Pediatric Diffuse Midline Glioma H3K27-Altered: From Developmental Origins to Therapeutic Challenges.

Diffuse intrinsic pontine glioma (DIPG), now referred to as diffuse midline glioma (DMG), is a highly aggressive pediatric cancer primarily affecting children aged 4 to 9 years old. Despite the research and clinical trials conducted to identify a possible treatment for DIPG, no effective drug is currently available. These tumors often affect deep midline brain structures in young children, suggesting a connection to early brain development's epigenetic regulation targets, possibly affecting neural progenitor functions and differentiation. The H3K27M mutation is a known DIPG trigger, but the exact mechanisms beyond epigenetic regulation remain unclear. After thoroughly examining the available literature, we found that over 85% of DIPG tumors contain a somatic missense mutation, K27M, in genes encoding histone H3.3 and H3.1, leading to abnormal gene expression that drives tumor growth and spread. This mutation impacts crucial brain development processes, including the epithelial-mesenchymal transition (EMT) pathway, and may explain differences between H3K27M and non-K27M pediatric gliomas. Effects on stem cells show increased proliferation and disrupted differentiation. The genomic organization of H3 gene family members in the developing brain has revealed variations in their expression patterns. All these observations suggest a need for global efforts to understand developmental origins and potential treatments.

Luminescent Alendronic Acid-Conjugated Micellar Nanostructures for Potential Application in the Bone-Targeted Delivery of Cholecalciferol.

Vitamin D, an essential micronutrient crucial for skeletal integrity and various non-skeletal physiological functions, exhibits limited bioavailability and stability in vivo. This study is focused on the development of polyethylene glycol (PEG)-grafted phospholipid micellar nanostructures co-encapsulating vitamin D3 and conjugated with alendronic acid, aimed at active bone targeting. Furthermore, these nanostructures are rendered optically traceable in the UV-visible region of the electromagnetic spectrum via the simultaneous encapsulation of vitamin D3 with carbon dots, a newly emerging class of fluorescents, biocompatible nanoparticles characterized by their resistance to photobleaching and environmental friendliness, which hold promise for future in vitro bioimaging studies. A systematic investigation is conducted to optimize experimental parameters for the preparation of micellar nanostructures with an average hydrodynamic diameter below 200 nm, ensuring colloidal stability in physiological media while preserving the optical luminescent properties of the encapsulated carbon dots. Comprehensive chemical-physical characterization of these micellar nanostructures is performed employing optical and morphological techniques. Furthermore, their binding affinity for the principal inorganic constituent of bone tissue is assessed through a binding assay with hydroxyapatite nanoparticles, indicating significant potential for active bone-targeting. These formulated nanostructures hold promise for novel therapeutic interventions to address skeletal-related complications in cancer affected patients in the future.

Bioaerosol Sampling Devices and Pretreatment for Bacterial Characterization: Theoretical Differences and a Field Experience in a Wastewater Treatment Plant.

Studies on bioaerosol bacterial biodiversity have relevance in both ecological and health contexts, and molecular methods, such as 16S rRNA gene-based barcoded sequencing, provide efficient tools for the analysis of airborne bacterial communities. Standardized methods for sampling and analysis of bioaerosol DNA are lacking, thus hampering the comparison of results from studies implementing different devices and procedures. Three samplers that use gelatin filtration, swirling aerosol collection, and condensation growth tubes for collecting bioaerosol at an aeration tank of a wastewater treatment plant in Trieste (Italy) were used to determine the bacterial biodiversity. Wastewater samples were collected directly from the untreated sewage to obtain a true representation of the microbiological community present in the plant. Different samplers and collection media provide an indication of the different grades of biodiversity, with condensation growth tubes and DNA/RNA shieldTM capturing the richer bacterial genera. Overall, in terms of relative abundance, the air samples have a lower number of bacterial genera (64 OTUs) than the wastewater ones (75 OTUs). Using the metabarcoding approach to aerosol samples, we provide the first preliminary step toward the understanding of a significant diversity between different air sampling systems, enabling the scientific community to orient research towards the most informative sampling strategy.

Fluorescent imaging probes for in vivo ovarian cancer targeted detection and surgery.

