Lassa fever virus (LASV) and other members of the arenavirus family are important human pathogens that can cause hemorrhagic fevers with mortality rates over 20%. Because their aerosol transmissibility and elevated morbidity and mortality, LASV and pathogenic strains of Junin virus (JUNV) pose a bioterrorism risk and are in the list of high priority Category A agents from the department of Health and Human Services (DHHS). Underscoring the national security threat posed by these viruses, there are no licensed preventive vaccines to protect the population. Current anti-arenavirus therapies are limited to the use of the non-specific antiviral ribavirin (Rib), which is only partially efficient and associted with significant side effects. The lack of assays to rapidly and quantitatively detect multiplicatin of LASV and JUNV pathogenic strains together with the requirement of bio-safety containment level 4 (BSL4) laboratory conditions to handle them pose great obstacles for the development of HTS campaigns to identify candidate antiviral drugs. To overcome this problem, we have generated recombinant lymphocytic choriomeningitis viruses (rLCMV) expressing reporter genes of interest. These viruses can be safely used in cell-based screens in which virus multiplication can be readily assessed based on reporter gene activity under relaxed bio-safety conditions. We propose to use this approach to first screen for inhibitors of LCMV multiplication, and then identify compounds with antiviral activity against the highly pathogenic LASV and JUNV.
The specific aims of this proposal are:
Specific Aim 1. To develop an HT cell-based assay to identify inhibitors of LCMV replication. We will use a tri-segmented LCMV carrying a GFP reporter gene (r3LCMV/GFP) to develop a cell-based assay amenable for HTS. Assay parameters will be optimized to achieve Z'values compatible for the development of HTS. Known anti-arena viral drugs will be used to validate the assay and ensure that our screening platform can identify inhibitors of replication acting at different steps of the virus cycle. Speciic Aim 2. Screening of a library of 50,600 small-molecules. We will use the optimized assay to screen a highly diverse chemical library, which will include molecules disrupting protein-protein interactions, pharmacophores optimized for targeting kinases, and a library of compounds with known pharmacological profiles (Lopac). Candidates with EC50 values d 5 ?M and therapeutic indices (TI) e 30 will be advanced to SA3.
Specific Aim 3. Validation and characterization of hit potency and specificity. Hits will be evaluated with a battery of secondary tests to confirm activity, determine specificity and gather important information on the mechanism of action of these inhibitors. Most potent hits will be assessed to determine the stage at which they block LCMV replication. A small set of compounds (<30) will be tested in a virus yield reduction assay to identify compounds with antiviral activity against JUNV and LASV under BSL4 laboratory settings. In a Phase II proposal, chemical leads with the best properties will be used for medicinal chemistry SAR optimization for potency, specificity and oral bioavailability. Improved compounds will be tested in a guinea pig model of arenavirus infection and drug-resistant strains will be generated to determine mechanism of action. The best compounds will be advanced into preclinical development to support future human trials. These studies may lead to the development of much needed antivirals for the treatment of highly pathogenic arenavirus infections. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page
Several arenaviruses cause hemorrhagic fever disease in humans and due to their high morbidity and significant mortality pose main public health problems within their endemic geographic areas. Thus Lassa virus (LASV), the causative agent of Lassa fever (LF) infects over 300,000 individuals per year in West Africa resulting in over 20,000 deaths annually. Moreover, LASV and several other arenaviruses have ideal features for their development as potential as a weapon of biowarfare, which has been a source of national concern and has caused it to be placed on the Category A list of microbial agents (DHHS). Currently, there are no licensed LASV vaccines and LASV therapy is limited to use of the nucleoside analogue ribavirin, which is only partially effective and associated with significant side effects. The impact of LASV on public health and biodefense readiness, together with the fact that the armamentarium available to combat LASV infections is limited, underscore the importance of developing novel and effective strategies to combat LASV. We propose to identify and characterize anti-arenaviral compounds that block virus multiplication at different steps of their life cycle.