Dr. Iris Salgado
Institution: Universidad Central del Caribe
Potential mechanism of action of selected lead compounds with influenza antiviral activity
Influenza viruses are implicated in all seasonal influenza epidemics and sporadic pandemics affecting humans. Limited therapeutic options and the emergence of resistant strains highlight the need for new and effective treatments. Our long-term goal is to provide novel “broad-spectrum” antiviral agents effective against all influenza virus infections and with reduced resistance development potential. We focus on the influenza polymerase complex as a new target for structure-guided drug design and development. Following a novel in silico drug discovery approach targeting this well-defined and highly conserved protein-protein interaction (PPI) area between PA and PB1 subunits resulted in the identification of a group of small (MW <350Da) quinoline-morpholinyl compounds. We measured the activity of this group of compounds to inhibit the INF viral proliferation in infected MDCK cells resulted in the identification of 5 highly potent compounds. In our previous INBRE-Pilot Project, we demonstrated using Western Blot, ELISA, and Alamar Blue analysis that these selected 5 morpholinyl quinoline lead compounds are highly potent, efficient, and relatively safe compounds. Precisely, this group of compounds decreased INF viral proliferation by 90% with concentrations as low as 100 nM. Results from cytotoxicity assays in MDCK cells revealed a mean toxic dose (TD50) greater than 100 uM suggesting the possibility of these compounds to exhibit a wide therapeutic window. Armed with the results from the in-silico and in-vitro analysis, we proposed for this one-year extension of the INBREPilot project to demonstrate that the most possible mechanism of action of our small group of lead compounds is inhibition of the PPI between the PA-PB1 subunit of the viral polymerase. Working hypothesis: Morpholinyl quinoline lead compounds are specific inhibitors of influenza viruses through the disruption of the interaction between the PA-PB1 subunit of the viral polymerase. To test this hypothesis, we establish the following Specific Aim: To evaluate/determine the mechanism (s) of action responsible for the observed influenza antiviral activity of our selected lead compounds. Several approaches will be employed to meet this specific aim. First, this study will evaluate the potential of our “lead
compounds” to inhibit the protein-protein interaction (PPI) area between PA and PB1 subunits of the INF virus polymerase performing ELISA technology. This technology will employ a His-Halo double-tagged PA and PB-1 full-length purified proteins developed in our laboratory. Second, the specificity of our compounds for INF-V will be determined by evaluating their activity against a group of cell-adapted viruses (human cytomegalovirus, herpes simplex virus, dengue virus, human coronavirus 229E, and human coronavirus OC43). Depending on the type of virus, the assays to measure the activity of our lead compounds will include RT-PCR, plaque, and ELISA tests. Finally, the effect of our compounds on inhibiting the early stages of influenza virus infection will be evaluated by performing viral attachment and entry/fusion tests. After each assay, viral proliferation will be measured through RT-qPCR and plaque assay methodology. Expected outcome: To clearly demonstrate the mechanism of action of our morpholinyl quinoline “lead compounds”. The long-term strategic plan for this project is to take this group of efficient, potent, and safe compounds into the pre-clinical phase of development.