Combating Flaviviral Infections with Novel Broad-Spectrum Macrocyclic Therapeutic and Prophylactic Agents

Background: Many of the 70+ members of the flavivirus family are important human pathogens, which cause an unsettlingly wide variety of emerging infectious diseases (a Fiscal Year 2019 Peer Reviewed Medical Research Program Topic Area) and exert a significant healthcare burden on both military and civilian populations with the most urgent need for effective treatments/preventatives for dengue (DENV) and Zika (ZIKV) viruses. Globally, the negative impact of flaviviral infections is substantial, resulting in tens of thousands of deaths and millions of illnesses annually. The increasing spread of DENV and the recent outbreaks of ZIKV infections pose serious and accelerating threats that are now affecting North America and Europe in addition to their typical endemic tropical and subtropical regions. Pharmacological strategies against flaviviral infections have proven woefully inadequate to date, with vaccines, the only current option, having been found to possess significant limitations. Hypothesis and Rationale: To remedy this acute deficiency, the project is directed at inhibition of the viral NS2BNS3 protease, a vital component for pathogen replication possessing substantial homology between family members. However, despite notable success with other viral proteases, flaviviral protease inhibitors have proven particularly elusive. Since prior attempts with traditional small molecules have failed to provide a route to a viable pharmacotherapy, it is hypothesized that unconventional and underexplored compounds, small molecule macrocycles, could provide a novel strategy to succeed in this endeavor. Initial discovery efforts using a library of these unique molecules have identified a novel lead series displaying nanomolar level potency against the proteases from DENV, ZIKV, and other flaviviruses, ranking among the most active such inhibitors to date. Objective and Specific Aims: The project objective is to discover and develop a first-in-class, oral, broad-spectrum antiviral agent to prophylactically protect patients from, and therapeutically resolve, flaviviral infections, and their associated diseases. The proposal outlines progression through the following three partially overlapping aims: (1) improve in vitro pharmacokinetic (PK-ADME) properties of the existing lead compounds to enable cellular uptake and blood-brain barrier (BBB) penetration; (2) achieve proof-of-efficacy (PoE) in relevant cellular models as well as oral bioavailability and brain penetration in vivo; (3) obtain preclinical proof-of-concept (PoC) in relevant animal infection models and select a clinical candidate with suitable pharmacodynamic (PD), PK-ADME and safety profiles for progression into formal investigational new drug (IND)-enabling studies. Study Design: These aims will be accomplished through iterative cycles of compound library design and synthesis utilizing the applicants' proprietary technology for multi-step parallel synthesis of complex macrocycles, followed by in-depth in vitro and in vivo characterization of their properties, which will guide subsequent molecule designs. Specifically, these macrocycles will be evaluated for their potency against DENV, ZIKV, West Nile (WNV), and Japanese encephalitis (JEV) proteases, selectivity vs. mammalian proteases, cellular activity against viral replication, PK-ADME parameters and safety profile. Critical aspects of this screening cascade are the inclusion of a subgenomic reporter replicon assay and an in vitro BBB model derived from inducible pluripotent stem cells, each of which has demonstrated excellent translatability, and studying the emergence of resistance to these inhibitors. Testing of highly potent analogues with suitable PK-ADME properties in relevant animal models of flavivirus infection will verify that the in vitro results are predictive of efficacious antiviral action in vivo. At the conclusion of the 3-year project, one or more macrocyclic candidates suitable for advancement into IND-enabling studies are expected to be obtained. Impact: These safe and potent broad-spectrum inhibitors would provide a reliable, effective, orally administered, prophylactic, and therapeutic intervention that covers infections by the most important pathogenic flaviviruses and, as such, will generate an immediate and profound health benefit for the general public in endemic regions, as well as in the areas to which these infections have spread. That positive impact is even more critical for the military, as it will directly affect operational readiness. Indeed, DENV infections have exerted adverse effects on the U.S. military since at least the Spanish-American War, resulting in substantial lost duty time and erosion of mission capabilities. These agents would provide a unique first-line treatment modality to effectively fight existing and epidemic outbreaks of viral infections. Critically, drugs from this project will be able to fight, as well as protect against, infections from multiple pathogens with a single agent. This offers substantial advantages over vaccines where separate product development is necessary for each microorganism and the risk from antigen-dependent enhancement is omnipresent for new serotypes that can arise. Thus, success in this project will ultimately lead to pan-flaviviral inhibitor pharmaceuticals that finally address the imperative dual-use need in military and civilian populations with the potential to save thousands of lives and billions of dollars in healthcare costs and economic losses currently attributable to flaviviruses and their diseases. Less