Novel antiviral strategies against emerging viral diseases

Novel antiviral strategies against emerging viral diseases Doctoral Research School of Biochemistry and Molecular Biology-Cycle XXXII; Tutor: Luisa Bracci; Supervisor: Maurizio Zazzi; Candidate: Adele Boccuto Background Zika virus (ZIKV) Dengue virus (DENV) and West Nile virus (WNV) belong to the Flaviviridae family. The World Health Organization has ranked DENV as the most critical mosquito-borne viral disease and declared ZIKV an international public health emergency. Indeed, WNV is widely established in USA and some European areas and is associated to neuroinvasive disease. Despite intensive work, no specific antiviral therapy is available for ZIKV, DENV or WNV. In addition, increasing co-infections with different flaviviruses or serotypes cocirculating within the same area complicate the clinical outcome and treatment options. The high degree of conservation of some viral enzymes or host factor essential for Flaviviridae replication makes it reasonable to search for “broad-spectrum” antivirals. Materials and Methods The half-maximal cytotoxic concentration (CC50) of candidate inhibitors was calculated by Cell Titer-Glo cell viability assay. We determined the drug concentration inhibiting 50% of viral replication (IC50) of ZIKV, DENV and/or WNV by infection of Huh7 (hepatoma cell line) at a multiplicity of infection (MOI) of 0.005 (DENVZIKV) or 0.0025 (WNV) for 1 h and at the end of incubation treating Huh7 cells with serial dilutions of candidate compound at 37°C and 5% CO2. After 72 h (DENV-ZIKV) or 48h (WNV), the IC50 of compounds interfering with the virus life cycle up to protein production, but not at later steps, was directly measured by immune-detection assay (direct yield reduction assay, DYRA). Otherwise, to determine the impairment of viral replicative capacity exerted at late steps of viral replication, e.g. viral assembly and budding, we performed an additional replicative cycle or by infection of pre-seeded Huh7 (Secondary yield reduction assay, SYRA) with supernatants obtained by first round of infection or by Plaque reduction assay (PRA) as previously described (Vicenti et al., 2018). Selectivity indexes (SI) were calculated as ratio between CC50 and IC50. For in vitro ZIKV resistance selection to sofosbuvir, ZIKV was propagated in the Huh-7 cell line with increasing concentrations of sofosbuvir. Viral clones replicating at each drug increment step were collected and sequenced to detect emergent mutations in the polymerase (NS5) region and sofosbuvir IC50 was measured by DYRA. Results and discussion Evaluation of anti-DENV compounds targeting cellular DEAD-box 3 RNA helicase. Asp-Glu-Ala-Asp (DEAD)-box polypeptide 3 (DDX3) belongs to a family of ATP-dependent RNA helicases and is involved in many aspects of RNA metabolism as well as potentially participating to the antiviral innate immune signaling pathways. On the other hand, many studies highlight DDX3 as an essential host factor for the replication of clinically relevant viruses. As part of the activities planned in the UNAVIR and PANVIR project and in collaboration with First Health Pharmaceuticals, we evaluated the effect of DDX3 helicase inhibitors focusing on antiviral activity against DENV. Overall we tested 51 compounds, 31 showed anti-DENV activity and among these 11 molecules showed promising antiviral activity with median SI 66 (IQR 59-74). Overall, the preliminary data obtained so far support targeting DDX3 protein as a potentially effective strategy for inhibition of DENV replication and feasible broad-spectrum option to minimize the possibilities of generating resistance. The evaluation of the mechanism of action of a DDX3 inhibitor compound are currently under evaluation. Evaluation of candidate antivirals agents targeting viral entry/fusion. Virus entry by enveloped viruses is dependent not only on cellular receptors but also on cellular lipids playing multifaceted roles in viral infections as structural components and as necessary cofactors for viral replication. In this scenario, the possibility to identify broad-spectrum antiviral molecules acting on viral envelopes is of particular interest. We tested derivatives entry inhibitors of the previously identified MAS family (Cagno et al., 2018; Tintori et al., 2018) expected to affect virus-cell membrane fusion during DENV and ZIKV infection. We evaluated the antiviral activity of MAS compounds adapting DYRA and incubating Huh7 cells with serial dilution of MAS molecules for 30 minutes before infection with ZIKV or DENV. Of