Suppression of lipopolysaccharide-induced COX-2 expression via p38MAPK, JNK, and C/EBPß phosphorylation inhibition by furomagydarin A, a benzofuran glycoside from Magydaris pastinacea.

The phytochemical investigation of the methanol extract of the seeds of Magydaris pastinacea afforded two undescribed benzofuran glycosides, furomagydarins A-B (1, 2), together with three known coumarins. The structures of the new isolates were elucidated after extensive 1D and 2D NMR experiments as well as HR MS. Compound 1 was able to inhibit the COX-2 expression in RAW264.7 macrophages exposed to lipopolysaccharide, a pro-inflammatory stimulus. RT-qPCR and luciferase reporter assays suggested that compound 1 reduces COX-2 expression at the transcriptional level. Further studies highlighted the capability of compound 1 to suppress the LPS-induced p38MAPK, JNK, and C/EBPß phosphorylation, leading to COX-2 down-regulation in RAW264.7 macrophages.

2H-chromene and 7H-furo-chromene derivatives selectively inhibit tumour associated human carbonic anhydrase IX and XII isoforms.

Tumour associated carbonic anhydrases (CAs) IX and XII have been recognised as potential targets for the treatment of hypoxic tumours. Therefore, considering the high pharmacological potential of the chromene scaffold as selective ligand of the IX and XII isoforms, two libraries of compounds, namely 2H-chromene and 7H-furo-chromene derivatives, with diverse substitution patterns were designed and synthesised. The structure of the newly synthesised compounds was characterised and their inhibitory potency and selectivity towards human CA off target isoforms I, II and cancer-associated CA isoforms IX and XII were evaluated. Most of the compounds inhibit CA isoforms IX and XII with no activity against the I and II isozymes. Thus, while the potency was influenced by the substitution pattern along the chromene scaffold, the selectivity was conserved along the series, confirming the high potential of both 2H-chromene and 7H-furo-chromene scaffolds for the design of isozyme selective inhibitors.

5-Nitro-3-(2-(4-phenylthiazol-2-yl)hydrazineylidene)indolin-2-one derivatives inhibit HIV-1 replication by a multitarget mechanism of action.

In the effort to identify and develop new HIV-1 inhibitors endowed with innovative mechanisms, we focused our attention on the possibility to target more than one viral encoded enzymatic function with a single molecule. In this respect, we have previously identified by virtual screening a new indolinone-based scaffold for dual allosteric inhibitors targeting both reverse transcriptase-associated functions: polymerase and RNase H. Pursuing with the structural optimization of these dual inhibitors, we synthesized a series of 35 new 3-[2-(4-aryl-1,3-thiazol-2-ylidene)hydrazin-1-ylidene]1-indol-2-one and 3-[3-methyl-4-arylthiazol-2-ylidene)hydrazine-1-ylidene)indolin-2-one derivatives, which maintain their dual inhibitory activity in the low micromolar range. Interestingly, compounds 1a, 3a, 10a, and 9b are able to block HIV-1 replication with EC50 < 20 µM. Mechanism of action studies showed that such compounds could block HIV-1 integrase. In particular, compound 10a is the most promising for further multitarget compound development.

Ebola virus disease: In vivo protection provided by the PAMP restricted TLR3 agonist rintatolimod and its mechanism of action.

Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks.

Cynarin blocks Ebola virus replication by counteracting VP35 inhibition of interferon-beta production.

Ebola virus (EBOV) is one of the deadliest infective agents whose lethality is linked to the ability to efficiently bypass the host's innate antiviral response. EBOV multifunctional protein VP35 plays a major role in viral replication both as polymerase cofactor and interferon (IFN) antagonist. By hiding the non-self 5'-ppp dsRNA from the cellular receptor RIG-I, VP35 prevents its activation and inhibits IFN-ß production. Blocking VP35-dsRNA interaction and IFN-ß suppression is a validated drug target. We screened a library of natural extracts and found that cynarin inhibits dsRNA-VP35 binding with an IC50 value of 8.5 µM. It reverts the EBOV VP35 inhibition of IFN-ß production, while it does not induce IFN production by itself. Docking experiments suggest that the molecule can bind on the end-capping pocket of VP35 C-terminal Interferon Inhibitory domain (IID), and differential scanning fluorimetry confirmed that cynarin interacts with VP35-IID with a KD of 12 µM. Cynarin was further tested in an EBOV minigenome assay but did not inhibit VP35 polymerase cofactor activity. When evaluated during challenge of IFN-susceptible A549 cells with EBOV isolate derived from the 2014 West African outbreak, cynarin was able to inhibit viral replication with an EC50 value of 9.1 µM, showing no significant cytotoxicity. Our findings show that cynarin blocks EBOV replication by acting directly on VP35 and subverting its IFN antagonism function but not cofactor function, and as such identify the first EBOV inhibitor with this mode of action.

