Session: Parallel session 9 - Complex mixtures - polymers - microplastics

Identification of peptide inhibitors of the coronavirus 3CL protease from a Fucus ceranoides L. hydroalcoholic extract using a Ligand-fishing strategy

Luiz ANTONIO MIRANDA DE SOUZA DUARTE FILHO¹, Claudio VIEGAS JR⁵, Nathalie BOURGOUGNON⁶, Laurent PICOT¹, Cintia EMI YANAGUIBASHI LEAL ², Pierre-Edouard BODET¹, Edilson BESERRA DE ALENCAR FILHO ³, Jackson GUEDES DA SILVA ALMEIDA⁴, Manon PORTA¹, Oussama ACHOUR¹, Hugo GROULT¹, Carlos ARTHUR GOUVEIA VELOSO¹

¹La Rochelle University, La Rochelle, France
²Unité de Génie Enzymatique et Cellulaire, UMR CNRS 7025, Université de Picardie Jules Verne, Amiens, France
³Department of Pharmacy, Universidade Federal do Vale do São Francisco, Petrolina, Brazil
⁴NEPLAME, Universidade Federal do Vale do São Francisco, Petrolina, Brazil
⁵Federal University of Alfenas, Institute of chemistry, Alfenas, Brazil
⁶Laboratoire de Biotechnologie et Chimie Marines, LBCM, Université Bretagne Sud, EMR CNRS 6076, IUEM, Vannes, France

Introduction: Brown seaweeds of the Fucus genus represent a rich source of antiviral natural products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CLprotease (3CLpro), an enzyme playing a pivotal role in polyprotein processing during coronavirus replication and identified as a relevant drug discovery target for SARS- and MERS-CoVs infections.

Methodology: To purify and identify in a one-step approach 3CLpro ligands with potential inhibitory activity, we immobilized the enzyme onto magnetic microbeads (3CLpro-MP), checked that the enzymatic activity was maintained after grafting and used this bait for a ligand fishing strategy followed by high-resolution mass spectrometry analysis of the fished-out molecules.

Results: Proof-of-concept for the ligand fishing capacity of 3CLpro-MP was demonstrated by doping the FCHE extract with the TSAVLQ-pNA peptide, resulting in the preferential capture of this high-affinity substrate. Ligand fishing in the FCHE alone led to the purification and HRMS identification of hepta-, octa-, and decapeptides, in an eluate mix that significantly inhibited free 3CLpro, more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between seven of these peptides and 3CLpro suggested a high affinity for the enzyme's proteolytic active site, surpassing that of the co-crystallographic ligand. Testing of the corresponding synthetic peptides demonstrated that six out of the seven highly inhibited free 3CLpro.

Conclusion: This study is the first to report peptides from Fucus ceranoides with high inhibitory activity against SARS-CoV-2 3CLprotease, showing a strong binding affinity for the protease's active site. It also confirms the effectiveness of the ligand fishing strategy associated to HRMS analysis for the single-step purification of enzyme inhibitors from complex matrices.