Ovarian cancer is the most lethal gynecological cancer, with a survival rate of approximately 40% at five years from the diagno. The first-line treatment consists of cytoreductive surgery combined with chemotherapy (platinum- and taxane-based drugs). To date, the main prognostic factor is related to the complete surgical resection of tumor lesions, including occult micrometastases. The presence of minimal residual diseases not detected by visual inspection and palpation during surgery significantly increases the risk of disease relapse. Intraoperative fluorescence imaging systems have the potential to improve surgical outcomes. Fluorescent tracers administered to the patient may support surgeons for better real-time visualization of tumor lesions during cytoreductive procedures. In the last decade, consistent with the discovery of an increasing number of ovarian cancer-specific targets, a wide range of fluorescent agents were identified to be employed for intraoperatively detecting ovarian cancer. Here, we present a collection of fluorescent probes designed and developed for fluorescence-guided ovarian cancer surgery. Original articles published between 2011 and November 2022 focusing on fluorescent probes, currently under preclinical and clinical investigation, were searched in PubMed. The keywords used were targeted detection, ovarian cancer, fluorescent probe, near-infrared fluorescence, fluorescence-guided surgery, and intraoperative imaging. All identified papers were English-language full-text papers, and probes were classified based on the location of the biological target: intracellular, membrane, and extracellular.

A further pocket or conformational plasticity by mapping COX-1 catalytic site through modified-mofezolac structure-inhibitory activity relationships and their antiplatelet behavior.

Cyclooxygenase enzymes have distinct roles in cardiovascular, neurological, and neurodegenerative disease. They are differently expressed in different type of cancers. Specific and selective COXs inhibitors are needed to be used alone or in combo-therapies. Fully understand the differences at the catalytic site of the two cyclooxygenase (COX) isoforms is still opened to investigation. Thus, two series of novel compounds were designed and synthesized in fair to good yields using the highly selective COX-1 inhibitor mofezolac as the lead compound to explore a COX-1 zone formed by the polar residues Q192, S353, H90 and Y355, as well as hydrophobic amino acids I523, F518 and L352. According to the structure of the COX-1:mofezolac complex, hydrophobic amino acids appear to have free volume eventually accessible to the more sterically hindering groups than the methoxy linked to the phenyl groups of mofezolac, in particular the methoxyphenyl at C4-mofezolac isoxazole. Mofezolac bears two methoxyphenyl groups linked to C3 and C4 of the isoxazole core ring. Thus, in the novel compounds, one or both methoxy groups were replaced by the higher homologous ethoxy, normal and isopropyl, normal and tertiary butyl, and phenyl and benzyl. Furthermore, a major difference between the two sets of compounds is the presence of either a methyl or acetic moiety at the C5 of the isoxazole. Among the C5-methyl series, 12 (direct precursor of mofezolac) (COX-1 IC50 = 0.076 µM and COX-2 IC50 = 0.35 µM) and 15a (ethoxy replacing the two methoxy groups in 12; COX-1 IC50 = 0.23 µM and COX-2 IC50 > 50 µM) were still active and with a Selectivity Index (SI = COX-2 IC50/COX-1 IC50) = 5 and 217, respectively. The other symmetrically substituted alkoxyphenyl moietis were inactive at 50 µM final concentration. Among the asymmetrically substituted, only the 16a (methoxyphenyl on C3-isoxazole and ethoxyphenyl on C4-isoxazole) and 16b (methoxyphenyl on C3-isoxazole and n-propoxyphenyl on C4-isoxazole) were active with SI = 1087 and 38, respectively. Among the set of compounds with the acetic moiety, structurally more similar to mofezolac (SI = 6329), SI ranged between 1.4 and 943. It is noteworthy that 17b (n-propoxyphenyl on both C3- and C4-isoxazole) were found to be a COX-2 slightly selective inhibitor with SI = 0.072 (COX-1 IC50 > 50 µM and COX-2 IC50 = 3.6 µM). Platelet aggregation induced by arachidonic acid (AA) can be in vitro suppressed by the synthesized compounds, without affecting of the secondary hemostasia, confirming the biological effect provided by the selective inhibition of COX-1. A positive profile of hemocompatibility in relation to erythrocyte and platelet toxicity was observed. Additionally, these compounds exhibited a positive profile of hemocompatibility and reduced cytotoxicity. Quantitative structure activity relationship (QSAR) models and molecular modelling (Ligand and Structure based virtual screening procedures) provide key information on the physicochemical and pharmacokinetic properties of the COX-1 inhibitors as well as new insights into the mechanisms of inhibition that will be used to guide the development of more effective and selective compounds. X-ray analysis was used to confirm the chemical structure of 14 (MSA17).

