Project 1: Identification and Development of Anti-Flavivirus Lead Drug Candidates

PI: Jay A. Nelson, Oregon Health & Science University

The flaviviruses are mosquito-borne viruses that are associated with significant worldwide morbidity and mortality. The dengue viruses (DENV) are members of this group associated with dengue fever, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). These viruses are endemic in most of the tropical and sub-tropical world, and roughly one-third of the Earth’s populace lives in areas at risk for dengue transmission. A second member of the flaviviruses, West Nile virus (WNV), is a neurotropic virus; infection can result in encephalitis/meningitis and subsequent long-term neurologic complications or death. Despite this threat, no specific therapy exists for either virus. Treatment is largely supportive, and in the case of DHF and DSS or neuroinvasive WNV, generally requires hospitalization. In this project, we will identify and develop small molecule inhibitors of these viruses. We will emphasize the development of molecules that show broad-spectrum activity against multiple flaviviruses, and potentially members of other virus families as well. Initial screening of multiple compound libraries comprising >100,000 compounds will be carried out in collaboration with the Southern Research Institute (SRI). In additional arms of AD3C, the same libraries will be screened for activity against other virus families, including influenza, alphaviruses, and coronaviruses. Therefore, AD3C has the potential to identify compounds that are not only active against flaviviruses, but other medically important viruses as well. Multiple techniques will be used to characterize mechanisms of action and targets of antiviral compounds. We will focus on compounds that target one of the following important enzymatic activities of the flavivirus NS5 protein: the RNA-dependent RNA polymerase, which is essential for replication of the viral RNA genome, and the 2’-O-methyltransferase, which is required for the virus to avoid the host innate immune response. Compounds with activities targeting these enzymes will be further developed through the synthesis of structural analogs, analysis of structure-activity relationship (SAR), and testing in in vivo (mouse) models of virus infection.

Project 2: Inhibitors of Coronavirus Fidelity and Cap Methylation as Broadly Applicable Therapeutics

PI: Mark R. Denison, Vanderbilt University

Both the emergence and subsequent human-to-human transmission of SARS-CoV in 2002-2003, and of the highly virulent human coronavirus HCoV-EMC in the Middle East and Europe in 2012-2013 exemplifies CoV movement potential and transmissibility, and underscores the urgent and critical need for a broadly efficacious therapeutics. The overall goal of Project 2 is to identify inhibitors of two highly conserved CoV processes, replication fidelity and RNA capping, that are essential for SARS-CoV virulence and survival in vivo. Multiple viral proteins and enzymatic activities are critical for these processes, including CoV 3’-to-5’ exoribonuclease (fidelity; nsp14-ExoN) and 2’-O-methyltransferase (capping; nsp16-OMTase) activities. Consistent with the importance of these processes, we have shown that decreased replication fidelity and ablation of RNA capping through genetic inactivation of either ExoN or OMTase, respectively, results in replication competent viruses that are profoundly attenuated in vivo. In Aims 1 and 2, we will work with the Screening Core (Core B) and the Medicinal Chemistry lead Development Core (Core C) to identify, characterize, and optimize small molecule inhibitors of SARS-CoV fidelity and RNA capping. Once active compounds are identified, we will define their mechanism of action, test for the development of virus resistance, and determine their activity across the CoV family. In Aim 3, we will work with Core C to chemically optimize and test the in vivo efficacy of lead compounds in progressively tiered models of SARS-CoV disease severity, and assess the development of drug resistance in vivo. The complementary expertise of the Denison and Baric Labs, extensive preliminary datasets, state-of-the-art technologies, and the expertise of SR in the areas of medicinal chemistry, high-throughput screening, and drug development will contribute significantly to the successful identification, confirmation, and in vivo testing of lead compounds. Ultimately, inhibiting these two conserved and distinct pathways required for in vivo pathogenesis will allow for the treatment of endemic and emerging CoVs and potentially reduce the emergence of viral resistance.

Project 3: Novel Therapeutic Strategies Targeting Re-emerging Alphaviruses

PI: Daniel N. Streblow, Oregon Health & Science University

The main goal of this project is to develop novel nucleoside and nucleotide inhibitors directed against Alphaviruses. There is a very important need for the development of treatments against these important human pathogens. We propose to identify potent inhibitors of CHIKV and VEEV replication by performing a high-throughput screen with several chemical libraries. Compounds displaying activity against the two viruses will be further validated and then characterized for specificity within the Alphavirus genus, efficacy and toxicity in multiple relevant cell-types, mode of action, and generation of viral mutants. Compounds that pass this second set of filters will undergo Hit to lead optimization and SAR with subsequent retesting in secondary screens. Last of all, we will perform in vivo efficacy testing in relevant mouse and non-human primate models of alphavirus infection and disease for compounds with good bioavailability and activity. Through these studies we will develop robust antiviral agents against multiple members of the Alphavirus genus.      

Project 4: Identification and characterization of novel drugs that target the Influenza virus polymerase functions

PI: Richard J. Whitley, University of Alabama at Birmingham

The overall goal of this project is to identify new therapies that target influenza virus replication. The global health burden of annual influenza epidemics coupled with the emergence of highly pathogenic strains of influenza virus has highlighted the urgent need for new effective treatments. A primary concern with the current drugs (amantadines and neuraminidase inhibitors) used to treat influenza is the development of resistance mutations that negate therapeutic benefit. Published evidence suggests that targeting the influenza virus RNA dependent RNA polymerase (RdRp) is a rational approach for antiviral therapy.  The RdRp is responsible for a number of functions including 5’cap recognition, endonuclease activity, replication, transcription, and polyadenylation. Recently, cryo-EM reconstitution studies identified branched-ribonucleoproteins (RNPs) structures as putative replication intermediates and suggested a mechanism for viral replication by a second polymerase activity on the RNP template. The second polymerase activity is believed to be a function of the polymerase complex. Clearly, the RdRp provides multiple functional domains that could be targets for antiviral drug therapy. Previous studies showed that mutations in the conserved regions of PB1 subunit of the polymerase complex produce inactive RNA polymerase. We hypothesize that compounds that specifically target the polymerase complex might reduce the frequency of escape mutations, or promote escape mutants that are unfit for replication. We have recently identified potential hit compounds from previous HTS screens that significantly inhibit the influenza virus polymerase activity in an RdRp transient assay. These hit compounds were effective against three different strains of influenza viruses in CPE assays. We propose to characterize these compounds and use high-throughput screening (HTS) of novel small molecule libraries to identify anti-polymerase candidate compounds, chemically optimize them, and establish their effectiveness in an influenza mouse model.