Exploring New Scaffolds for the Dual Inhibition of HIV-1 RT Polymerase and Ribonuclease Associated Functions.

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3-[(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide (EMAC2063) was the most potent towards RNaseH (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated functions.

Flavonoids and Acid-Hydrolysis derivatives of Neo-Clerodane diterpenes from Teucrium flavum subsp. glaucum as inhibitors of the HIV-1 reverse transcriptase-associated RNase H function.

Bioassay-guided fractionation of the ethyl acetate extract from Teucrium flavum subsp. glaucum, endowed with inhibitory activity towards the HIV-1 reverse transcriptase-associated RNase H function, led to the isolation of salvigenin (1), cirsimaritin (2) and cirsiliol (3) along with the neo-clerodanes teuflavin (4) and teuflavoside (5). Acid hydrolysis of the inactive teuflavoside provided three undescribed neo-clerodanes, flavuglaucins A-C (7-9) and one known neo-clerodane (10). Among all neo-clerodanes, flavuglaucin B showed the highest inhibitory activity towards RNase H function with a IC50 value of 9.1 µM. Molecular modelling and site-directed mutagenesis analysis suggested that flavuglaucin B binds into an allosteric pocket close to RNase H catalytic site. This is the first report of clerodane diterpenoids endowed with anti-reverse transcriptase activity. Neo-clerodanes represent a valid scaffold for the development of a new class of HIV-1 RNase H inhibitors.

Selective inhibition of carbonic anhydrase IX and XII by coumarin and psoralen derivatives.

A small library of coumarin and their psoralen analogues EMAC10157a-b-d-g and EMAC10160a-b-d-g has been designed and synthesised to investigate the effect of structural modifications on their inhibition ability and selectivity profile towards carbonic anhydrase isoforms I, II, IX, and XII. None of the new compounds exhibited activity towards hCA I and II isozymes. Conversely, both coumarin and psoralen derivatives were active against tumour associated isoforms IX and XII in the low micromolar or nanomolar range of concentration. These data further corroborate our previous findings on analogous derivatives, confirming that both coumarins and psoralens are interesting scaffolds for the design of isozyme selective hCA inhibitors.

2-(2-Methyl-2-nitrovinyl)furan but Not Furvina Interfere with Staphylococcus aureus Agr Quorum-Sensing System and Potentiate the Action of Fusidic Acid against Biofilms.

Quorum sensing (QS) plays an essential role in the production of virulence factors, in biofilm formation and antimicrobial resistance. Consequently, inhibiting QS is being considered a promising target for antipathogenic/anti-virulence therapies. This study aims to screen 2-nitrovinylfuran derivatives structurally related to Furvina (a broad-spectrum antibiotic already used for therapeutic purposes) for their effects on QS and in biofilm prevention/control. Furvina and four 2-nitrovinylfuran derivatives (compounds 1-4) were tested to assess the ability to interfere with QS of Staphylococcus aureus using bioreporter strains (S. aureus ALC1742 and ALC1743). The activity of Furvina and the most promising quorum-sensing inhibitor (QSI) was evaluated in biofilm prevention and in biofilm control (combined with fusidic acid). The biofilms were further characterized in terms of biofilm mass, viability and membrane integrity. Compound 2 caused the most significant QS inhibition with reductions between 60% and 80%. Molecular docking simulations indicate that this compound interacts preferentially with the protein hydrophobic cleft in the LytTR domain of AgrA pocket. Metabolic inactivations of 40% for S. aureus ALC1742 and 20% for S. aureus ALC1743 were reached. A 24 h-old biofilm formed in the presence of the QSI increased the metabolic inactivation by fusidic acid to 80%, for both strains. The overall results highlight the effects of compound 2 as well as the potential of combining QSI with in-use antibiotics for the management of skin and soft tissues infections.

Targeting HIV-1 RNase H: N'-(2-Hydroxy-benzylidene)-3,4,5-Trihydroxybenzoylhydrazone as Selective Inhibitor Active against NNRTIs-Resistant Variants.

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N'-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants.

