Mapping the SARS-CoV-2-Host Protein-Protein Interactome by Affinity Purification Mass Spectrometry and Proximity-Dependent Biotin Labeling: A Rational and Straightforward Route to Discover Host-Directed Anti-SARS-CoV-2 Therapeutics

Abstract

Protein-protein interactions (PPIs) are the vital engine of cellular machinery. After virus entry in host cells the global organization of the viral life cycle is strongly regulated by the formation of virus-host protein interactions. With the advent of high-throughput -omics platforms, the mirage to obtain a "high resolution" view of virus-host interactions has come true. In fact, the rapidly expanding approaches of mass spectrometry (MS)-based proteomics in the study of PPIs provide efficient tools to identify a significant number of potential drug targets. Generation of PPIs maps by affinity purification-MS and by the more recent proximity labeling-MS may help to uncover cellular processes hijacked and/or altered by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), providing promising therapeutic targets. The possibility to further validate putative key targets from high-confidence interactions between viral bait and host protein through follow-up MS-based multi-omics experiments offers an unprecedented opportunity in the drug discovery pipeline. In particular, drug repurposing, making use of already existing approved drugs directly targeting these identified and validated host interactors, might shorten the time and reduce the costs in comparison to the traditional drug discovery process. This route might be promising for finding effective antiviral therapeutic options providing a turning point in the fight against the coronavirus disease-2019 (COVID-19) outbreak.

Keywords: COVID-19; SARS-CoV-2; affinity-purification mass spectrometry; antiviral; drug repurposing; emerging; protein–protein interaction; proteomics; proximity-dependent biotin labeling (BioID); virus.

Figure 1

Schematic representation of (protein–protein interaction) PPI identification strategies. (A) In the Affinity Purification (AP) strategy, the sequence coding for the viral protein (shown in blu) is fused in frame, either at the N- or at the C-terminus, with a peptide tag (shown in purple) which, for the investigations here reviewed, can be Strep [17,18], FLAG [19,20] or HA [21]. In the proximity labeling strategy, the sequence coding for the viral protein is fused in frame, either at the N- or at the C-terminus, with a biotin protein ligase modifying enzyme (shown in red). In all cases, a promoter (green arrow) drives the expression of the fusion protein. (B) The constructs described in (A) are transferred to the test cell line which, for the investigations here reviewed, can be HEK-293 cells [17,18,19,20,23,24] or A549 cells [21,22] by mean of transfection (either transient [17,18,19,20] or stable [23,24]) or by lentiviral mediated transduction [21,22]. In the test cell line, the promoter of the construct drives the expression of the fusion protein. (C) In the AP strategy, following gentle cell lysis, the cellular preys (here shown in different colors and shapes) interacting, either directly or indirectly, with the viral bait are affinity purified, usually using a monoclonal antibody specific for the AP tag (shown in purple). If a cellular protein (indicated by a triangle) does not interact with the viral bait, it is not affinity purified at this stage. Finally, after AP, specifically bound proteins are digested and identified by mass spectrometry (MS). (D) In the proximity labeling approach, after expression of the fusion protein (here shown as a blue square, representing the viral protein, fused with a red circle, representing the modifying enzyme) in the test cell, the biotin protein ligase modifying enzyme mediates the “promiscuous” biotinylation (shown in red) of proteins (here shown in different colors and shapes) in close proximity (~5–10 nm) to the enzyme. If a cellular protein (indicated by a triangle) is not in proximity with the enzyme-viral bait fusion protein, it is not biotinylated. Following cell lysis, only biotinylated proteins are purified by streptavidin and then identified by MS.

Figure 2

Overview of PPIs-based drug repurposing. SARS-CoV-2 interactomes are generated by affinity purification Mass Spectrometry (AP-MS) or by Proximity-Dependent Biotin Labeling (BioID). Interactions between SARS-CoV-2 viral baits proteins and host proteins targetable by already approved drugs are selected. Next, druggable host targets are analysed by biochemical and cell-based assays for antiviral activity. Selected lead compounds with in vitro anti-viral efficacy are then tested for in vivo efficacy on COVID-19 patients.