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Severe acute respiratory syndrome-related Coronavirus 2 (SARS-CoV-2) has infected more than 762 million people to date and has caused approximately 7 million deaths all around the world, involving more than 187 countries. Although currently available vaccines show high efficacy in preventing severe respiratory complications in infected patients, the high number of mutations in the S proteins of the current variants is responsible for the high level of immune evasion and transmissibility of the virus and the reduced effectiveness of acquired immunity. In this scenario, the development of safe and effective drugs of synthetic or natural origin to suppress viral replication and treat acute forms of COVID-19 remains a valid therapeutic challenge. Given the successful history of flavonoids-based drug discovery, we developed esters of substituted cinnamic acids with quercetin to evaluate their in vitro activity against a broad spectrum of Coronaviruses. Interestingly, two derivatives, the 3,4-methylenedioxy 6 and the ester of acid 7, have proved to be effective in reducing OC43-induced cytopathogenicity, showing interesting EC50s profiles. The ester of synaptic acid 7 in particular, which is not endowed with relevant cytotoxicity under any of the tested conditions, turned out to be active against OC43 and SARS-CoV-2, showing a promising EC50. Therefore, said compound was selected as the lead object of further analysis. When tested in a yield reduction, assay 7 produced a significant dose-dependent reduction in viral titer. However, the compound was not virucidal, as exposure to high concentrations of it did not affect viral infectivity, nor did it affect hCoV-OC43 penetration into pre-treated host cells. Additional studies on the action mechanism have suggested that our derivative may inhibit viral endocytosis by reducing viral attachment to host cells.

Cancer is a disease that can affect any organ and spread to other nearby or distant organs [...]

Osteoarthritis (OA) is the most prevalent joint disease associated with chronic pain. OA pain is often accompanied by mood disorders. We addressed the role of the Prokineticin (PK) system in pain and mood alterations in a mice OA model induced with monosodium iodoacetate (MIA). The effect of a PK antagonist (PC1) was compared to that of diclofenac. C57BL/6J male mice injected with MIA in the knee joint were characterized by allodynia, motor deficits, and fatigue. Twenty-eight days after MIA, in the knee joint, we measured high mRNA of PK2 and its receptor PKR1, pro-inflammatory cytokines, and MMP13. At the same time, in the sciatic nerve and spinal cord, we found increased levels of PK2, PKR1, IL-1β, and IL-6. These changes were in the presence of high GFAP and CD11b mRNA in the sciatic nerve and GFAP in the spinal cord. OA mice were also characterized by anxiety, depression, and neuroinflammation in the prefrontal cortex and hippocampus. In both stations, we found increased pro-inflammatory cytokines. In addition, PK upregulation and reactive astrogliosis in the hippocampus and microglia reactivity in the prefrontal cortex were detected. PC1 reduced joint inflammation and neuroinflammation in PNS and CNS and counteracted OA pain and emotional disturbances.

Compounds combining dual inhibitory action against FAAH and cyclooxygenase (COX) may be potentially useful analgesics. Here, we describe a novel flurbiprofen analogue, N-(3-bromopyridin-2-yl)-2-(2-fluoro-(1,1'-biphenyl)-4-yl)propanamide (Flu-AM4). The compound is a competitive, reversible inhibitor of FAAH with a Ki value of 13 nM and which inhibits COX activity in a substrate-selective manner. Molecular modelling suggested that Flu-AM4 optimally fits a hydrophobic pocket in the ACB region of FAAH, and binds to COX-2 similarly to flurbiprofen. In vivo studies indicated that at a dose of 10 mg/kg, Flu-AM4 was active in models of prolonged (formalin) and neuropathic (chronic constriction injury) pain and reduced the spinal expression of iNOS, COX-2, and NFκB in the neuropathic model. Thus, the present study identifies Flu-AM4 as a dual-action FAAH/substrate-selective COX inhibitor with anti-inflammatory and analgesic activity in animal pain models. These findings underscore the potential usefulness of such dual-action compounds.

