PROJECT SUMMARY The World Health Organization (WHO) declared Mpox (MPX) a global health emergency in 2022 and in the United States, MPX was declared a Public Health Emergency with >30,500 cases confirmed so far. MPX is a zoonotic infectious disease caused by the orthopoxvirus Mpox virus (MPXV), which is related to the virus that causes smallpox. Vaccination against smallpox offers protection against MPXV; however, routine smallpox vaccination ended in 1972 in US and 1984 world-wide following the successful global eradication of smallpox in 1979. Hence, most of the world’s population is highly vulnerable. In March-June 2023, 40 Mpox cases were identified in Chicago, including many vaccinated patients, suggesting decreased vaccine effectiveness. There are currently no FDA-approved drugs for MPX. There is an Expanded Access Investigational New Drug (EA-IND) Protocol for use of tecovirimat (TPOXX), which targets VP37, a viral protein involved in the envelopment of intracellular mature virus. Cidofovir (CDV) and its lipid pro-drug brincidofovir have been reported to also inhibit multiple orthopoxviruses, albeit with reduced potency relative to their activity against herpesviruses. There are several reports that establish that the barriers to resistance for TOPXX and CDV are low. Hence, there is a lack of efficacious, well-tolerated drugs that can be broadly used for the treatment and prevention of MPXV infections. To address this public health challenge our multidisciplinary team will extend long standing collaborative efforts on other infectious diseases towards the discovery of innovative antivirals that target MPXV. This strategy is designed to leverage exceptional expertise in drug discovery, nucleoside chemistry, assay development, screening technologies, and mechanism of action studies, augmented by the expertise at the CDC. We hypothesize that direct antiviral agents (DAA) against MPXV can be efficiently discovered through informative assays for high throughput screening (HTS) and high content screening (HCS) and validated, thus leading into downstream lead candidate development. To address this hypothesis, we propose experiments that address the following aims: Specific Aim 1. To further develop and optimize antiviral assays Specific Aim 2. To implement screening campaigns for the discovery of low cytotoxicity antiviral hits Specific Aim 3. To determine the mechanisms of action and resistance (MOA and MOR) of select hits Specific Aim 4. To optimize potent, broad-spectrum, non-cytotoxic hits and identify a clinical candidate Given the team’s preliminary data, outstanding expertise, access to world-class state-of-the-art screening facilities (including BSL-3 labs), and the contributions to development of drugs for the treatment of HIV, HBV, HCV, and SARS-CoV-2 in humans, we expect to generate compound leads that can progress rapidly to advanced preclinical development.

PROJECT NARRATIVE Mpox is a zoonotic infectious disease caused by Mpox virus, which has been endemic in Africa and has recently caused an ongoing global health emergency involving 100 countries around the world. There are currently no FDA-approved drugs that target Mpox virus, and while there are some options available to use repurposed drugs, these antivirals have a low barrier to resistance, resulting in a lack of efficacious drugs for the treatment and prevention of Mpox virus. The goal of this project is to utilize exceptional expertise in drug discovery, high- throughput assay development, screening technologies, and mechanism of action studies towards the discovery of innovative antivirals that target Mpox virus.