The filoviruses Ebola (EBOV) and Marburg (MARV) cause the most severe hemorrhagic fever known, have no approved vaccines or treatments, are highly contagious and infectious In an aerosolized form, and can be used as weapons in acts of bioterrorism and biological warfare. The viruses have the RNA-dependent polymerase, which mediates both the transcription and replication of the genome. The EBOV polymerase complex consists of three subunits common for all Mononegavirales: nucleoprotein (N), phosphoprotein (VP35), and the large subunit of polymerase (L). In addition, the polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional, but not replication activity of EBOV polymerase, and can only support transcription In a dephosphorylated form. Our preliminary studies show that protein phosphatase 1 (PP1) dephosphorylates VP30 in cultured cells. Furthermore, PP1-targeted small molecule compound 1E7-03 that was developed through rounds of computational modeling followed by screening induced phosphorylation of VP30 and inhibited transcription of EBOV genes and replication of the virus. The 1E7-03 compound targeted a non-catalytic site of PP1 and had no toxicity or produced significant changes in cellular proteome. The compound inhibited the transcription but not replication of the viral genome, and completely suppressed replication of EBOV. Our findings suggest that targeting PP1 Is a feasible approach for development of a drug against filoviruses. Furthermore, a remarkable similarity in the phosphorylation sites at the N-termini of the VP30 proteins of all species of EBOV and MARV suggests that this PP1 targeting approach can be used for developing a pan-filovirus drug. The proposed study Is aimed at the detailed testing and optimization of the 1E7-03 compound as a drug against EBOV and MARV. The following Specific Aims w be pursued: 1) Continue optimization of the molecules for the inhibition of EBOV and MARV replication by using a medicinal chemistry approach; 2) Characterize the ability of the most potent small molecules to inhibit replication of EBOV and MARV in vitro and in vivo; 3) Non-clinical toxicology and PK research and development. The approach is innovative, because the developed compound targets PP1 without affecting Its general enzymatic activity, and this approach has never previously been used for any virus, with the exception of the Pi's recent experiments with EBOV and HIV-1. The proposed research is significant because It may open a new therapeutic avenue against multiple filovirus species.
The proposed research is relevant to public health because It Is aimed at development of a drug which will target the interaction of a viral protein with a host enzyme and which is likely to be active against all species of filoviruses. The proposed research Is relevant to the NIH mission that pertains to a reduced vulnerability of the population to biothreat agents. Including hemorrhagic fever viruses..
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