Discovery, study of the antiviral properties and mechanism of action of novel inhibitors of the infection of Flavi- and Alphaviruses

There is an urgent need for potent drugs for the treatment and/or prophylaxis of infections with flaviviruses (such as the dengue fever virus and the yellow fever virus) and alphaviruses (in particular the chikungunya virus). The aim of this thesis was to identify novel inhibitors of these viruses, to study their particular characteristics and mechanism of action; thereby contributing to the development of (novel) antiviral strategies against these viruses. An analogue of the antibiotic doxorubicin, designated SA-17, was identified as a selective inhibitor of invitro dengue virus (DENV) serotype 2 infection. SA-17 is markedly more potent in inhibiting viral infection as well as markedly less cytostatic than its parent compound. The molecule inhibits also the infection of the yellow fever virus 17D (YFV-17D) although less efficiently than DENV infection, but proved inactive against a variety of other enveloped and non-enveloped viruses. In addition, an aglycon analogue of yet another antibiotic, i.e. teicoplanin, designated LCTA-949, was also identified as an inhibitor of flavivirus infection. LCTA-949 inhibits DENV-induced cytopathic effect (CPE) in a dose-dependent manner. Antiviral activity was also observed against other flaviviruses including the YFV and inparticular potent activity was observed against the tick-borne encephalitis virus. T ime-of-drug-addition studies revealed that SA-17 and LCTA-949 act at the very early stages of the viral life cycle (i.e., virus attachment and/or virus entry). This was corroborated by the observationthat SA-17 and LCTA-949, unlike the nucleoside analogue ribavirin, do not inhibit the replication of DENV subgenomic replicons. Docking experiments revealed the capacity of SA-17 to bind in the β-OG pocket of the DENV E protein. Binding to this pocket would inhibit the fusion process that is necessary for the release of the viral RNA into the cytoplasm of the host cell. In addition, using a microscopy-based binding and fusion assay employing DiD-labeled viruses, it was shown that LCTA-949 targets the early stage (binding/entry) of the infection. Neither SA-17, nor LCTA-949 are virucidal. SA-17 was shown to completely annihilate infectivity of a high titered stock of virus, when preincbated for 30 min with the virus. LCTA-949 also efficiently inhibits infectivity of DENV particles pre-opsonized with antibodies, suggesting that such molecules may be effective during infections in which DENV enters the cell via an antibody mediated event [antibody-dependent enhancement (ADE)]. Thus, SA-17 and LCTA-949 exert in vitro activity against several flaviviruses and do so (as shown for DENV) by interfering with an early step in theviral life cycle. We wanted to further explore the β-OG pocket as a target for inhibition of viral infection. Small molecules were designed in silico to fit in the β-OG pocket of the E protein based on the crystal structure of this protein. A total of 60 structurally related molecules were synthesized and evaluated for their potential inhibitory effect on DENV-2 infection. One molecule (Compound 31) was identified as the most selective and potent inhibitor in its series. Compound31 also inhibited the infection of the three other DENV serotypes as well as that of YFV-17D, all with EC50 values in the low micromolar range.The antiviral effect was most pronounced when the virus and the inhibitor were preincubated for 2 hours at 37°C before infection was allowed toproceed. Further experiments are ongoing to provide evidence that the β -OG pocket is the compounds most likely target. A yetunexplored and unexploited protein site that is believed to be mechanistically involved in the catalytic activity of the flavivirus helicases was identified in collaboration with the group of Dr. Bolognesi (University of Milano, Italy). By means of i n silico docking his team identified a number of molecules with a highly predicted affinity for this site, among these, Ivermectin, a broadly used anti-helmintic drug. Ivermectin proved to be a highly potent inhibitor of YFV replication (EC50 values in the sub-nanomolar range). Moreover, Ivermectin inhibited,although less efficiently the replication of several other flaviviruses. We demonstrated that the molecule exerts its effect at a time point that coincides with the onset of intracellular viral RNA synthesis, as expected for a molecule that specifically targets the viral helicase. Unfortunately and much to our surprise, we found that Ivermectin did not protect against YFV-induced disease/mortality in hamsters; which can likely be explained by the fact that also in vitro , no antiviral activity isobserved in YFV-17D infected BHK cells. Potential activity in a mouse model will now be assessed. Considering the fact that Ivermectinhas been used for the treatment of a variety of parasitic disease in man for more than twenty years, assessing its potential for the treatment of life threatening flavivirus infections in clinical trials may requirea minimum effort. We identified the in vitro anti-flavivirus activity of 3 ,5 di-O -trityluridine (and certain analogues).The compound results in a dose-dependent inhibition of DENV- and YFV-induced cytopathic effect (EC50 values in the low micromolar range for all4 DENV serotypes). Antiviral activity was also demonstrated in DENV subgenomic replicons (which do not encode the structural viral proteins). The molecule inhibits the viral RNA-dependent RNA polymerase activity, but is inactive against the Coxsackie virus B3 (CVB3) polymerase. The latter observation corroborates the lack of antiviral activity of the molecule against CVB3 replication. Kinetic analysis of the inhibition of the DENV RdRp revealed that the molecule inhibits viral RNA elongation, without competing for UTP during this process when using DENV-specific templates. Obviously, this molecule does not act as a nucleoside analogue, since it cannot be phosphorylated. Understanding the precise mechanism by which 3 ,5 di-O -trityluridine inhibits the elongation process of the viral polymerase may allow designing highly specific non-nucleoside inhibitors of flavivirus RNA replication. Finally, a selected small molecule library was evaluated for a potential inhibitory effect on chikungunya virus (CHIKV) infection in a CPE-based assay. A small-molecule inhibitor, MADTP_0274, was identified that inhibits viral replication. Chemical validation of the bio-active sample revealed that not the originalcompound but a conversion product, (now identified as MADTP_0314) is responsible for the antiviral activity. To identify the viral protein(s) involved in the mechanism of action, drug-resistant virus was selected. One mutation was identified in the methyltransferase gene (nsP1) of all independently selected drug-resistant clones. The MADTP_0314-selected proline-to-serine mutation at position 34 of the nsP1 of CHIKV is located very close to the conserved histidine residue at position 37 of theenzyme of the alphavirus-like superfamily, which is critical for nsP1 enzymatic activity. Optimization of the structure-activity relationship of this scaffold is currently ongoing, as well as studies aiming to further unravel the precise molecular mechanism of action. These findings indicate that the methyltransferase of alphaviruses may be an excellent target for inhibitors of viral replication.status: publishe