Mosquito-borne members of the Flavivirus family including Zika virus (ZKV), Dengue virus (DNV) and West Nile virus (WNV), are classified as re-emerging pathogens due to the frequency and severity of recent epidemics. Also known as arboviruses, these viruses are the etiologic agents of many debilitating diseases affecting the human population worldwide. Consequently, vector borne diseases now account for 17% of all infections worldwide. DNV is the fastest growing arboviral disease currently affecting 400 million annually with 96 million cases manifesting into clinical severity and 22,000 deaths, mainly children. WNV is considered the most important causative agent of viral encephalitis worldwide. The recent ZKV infection outbreak has been associated with congenital microcephaly and intracranial calcification and, in adults, with GBS and severe thrombocytopenia. Currently there is no effective treatment for infections caused by these viruses, which highlights the urgent need to find preventive and therapeutic interventions. The Flaviviridae genome is translated into a single polyprotein which is processed to yield 3 structural and 7 nonstructural proteins. The correct processing of the polyprotein is essential for replication of all flaviviruses, which requires both host proteases and the highly conserved viral NS2B-NS3 protease (NS2B-NS3pro). Hence, the viral protease is a rational target for development of small molecule inhibitors that block flavivirus replication. Small molecule antivirals targeting HIV-1-encoded and HCV-encoded proteases have been successfully developed, which supports the concept of developing chemotherapeutic agents targeting the flavivirus NS2B-NS3pro. The innovation of our proposal is: (i) optimization of a highly-sensitive screening assay for the identification of low binding fragment hits; (ii) evolution of fragment and compound hits into broad-spectrum leads; (iii) the use of replicon and plaque assays to test for cellular efficacy and guide optimization. Our preliminary results and the use of multiple cell-based models supports the feasibility of the discovery of broad-spectrum anti-flaviviral therapeutics. The specific aims are: Aim 1: Complete the screening of fragment and compound libraries for the identification of broad-spectrum NS2B-NS3pro hits. Milestone 1: Identify 6-8 structurally distinct broad-spectrum NS2B-NS3pro hits with an IC50 ? 25?M. Aim 2a: Iterative 3D-structure and SAR-based discovery of three non-overlapping broad-spectrum NS2B-NS3pro inhibitor series, using a combination of: (i) commercial analogues and (ii) med-chem design and synthesis approach. Milestone 2: Identify three non-overlapping broad-spectrum NS2B- NS3pro inhibitor series with an IC50 ? 200nM. Aim 2b: Characterize biochemically potent inhibitors for: (a) mode of inhibition and (b) enzyme:ligand interactions and prioritize compounds with IC50 ? 200nM, for cellular efficacy studies. Aim 3a: In vitro evaluation and optimization of biochemically potent compounds for cellular efficacy (EC50) and cytotoxicity (CC50). Milestone 3: Identify 4-6 lead compounds from each series with EC50 ? 5?M and CC50 ? 200?M. Aim 3b: Conduct in vitro ADME-based lead optimization of compounds with broad spectrum in vitro activity and acceptable therapeutic indices. Phase I Milestone: Identify 2-4 lead compounds exhibiting high bioavailability, weak inhibitor of CYPP450s, optimum stability and are not hERG channel blockers.
Public Health Relevance Statement: Project Narrative The flavivirus family members, Dengue virus, West Nile virus, Zika virus, Japanese encephalitis virus and yellow fever virus are classified as NIAID Categories A and B Emerging Infectious Pathogens, representing the top two tiers posing the greatest risk to national security and public health. Also known as arboviruses due to their ability to be transmitted by the bite from an infected mosquito, these viruses are the etiologic agents of many debilitating diseases affecting the human population worldwide. Consequently, now account for 17% of all infections worldwide. Despite these serious risks, no specific therapy is available to treat or prevent the infections caused by these viruses. Therefore, there is an urgent unmet need to develop safe, effective and affordable treatment for the growing threat of arboviral infections and is the focus of our proposal.
Project Terms: abortion; absorption; acute toxicity; Adult; Affect; American; analog; Animal Model; Animals; anti-viral efficacy; Antiviral Agents; Arbovirus Infections; Arboviruses; Arthrogryposis; Aspartic Endopeptidases; base; Binding; Biochemical; Biological Assay; Biological Availability; Bite; calcification; Categories; Cell Line; Cells; Cerebral Calcification; Cessation of life; channel blockers; chemotherapeutic agent; Child; Clinical; congenital zika syndrome; Country; Culicidae; cytotoxicity; Dengue Vaccine; Dengue Virus; design; Development; Disease; Disease Outbreaks; Drug Kinetics; effective therapy; efficacy study; efficacy trial; Encephalitis; Enzymes; Epidemic; Etiology; Evaluation; Evolution; Exhibits; Family; Family member; Fever; Flaviviridae; Flavivirus; Frequencies; Genome; Geography; global health; Guillain-Barré Syndrome; Hepatitis C virus; HIV-1; Human; In Vitro; in vitro activity; in vivo; Incidence; Infant; Infection; Infection prevention; inhibitor/antagonist; innovation; Japanese encephalitis virus; Lead; lead optimization; Libraries; Ligands; Link; Liver; Luciferases; Measures; member; Meningitis; Metabolism; Microcephaly; Microsomes; Modeling; mortality; mosquito-borne; Mosquito-borne infectious disease; National Institute of Allergy and Infectious Disease; National Security; Neurons; Nonstructural Protein; novel; Paralysed; pathogen; Pathology; Patients; Peptide Hydrolases; Performance; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Plaque Assay; Polyproteins; Population; Pregnant Women; Prevention; Preventive Intervention; Process; Property; Protease Inhibitor; Public Health; Rattus; Replicon; Reporter; Risk; Safety; screening; Series; Serine Protease; Serotyping; Severities; small molecule; small molecule inhibitor; Solubility; stillbirth; Structure; success; System; targeted agent; Testing; Therapeutic; Therapeutic Index; Therapeutic Intervention; three dimensional structure; Thrombocytopenia; Translating; vaccine candidate; Vaccines; Vector-transmitted infectious disease; Vero Cells; Viral; Viral Encephalitis; Virus; West Nile viral infection; West Nile virus; World Health Organization; Yellow fever virus; Zika Virus; ZIKV infection
