Design and Synthesis of Novel Bis-Imidazolyl Phenyl Butadiyne Derivatives as HCV NS5A Inhibitors

In today’s global plan to completely eradicate hepatitis C virus (HCV), the essential list of medications used for HCV treatment are direct-acting antivirals (DAAs), as interferon-sparing regimens have become the standard-of-care (SOC) treatment. HCV nonstructural protein 5A (NS5A) inhibitors are a very common component of these regimens. Food and Drug Administration (FDA)- approved NS5A inhibitors, although very potent, do not have the same potency against all eight genotypes of HCV. Therefore, this study aims to synthesize NS5A inhibitor analogues with high potency pan-genotypic activity and high metabolic stability. Starting from an NS5A inhibitor scaffold previously identified by our research group, we made several modifications. Two series of compounds were created to test the effect of changing the length and spatial conformation (para-para vs. meta-metapositioned bis-imidazole-proline-carbamate), replacing amide groups in the linker with imidazole groups, as well as different end-cap compositions and sizes. The frontrunner inhibits genotype 1b (Con1) replicon, with an EC50 value in the picomolar range, and showed high genotypic coverage with nanomolar range EC50 values against four more genotypes. This together with its high metabolic stability (t1 ⁄2 > 120 min) makes it a potential preclinical candidate

Scaffold hybridization strategy towards potent hydroxamate-based inhibitors of Flaviviridae viruses and Trypanosoma species.

Infections with Flaviviridae viruses, such as hepatitis C virus (HCV) and dengue virus (DENV) pose global health threats. Infected individuals are at risk of developing chronic liver failure or haemorrhagic fever respectively, often with a fatal outcome if left untreated. Diseases caused by tropical parasites of the Trypanosoma species, T. brucei and T. cruzi, constitute significant socioeconomic burden in sub-Saharan Africa and continental Latin America, yet drug development is under-funded. Anti-HCV chemotherapy is associated with severe side effects and high cost, while dengue has no clinically approved therapy and antiparasitic drugs are outdated and difficult to administer. Moreover, drug resistance is an emerging concern. Consequently, the need for new revolutionary chemotherapies is urgent. By utilizing a molecular framework combination approach, we combined two distinct chemical entities with proven antiviral and trypanocidal activity into a novel hybrid scaffold attached by an acetohydroxamic acid group (CH2CONHOH), aiming at derivatives with dual activity. The novel spiro-carbocyclic substituted hydantoin analogues were rationally designed, synthesized and evaluated for their potency against three HCV genotypes (1b, 3a, 4a), DENV and two Trypanosoma species (T. brucei, T. cruzi). They exhibited significant EC50 values and remarkable selectivity indices. Several modifications were undertaken to further explore the structure activity relationships (SARs) and confirm the pivotal role of the acetohydroxamic acid metal binding group

Evaluation of new inhibitors against Hepatitis C virus and related viruses, as well as of therapeutic biomarkers