Inhibition of fatty acid amide hydrolase (FAAH) reduces the gastrointestinal damage produced by non-steroidal anti-inflammatory agents such as sulindac and indomethacin in experimental animals, suggesting that a dual-action FAAH-cyclooxygenase (COX) inhibitor could have useful therapeutic properties. Here, we have investigated 12 novel amide analogues of ibuprofen as potential dual-action FAAH/COX inhibitors. N-(3-Bromopyridin-2-yl)−2-(4-isobutylphenyl)propanamide (Ibu-AM68) was found to inhibit the hydrolysis of [3H]anandamide by rat brain homogenates by a reversible, mixed-type mechanism of inhibition with a Ki value of 0.26 µM and an α value of 4.9. At a concentration of 10 µM, the compound did not inhibit the cyclooxygenation of arachidonic acid by either ovine COX-1 or human recombinant COX-2. However, this concentration of Ibu-AM68 greatly reduced the ability of the COX-2 to catalyse the cyclooxygenation of the endocannabinoid 2-arachidonoylglycerol. It is concluded that Ibu-AM68 is a dual-acting FAAH/substrate-selective COX inhibitor.

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.

A series of 6- and 6,8-halocoumarin derivatives have been investigated as potential antiproliferative compounds against a panel of tumor and normal cell lines. Cytotoxic effects were determined by the MTT method. To investigate the potential molecular mechanism involved in the cytotoxic effect, apoptosis assay, cell cycle analysis, reactive oxygen species (ROS), and reduced glutathione analysis were performed. Among the screened compounds, coumarins 6,8-dibromo-2-oxo-2H-chromene-3-carbonitrile 2h and 6,8-diiodo-2-oxo-2H-chromene-3-carbonitrile 2k exhibited the most antiproliferative effect in thyroid cancer-derived cells TPC-1. The apoptosis assay showed that both 2h and 2k induced apoptosis in TPC-1 thyroid cancer cells. According to these experiments, both coumarins induced a slight increase in TPC-1 cells in the G2/M phase and a decrease in the S phase. A significant increase in ROS levels was observed in TPC-1 treated with diiodocoumarin 2k, while the dibromocoumarin 2h induced a decrease in ROS in a dose and time-dependent manner.

A small series of hydrazonobenzenesulfonamides was designed, synthesized and studied for their human carbonic anhydrase (hCA) inhibitory activity. The synthesized compounds were evaluated against hCA I, II, IX and XII isoforms using acetazolamide (AAZ) as the standard inhibitor. Various hydrazonosulfonamide derivatives showed inhibitory activity at low nanomolar levels with selectivity against the cytosolic hCA II isoform, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The most potent and selective hydrazones 8, 9, 10, 11, 19 and 24 were docked into isoforms I, II, IX and XII to better understand their activity and selectivity for the different CA isoforms.

Abstract Background Neuropathy is a dose-limiting side effect of many chemotherapeutics, including bortezomib. The mechanisms underlying this condition are not fully elucidated even if a contribution of neuroinflammation was suggested. Here, we investigated the role of a chemokine family, the prokineticins (PKs), in the development of bortezomib-induced peripheral neuropathy (BIPN), and we used a PK receptor antagonist to counteract the development and progression of the pathology. Methods Neuropathy was induced in male C57BL/6J mice by using a protocol capable to induce a detectable neuropathic phenotype limiting systemic side effects. The presence of allodynia (both mechanical and thermal) and thermal hyperalgesia was monitored over time. Mice were sacrificed at two different time points: 14 and 28 days after the first bortezomib (BTZ) injection. At these times, PK system activation (PK2 and PK-Rs), macrophage and glial activation markers, and cytokine production were evaluated in the main station involved in pain transmission (sciatic nerve, DRG, and spinal cord), and the effect of a PK receptors antagonist (PC1) on the same behavioral and biochemical parameters was assessed. Structural damage of DRG during BTZ treatment and an eventual protective effect of PC1 were also evaluated. Results BTZ induces in mice a dose-related allodynia and hyperalgesia and a progressive structural damage to the DRG. We observed a precocious increase of macrophage activation markers and unbalance of pro- and anti-inflammatory cytokines in sciatic nerve and DRG together with an upregulation of GFAP in the spinal cord. At higher BTZ cumulative dose PK2 and PK receptors are upregulated in the PNS and in the spinal cord. The therapeutic treatment with the PK-R antagonist PC1 counteracts the development of allodynia and hyperalgesia, ameliorates the structural damage in the PNS, decreases the levels of activated macrophage markers, and prevents full neuroimmune activation in the spinal cord. Conclusions PK system may be a strategical pharmacological target to counteract BTZ-induced peripheral neuropathy. Blocking PK2 activity reduces progressive BTZ toxicity in the DRG, reducing neuroinflammation and structural damage to DRG, and it may prevent spinal cord sensitization.

