Abstract Rationale PAV-104 is a novel small molecule that has been shown robustly active on multiple strains of SARS-CoV-2 including in primary human bronchial epithelial cells cultured to an air-liquid interface (HBE@ALI). PAV-104 targets the protein 14-3-3 zeta at a host-viral interface with the viral nucleoprotein, within a large host multi-protein complex. In silico analysis is consistent with the hypothesis that PAV-104 binds with high affinity to an allosteric site. The target multi-protein complex is highly unusual in that it: 1) is assembled only transiently, 2) is energy-dependent for both its formation and action, 3) is modified in protein composition upon respiratory viral infection, and 4) involves a unique subfraction (5%) of the total of its component proteins present in the cell, that is not in equilibrium with the remaining 95%. For these reasons it is extremely difficult to detect by genomic or conventional proteomic methods. Earlier compounds in this lead series were found active on all six families of viruses causing 98% of human viral respiratory disease but also had significant animal toxicity with a narrow therapeutic window. However, PAV-104 is strikingly non-toxic. Subsequent drug development has demonstrated specificity is inversely related to toxicity suggesting PAV-104 is selective for virally infected cells. We find that PAV-104 retains the pan-respiratory antiviral feature of the earlier compounds including activity against RFP-labelled influenza virus infection in MRC-5 cells. Cy2 fluorophore-labelled analogs of earlier analog PAV-431 and PAV-104 confirm the infected cell selectivity of the latter. We hypothesize that PAV-104 is not only anti-viral against influenza virus, but is selective for only the virally-infected cells. Methods Human MRC-5 cells were infected with RFP-labelled influenza virus without and with subsequent treatment with PAV-431 or PAV-104 and their cy-2 labelled analogs. EC50 was determined by Western blot for viral nucleoprotein and TCID50. Results Fluorescence imaging confirmed a reduction in infectivity with treatment of either PAV analog. Imaging of the cy-2 label demonstrated selectivity of PAV-104 for only the infected cells, providing further explanation for its remarkable non-toxicity including in rodents (see Figure). Conclusions PAV-104 targets an allosteric site within a selective subset of 14-3-3 zeta and does so in a manner selective only for virus-infected cells. This abstract is funded by: Prosetta Biosciences
Zeki et al. (Fri,) studied this question.