Work

Identification of protease inhibitors from the human peptidome to develop novel antivirals

Year: 2024

Type: conference-abstract

Abstract: Protease inhibitors are potent modulators of the viral replication cycle by selectively interfering with host cell and viral protease functions, thus effectively preventing the propagation of infectious progeny virions.The human body produces a myriad of peptides and proteins of physiological relevance1; some play critical roles in controlling viral pathogens.Exploration of the human peptidome by body fluid analyses has revealed new therapeutic candidates against bacteria and viruses 1 .Protease inhibitors have also been found in the search for antiviral agents by bioassay-guided purification from body fluids 2,3 .However, the isolation and further analysis of low-abundance protease inhibitors can be challenging, and a different approach may often be needed to remove the impurities and concentrate the active molecules.In the present work, we used affinity chromatography and mass spectrometry to discover the main serine-protease inhibitors from a human hemofiltrate pool.Chromatographic fractionation of 1000L HF using pH-gradient cation-exchange chromatography yielded four pools by stepwise elution in increasing order of pH.A sample of each pool and the non-retained fraction were subjected to affinity chromatography on an immobilized-Trypsin gel based on a cheap and available protease used as a prototype to capture serine-protease inhibitors.Subsequently, the eluates were analyzed using a bottom-up mass spectrometry approach to sequence the peptides/proteins.Several serine-protease inhibitors were identified, with Kazal-type 1 showing the highest abundance in HF.Although much less abundant, serine-protease inhibitor Kazal-type 5 and the cysteine-protease Cystatin C3 were also found among the major inhibitors.Further analyses will reveal their antiviral activity and whether these molecules are composed of full-length proteins or fragments that can display antiviral activity. We expect this approach to be applied not only to serine-protease inhibitors but also extended to other relevant proteases for discovering novel protease inhibitors with antiviral activity.

Source: European Peptide Symposium (EPS)

DOI: 10.17952/37eps.2024.p1276