Despite the effective treatments against Hepatitis C virus (HCV), problems still exist, such as high treatment costs, the emergence of resistant viral strains, and the co-infections with Hepatitis B Virus (HBV) and the related to HCV Dengue (DENV), Yellow Fever (YFV) and Zika (ZIKV) viruses. Moreover, the cellular mechanisms that regulate viral replication are not fully understood. Therefore, in this project a) new inhibitors against viruses of Flaviviridae family (HCV, DENV, YFV, ZIKV) and the molecular mechanisms of action were studied, and b) the role of cellular factors in viral replication and pathogenesis was investigated, to highlight new biomarkers and therapeutic targets against Flaviviridae viruses, by focusing on the signaling pathways of hypoxia, PI3K/AKT and a PI3K-binding factor, L-Dopa decarboxylase (DDC). Concerning the first objective, we tested against Flaviviridae viruses: 1) plant extracts and isolated compounds, 2) nucleoside and peptidomimetic synthetic analogues based on approved anti-HCV drugs, and 3) analogues of N-hydroxyimide and acetohydroxamic acid, which chelate divalent metal ions in the active site of viral enzymes. Specifically, we found compounds broadly active against HCV, DENV and YFV, while the flavonoid 2'-hydroxygensitein-7-O-glucopyranoside was effective against DENV (median effective concentration-EC50=6.38 μM). Nucleoside analogue 15d inhibited HCV 1b (EC50=40.22 μM), with high barrier to resistance. The resistance mutations suggest that it inhibits the interactions of NS5A. The N-hydroxyimide ZF66 had EC50=3.08 μM against HCV 1b, while exhibiting a high barrier to resistance, and possibly chelates the Mg2+ ions in the active site of NS5B polymerase. Finally, acetohydroxamic acid analogues bearing an adamantane group as a substituent, have broad activity against HCV, DENV, YFV and ZIKV), with ZB5 (HCV 1b: EC50=0.08 μM) and ZF64 (DENV: EC50=0.07 μM) being the most active. They also showed a high barrier to resistance and possibly target viral NS3 protein. Concerning the second objective, hypoxia (3% v/v O2) increased DENV RNA replication, through factors that activate under low oxygen, specifically HIF and AKT, but not PI3K. In addition, hypoxia reduced the activity of HCV and DENV inhibitors. Inversely, DENV induces HIF under atmospheric oxygen, causing a metabolic reprogramming in the host cell that favors viral proliferation. In addition, DENV and HCV infection negatively affect the expression of DDC, while DDC negatively affects viral replication. The negative correlation between HCV RNA and DDC mRNA was confirmed in liver biopsies from patients with chronic HCV infection. Overall, broadly effective inhibitors targeting Flaviviridae viruses were detected, which could be developed as antivirals, or against co-infections and emerging viruses. In addition, factors regulated by hypoxia and PI3K/AKT pathways were found to play an important role in the replication of Flaviviridae viruses, such as HIF (DENV) and DDC (HCV, DENV), which could be used as antiviral targets, or biomarkers of disease progression.