Harmaline to Human Mitochondrial Caseinolytic Serine Protease Activation for Pediatric Diffuse Intrinsic Pontine Glioma Treatment.

Diffuse intrinsic pontine glioma (DIPG), affecting children aged 4-7 years, is a rare, aggressive tumor that originates in the pons and then spreads to nearby tissue. DIPG is the leading cause of death for pediatric brain tumors due to its infiltrative nature and inoperability. Radiotherapy has only a palliative effect on stabilizing symptoms. In silico and preclinical studies identified ONC201 as a cytotoxic agent against some human cancer cell lines, including DIPG ones. A single-crystal X-ray analysis of the complex of the human mitochondrial caseinolytic serine protease type C (hClpP) and ONC201 (PDB ID: 6DL7) allowed hClpP to be identified as its main target. The hyperactivation of hClpP causes damage to mitochondrial oxidative phosphorylation and cell death. In some DIPG patients receiving ONC201, an acquired resistance was observed. In this context, a wide program was initiated to discover original scaffolds for new hClpP activators to treat ONC201-non-responding patients. Harmaline, a small molecule belonging to the chemical class of ß-carboline, was identified through Fingerprints for Ligands and Proteins (FLAP), a structure-based virtual screening approach. Molecular dynamics simulations and a deep in vitro investigation showed interesting information on the interaction and activation of hClpP by harmaline.

Tetrazole and oxadiazole derivatives as bioisosteres of tariquidar and elacridar: New potent P-gp modulators acting as MDR reversers.

New 2,5- and 1,5-disubstituted tetrazoles, and 2,5-disubstituted-1,3,4-oxadiazoles were synthesized as tariquidar and elacridar derivatives and studied as multidrug resistance (MDR) reversers. Their behaviour on the three ABC transporters P-gp, MRP1 and BCRP was investigated. All compounds inhibited the P-gp transport activity in MDCK-MDR1 cells overexpressing P-gp, showing EC50 values even in the low nanomolar range (compounds 15, 22). Oxadiazole derivatives were able to increase the antiproliferative effect of doxorubicin in MDCK-MDR1 and in HT29/DX cells confirming their nature of P-gp modulators, with derivative 15 being the most potent in these assays. Compound 15 also displayed a dual inhibitory effect showing good activities towards both P-gp and BCRP. A computational study suggested a common interaction pattern on P-gp for most of the potent compounds. The bioisosteric substitution of the amide group of lead compounds allowed identifying a new set of potent oxadiazole derivatives that modulate MDR through inhibition of the P-gp efflux activity. If compared to previous amide derivatives, the introduction of the heterocycle rings greatly enhances the activity on P-gp, introduces in two compounds a moderate inhibitory activity on MRP1 and maintains in some cases the effect on BCRP, leading to the unveiling of dual inhibitor 15.

m-Terphenylamines, Acting as Selective COX-1 Inhibitors, Block Microglia Inflammatory Response and Exert Neuroprotective Activity.

Inhibition of cyclooxygenase-2 (COX-2) has been extensively studied as an approach to reduce proinflammatory markers in acute brain diseases, but the anti-neuroinflammatory role of cyclooxygenase-1 (COX-1) inhibition has been rather neglected. We report that m-terphenylamine derivatives are selective COX-1 inhibitors, able to block microglia inflammatory response and elicit a neuroprotective effect. These compounds were synthesized via a three-component reaction of chalcones, ß-ketoesters, and primary amines, followed by hydrolysis/decarboxylation of the ester group. Together with their synthetic intermediates and some urea derivatives, they were studied as inhibitors of COX-1 and COX-2. The m-terphenylamine derivatives, which were selective COX-1 inhibitors, were also analyzed for their ability to block microglia inflammatory and oxidative response. Compound 3b presented an interesting anti-inflammatory and neuroprotective profile by reducing nitrite release, ROS overproduction, and cell death in organotypic hippocampal cultures subjected to LPS. We thus show that COX-1 inhibition is a promising approach to provide enhanced neuroprotection against acute inflammatory processes, which are crucial in the development of a plethora of acute neurodegenerative injuries.