the ten compounds investigated, five showed antiviral activity both against ZIKV (median SI 16 µM; IQR 12-26) and DENV (median SI 7; IQR 6-14), 3 showed antiviral activity against ZIKV in DYRA (median SI 8; IQR 6.5-17) and anti-DENV activity in SYRA (median SI 11; IQR 9-14), while 2 compounds were completely inactive . Interestingly, the three compound active only against ZIKV were found active also against DENV in SYRA (median SI 11; IQR 9-14). We further evaluated the antiviral activity of MAS9 showing IC50 values of 2.1±1.6 (SI 14) and 2.6±2.2 (SI 12) for DENV and ZIKV, respectively. The evaluation of the mechanism of action confirmed the antiviral activity of MAS9 in viral entry/fusion step of viral replication and through a higher selectivity for viral membranes. These preliminary results support the strategy to target selectively the viral membrane, possibly based on the efficiency of the repair activity of host cell, but not virus, membranes. This class of compounds warrants further investigations to prove and characterize the mechanism of action and the ability to inhibit different emerging enveloped viruses. Evaluation of candidate anti-flaviviral agents targeting the NS3-NS5 interaction. A family of 2,6-diaminopurine derivatives targeting a conserved allosteric pocket on DENV/ZIKV NS5 polymerase, which is required to bind NS3 and generate the functional replication complex, has been recently discovered (Vincettiet al., 2019, 2015). Herein, we analysed a new series of derivatives molecules structurally modified to increase the affinity for the allosteric pocket on NS5 and reduce their cellular toxicity. Of the 21 derivatives compounds investigated against ZIKV and DENV, 5 were cytotoxic, 16 showed anti-ZIKV activity (median SI 9 [IQR 7-26.5]) and 13 showed anti-DENV activity (median SI 11 [IQR 6-13]) in SYRA. The best SI was obtained for compound MR333 with SI of 182 and 77 for ZIKV and DENV, respectively. Moreover, MR333 was found to be active also against WNV with comparable antiviral activity in PRA and SI of 55. This class of compounds appears to be attractive and warrants further evaluation of the mechanism of action and the genetic barrier to the emergence of resistance mutations. Biochemical assays investigating MR333 are underway to confirm and characterize the expected inhibition of multiple viruses based on binding to the highly conserved pocket on the thumb of Flavivirus polymerases. Characterization of in vitro ZIKV resistance to sofosbuvir. Given the high degree of NS5 homology observed among members of the Flaviviridae family (Boldescu et al., 2017; Lim et al., 2013), sofosbuvir has been recently evaluated as an anti-Flavivirus lead candidate. Indeed, the anti-ZIKV activity of sofosbuvir has been showed in vitro with cell-based assay and in animal models. However, sofosbuvir drug genetic barrier has been not yet evaluated with ZIKV. The virus breakthrough time grew with increasing sofosbuvir concentration of 5, 10, 20 and 40 µM (5, 15, 22 and 40±9 days, respectively) with exception of 80 µM (20±2 days). No sequence variations were found up to 20 µM sofosbuvir, while the mutations V360L and V607I were selected in presence of 40 µM sofosbuvir and 80 µM. The mutations C269Y and H289T were individually selected in two experiments with 80 µM sofosbuvir. Globally, majority of mutations observed in Sanger sequencing were confirmed by NGS with addition of N407T, T833I, K834C and W835R mutations which were not associated to sofosbuvir drugpressure. By DYRA we demonstrated the reduced susceptibility to sofosbuvir of viruses carrying the C269Y, V360L and V607I mutations. Moreover, the analysis of NS5 gene showed that the V607I is located near the well-known mutation S282T in SGxxxT consensus sequence of HCV NS5B, within the motif B, and is implicated in drug resistance of HCV toward nucleotide inhibitors (Gane et al., 2016). Interestingly, the V360L mutation is located in a well-known stretch of amino acids termed β-nuclear localization sequence (positions 320-368), whereas, the C269Y is located in the poorly conserved 10-residue linker domain (aminoacidic positions 263 to 272) which is essential for the adequate NS5 conformation. Therefore, the mutations detected are probably related to emergence of ZIKV resistance to sofosbuvir in vitro and, certainly, require further biochemical investigations. References Boldescu, V., Behnam, M.A.M., Vasilakis, N., Klein, C.D., 2017. Broad-spectrum agents for flaviviral infections: Dengue, Zika and beyond. Nat. Rev. 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