Receptor-directed small-molecule inhibitors of New World hemorrhagic fever mammarenavirus entry

PROJECT SUMMARY Mammarenaviruses are endemic in rodent populations worldwide and zoonotic transmission can lead to severe life-threatening hemorrhagic fever. In the Americas, five mammarenavirus species, including Junín and Machupo viruses (JUNV and MACV, respectively), cause viral hemorrhagic fever. In the absence of FDA-licensed antiviral therapies or vaccines, these viruses pose a significant public health concern and threaten national security. The pathogenic New World mammarenaviruses (NWMs) utilize the human transferrin receptor 1 (hTfR1) for entry into human cells. The GP1 subunit of the virus envelope glycoprotein binds to the apical domain of hTfR1, a region that is not involved in the binding to the major hTfR1 ligands. This interaction between GP1 and hTfR1 therefore represents a potential therapeutic target for broad inhibition of NWM infection. Based on the structure of the MACV envelope glycoprotein GP1 subunit in complex with hTfR1, we identified a novel druggable site in the apical domain of hTfR1. A docking-based virtual screening campaign identified 28 hits that were subsequently characterized to determine antiviral activity against JUNV infection in cell culture. Based on this assessment, we selected two chemically distinct molecules displaying strong activity against JUNV for further consideration. We hypothesize that targeting the apical domain pocket of hTfR1 that interacts with viral GP1 will broadly inhibit infection by all known pathogenic NWMs and thereby protect mice expressing hTfR1 from lethal disease associated with JUNV infection. To explore this hypothesis, we will pursue the following specific aims. Aim 1. Identify potent and broadly active compounds targeting the druggable hTfR1 apical domain site that interacts with NWM GP1. The two distinct chemical scaffolds identified in our preliminary studies will serve as the foundation for the design of analogs to define structure-activity relationships (SARs) important for broad antiviral activity. We will implement an iterative campaign involving molecular modeling, medicinal chemistry and in vitro antiviral testing against native JUNV and pseudotyped viruses displaying the envelope glycoprotein of NWMs. Our strategy will include optimization for drug-like properties. Aim 2. Evaluate pharmacokinetics (PK) and efficacy of optimized lead candidates in hTfR1 mice. Two optimized compounds from each chemical scaffold will be chosen for evaluation in animals based on their in vitro potency, selectivity, breadth of inhibition and drug-like properties. We will assess oral bioavailability, determine the maximum tolerated dose and establish PK profiles for the lead candidates in mice. Results from these studies will guide decisions regarding dosing levels and the frequency and duration of treatment in subsequent efficacy studies in a validated hTfR1 mouse model of lethal JUNV infection. In addition to improved survival outcome in JUNV-challenged hTfR1 mice, we will measure the impact of treatments on viral loads, histopathology, and disease severity and duration. Our long-term goal is to develop a novel host-directed small-molecule therapeutic agent that could be deployed to treat NWM hemorrhagic fevers.