The multiple combination of Paclitaxel, Ramucirumab and Elacridar reverses the paclitaxel-mediated resistance in gastric cancer cell lines.

Introduction: Paclitaxel (PTX) interferes with microtubule architecture by binding to ß-tubulin, thereby blocking progression at the G2/M phase and inducing apoptosis. This study aimed to investigate molecular processes underlying PTX-mediated resistance in gastric cancer (GC) cells. Methods: PTX-mediated resistance involves many processes, and in this work some of the factors involved in the resistance mechanism were identified by comparing two GC lines with PTX induced resistance to their sensitive counterparts. Results: Thus, the key feature of PTX-resistant cells was the overexpression of pro-angiogenic factors such as VEGFA, VEGFC, and Ang2, known to support tumor cell growth. A second relevant change detected in PTX-resistant lines was the elevated level of TUBßIII, a tubulin isoform that opposes microtubule stabilization. A third identified factor contributing to PTX-resistance was P-glycoprotein (P-gp), a transporter responsible for chemotherapy efflux from the cells, highly expressed in PTX-resistant lines. Discussion: These findings were in line with a greater sensitivity of resistant cells to treatment with both Ramucirumab and Elacridar. Ramucirumab significantly reduced the expression of angiogenic molecules and TUBßIII, while Elacridar restored the access of chemotherapy, recovering its anti-mitotic and pro-apoptotic effects. Finally, this study highlighted the role played by exosomes in spreading factors responsible for resistance in the tumor microenvironment.

Zoledronic Acid Blocks Overactive Kir6.1/SUR2-Dependent KATP Channels in Skeletal Muscle and Osteoblasts in a Murine Model of Cantú Syndrome.

Cantú syndrome (CS) is caused by the gain of function mutations in the ABCC9 and KCNJ8 genes encoding, respectively, for the sulfonylureas receptor type 2 (SUR2) and the inwardly rectifier potassium channel 6.1 (Kir6.1) of the ATP-sensitive potassium (KATP) channels. CS is a multi-organ condition with a cardiovascular phenotype, neuromuscular symptoms, and skeletal malformations. Glibenclamide has been proposed for use in CS, but even in animals, the drug is incompletely effective against severe mutations, including the Kir6.1wt/V65M. Patch-clamp experiments showed that zoledronic acid (ZOL) fully reduced the whole-cell KATP currents in bone calvaria cells from wild type (WT/WT) and heterozygous Kir6.1wt/V65MCS mice, with IC50 for ZOL block < 1 nM in each case. ZOL fully reduced KATP current in excised patches in skeletal muscle fibers in WT/WT and CS mice, with IC50 of 100 nM in each case. Interestingly, KATP currents in the bone of heterozygous SUR2wt/A478V mice were less sensitive to ZOL inhibition, showing an IC50 of ~500 nM and a slope of ~0.3. In homozygous SUR2A478V/A478V cells, ZOL failed to fully inhibit the KATP currents, causing only ~35% inhibition at 100 µM, but was responsive to glibenclamide. ZOL reduced the KATP currents in Kir6.1wt/VMCS mice in both skeletal muscle and bone cells but was not effective in the SUR2[A478V] mice fibers. These data indicate a subunit specificity of ZOL action that is important for appropriate CS therapies.

Olive Mill Wastewater Fermented with Microbial Pools as a New Potential Functional Beverage.

