Screening of Medicines for Malaria Venture Open Boxes Identifies Potent SARS-CoV-2 Papain-like Protease (PLpro) Inhibitors

The COVID-19 pandemic, caused by SARS-CoV-2, has led to more than 760 million infections and 6.9 million deaths worldwide. While vaccines have played a crucial role in controlling the disease, emerging viral variants pose a threat to their long-term efficacy, highlighting the need for antiviral therapies. To accelerate drug discovery, Medicines for Malaria Venture (MMV) developed open-access compound libraries, including the malaria box, pathogen box, COVID box and, with the drugs for neglected disease initiative, pandemic response box, comprising nearly 1400 drug-like molecules. These resources have been widely used in phenotypic screens to identify potential SARS-CoV-2 inhibitors, but target-based screening remained unexplored, especially for targets such as papain-like protease (PL^pro). Here, we report a target-based screening campaign against SARS-CoV-2 proteases, focusing on both the main protease (M^pro) and PL^pro. From this effort, MMV1634397 emerged as a promising PL^pro inhibitor (IC₅₀ = 0.7 μM). Further optimization led to analogs with improved activity such as 5, 10, and 13, with IC₅₀ values of 0.16, 0.34, and 0.06 μM, respectively. The most potent compound and its isomer also exhibited antiviral activity in ACE2-HeLa cells (EC₅₀ = 2.9 and 3.7 μM, respectively) with favorable pharmacokinetic properties. Our findings highlight the value of the MMV open-access libraries in accelerating target-based antiviral drug discovery against SARS-CoV-2 and other emerging pathogens.