Dissertation committee:
Αικατερίνη-Μαρία Παππά, Επίκουρη Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Νίκη Βασιλάκη, Εντεταλμένη Ερευνήτρια, Τμήμα Μικροβιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Πηνελόπη Μαυρομαρά, Καθηγήτρια, Τμήμα Μοριακής Βιολογίας και Γενετικής, Δημοκρίτειο Πανεπιστήμιο Θράκης
Παναγιώτης Μαράκος, Καθηγητής, Τμήμα Φαρμακευτικής, ΕΚΠΑ
Χαραλαμπία Μπολέτη, Κύρια Ερευνήτρια, Τμήμα Μικροβιολογίας, Ελληνικό Ινστιτούτο Παστέρ
Διδώ Βασιλακοπούλου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Κλεονίκη Λάμνησου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, ΕΚΠΑ
Summary:
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.