Olive mill wastewater (OMWW) represents a by-product but also a source of biologically active compounds, and their recycling is a relevant strategy to recover income and to reduce environmental impact. The objective of the present study was to obtain a new functional beverage with a health-promoting effect starting from OMWW. Fresh OMWW were pre-treated through filtration and/or microfiltration and subjected to fermentation using strains belonging to Lactiplantibacillus plantarum, Candida boidinii and Wickerhamomyces anomalus. During fermentation, phenolic content and hydroxytyrosol were monitored. Moreover, the biological assay of microfiltered fermented OMWW was detected versus tumor cell lines and as anti-inflammatory activity. The results showed that in microfiltered OMWW, fermentation was successfully conducted, with the lowest pH values reached after 21 days. In addition, in all fermented samples, an increase in phenol and organic acid contents was detected. Particularly, in samples fermented with L. plantarum and C. boidinii in single and combined cultures, the concentration of hydroxytyrosol reached values of 925.6, 902.5 and 903.5 mg/L, respectively. Moreover, biological assays highlighted that fermentation determines an increase in the antioxidant and anti-inflammatory activity of OMWW. Lastly, an increment in the active permeability on Caco-2 cell line was also revealed. In conclusion, results of the present study confirmed that the process applied here represents an effective strategy to achieve a new functional beverage.

Genome-Wide Identification and Validation of Gene Expression Biomarkers in the Diagnosis of Ovarian Serous Cystadenocarcinoma.

Ovarian cancer is the second most prevalent gynecologic malignancy, and ovarian serous cystadenocarcinoma (OSCA) is the most common and lethal subtype of ovarian cancer. Current screening methods have strong limits on early detection, and the majority of OSCA patients relapse. In this work, we developed and cross-validated a method for detecting gene expression biomarkers able to discriminate OSCA tissues from healthy ovarian tissues and other cancer types with high accuracy. A preliminary ranking-based approach was applied, resulting in a panel of 41 over-expressed genes in OSCA. The RNA quantity gene expression of the 41 selected genes was then cross-validated by using NanoString nCounter technology. Moreover, we showed that the RNA quantity of eight genes (ADGRG1, EPCAM, ESRP1, MAL2, MYH14, PRSS8, ST14 and WFDC2) discriminates each OSCA sample from each healthy sample in our data set with sensitivity of 100% and specificity of 100%. For the other three genes (MUC16, PAX8 and SOX17) in combination, their RNA quantity may distinguish OSCA from other 29 tumor types.

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.

Definition of an Indoor Air Sampling Strategy for SARS-CoV-2 Detection and Risk Management: Case Study in Kindergartens.

In the last two years, the world has been overwhelmed by SARS-CoV-2. One of the most important ways to prevent the spread of the virus is the control of indoor conditions: from surface hygiene to ventilation. Regarding the indoor environments, monitoring the presence of the virus in the indoor air seems to be promising, since there is strong evidence that airborne transmission through infected droplets and aerosols is its dominant transmission route. So far, few studies report the successful detection of SARS-CoV-2 in the air; moreover, the lack of a standard guideline for air monitoring reduces the uniformity of the results and their usefulness in the management of the risk of virus transmission. In this work, starting from a critical analysis of the existing standards and guidelines for indoor air quality, we define a strategy to set-up indoor air sampling plans for the detection of SARS-CoV-2. The strategy is then tested through a case study conducted in two kindergartens in the metropolitan city of Milan, in Italy, involving a total of 290 children and 47 teachers from 19 classrooms. The results proved its completeness, effectiveness, and suitability as a key tool in the airborne SARS-CoV-2 infection risk management process. Future research directions are then identified and discussed.

Bisphosphonates Targeting Ion Channels and Musculoskeletal Effects.

