Identification of motif-based interactions between SARS-CoV-2 protein domains and human peptide ligands pinpoint antiviral targets

Abstract

The infection and replication cycle of all viruses depend on interactions between viral and host proteins. Each of these protein-protein interactions is therefore a potential drug target. These host-virus interactions often involve a disordered protein region on one side of the interface and a folded protein domain on the other. Here, we used proteomic peptide phage display (ProP-PD) to identify peptides from the intrinsically disordered regions of the human proteome that bind to folded protein domains encoded by the SARS-CoV-2 genome. Eleven folded domains of SARS-CoV-2 proteins were found to bind peptides from human proteins. Of 281 high/medium confidence peptides, 23 interactions involving eight SARS-CoV-2 protein domains were tested by fluorescence polarization, and binding was observed with affinities spanning the whole micromolar range. The key specificity determinants were established for six of these domains, two based on ProP-PD and four by alanine scanning SPOT arrays. Finally, two cell-penetrating peptides, targeting Nsp9 and Nsp16, respectively, were shown to function as inhibitors of viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously provide information on potential host-virus interactions and identify ligands with antiviral properties.

Defining short linear motif binding determinants by phage-based multiplexed deep mutational scanning

“…The results obtained using the DMS-CoV library were less informative than the results obtained using DMS-BM library (Fig. 3D), partially due to the lower coverage but likely also due to the fact that the interactions probed were of lower affinity (Mihalic, Benz et al 2023) and due to some traits of the motifs as described below. In several cases, only one of the two overlapping parental peptides returned sufficient data, which may indicate that parts of the motifs were truncated in the shifted peptides.…”

“…3A). The interactions were previously found through proteomic peptide phage display (Kruse, Benz et al 2021, Mihalic, Benz et al 2023 or predicted based on consensus binding motif (i.e. the SH3 binding PxxP motif in the SARS-CoV-2 N binding to ABL1 SH3 domain).…”

“…Furthermore, viruses also exploit SLiM-based interactions to outcompete the endogenous interactions and take over the host cell machinery. Viral SLiMs may bind to host proteins (Davey, Trave et al 2011, Mihalic, Simonetti et al 2023, and folded viral proteins may bind to host SLiMs (Madhu, Davey et al 2022, Mihalic, Benz et al 2023. Both scenarios offer the possibility to inhibit viral infection by blocking the SLiM-binding pockets (Kruse, Benz et al 2021, Mihalic, Benz et al 2023.…”

“…Viral SLiMs may bind to host proteins (Davey, Trave et al 2011, Mihalic, Simonetti et al 2023, and folded viral proteins may bind to host SLiMs (Madhu, Davey et al 2022, Mihalic, Benz et al 2023. Both scenarios offer the possibility to inhibit viral infection by blocking the SLiM-binding pockets (Kruse, Benz et al 2021, Mihalic, Benz et al 2023. Finding and optimizing SLiM-based interactions between viral and human proteins thus offer potential strategies to develop antivirals.…”

“…1). We first evaluate the performance of phage based DMS by benchmarking the approach using a set of well-studied SLiMbased interactions (Benz, Ali et al 2022, Kumar, Michael et al 2024) and then applying it to a set of poorly characterized interactions involving peptides or folded domains from SARS-CoV-2 proteins (Kruse, Benz et al 2021, Mihalic, Benz et al 2023. We find that the DMS by phage display approach can be easily multiplexed and accurately determines binding specificities, pinpoints mutations that increase or decrease binding affinity, and provides directions for how to optimize the affinity of a given interaction.…”