Παρά τις αποτελεσματικές θεραπείες έναντι του ιού της Ηπατίτιδας C (HCV), παραμένουν ακόμη προβλήματα, όπως το υψηλό κόστος, η εμφάνιση ανθεκτικότητας και οι συμμολύνσεις με τον ιό της Ηπατίτιδας Β (HBV) και τους συγγενικούς στον HCV ιοί Δάγκειος (DENV), Κίτρινου πυρετού (YFV) και Ζίκα (ZIKV). Ακόμη, είναι πολλά άγνωστα για τους κυτταρικούς μηχανισμούς ρύθμισης της ιικής αντιγραφής. Επομένως, στα πλαίσια της εργασίας αυτής μελετήθηκαν α) νέοι αναστολείς έναντι ιών της οικογένειας Flaviviridae (HCV, DENV, YFV, ZIKV) και ο μηχανισμός δράσης τους, και β) η σημασία κυτταρικών παραγόντων στην ιική αντιγραφή και παθογένεια για την ανάδειξη νέων βιοδεικτών και θεραπευτικών στόχων έναντι των ιών Flaviviridae, εστιάζοντας στα σηματοδοτικά μονοπάτια υποξίας, PI3K/ΑΚΤ και στην αποκαρβοξυλάση της L-Dopa (DDC) η οποία εντοπίσαμε να προσδένεται στην PI3K. Για τον πρώτο στόχο, ελέγχθηκαν έναντι των ιών Flaviviridae: 1) φυτικά εκχυλίσματα και απομονωμένες ενώσεις, 2) νουκλεοσιδικά και πεπτιδομιμητικά συνθετικά ανάλογα, βασισμένα σε εγκεκριμένους αντι-HCV αναστολείς, και 3) ανάλογα Ν-υδροξυϊμιδίου και ακετοϋδροξαμικού οξέος, που δεσμεύουν χηλικά δισθενή μεταλλοϊόντα στο ενεργό κέντρο ιικών ενζύμων. Συγκεκριμένα, βρέθηκαν φυτικές ενώσεις με ευρεία αντι-ιική δράση, ενώ το φλαβονοειδές 2’-υδροξυγενιστεΐνη-7-O-γλυκοπυρανοσίδιο, ήταν αποτελεσματικό έναντι του DENV (μέση δραστική συγκέντρωση-EC50=6,38 μΜ. Το νουκλεοσιδικό ανάλογο 15d ανέστειλε τον HCV 1b (EC50=40,22 μΜ), με υψηλό φραγμό στην ανάπτυξη ανθεκτικότητας. Οι μεταλλαγές ανθεκτικότητας υποδεικνύουν πως αναστέλλει αλληλεπιδράσεις της NS5A. Το Ν-υδροξυμίδιo ZF66 είχε EC50=3,08 μΜ έναντι του HCV 1b, με υψηλό φραγμό στην ανάπτυξη ανθεκτικότητας, και πιθανά προσδένεται χηλικά στο ενεργό κέντρο της NS5B πολυμεράσης. Τέλος, τα ακετοϋδροξαμικά ανάλογα με υποκαταστάτη αδαμαντάνη, έχουν ευρεία δράση έναντι των HCV, DENV, YFV και ZIKV, με δραστικότερα τα ΖΒ5 (HCV 1b: EC50=0,08 μΜ) και ΖΡ64 (DENV: EC50=0,07 μΜ), παρουσιάζουν υψηλό φραγμό στην ανάπτυξη ανθεκτικότητας και πιθανόν στοχεύουν την ιική πρωτεΐνη NS3. Για τον δεύτερο στόχο, δείχθηκε πως η υποξία (3% v/v Ο2) αυξάνει την RNA αντιγραφή του DENV, μέσω παραγόντων που ενεργοποιούνται στο χαμηλό οξυγόνο, όπως oι ΗΙF και AKT, αλλά όχι η PI3K. Επιπλέον, μειώνει τη δράση αναστολέων των HCV και DENV. Αντίστροφα, ο DENV επάγει τον HIF στο ατμοσφαιρικό οξυγόνο, προκαλώντας προς ώφελός του έναν μεταβολικό επαναπρογραμματισμό στο κύτταρο-ξενιστή. Επιπλέον, η μόλυνση από DENV και HCV επηρεάζει αρνητικά την έκφραση της DDC, ενώ η DDC επηρεάζει αρνητικά την ιική αντιγραφή. H αρνητική συσχέτιση μεταξύ HCV RNA και mRNA της DDC επιβεβαιώθηκε σε ηπατικές βιοψίες ασθενών με χρόνια HCV μόλυνση. Συνολικά, βρέθηκαν αναστολείς με ευρεία δράση έναντι των ιών Flaviviridae, που θα μπορούσαν να αξιοποιηθούν ως αντι-ιικά φάρμακα, καθώς και για την αντιμετώπιση συλλοιμώξεων ή αναδυόμενων ιών. Επιπλέον, βρέθηκαν παράγοντες που ρυθμίζονται από τα μονοπάτια υποξίας και PI3K/AKT και παίζουν σημαντικό ρόλο στην αντιγραφή των ιών Flaviviridae, όπως οι HIF (DENV) και DDC (HCV, DENV), που θα μπορούσαν να χρησιμοποιηθούν ως αντι-ιικοί στόχοι, ή βιοδείκτες της εξέλιξης της νόσου