Bisphosphonates (BPs) are the most used bone-specific anti-resorptive agents, often chosen as first-line therapy in several bone diseases characterized by an imbalance between osteoblast-mediated bone production and osteoclast-mediated bone resorption. BPs target the farnesyl pyrophosphate synthase (FPPS) in osteoclasts, reducing bone resorption. Lately, there has been an increasing interest in BPs direct pro-survival/pro-mineralizing properties in osteoblasts and their pain-relieving effects. Even so, molecular targets involved in these effects appear now largely elusive. Ion channels are emerging players in bone homeostasis. Nevertheless, the effects of BPs on these proteins have been poorly described. Here we reviewed the actions of BPs on ion channels in musculoskeletal cells. In particular, the TRPV1 channel is essential for osteoblastogenesis. Since it is involved in bone pain sensation, TRPV1 is a possible alternative target of BPs. Ion channels are emerging targets and anti-target for bisphosphonates. Zoledronic acid can be the first selective musculoskeletal and vascular KATP channel blocker targeting with high affinity the inward rectifier channels Kir6.1-SUR2B and Kir6.2-SUR2A. The action of this drug against the overactive mutants of KCNJ9-ABCC9 genes observed in the Cantu' Syndrome (CS) may improve the appropriate prescription in those CS patients affected by musculoskeletal disorders such as bone fracture and bone frailty.

Overcoming Multidrug Resistance (MDR): Design, Biological Evaluation and Molecular Modelling Studies of 2,4-Substituted Quinazoline Derivatives.

Some 2,4-disubstituted quinazolines were synthesized and studied as multidrug resistance (MDR) reversers. The new derivatives carried the quinazoline-4-amine scaffold found in modulators of the ABC transporters involved in MDR, as the TKIs gefitinib and erlotinib. Their behaviour on the three ABC transporters, P-gp, MRP1 and BCRP, was investigated. Almost all compounds inhibited the P-gp activity in MDCK-MDR1 cells overexpressing P-gp, showing EC50 values in the nanomolar range (1 d, 1 e, 2 a, 2 c, 2 e). Some compounds were active also towards MRP1 and/or BCRP. Docking results obtained by in silico studies on the P-gp crystal structure highlighted common features for the most potent compounds. The P-gp selective compound 1 e was able to increase the doxorubicin uptake in HT29/DX cells and to restore its antineoplastic activity in resistant cancer cells in the same extent of sensitive cells. Compound 2 a displayed a dual inhibitory effect showing good activities towards both P-gp and BCRP.

Zoledronic Acid as a Novel Dual Blocker of KIR6.1/2-SUR2 Subunits of ATP-Sensitive K+ Channels: Role in the Adverse Drug Reactions.

Zoledronic acid (ZOL) is used as a bone-specific antiresorptive drug with antimyeloma effects. Adverse drug reactions (A.D.R.) are associated with ZOL-therapy, whose mechanics are unknown. ZOL is a nitrogen-containing molecule whose structure shows similarities with nucleotides, ligands of ATP-sensitive K+ (KATP) channels. We investigated the action of ZOL by performing in vitro patch-clamp experiments on native KATP channels in murine skeletal muscle fibers, bone cells, and recombinant subunits in cell lines, and by in silico docking the nucleotide site on KIR and SUR, as well as the glibenclamide site. ZOL fully inhibited the KATP currents recorded in excised macro-patches from Extensor digitorum longus (EDL) and Soleus (SOL) muscle fibers with an IC50 of 1.2 ± 1.4 × 10-6 and 2.1 ± 3.7 × 10-10 M, respectively, and the KATP currents recorded in cell-attached patches from primary long bone cells with an IC50 of 1.6 ± 2.8 × 10-10 M. ZOL fully inhibited a whole-cell KATP channel current of recombinant KIR6.1-SUR2B and KIR6.2-SUR2A subunits expressed in HEK293 cells with an IC50 of 3.9 ± 2.7 × 10-10 M and 7.1 ± 3.1 × 10-6 M, respectively. The rank order of potency in inhibiting the KATP currents was: KIR6.1-SUR2B/SOL-KATP/osteoblast-KATP > KIR6.2-SUR2A/EDL-KATP >>> KIR6.2-SUR1 and KIR6.1-SUR1. Docking investigation revealed that the drug binds to the ADP/ATP sites on KIR6.1/2 and SUR2A/B and on the sulfonylureas site showing low binding energy <6 Kcal/mol for the KIR6.1/2-SUR2 subunits vs. the <4 Kcal/mol for the KIR6.2-SUR1. The IC50 of ZOL to inhibit the KIR6.1/2-SUR2A/B channels were correlated with its musculoskeletal and cardiovascular risks. We first showed that ZOL blocks at subnanomolar concentration musculoskeletal KATP channels and cardiac and vascular KIR6.2/1-SUR2 channels.