New Dihydrothiazole Benzensulfonamides: Looking for Selectivity toward Carbonic Anhydrase Isoforms I, II, IX, and XII.

In the present study we investigated the structure-activity relationships of a new series of 4-[(3-ethyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10101a-m). All synthesized compounds, with the exception of compound EMAC10101k, preferentially inhibit off-target hCA II isoform. Within the series, compound EMAC10101d, bearing a 2,4-dichorophenyl substituent in position 4 of the dihydrothiazole ring, was the most potent and selective toward hCA II with an inhibitory activity in the low nanomolar range.

Quercetin Blocks Ebola Virus Infection by Counteracting the VP24 Interferon-Inhibitory Function.

Ebola virus (EBOV) is among the most devastating pathogens causing fatal hemorrhagic fever in humans. The epidemics from 2013 to 2016 resulted in more than 11,000 deaths, and another outbreak is currently ongoing. Since there is no FDA-approved drug so far to fight EBOV infection, there is an urgent need to focus on drug discovery. Considering the tight correlation between the high EBOV virulence and its ability to suppress the type I interferon (IFN-I) system, identifying molecules targeting viral protein VP24, one of the main virulence determinants blocking the IFN response, is a promising novel anti-EBOV therapy approach. Hence, in the effort to find novel EBOV inhibitors, a screening of a small set of flavonoids was performed; it showed that quercetin and wogonin can suppress the VP24 effect on IFN-I signaling inhibition. The mechanism of action of the most active compound, quercetin, showing a half-maximal inhibitory concentration (IC50) of 7.4 µM, was characterized to significantly restore the IFN-I signaling cascade, blocked by VP24, by directly interfering with the VP24 binding to karyopherin-α and thus restoring P-STAT1 nuclear transport and IFN gene transcription. Quercetin significantly blocked viral infection, specifically targeting EBOV VP24 anti-IFN-I function. Overall, quercetin is the first identified inhibitor of the EBOV VP24 anti-IFN function, representing a molecule interacting with a viral binding site that is very promising for further drug development aiming to block EBOV infection at the early steps.

Coumarins from Magydaris pastinacea as inhibitors of the tumour-associated carbonic anhydrases IX and XII: isolation, biological studies and in silico evaluation.

In an in vitro screening for human carbonic anhydrase (hCA) inhibiting agents from higher plants, the petroleum ether and ethyl acetate extracts of Magydaris pastinacea seeds selectively inhibited hCA IX and hCA XII isoforms. The phytochemical investigation of the extracts led to the isolation of ten linear furocoumarins (1-10), four simple coumarins (12-15) and a new angular dihydrofurocoumarin (11). The structures of the isolated compounds were elucidated based on 1 D and 2 D NMR, MS, and ECD data analysis. All isolated compounds were inactive towards the ubiquitous cytosolic isoform hCA I and II (Ki > 10,000 nM) while they were significantly active against the tumour-associated isoforms hCA IX and XII. Umbelliprenin was the most potent coumarin inhibiting hCA XII isoform with a Ki of 5.7 nM. The cytotoxicity of the most interesting compounds on HeLa cancer cells was also investigated.

Exploring new structural features of the 4-[(3-methyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzenesulphonamide scaffold for the inhibition of human carbonic anhydrases.

A library of 4-[(3-methyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulphonamides (EMAC8002a-m) was designed and synthesised to evaluate the effect of substituents in the positions 3 and 4 of the dihydrothiazole ring on the inhibitory potency and selectivity toward human carbonic anhydrase isoforms I, II, IX, and XII. Most of the new compounds preferentially inhibit the isoforms II and XII. Both electronic and steric features on the aryl substituent in the position 4 of the dihydrothiazole ring concur to determine the overall biological activity of these new derivatives.

Ferulic Acid Esters and Withanolides: In Search of Withania somnifera GABAA Receptor Modulators.

Nine compounds, including two undescribed withanolides, withasomniferolides A and B (1 and 2), three known withanolides (3-5), a ferulic acid dimeric ester (6), and an inseparable mixture of three long alkyl chain ferulic acid esters (7-9), were isolated from a GABAA receptor positive activator methanol extract of the roots of Withania somnifera. The structures of the isolated compounds were elucidated based on NMR, MS, and ECD data analysis. In order to bioassay the single ferulic acid derivatives, compounds 6-9 were also synthesized. The most active compound, docosanyl ferulate (9), was able to enhance the GABAA receptor inhibitory postsynaptic currents with an IC50 value of 7.9 µM. These results, by showing an ability to modulate the GABAA receptor function, cast fresh light on the biological activities of the secondary metabolites of W. somnifera roots.

