# Metabolites from South African Medicinal Plants as Dual-Function Inhibitors of the SARS-CoV-2 Papain-like Protease (PLpro)

## TL;DR

This study identifies natural compounds from South African plants that can inhibit a key SARS-CoV-2 enzyme, suggesting potential for antiviral drug development.

## Contribution

The study discovers and validates natural dual-function inhibitors of SARS-CoV-2 PLpro from Lippia javanica and Acorus calamus using a multi-omics approach.

## Key findings

- Catechin-7-glucoside is a promising lead compound with strong binding to PLpro and inhibitory activity.
- Both plant extracts and isolated compounds show concentration-dependent inhibition of PLpro activity.
- The compounds maintain cell viability in HEK293T cells, indicating a favorable safety profile.

## Abstract

The SARS-CoV-2 papain-like protease (PLpro) is an essential viral enzyme that promotes viral polyprotein processing while simultaneously suppressing the host innate immune response, which makes it a primary target for developing antiviral drugs. The present study employs a comprehensive approach integrating untargeted metabolomic profiling, in silico molecular docking and dynamics simulations, Molecular Mechanics Generalized Born Surface Area (MM-GBSA) energetic assessments, and biochemical enzyme assays. This integrated method aims to discover natural PLpro inhibitors from two ethnomedicinal plants, Lippia javanica and Acorus calamus, which have long been utilized in African traditional medicine to treat respiratory diseases. Comprehensive metabolite profiling using untargeted Ultra-Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS) and Global Natural Products Social (GNPS) molecular networking revealed flavonoid glucuronides and phenylpropanoid derivatives as the major constituents in both plant species. In situ histochemical staining further offered spatial validation of phenolic- and lignin-associated tissues, supporting the phenolic-dominated molecular families detected by GNPS molecular networking. In silico evaluation of six selected compounds demonstrated spontaneous and thermodynamically favorable binding to PLpro, with ΔG_bind values ranging from −5.63 to −6.43 kcal/mol. Catechin-7-glucoside emerged as the lead compound, establishing multiple hydrogen bond networks with Asp164, Gln269, Tyr264, and Asn267, supplemented by hydrophobic engagement with Pro247 and Pro248, and π-π stacking with the blocking loop 2 (BL2 loop). Molecular dynamics simulations confirmed the stability of the protein–ligand complexes. Biochemical enzyme assays confirmed concentration-dependent inhibition of PLpro proteolytic and deubiquitinating activity by both crude plant extracts and isolated bioactive compounds. However, S-adenosyl-methionine showed comparatively high PLpro proteolytic activity (IC50 5.872 µM) compared to catechin-7-glucoside, with an IC50 of 7.493 µM, exhibiting efficacy similar to the reference inhibitor GRL0617. Both the extracts of L. javanica and A. calamus have shown significant inhibitory activity while maintaining cell viability in Human embryonic kidney 293T cell (HEK293T) culture models, indicating a favorable safety profile of the tested concentrations. Based on these results, catechin-based polyphenols and phenylpropanoid derivatives appear as promising lead compounds for the development of PLpro inhibitors. To progress toward therapeutic use, further work is necessary in pharmacokinetics, structural optimization, and antiviral validation in cell models.