IC50 for RR11 is 2.4 µM while 23 (MSA14) did not inhibit COX-2 even at a 50 µM concentration. Together, this represented selectivity index = 75 and 874, respectively. Structure-based virtual screening (SBVS) performed with the Fingerprints for Ligands and Proteins (FLAP) software allowed both to differentiate highly active compounds from less active and inactive structures and to define their interactions inside the substrate-binding cavity of hCOX1. Fluorescent probes RR11 and 23 (MSA14), were used for preliminary near-infrared (NIR) fluorescent imaging (FLI) in human ovarian cancer (OVCAR-3 and SKOV-3) xenograft models. Surprisingly, a tumor-specific signal was observed for both tested fluorescent probes, even though this signal is not linked to the presence of COX-1.publishedVersio

Three-dimensional structure of human cyclooxygenase (hCOX)-1.

The beneficial effects of Cyclooxygenases (COX) inhibitors on human health have been known for thousands of years. Nevertheless, COXs, particularly COX-1, have been linked to a plethora of human diseases such as cancer, heart failure, neurological and neurodegenerative diseases only recently. COXs catalyze the first step in the biosynthesis of prostaglandins (PGs) and are among the most important mediators of inflammation. All published structural work on COX-1 deals with the ovine isoenzyme, which is easier to produce in milligram-quantities than the human enzyme and crystallizes readily. Here, we report the long-sought structure of the human cyclooxygenase-1 (hCOX-1) that we refined to an R/Rfree of 20.82/26.37, at 3.36 Å resolution. hCOX-1 structure provides a detailed picture of the enzyme active site and the residues crucial for inhibitor/substrate binding and catalytic activity. We compared hCOX-1 crystal structure with the ovine COX-1 and human COX-2 structures by using metrics based on Cartesian coordinates, backbone dihedral angles, and solvent accessibility coupled with multivariate methods. Differences and similarities among structures are discussed, with emphasis on the motifs responsible for the diversification of the various enzymes (primary structure, stability, catalytic activity, and specificity). The structure of hCOX-1 represents an essential step towards the development of new and more selective COX-1 inhibitors of enhanced therapeutic potential

Neuroinflammation and Microglial Constitutive COX-1 Inhibition

Neuroinflammation, as the erliest stage of neurological and neurodegenerative diseases, takes place about 15-20 years before the appearance of specific neurodegenerative clinical symptoms. Among the known mechanisms involved into the neuroinflammatory complex network, the cyclooxygenase-1 (COX-1) plays a previously unrecognized role in the neuroinflammation as demonstrated by the attenuation of the inflammatory response and neuronal loss due to the genetic ablation or pharmacological inhibition of COX-1 activity. The lack of drugs to treat diseases involving the central nervous system also resides into the shield exerted by the blood brain barrier matrix. BBB has also a low permeability, and the development of drugs able to penetrate through its network is one of the challenges of all scientists involved in projecting medicines having active principle ingredients targeting the CNS diseases. A commonly used strategy to overcome this drawback consists to incorporate into the pharmacological active molecule a sugar moiety (i.e. glucose or galactose), in turn capable to carry the entire molecule into the CNS by the GLUT-1 carrier, which is located on the membrane of the endothelial cells. In this context, a set of novel compounds endowed with inhibitory activity with cyclooxygenase-1 and GLUT-1 substrate will be presented. Specifically, their design rationale and biological activity will particularly detailed

A Simplified Direct O2 Consumption-Based Assay to Test COX Inhibition

Background: Cyclooxygenase is a well-known oxidoreductase that catalyzes the uptake of two moles of O2 by arachidonic acid (AA), producing the hydroperoxide Prostaglandin G2 (PGG2), then reduced to the prostaglandin precursor Prostaglandin H2 (PGH2). O2 consumption during such reactions is a measure of cyclooxygenase activity. O2 involved is generally measured by indirect methods, accomplished in the presence of the substrate AA and/or inhibitors. Methods: We developed a new simplified and easy to be carried out protocol for O2 consumption measurement by using disrupted HEK293-derived adherent cells, stably transfected either with COX-1 or COX-2 genes, as a source of the COX enzymes. The Clark electrode is used to measure the O2 concentration variation during the enzyme-catalyzed reactions. Results and Discussion: The novel assay was validated by determining the IC50 values of the known inhibitors such as indomethacin, ibuprofen, SC560, and celecoxib. Indomethacin and ibuprofen are two traditional non-steroidal anti-inflammatory drugs (tNSAIDs). SC560 is a commercially available reference compound used for COX-1 inhibition investigations. Celecoxib is a clinically used COXIBs. The assay was also applied to measure the kinetics and IC50 of mofezolac and P6. Mofezolac is the most potent selective COX-1 inhibitor, and active principle ingredient of Disopain® used to treat rheumatoid arthritis in Japan. P6, uncovered by us, is used together with mofezolac as a reference in in vitro and in vivo COX inhibition investigations and as a scaffold for structure-inhibition activity relationship studies. Conclusion: The obtained results showed the suitability of the newly developed assay to measure COXs activity in the presence of inhibitors as well as the kinetics of the inhibition (i.e., Vmax and Km)

