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

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)

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

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

Comparative Structural Analysis of hCOX‐1 and oCOX‐1 Isoenzymes

The different specific beneficial effects of Cyclooxygenase (COX)-1 and -2 inhibitors on human health have been known since the Nobel Laureate Prof. John Vane proven the existence of the COX-2. COXs are individually expressed in neurological and neurodegenerative diseases, and different types of cancer as well. More in-depth knowledge of the biology, structure and function of cyclooxygenase is required to plan pre-clinical studies. All the published structural works concern ovine (o) COX-1 (PDB ID: 3N8V), exploited for decades as a surrogate for the human (h) enzyme, more difficult to produce in milligrams quantities and recalcitrant to crystallization1. Herein, we present the 3.36 Å resolution X-ray crystal structure of the long-sought structure of hCOX-1 (PDB ID: 6Y3C) and relevant difference of the crystal structure of hCOX-1 and oCOX-1. Specifically, we also determined the amino acidic residues that, by changing their orientation, induce large variations in the position of neighboring residues due to the movement of the hinges. Conformations of Thr322, Pro218 and Ser126 in hCOX-1 and in the crystal structure of oCOX-1 are quite different, inducing the respective inhibitors to have different chemical characteristics. In summary, the 3D structure of hCOX-1 presented here will support, as main aim of this study, the development of probe targeting hCOX-1 for unmet clinical diagnosis of disease in which hCOX-1 is a biomarker

Repurposing of natural origin compounds as HsClpP inducers to treat diffuse midline glioma (DMG)

Diffuse midline glioma (DMG) is a fatal childhood CNS tumor whose treatment is still a challenge. DMG is has extremely poor prognosis due to localization and inoperability. Current treatments are limited to irradiation which only prolongs survival by a few months. Preclinical studies identified drug candidates, but their translation into the clinic remains a major obstacle. Over the past decade, ONC201, a known dopamine receptor antagonist, was discovered from a high-throughput screening of public libraries of compounds, to be endowed with cytotoxic activity against several tumor cell lines including glioma. Efforts to discover the true ONC201 target allow to identify Homo sapiens P caseinolytic mitochondrial protease (HsClpP) as responsible for its antiproliferative effect. Enhancement of HsClpP activity leads to its dysregulation useful in cancer therapeutics. HsClpP is overexpressed in multiple tumor cell types, such as DMG, to be considered a promising therapeutic target for its treatment. ONC201 reached DMG Phase III clinical trial. ONC201 and HsClpP co-crystal provided information on the aminoacids involved in the binding and structural features of HsClpP inducers. The identification of new therapeutic indications for marketed drugs represents one of the strategies (repurposing) to find treatments more quickly for diseases still lacking medications. Active principle ingredients (APIs) of natural origin present in known drugs can be used as they are (repurposing) or structurally ‘optimized’ by evolution to serve biological functions, including the regulation of endogenous defense mechanisms and the interaction with other organisms, which explains their high relevance for infectious diseases and cancer. APIs are characterized by enormous scaffold diversity and structural complexity. Thus, a Structure Based Virtual Screening of natural products (NPs), based on the Fingerprints for Ligands and Proteins (FLAP) algorithm, has been carrying out to identify a novel original scaffold as HsClpP inducers. The result of this screening, as well of the biological evaluation as protease inducers will be presented. [1] Perrone M.G. et al. Diffuse Intrinsic Pontine Glioma (DIPG): Breakthrough and Clinical Perspective Curr. Med.Chem. 2020, 27, 1-30

CHALLENGE TRANSABILITY OF “IN VITRO” TO “IN VIVO”: GENE-EXPRESSION BIOMARKERS AND FLUORESCENT IMAGE-GUIDED SURGERY PROBES IDENTIFICATION FOR OVARIAN CANCER

Ovarian cancer is the second most prevalent gynecologic malignancy, and the Ovarian Serous Cystadenocarcinoma (OSCA) is the most common and lethal subtype of ovarian cancer. Current screening methods have strong limits in early detection and, despite surgery and chemotherapy, the majority of OSCA patients relapse and eventually succumb to their disease, suggesting that further efforts are required to improve early diagnosis. Furthermore, 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. In this presentation, it will be described the development and cross-validated method for detecting gene expression biomarkers able to discriminate OSCA tissues from healthy ovarian tissues and from other cancer types with very high accuracy. Then, it will be described the synthesis (Figure), “in vitro” and “in vivo xenograft model” evaluation of ad hoc developed probes for fluorescent image-guided surgery, particularly useful in the resection of not palpable and not visible at naked eye lesions. The probes bearing fluorochromes with different fluorescent properties were used to challenge the transability of “in vitro “results to “in vivo” pre-cilinical model of ovarian cancer built by implanted human ovarian cancer cells in mice

Human Caseinolytic Protease P (hClpP) Activity Modulating Agents For The Treatment Of DIPG/DMG

Human Caseinolytic peptidase P (hClpP) is a key inducer of mitochondrial protein quality control for the clearance of misfolded or damaged proteins which are necessary for maintaining mitochondrial functions. Alterated activities of this protease are correlated with tumor development and progression. HClpP modulates cancer cell viability, reactive oxygen species (ROS) levels, and metabolic reprogramming under hypoxia, oxidative stress, or starvation. Consequently, inhibition or hyperactivation of this protease may be a potential therapeutic strategy for cancer treatment. The involvement of hClpP in cancer, is corroborated by the results obtained by its activator, named ONC201, an imipridone, that disrupts the ClpXP complex, by physically displacing ClpX from ClpP while keeping hClpP in its activated state. ONC201 was discovered from a high-throughput screening of public libraries of compounds, to be endowed with cytotoxic activity against several tumor cell lines, including glioma, and the Food and Drug Administration granted Fast Track designation to ONC201 for the treatment of highgrade glioma with recurrent H3-K27M mutation in adults. Diffuse Intrinsic Pontine Glioma (DIPG) is a subgroup of DMG, an astrocytoma involving the pons, a region of the brain stem, consisting in a broad horseshoeshaped mass of transverse nerve fibres that connect the medulla with the cerebellum. The pons, while involved in the regulation of functions carried out by the cranial nerves it houses, works together with the medulla oblongata to serve an especially critical role in generating the respiratory rhythm of breathing. Active functioning of the pons may also be fundamental to rapid eye movement (REM) sleep. The sensitivity of the affected region of the pons makes DIPG unsuitable for surgical resection and its current standard care is limited to focal radiotherapy providing only a limited and temporary benefit for patient survival. In the last decade, advances in genomic knowledge have made it possible to establish that in most cases (80%) of DIPG there is a K27M mutation of histone H3: hence the definition of "Diffuse gliomas of the midline H3K27M mutated”. ONC201 and hClpP co-crystal provided information on the amino acids involved in the binding and structural features of its activators. The identification of new therapeutic indications for marketed drugs represents one of the strategies (repurposing) to find treatments more quickly for diseases still lacking medications. Active principle ingredients (APIs) of natural origin present in known drugs can be used as they are (repurposing) or structurally ‘optimized’ by evolution to serve biological functions, including the regulation of endogenous defense mechanisms and the interaction with other organisms, which explains their high relevance for infectious diseases and cancer. APIs are characterized by enormous scaffold diversity and structural complexity. Thus, a Structure Based Virtual Screening of natural products (NPs), based on the Fingerprints for Ligands and Proteins (FLAP) algorithm, has been carrying out to identify a novel original scaffold as hClpP inducers. The result of this screening, as well of the biological evaluation as protease inducers will be presented