Insights Into Drug Repurposing, as Well as Specificity and Compound Properties of Piperidine-Based SARS-CoV-2 PLpro Inhibitors

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.

Allosteric Inhibitors of the SARS-COV-2 Papain-like Protease Domain Induce Proteasomal Degradation of Its Parent Protein NSP3

“…Interestingly, this region of the protein is somewhat remote with respect to the binding site, suggesting that molecule binding induces some long-range effects on the protein structure. This change correlates with slight structural shifts between aligned crystal structures for 1 -bound (7TZJ) and apo (6W9C) PL pro , where residue shifts of up to 2.9 Å can be observed within this region (alignment data is available in Supporting Information).…”

“…Parental A549 was used for comparison. (B–D) Chemical structures of PL pro inhibitors and their reported crystal structure binding conformations within SARS-COV-2 PL pro ; (C) 1 , (D) 2 , (E) 3 . Key hydrogen bonding (yellow) and π–π interactions (blue) are depicted as dashed lines.…”

Synthesis and Chemoinformatic Analysis of Fluorinated Piperidines as 3D Fragments for Fragment-Based Drug Discovery

“…The importance of the fluorinated moiety for the interaction within the 3CL Pro cavity has been confirmed also by others in the literature, 31 such as for example in the structure of S-217622, a noncovalent oral SARS-CoV-2 3CL Pro inhibitor possessing a 3,4,5-trifluorobenzene moiety that interacts with the side chain of His41 near the S2 pocket (PDB code: 7VTH), 32 or in the case of the fluorine containing compounds from a piperidine-based library (ReFRAME library). 33…”

Peptide Mold: A Novel Strategy for Mapping Potential Binding Sites in Protein Targets

“…21 Significant number of drugs have been studied for their potential to inhibit PLpro, including HIV protease inhibitors, repurposed medications like sitagliptin and daclatasvi, and investigational drugs like tropifexor, ebselen, disulfiram, tanshinone, cryptotanshinone, asunaprevir, simeprevir, famotidine, carmofur, PX12, and tideglusib. [22][23][24] Several new chemical entities (NCEs) belonging to varied chemical series have been reported as potent or moderately potent inhibitors of PLpro 25,26 (reviewed in Ref. 25).…”