COX-1 INHIBITORS AS ANTI-PLATELET AGENTS IN COVID-19

Coronavirus Disease 19 (COVID-19) is primarily a lung disease which frequently leads to major cardiovascular complications and a poor prognosis due to excessive platelet activation, uncontrolled immune/inflammatory reactions ("cytokine storm"), endothelial dysfunction, and coagulopathy.1 Aspirin, due to its anti-inflammatory and anti-platelet aggregation properties, has been evaluated as a potential therapeutic agent for COVID-19. Low-doses Aspirin (typically 75–81 mg/day) irreversibly inhibits platelet cyclooxygenase-1 (COX-1) by Ser530 acetylation preventing conversion of arachidonic acid into PGG2/PGH2, the latter in turn transformed by thromboxane synthase in thromboxane A2, thus resulting in an antithrombotic effect. Unfortunately, its use is limited by gastrointestinal side effects and aspirin resistance.2 Therefore, novel COX-1 inhibitors are needed. Mofezolac (Figure) is the most potent and selective COX-1 inhibitor administrated to humans as an anti-arthritis drug (DisopainTM). It belongs to the diarylisoxazoles chemical class and used as a “hit compound” for Structure Activity Relationship (SAR) studies to design novel leads with antithrombotic activity. Replacing one or both mofezolac methoxyl with chemical groups with either increasing steric hindrance and capable to establish different interactions inside the COX-1 active site allowed the identification of novel COX-1 inhibitors. Evaluation of their effects on the blood coagulation cascade is ongoing

Design and synthesis of new [4,5-b] and [4,5-c] imidazopyridines as potential fluorophores/inhibitors for Human Cyclooxygenase-1.

In recent years, a great deal of research effort has been devoted to identifying targeted fluorophores, for intraoperative detection of neoplastic bodies and real-time assessment of tumor borders to achieve complete tumor removal [1]. The aim of this work is to design, synthesize new aryl derivatives of [4,5-b] and [4,5-c] imidazopyridines as potential ligand/inhibitors of Cyclooxygenase-1, isoform definitively ascertained as a tumor-associated target in ovarian cancer. In this regard, it was possible to carry out a Structure-based virtual screening (SBVS) performed with the Fingerprints for Ligands and Proteins (FLAP) software, by taking advantage of the recently published crystallographic structure of human Cyclooxygenase-1 (hCOX1) [2]. In this preliminary step, we designed different imidazopyridine derivates, substituted on both the imidazole ring and the aromatic ring, as can be seen from Figure 1. Then, identified the pocket of hCOX1, it was possible to calculate binding poses of our compounds and differentiate highly active structures from less active or inactive structures. Based on the results of the SBVS, we select the most promising candidates for the synthetic phase: the strategy adopted involves the direct condensation between different benzaldehydes, previously treated with Na2S2O5, and the appropriate diaminopyridine. The products obtained were isolated, purified and characterized by NMR Spectroscopy and Mass Spectrometry. The synthesized compounds were finally tested to evaluate their binding/inhibitory activity and selectivity towards COX-1 enzyme

Structure-activity relationships of piperazine-2-one derivatives as novel human ClpP activators

The human caseinolytic protease P (hClpP) is a serine protease localized at the mitochondrial matrix. It is involved in maintaining the homeostasis of the mitochondrial proteome. hClpP, together with the AAA+ unfoldase protein (ClpX), forms the complex hClpXP, which primary function is to facilitate the turnover of degraded proteins to prevent their accumulation, causing disruption of normal cellular function. Although, the pathological role of hClpP in cancer is not yet fully understood, its expression is upregulated in several types of solid tumors, such as lung, stomach, liver, thyroid, bladder, breast, ovary, prostate, testis, and brain. Therefore, chemical modulators of hClpP proteolytic activity might have potential antitumor use. hClpP activators act by disrupting the hClpXP complex, generating a physical detachment of ClpX from ClpP still maintaining ClpP in its active state. Several hClpP activators are known: imipridones, macrocyclic acyldepsipeptides (ADEPs), and oxadiazonocarboxyamides. The most successful ClpP activators belong to the imipridone class and its protype is ONC201, then through in silico techniques a new scaffold tetrahydropyridopyrimidinone (THPPD) was identified, and more potent activators of hClpP were obtained (eg TR57). Such a scaffold was further simplified to 1,4-dibenzylpiperazine-2-one derivatives (DA series) and preliminary information obtained from a structure-activity relationship study aimed at identifying the best isosteric chemical structure modifications to produce potent hClpP activators (nM scale) will be presented

Ovine COX-1 Isoenzyme Bio-production

Background: Recent findings enlightened the pivotal role of cyclooxygenases-1 and -2 (COX-1 and COX-2) in human diseases with inflammation as the committed earliest stage, such as cancer and neurodegenerative diseases. COXs are the main targets of nonsteroidal anti-inflammatory drugs and catalyze the bis-oxygenation of arachidonic acid into prostaglandin PGH2, then converted into prostaglandins, thromboxane, and prostacyclin by tissue-specific isomerases. A remarkable amount of pure COX-1 is necessary to investigate COX-1 structure and function, as well as for in vitro disease biochemical pathway investigations. Methods: Spodoptera frugiperda cells were infected with Baculovirus that revealed to be an efficient expression system to obtain a high amount of ovine(o)COX-1. Protein solubilization time in the presence of a non-ionic detergent was modified, and a second purification step was introduced. Results and Discussion: An improvement of a previously reported method for pure recombinant oCOX-1 production and isolation has been achieved, leading to a lower starting volume of infected cells for each purification, an increased cell density, an increased number of viral particles per cell, and a shortened infection period. The protocol for the recombinant oCOX-1 expression and purification has been in-depth elaborated to obtain 1 mg/L of protein. Conclusion: The optimized procedure could be suitable for producing other membrane proteins as well, for which an improvement in the solubilization step is necessary to have the availability of high concentration proteins