Investigating the Anticancer Activity of Isatin/Dihydropyrazole Hybrids.

A series of isatin-dihydropyrazole hybrids have been synthesized in order to assess their potential as anticancer agents. In particular, 12 compounds were evaluated for their antiproliferative activity toward A549, IGR39, U87, MDA-MB-231, MCF-7, BT474, BxPC-3, SKOV-3, and H1299 cell lines, and human foreskin fibroblasts. Four compounds exhibited interesting antiproliferative activity and were further examined to determine their EC50 values toward a panel of selected tumor cell lines. The best compounds were then investigated for their induced mechanism of cell death. Preliminary structure-activity relationship indicates that the presence of a substituent such as a chlorine atom or a methyl moiety in position 5 of the isatin nucleus is beneficial for the antitumor activity. EMAC4001 proved the most promising compound within the studied series with EC50 values ranging from 0.01 to 0.38 µM.

Novel natural non-nucleoside inhibitors of HIV-1 reverse transcriptase identified by shape- and structure-based virtual screening techniques.

In this work we report a parallel application of both docking- and shape-based virtual screening (VS) methods, followed by Molecular Dynamics simulations (MDs), for discovering new compounds able to inhibit the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) RNA-dependent DNA polymerase activity. Specifically, we screened more than 143000 natural compounds commercially available in the ZINC database against the best five RT crystallographic models, taking into account the five approved NNRTIs as query compounds. As a result, 20 hit molecules were selected and tested on biochemical assays for the inhibition of the RNA dependent DNA polymerase RT function and, among them, an indoline pyrrolidine (hit1), an indonyl piperazine (hit2) and an indolyl indolinone (hit3) derivatives were identified as novel non-nucleoside RT inhibitors in the low micromolar range.

From cycloheptathiophene-3-carboxamide to oxazinone-based derivatives as allosteric HIV-1 ribonuclease H inhibitors.

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC50s of 0.53 and 2.90 µM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre.

Tuning the Dual Inhibition of Carbonic Anhydrase and Cyclooxygenase by Dihydrothiazole Benzensulfonamides.

A novel series of of 4-[(3-phenyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides (EMAC10111a-g) was synthesized and assayed toward both human carbonic anhydrase isozymes I, II, IX, and XII and cyclooxygenase isoforms. The majority of these derivatives preferentially inhibit hCA isoforms II and XII and hCOX-2 isozyme, indicating that 2,3,4-trisubstituted 2,3-dihydrothiazoles are a promising scaffold for the inhibition of hCA isozymes and of hCOX-2 enzyme. The nature of the substituent at the dihydrothiazole ring position 4 influenced the activity and selectivity toward both enzyme families. EMAC10111g resulted as the best performing compound toward both enzyme families and exhibited preferential activity toward hCA XII and hCOX-2 isozymes.

Identification of Myricetin as an Ebola Virus VP35-Double-Stranded RNA Interaction Inhibitor through a Novel Fluorescence-Based Assay.

Ebola virus (EBOV) is a filovirus that causes a severe and rapidly progressing hemorrhagic syndrome; a recent epidemic illustrated the urgent need for novel therapeutic agents because no drugs have been approved for treatment of Ebola virus. A key contribution to the high lethality observed during EBOV outbreaks comes from viral evasion of the host antiviral innate immune response in which viral protein VP35 plays a crucial role, blocking interferon type I production, first by masking the viral double-stranded RNA (dsRNA) and preventing its detection by the pattern recognition receptor RIG-I. Aiming to identify inhibitors of the interaction of VP35 with the viral dsRNA, counteracting the VP35 viral innate immune evasion, we established a new methodology for high-yield recombinant VP35 (rVP35) expression and purification and a novel and robust fluorescence-based rVP35-RNA interaction assay ( Z' factor of 0.69). Taking advantage of such newly established methods, we screened a small library of Sardinian natural extracts, identifying Limonium morisianum as the most potent inhibitor extract. A bioguided fractionation led to the identification of myricetin as the component that can inhibit rVP35-dsRNA interaction with an IC50 value of 2.7 µM. Molecular docking studies showed that myricetin interacts with the highly conserved region of the VP35 RNA binding domain, laying the basis for further structural optimization of potent inhibitors of VP35-dsRNA interaction.