Learning from Nature: From a Marine Natural Product to Synthetic Cyclooxygenase-1 Inhibitors by Automated De Novo Design.

The repertoire of natural products offers tremendous opportunities for chemical biology and drug discovery. Natural product-inspired synthetic molecules represent an ecologically and economically sustainable alternative to the direct utilization of natural products. De novo design with machine intelligence bridges the gap between the worlds of bioactive natural products and synthetic molecules. On employing the compound Marinopyrrole A from marine Streptomyces as a design template, the algorithm constructs innovative small molecules that can be synthesized in three steps, following the computationally suggested synthesis route. Computational activity prediction reveals cyclooxygenase (COX) as a putative target of both Marinopyrrole A and the de novo designs. The molecular designs are experimentally confirmed as selective COX-1 inhibitors with nanomolar potency. X-ray structure analysis reveals the binding of the most selective compound to COX-1. This molecular design approach provides a blueprint for natural product-inspired hit and lead identification for drug discovery with machine intelligence.

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.

An attempt to chemically state the cross-talk between monomers of COX homodimers by double/hybrid inhibitors mofezolac-spacer-mofezolac and mofezolac-spacer-arachidonic acid.

Cardiovascular diseases (CVDs) account for over 17 million death globally each year, including arterial thrombosis. Platelets are key components in the pathogenesis of this disease and modulating their activity is an effective strategy to treat such thrombotic events. Cyclooxygenase-1 (COX-1) isoenzyme is involved in platelet activation and is the main target of non-steroidal anti-inflammatory drugs (NSAIDs) and new selective inhibitor research. Inhibitors of general formula mofezolac-spacer-mofezolac (mof-spacer-mof) and mofezolac-spacer-arachidonic acid (mof-spacer-AA) were projected to investigate the possible cross-talk between the two monomers (Eallo and Ecat) forming the COX-1 homodimer. Mofezolac was chosen as either one or two moieties of these molecules being the known most potent and selective COX-1 inhibitor and administrated to humans as Disopain™, then arachidonic acid (AA) was used to develop molecules bearing, in the same compound, in addition to the inhibitor moiety (mofezolac) also the natural COX substrate. Depending on the nature of the spacer, COX-1 and COX-2 activity was differently inhibited by mof-spacer-mof set with a preferential COX-1 inhibition. The highest COX-1 selectivity was exhibited by the compound in which the spacer was the benzidine [N,N'-(biphenyl-4,4'-di-yl)bis (2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamide) (15): COX-1 IC50 = 0.08 µM, COX-2 IC50 > 50 µM, Selectivity Index (SI) > 625]. In the case of mof-spacer-AA set, the COX inhibitory potency and also the isoform preference changed. (5Z, 8Z, 11Z, 14Z)-N-(4-{2-[3,4-Bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}butyl)icosa-5,8,11,14-tetraenamide (19) and (5Z, 8Z, 11Z, 14Z)-N-(4'-{2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}-[1,1'-biphenyl]-4-yl)icosa-5,8,11,14-tetraenamide (21), in which the spacer is the 1,2-diaminobutane or benzidine, respectively, selectively inhibited the COX-2, whereas when the spacer is the 1,4-phenylendiamine [(5Z, 8Z, 11Z, 14Z)-N-(4-{2-[3,4-bis(4-methoxyphenyl)isoxazol-5-yl]acetamido}phenyl)icosa-5,8,11,14-tetraenamide) (20) the COX preference is COX-1 (COX-1 IC50 = 0.05 µM, COX-2 IC50 > 50 µM, with a COX-1 selectivity > 1000). Molecular modelling by using FLAP algorithm shows fundamental interactions of the novel compounds at the entry channel of COX and inside its catalytic cavity. The effect of these mof-spacer-mof and mof-spacer-AA in inhibiting in vitro free arachidonic acid-induced platelet aggregation was also determined. A positive profile of hemocompatibility in relation to their influence on the blood coagulation cascade and erythrocyte toxicity was observed. Cytotoxicity and genotoxicity safety were also found for these two novel sets of compounds.