Fatty Acid Amide Hydrolase (FAAH) is a serine hydrolase that plays a key role in controlling endogenous levels of endocannabinoids. FAAH inhibition is considered a powerful approach to enhance the endocannabinoid signalling, and therefore it has been largely studied as a potential target for the treatment of neurological disorders such as anxiety or depression, or of inflammatory processes. We present two novel series of amide derivatives of ibuprofen designed as analogues of our reference FAAH inhibitor Ibu-AM5 to further explore its structure-activity relationships. In the new amides, the 2-methylpyridine moiety of Ibu-AM5 was substituted by benzylamino and piperazinoaryl moieties. The obtained benzylamides and piperazinoarylamides showed FAAH inhibition ranging from the low to high micromolar potency. The binding of the new amides in the active site of FAAH, estimated using the induced fit protocol, indicated arylpiperazinoamides binding the ACB channel and the cytosolic port, and benzylamides binding the ACB channel.

Here we report a small library of hydrazinocarbonyl-ureido and thioureido benzenesulfonamide derivatives, designed and synthesized as potent and selective human carbonic anhydrase inhibitors (hCAIs). The synthesized compounds were evaluated against isoforms hCA I, II, IX and XII using acetazolamide (AAZ) as standard inhibitor. Several urea and thiourea derivatives showed inhibitory activity at low nanomolar levels with selectivity against the cytosolic hCA II isoform, as well as the transmembrane, tumor-associated enzymes hCA IX and XII. The thiourea derivatives showed enhanced potency as compared to urea analogues. Additionally, eight compounds 5g, 5m, 5o, 5q, 6l, 6j, 6o and 6u were selected for docking analysis on isoform I, II, IX, XII to illustrate the potential interaction with the enzyme to better understand the activity against the different isoforms.

The Enterovirus genus includes many viruses that are pathogenic in humans, including Coxsackie viruses and rhinoviruses, as well as the emerging enteroviruses D68 and A71. Currently, effective antiviral agents are not available for the treatment or prevention of enterovirus infections, which remain an important threat to public health. We recently identified a series of quinoxaline derivatives that were provento be potent inhibitors of coxsackievirus B5, the most common and a very important human pathogen belonging to the enterovirus genus. We have shown how most active derivatives interfere with the earliest stages of viral replication, blocking infection. Considering the broad antiviral spectrum, a very attractive property for an antiviral drug, we aimed to investigate the antiviral activity of the most promising compounds against other Enterovirus species. Here, we investigated the susceptibility of a panel of representatives of Enterovirus genus (enterovirus A71, belonging to A species; coxsackieviruses B4 and B3;echovirus 9, belonging to B species; and enterovirus D68, belonging to D species) to quinoxaline inhibitors. We also tested cytotoxicity and selectivity indices of the selected compounds, as well as their effects on virus yield.We also investigated their potential mechanism of action by a time course assay. In addition, a bioinformatic analysis was carried out to discover potential new conserved motifs in CVB3 and CVB4 compared to the other enterovirus species that can be used as new targets.

The tumor microenvironment is crucial for the growth of cancer cells, triggering particular biochemical and physiological changes, which frequently influence the outcome of anticancer therapies. The biochemical rationale behind many of these phenomena resides in the activation of transcription factors such as hypoxia-inducible factor 1 and 2 (HIF-1/2). In turn, the HIF pathway activates a number of genes including those involved in glucose metabolism, angiogenesis, and pH regulation. Several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, such as CA IX and XII, actively participate in these processes and were validated as antitumor/antimetastatic drug targets. Here, we review the field of CA inhibitors (CAIs), which selectively inhibit the cancer-associated CA isoforms. Particular focus was on the identification of lead compounds and various inhibitor classes, and the measurement of CA inhibitory on-/off-target effects. In addition, the preclinical data that resulted in the identification of SLC-0111, a sulfonamide in Phase Ib/II clinical trials for the treatment of hypoxic, advanced solid tumors, are detailed.