Αξιολόγηση νέων αναστολέων του ιού της Ηπατίτιδας C και συγγενικών ιών, καθώς και βιοδεικτών απόκρισης σε θεραπευτικές προσεγγίσεις

Παρά τις αποτελεσματικές θεραπείες έναντι του ιού της Ηπατίτιδας C (HCV), παραμένουν ακόμη προβλήματα, όπως το υψηλό κόστος, η εμφάνιση ανθεκτικότητας και οι συμμολύνσεις με τον ιό της Ηπατίτιδας Β (HBV) και τους συγγενικούς στον HCV ιοί Δάγκειος (DENV), Κίτρινου πυρετού (YFV) και Ζίκα (ZIKV). Ακόμη, είναι πολλά άγνωστα για τους κυτταρικούς μηχανισμούς ρύθμισης της ιικής αντιγραφής. Επομένως, στα πλαίσια της εργασίας αυτής μελετήθηκαν α) νέοι αναστολείς έναντι ιών της οικογένειας Flaviviridae (HCV, DENV, YFV, ZIKV) και ο μηχανισμός δράσης τους, και β) η σημασία κυτταρικών παραγόντων στην ιική αντιγραφή και παθογένεια για την ανάδειξη νέων βιοδεικτών και θεραπευτικών στόχων έναντι των ιών Flaviviridae, εστιάζοντας στα σηματοδοτικά μονοπάτια υποξίας, PI3K/ΑΚΤ και στην αποκαρβοξυλάση της L-Dopa (DDC) η οποία εντοπίσαμε να προσδένεται στην PI3K. Για τον πρώτο στόχο, ελέγχθηκαν έναντι των ιών Flaviviridae: 1) φυτικά εκχυλίσματα και απομονωμένες ενώσεις, 2) νουκλεοσιδικά και πεπτιδομιμητικά συνθετικά ανάλογα, βασισμένα σε εγκεκριμένους αντι-HCV αναστολείς, και 3) ανάλογα Ν-υδροξυϊμιδίου και ακετοϋδροξαμικού οξέος, που δεσμεύουν χηλικά δισθενή μεταλλοϊόντα στο ενεργό κέντρο ιικών ενζύμων. Συγκεκριμένα, βρέθηκαν φυτικές ενώσεις με ευρεία αντι-ιική δράση, ενώ το φλαβονοειδές 2’-υδροξυγενιστεΐνη-7-O-γλυκοπυρανοσίδιο, ήταν αποτελεσματικό έναντι του DENV (μέση δραστική συγκέντρωση-EC50=6,38 μΜ. Το νουκλεοσιδικό ανάλογο 15d ανέστειλε τον HCV 1b (EC50=40,22 μΜ), με υψηλό φραγμό στην ανάπτυξη ανθεκτικότητας. Οι μεταλλαγές ανθεκτικότητας υποδεικνύουν πως αναστέλλει αλληλεπιδράσεις της NS5A. Το Ν-υδροξυμίδιo ZF66 είχε EC50=3,08 μΜ έναντι του HCV 1b, με υψηλό φραγμό στην ανάπτυξη ανθεκτικότητας, και πιθανά προσδένεται χηλικά στο ενεργό κέντρο της NS5B πολυμεράσης. Τέλος, τα ακετοϋδροξαμικά ανάλογα με υποκαταστάτη αδαμαντάνη, έχουν ευρεία δράση έναντι των HCV, DENV, YFV και ZIKV, με δραστικότερα τα ΖΒ5 (HCV 1b: EC50=0,08 μΜ) και ΖΡ64 (DENV: EC50=0,07 μΜ), παρουσιάζουν υψηλό φραγμό στην ανάπτυξη ανθεκτικότητας και πιθανόν στοχεύουν την ιική πρωτεΐνη NS3. Για τον δεύτερο στόχο, δείχθηκε πως η υποξία (3% v/v Ο2) αυξάνει την RNA αντιγραφή του DENV, μέσω παραγόντων που ενεργοποιούνται στο χαμηλό οξυγόνο, όπως oι ΗΙF και AKT, αλλά όχι η PI3K. Επιπλέον, μειώνει τη δράση αναστολέων των HCV και DENV. Αντίστροφα, ο DENV επάγει τον HIF στο ατμοσφαιρικό οξυγόνο, προκαλώντας προς ώφελός του έναν μεταβολικό επαναπρογραμματισμό στο κύτταρο-ξενιστή. Επιπλέον, η μόλυνση από DENV και HCV επηρεάζει αρνητικά την έκφραση της DDC, ενώ η DDC επηρεάζει αρνητικά την ιική αντιγραφή. H αρνητική συσχέτιση μεταξύ HCV RNA και mRNA της DDC επιβεβαιώθηκε σε ηπατικές βιοψίες ασθενών με χρόνια HCV μόλυνση. Συνολικά, βρέθηκαν αναστολείς με ευρεία δράση έναντι των ιών Flaviviridae, που θα μπορούσαν να αξιοποιηθούν ως αντι-ιικά φάρμακα, καθώς και για την αντιμετώπιση συλλοιμώξεων ή αναδυόμενων ιών. Επιπλέον, βρέθηκαν παράγοντες που ρυθμίζονται από τα μονοπάτια υποξίας και PI3K/AKT και παίζουν σημαντικό ρόλο στην αντιγραφή των ιών Flaviviridae, όπως οι HIF (DENV) και DDC (HCV, DENV), που θα μπορούσαν να χρησιμοποιηθούν ως αντι-ιικοί στόχοι, ή βιοδείκτες της εξέλιξης της νόσου.Despite the effective treatments against Hepatitis C virus (HCV), problems still exist, such as high treatment costs, the emergence of resistant viral strains, and the co-infections with Hepatitis B Virus (HBV) and the related to HCV Dengue (DENV), Yellow Fever (YFV) and Zika (ZIKV) viruses. Moreover, the cellular mechanisms that regulate viral replication are not fully understood. Therefore, in this project a) new inhibitors against viruses of Flaviviridae family (HCV, DENV, YFV, ZIKV) and the molecular mechanisms of action were studied, and b) the role of cellular factors in viral replication and pathogenesis was investigated, to highlight new biomarkers and therapeutic targets against Flaviviridae viruses, by focusing on the signaling pathways of hypoxia, PI3K/AKT and a PI3K-binding factor, L-Dopa decarboxylase (DDC). Concerning the first objective, we tested against Flaviviridae viruses: 1) plant extracts and isolated compounds, 2) nucleoside and peptidomimetic synthetic analogues based on approved anti-HCV drugs, and 3) analogues of N-hydroxyimide and acetohydroxamic acid, which chelate divalent metal ions in the active site of viral enzymes. Specifically, we found compounds broadly active against HCV, DENV and YFV, while the flavonoid 2'-hydroxygensitein-7-O-glucopyranoside was effective against DENV (median effective concentration-EC50=6.38 μM). Nucleoside analogue 15d inhibited HCV 1b (EC50=40.22 μM), with high barrier to resistance. The resistance mutations suggest that it inhibits the interactions of NS5A. The N-hydroxyimide ZF66 had EC50=3.08 μM against HCV 1b, while exhibiting a high barrier to resistance, and possibly chelates the Mg2+ ions in the active site of NS5B polymerase. Finally, acetohydroxamic acid analogues bearing an adamantane group as a substituent, have broad activity against HCV, DENV, YFV and ZIKV), with ZB5 (HCV 1b: EC50=0.08 μM) and ZF64 (DENV: EC50=0.07 μM) being the most active. They also showed a high barrier to resistance and possibly target viral NS3 protein. Concerning the second objective, hypoxia (3% v/v O2) increased DENV RNA replication, through factors that activate under low oxygen, specifically HIF and AKT, but not PI3K. In addition, hypoxia reduced the activity of HCV and DENV inhibitors. Inversely, DENV induces HIF under atmospheric oxygen, causing a metabolic reprogramming in the host cell that favors viral proliferation. In addition, DENV and HCV infection negatively affect the expression of DDC, while DDC negatively affects viral replication. The negative correlation between HCV RNA and DDC mRNA was confirmed in liver biopsies from patients with chronic HCV infection. Overall, broadly effective inhibitors targeting Flaviviridae viruses were detected, which could be developed as antivirals, or against co-infections and emerging viruses. In addition, factors regulated by hypoxia and PI3K/AKT pathways were found to play an important role in the replication of Flaviviridae viruses, such as HIF (DENV) and DDC (HCV, DENV), which could be used as antiviral targets, or biomarkers of disease progression

