ABSTRACT
The COVID-19 pandemic continues unabated, emphasizing the need for additional antiviral treatment options to prevent hospitalization and death of patients infected with SARS-CoV-2. The papain-like protease (PLpro) domain is part of the SARS-CoV-2 non-structural protein (nsp)-3, and represents an essential protease and validated drug target for preventing viral replication. PLpro moonlights as a deubiquitinating (DUB) and deISGylating enzyme, enabling adaptation of a DUB high throughput (HTS) screen to identify PLpro inhibitors. Drug repurposing has been a major focus through the COVID-19 pandemic as it may provide a fast and efficient route for identifying clinic-ready, safe-in-human antivirals. We here report our effort to identify PLpro inhibitors by screening the ReFRAME library of 11,804 compounds, showing that none inhibit PLpro with any reasonable activity or specificity to justify further progression towards the clinic. We also report our latest efforts to improve piperidine-scaffold inhibitors, 5c and 3k, originally developed for SARS-CoV PLpro. We report molecular details of binding and selectivity, as well as in vitro absorption, distribution, metabolism and excretion (ADME) studies of this scaffold. A co-crystal structure of SARS-CoV-2 PLpro bound to inhibitor 3k guides medicinal chemistry efforts to improve binding and ADME characteristics. We arrive at compounds with improved and favorable solubility and stability characteristics that are tested for inhibiting viral replication. Whilst still requiring significant improvement, our optimized small molecule inhibitors of PLpro display decent antiviral activity in an in vitro SARS-CoV-2 infection model, justifying further optimization.
ABSTRACT
The SARS-CoV-2 coronavirus encodes an essential papain-like protease domain as part of its non-structural protein (nsp)-3, namely SARS2 PLpro, that cleaves the viral polyprotein, but also removes ubiquitin-like ISG15 protein modifications as well as, with lower activity, Lys48-linked polyubiquitin. Structures of PLpro bound to ubiquitin and ISG15 reveal that the S1 ubiquitin-binding site is responsible for high ISG15 activity, while the S2 binding site provides Lys48 chain specificity and cleavage efficiency. To identify PLpro inhibitors in a repurposing approach, screening of 3,727 unique approved drugs and clinical compounds against SARS2 PLpro identified no compounds that inhibited PLpro consistently or that could be validated in counterscreens. More promisingly, non-covalent small molecule SARS PLpro inhibitors also target SARS2 PLpro, prevent self-processing of nsp3 in cells and display high potency and excellent antiviral activity in a SARS-CoV-2 infection model.
Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus 3C Proteases/metabolism , SARS-CoV-2/metabolism , Ubiquitin/metabolism , Animals , Binding Sites , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/genetics , Crystallography, X-Ray , Cytokines/genetics , Drug Evaluation, Preclinical/methods , Drug Repositioning , Fluorescence Polarization , HEK293 Cells , Humans , Kinetics , Models, Molecular , Protease Inhibitors/pharmacology , Protein Conformation , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Ubiquitins/genetics , Vero CellsABSTRACT
A persistent latent reservoir of virus in CD4+ T cells is a major barrier to cure HIV. Activating viral transcription in latently infected cells using small molecules is one strategy being explored to eliminate latency. We previously described the use of a FlpIn.FM HEK293 cellular assay to identify and then optimize the 2-acylaminothiazole class to exhibit modest activation of HIV gene expression. Here, we implement two strategies to further improve the activation of viral gene expression and physicochemical properties of this class. Firstly, we explored rigidification of the central oxy-carbon linker with a variety of saturated heterocycles, and secondly, investigated bioisosteric replacement of the 2-acylaminothiazole moiety. The optimization process afforded lead compounds (74 and 91) from the 2-piperazinyl thiazolyl urea and the imidazopyridine class. The lead compounds from each class demonstrate potent activation of HIV gene expression in the FlpIn.FM HEK293 cellular assay (both with LTR EC50s of 80 nM) and in the Jurkat Latency 10.6 cell model (LTR EC50 220 and 320 nM respectively), but consequently activate gene expression non-specifically in the FlpIn.FM HEK293 cellular assay (CMV EC50 70 and 270 nM respectively) manifesting in cellular cytotoxicity. The lead compounds have potential for further development as novel latency reversing agents.