Induction of apoptosis is a common chemotherapeutic mechanism to kill cancer cells The thiazole system has been reported over the past decades as a building block for the preparation of anticancer agents. A novel series of 2-arylalkylamino-4-amino-5-(3′,4′,5′-trimethoxybenzoyl)-thiazole derivatives designed as dual inhibitors of tubulin and cyclin-dependent kinases (CDKs) were synthesized and evaluated for their antiproliferative activity in vitro against two cancer cell lines and, for selected highly active compounds, for interactions with tubulin and cyclin-dependent kinases and for cell cycle and apoptosis effects. Structure-activity relationships were elucidated for various substituents at the 2-position of the thiazole skeleton. Among the synthesized compounds, the most active analogues were found to be the p-chlorobenzylamino derivative 8e as well as the p-chloro and p-methoxyphenethylamino analogues 8f and 8k, respectively, which inhibited the growth of U-937 and SK-MEL-1 cancer cell lines with IC50 values ranging from 5.7 to 12.2 μM. On U-937 cells, the tested compounds 8f and 8k induced apoptosis in a time and concentration dependent manner. These two latter molecules did not affect tubulin polymerization (IC50 > 20 μM) nor CDK activity at a single concentration of 10 μM, suggesting alternative targets than tubulin and CDK for the compounds.

In the search of multifunctional compounds we designed benzimidazole derivatives endowed with phenolic hydroxy groups and a hydrazone moiety as potential radical-scavenger and the antioxidant agents. The target molecules have been prepared by a simple synthetic procedure and tested for their antioxidant activity by DPPH, FRAP, and ORAC test, for photoprotective activity against UV rays and for antiproliferative activity against Colo-38 melanoma cells. Furthermore, two different dermocosmetic formulations were prepared with the compounds endowed with the best antioxidant and photoprotective profile and their release from formulation evaluated using Franz Cells system. High antioxidant activity is related to the presence of at least two hydroxy groups on arylidene moiety of benzimidazoles. Structure activity analysis revealed that the position of hydroxy groups is crucial for antioxidant activity as well as the presence of a 2-hydroxy-4-(diethylamino)arylidene group. The same correlation pattern was found to be related to photoprotective activity resulting in an UVA Protection Factor better than the commercial solar filter PBSA and antiproliferative activity against melanoma cells without producing cytotoxicity on normal keratinocytes. The release analysis indicated that high antioxidant activities are achieved with limited release at concentration compatible with the use as UV sunscreen filter.

The persistence of the AIDS epidemic, and the life-long treatment required, indicate the constant need of novel HIV-1 inhibitors. In this scenario the HIV-1 Reverse Transcriptase (RT)-associated ribonuclease H (RNase H) function is a promising drug target. Here we report a series of compounds, developed on the 2-amino-6-(trifluoromethyl)nicotinic acid scaffold, studied as promising RNase H dual inhibitors. Among the 44 tested compounds, 34 inhibited HIV-1 RT-associated RNase H function in the low micromolar range, and seven of them showed also to inhibit viral replication in cell-based assays with a selectivity index up to 10. The most promising compound, 21, inhibited RNase H function with an IC50 of 14 µM and HIV-1 replication in cell-based assays with a selectivity index greater than 10. Mode of action studies revealed that compound 21 is an allosteric dual-site compound inhibiting both HIV-1 RT functions, blocking the polymerase function also in presence of mutations carried by circulating variants resistant to non-nucleoside inhibitors, and the RNase H function interacting with conserved regions within the RNase H domain. Proving compound 21 as a promising lead for the design of new allosteric RNase H inhibitors active against viral replication with not significant cytotoxic effects.

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC50 values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D2 and E2 in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.

Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here.FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH(T488A)-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 μM) was more potent than the (R)-enantiomer (IC50 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH.The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.

The synthesis and antiproliferative activity of new benzimidazole derivatives bearing an hydrazone mojety at the 2-position is described. The new N′-(4-arylidene)-1H-benzo[d]imidazole-2-carbohydrazides were evaluated for their cytostatic activity toward the murine leukemia (L1210), human T-cell leukemia (CEM), human cervix carcinoma (HeLa) and human pancreas carcinoma cells (Mia Paca-2). A preliminary structure-activity relationship could be defined. Some of the compounds possess encouraging and consistent antiproliferative activity, having IC50 values in the low micromolar range.