A Novel Cis-Acting RNA Structural Element Embedded in the Core Coding Region of the Hepatitis C Virus Genome Directs Internal Translation Initiation of the Overlapping Core+1 ORF

Hepatitis C virus (HCV) genome translation is initiated via an internal ribosome entry site (IRES) embedded in the 5′-untranslated region (5′UTR). We have earlier shown that the conserved RNA stem-loops (SL) SL47 and SL87 of the HCV core-encoding region are important for viral genome translation in cell culture and in vivo. Moreover, we have reported that an open reading frame overlapping the core gene in the +1 frame (core+1 ORF) encodes alternative translation products, including a protein initiated at the internal AUG codons 85/87 of this frame (nt 597–599 and 603–605), downstream of SL87, which is designated core+1/Short (core+1/S). Here, we provide evidence for SL47 and SL87 possessing a novel cis-acting element that directs the internal translation initiation of core+1/S. Firstly, using a bicistronic dual luciferase reporter system and RNA-transfection experiments, we found that nucleotides 344–596 of the HCV genotype-1a and -2a genomes support translation initiation at the core+1 frame AUG codons 85/87, when present in the sense but not the opposite orientation. Secondly, site-directed mutagenesis combined with an analysis of ribosome–HCV RNA association elucidated that SL47 and SL87 are essential for this alternative translation mechanism. Finally, experiments using cells transfected with JFH1 replicons or infected with virus-like particles showed that core+1/S expression is independent from the 5′UTR IRES and does not utilize the polyprotein initiation codon, but it requires intact SL47 and SL87 structures. Thus, SL47 and SL87, apart from their role in viral polyprotein translation, are necessary elements for mediating the internal translation initiation of the alternative core+1/S ORF

Emerging Role of l-Dopa Decarboxylase in Flaviviridae Virus Infections

l-dopa decarboxylase (DDC) that catalyzes the biosynthesis of bioactive amines, such as dopamine and serotonin, is expressed in the nervous system and peripheral tissues, including the liver, where its physiological role remains unknown. Recently, we reported a physical and functional interaction of DDC with the major signaling regulator phosphoinosite-3-kinase (PI3K). Here, we provide compelling evidence for the involvement of DDC in viral infections. Studying dengue (DENV) and hepatitis C (HCV) virus infection in hepatocytes and HCV replication in liver samples of infected patients, we observed a negative association between DDC and viral replication. Specifically, replication of both viruses reduced the levels of DDC mRNA and the ~120 kDa SDS-resistant DDC immunoreactive functional complex, concomitant with a PI3K-dependent accumulation of the ~50 kDa DDC monomer. Moreover, viral infection inhibited PI3K-DDC association, while DDC did not colocalize with viral replication sites. DDC overexpression suppressed DENV and HCV RNA replication, while DDC enzymatic inhibition enhanced viral replication and infectivity and affected DENV-induced cell death. Consistently, we observed an inverse correlation between DDC mRNA and HCV RNA levels in liver biopsies from chronically infected patients. These data reveal a novel relationship between DDC and Flaviviridae replication cycle and the role of PI3K in this process

New Affordable Methods for Large-Scale Isolation of Major Olive Secoiridoids and Systematic Comparative Study of Their Antiproliferative/Cytotoxic Effect on Multiple Cancer Cell Lines of Different Cancer Origins

Olive oil phenols (OOPs) are associated with the prevention of many human cancers. Some of these have been shown to inhibit cell proliferation and induce apoptosis. However, no systematic comparative study exists for all the investigated compounds under the same conditions, due to difficulties in their isolation or synthesis. Herein are presented innovative methods for large-scale selective extraction of six major secoiridoids from olive oil or leaves enabling their detailed investigation. The cytotoxic/antiproliferative bioactivity of these six compounds was evaluated on sixteen human cancer cell lines originating from eight different tissues. Cell viability with half-maximal effective concentrations (EC50) was evaluated after 72 h treatments. Antiproliferative and pro-apoptotic effects were also assessed for the most bioactive compounds (EC50 ≤ 50 μM). Oleocanthal (1) showed the strongest antiproliferative/cytotoxic activity in most cancer cell lines (EC50: 9–20 μM). The relative effectiveness of the six OOPs was: oleocanthal (1) > oleuropein aglycone (3a,b) > ligstroside aglycone (4a,b) > oleacein (2) > oleomissional (6a,b,c) > oleocanthalic acid (7). This is the first detailed study comparing the bioactivity of six OOPs in such a wide array of cancer cell lines, providing a reference for their relative antiproliferative/cytotoxic effect in the investigated cancers

New Affordable Methods for Large-Scale Isolation of Major Olive Secoiridoids and Systematic Comparative Study of Their Antiproliferative/Cytotoxic Effect on Multiple Cancer Cell Lines of Different Cancer Origins

