key: cord-0825362-9lvklxa2
authors: Chatterjee, Pranam; Ponnapati, Manvitha; Jacobson, Joseph M.
title: Targeted Intracellular Degradation of SARS-CoV-2 RBD via Computationally-Optimized Peptide Fusions
date: 2020-06-01
journal: bioRxiv
DOI: 10.1101/2020.06.01.127829
sha: b4bbbeca2013160d891e0f37d51e04c71a5de2c5
doc_id: 825362
cord_uid: 9lvklxa2

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has elicited a global health crisis of catastrophic proportions. With no approved cure or vaccine currently available, there is a critical need for effective antiviral strategies. In this study, we report a novel antiviral platform, through computational design of ACE2-derived peptides which both target the viral spike protein receptor binding domain (RBD) and recruit E3 ubiquitin ligases for subsequent intracellular degradation of SARS-CoV-2 in the proteasome. Our engineered peptide fusions demonstrate robust RBD degradation capabilities in human cells, thus prompting their further experimental characterization and therapeutic development.

tolerance to viral evolution ( Figure 1A ).

To do this, we retrieved a structure of the SARS-CoV-2 RBD bound to sACE2 from 70 the Protein Data Bank (PDB 6M0J) (16). We first utilized the PeptiDerive protocol (17) 71 in the Rosetta protein modeling software (18) to generate truncated linear sACE2 peptide 72 segments between 10 to 150 amino acids with significant binding energy compared to that Recently, the Trim-Away technique was developed for acute and rapid degradation of 87 endogenous proteins, by co-expressing TRIM21 with an anti-target antibody (23). We 88 thus hypothesized that by fusing the Fc domain to the C-terminus of candidate peptides 89 and co-expressing TRIM21, we can mediate degradation of the RBD fused to a stable 90 fluorescent marker, such as superfolder GFP (8) (RBD-sfGFP), in human HEK293T cells 91 using a simple plasmid-based assay (Figure 2A and 2B). We chose the two most compact 92 candidate peptides, an 18-mer and 23-mer derived from the ACE2 peptidase domain α1 93 helix, which is composed entirely of proteinogenic amino acids, as well as the candidate 94 peptide computationally predicted to have highest binding affinity to the RBD (a 148-95 mer), for testing alongside sACE2 ( Figure 1C) . We also tested a recently-engineered 23- (Figure 3A .iii). Our results in the subsequent TRIM21 assay identi-to divergent RBD sequences in the case of future mutation as SARS-CoV-2 continues on 140 its evolutionary trajectory. We also predict that the peptide will avoid binding to integrin 141 α5β1, thus preventing interference of critical cellular pathways. In total, we envision that the strategy of utilizing a computationally-designed peptide A) Architecture and mechanism of TRIM21-based degradation system. The Fc domain is fused to the C-terminus of RBDtargeting peptides. TRIM21 recognizes the Fc domain and tags SARS-CoV-2 for ubiquitin-mediated degradation in the proteasome. B) Three plasmid assay used to experimentally validate degradation architecture in human HEK293T cells. All CDS are inserted into the pcDNA3.1 backbone. C) Analysis of RBD-sfGFP degradation by flow cytometry, in the absence or presence of Fc (in cis), TRIM21 (in trans), or both. All samples were performed in independent transfection duplicates (n=2) and gated on GFP+ fluorescence. Mean percentage of GFP+ cell depletion was calculated in comparison to the RBD-sfGFP only control. Standard deviation was used to calculate error bars.

An-267 giotensin converting enzyme (ACE) and ACE2 bind integrins and ACE2 regulates 268 integrin signalling

The 270 proximal origin of SARS-CoV-2

Structure of the SARS-CoV-2 spike receptor-binding domain bound to 272 the ACE2 receptor

All binding affinity scores have been re-scaled to 0 (highest) to 1 (lowest) for visualization. Original amino acids are indicated on the y-axes. B) Analysis of RBD-sfGFP degradation by flow cytometry. All samples were performed in independent transfection duplicates (n=2) and gated on GFP+ fluorescence. All indicated samples were co-transfected with RBD-sfGFP and TRIM21 in trans, and mean percentage of GFP+ cell depletion was calculated in comparison to the RBD-sfGFP-only control. Standard deviation was used to calculate error bars. 23-mer mutations are underlined according to origin. C) Architecture and mechanism of CHIP∆TPR-based degradation system. CHIP∆TPR is fused to the C-terminus of RBD-targeting petpides. CHIP∆TPR can thus tag SARS-CoV-2 for ubiquitin-mediated degradation in the proteasome. D) Analysis of RBD-sfGFP degradation by flow cytometry in the presence of Fc (in cis) and TRIM21 (in trans) or CHIP∆TPR (in cis)