Olive oil phenols (OOPs) are associated with the prevention of many human cancers. Some of these have been shown to inhibit cell proliferation and induce apoptosis. However, no systematic comparative study exists for all the investigated compounds under the same conditions, due to difficulties in their isolation or synthesis. Herein are presented innovative methods for large-scale selective extraction of six major secoiridoids from olive oil or leaves enabling their detailed investigation. The cytotoxic/antiproliferative bioactivity of these six compounds was evaluated on sixteen human cancer cell lines originating from eight different tissues. Cell viability with half-maximal effective concentrations (EC50) was evaluated after 72 h treatments. Antiproliferative and pro-apoptotic effects were also assessed for the most bioactive compounds (EC50 ≤ 50 μM). Oleocanthal (1) showed the strongest antiproliferative/cytotoxic activity in most cancer cell lines (EC50: 9–20 μM). The relative effectiveness of the six OOPs was: oleocanthal (1) > oleuropein aglycone (3a,b) > ligstroside aglycone (4a,b) > oleacein (2) > oleomissional (6a,b,c) > oleocanthalic acid (7). This is the first detailed study comparing the bioactivity of six OOPs in such a wide array of cancer cell lines, providing a reference for their relative antiproliferative/cytotoxic effect in the investigated cancers

Design and Synthesis of Novel Bis-Imidazolyl Phenyl Butadiyne Derivatives as HCV NS5A Inhibitors

In today’s global plan to completely eradicate hepatitis C virus (HCV), the essential list of medications used for HCV treatment are direct-acting antivirals (DAAs), as interferon-sparing regimens have become the standard-of-care (SOC) treatment. HCV nonstructural protein 5A (NS5A) inhibitors are a very common component of these regimens. Food and Drug Administration (FDA)-approved NS5A inhibitors, although very potent, do not have the same potency against all eight genotypes of HCV. Therefore, this study aims to synthesize NS5A inhibitor analogues with high potency pan-genotypic activity and high metabolic stability. Starting from an NS5A inhibitor scaffold previously identified by our research group, we made several modifications. Two series of compounds were created to test the effect of changing the length and spatial conformation (para-para vs. meta-meta-positioned bis-imidazole-proline-carbamate), replacing amide groups in the linker with imidazole groups, as well as different end-cap compositions and sizes. The frontrunner inhibits genotype 1b (Con1) replicon, with an EC50 value in the picomolar range, and showed high genotypic coverage with nanomolar range EC50 values against four more genotypes. This together with its high metabolic stability (t½ > 120 min) makes it a potential preclinical candidate

Dengue Virus Replication Is Associated with Catecholamine Biosynthesis and Metabolism in Hepatocytes

Previously, the association between the catecholamine biosynthetic enzyme L-Dopa decarboxylase (DDC) and Dengue virus (DV) replication was demonstrated in liver cells and was found to be mediated at least by the interaction between DDC and phosphoinositide 3-kinase (PI3K). Here, we show that biogenic amines production and uptake impede DV replication in hepatocytes and monocytes, while the virus reduces catecholamine biosynthesis, metabolism, and transport. To examine how catecholamine biosynthesis/metabolism influences DV, first, we verified the role of DDC by altering DDC expression. DDC silencing enhanced virus replication, but not translation, attenuated the negative effect of DDC substrates on the virus and reduced the infection related cell death. Then, the role of the downstream steps of the catecholamine biosynthesis/metabolism was analyzed by chemical inhibition of the respective enzymes, application of their substrates and/or their products; moreover, reserpine, the inhibitor of the vesicular monoamine transporter 2 (VMAT2), was used to examine the role of uptake/storage of catecholamines on DV. Apart from the role of each enzyme/transporter, these studies revealed that the dopamine uptake, and not the dopamine-signaling, is responsible for the negative effect on DV. Accordingly, all treatments expected to enhance the accumulation of catecholamines in the cell cytosol suppressed DV replication. This was verified by the use of chemical inducers of catecholamine biosynthesis. Last, the cellular redox alterations due to catecholamine oxidation were not related with the inhibition of DV replication. In turn, DV apart from its negative impact on DDC, inhibits tyrosine hydroxylase, dopamine beta-hydroxylase, monoamine oxidase, and VMAT2 expression