Cyclic peptides targeting the SARS-CoV-2 programmed ribosomal frameshifting RNA from a multiplexed phage display library

Abstract

RNA provides the genetic blueprint for many pathogenic viruses, including SARS-CoV-2. The propensity of RNA to fold into specific tertiary structures enables the biomolecular recognition of cavities and crevices suited for the binding of drug-like molecules. Despite increasing interest in RNA as a target for chemical biology and therapeutic applications, the development of molecules that recognize RNA with high affinity and specificity represents a significant challenge. Here, we report a strategy for the discovery and selection of RNA-targeted macrocyclic peptides derived from combinatorial libraries of peptide macrocycles displayed by bacteriophages. Specifically, a platform for phage display of macrocyclic organo-peptide hybrids (MOrPH-PhD) was combined with a diverse set of non-canonical amino acid-based cyclization modules to produce large libraries of 10⁷ structurally diverse, genetically encoded peptide macrocycles. These libraries were panned against the -1 programmed ribosomal frameshifting stimulatory sequence (FSS) RNA pseudoknot of SARS-CoV-2, which revealed specific macrocyclic peptide sequences that bind this essential motif with high affinity and selectivity. Peptide binding localizes to the FSS dimerization loop based on chemical modification analysis and binding assays and the cyclic peptides show specificity toward the target RNA over unrelated RNA pseudoknots. This work introduces a novel system for the generation and high-throughput screening of topologically diverse cyclopeptide scaffolds (multiplexed MOrPH-PhD), and it provides a blueprint for the exploration and evolution of genetically encoded macrocyclic peptides that target specific RNAs.

Fig. 1. Overview of the −1 programmed ribosomal frameshifting stimulatory sequence (FSS) of SARS-CoV-2. (a) Genomic organization of the SARS-CoV-2 open reading frames (ORFs) whose expression is controlled by the FSS element. (b) Secondary structure of the FSS pseudoknot (PK) of SARS-CoV-2 with the dimerization loop hairpin (DL) boxed (orange). The PK sequence used for this study began at C13476 and ended at U13543. (c) Crystal structure of the FSS PK variant (PDB 7mky). The GNRA tetra loop in the original structure was replaced with the actual dimerization loop sequence.

Fig. 2. Multiplexed MOrPH-PhD system for the selection of SARS-CoV-2 FSS-targeting cyclic peptides. MOrPH-PhD libraries were diversified through cyclization of 11-mer (i/i ± 10) and 9-mer (i/i ± 8) peptide sequences using four different eUAAs and two different orientations for the eUAA/Cys linkage (X = randomized amino acid position, C = cysteine residue, Y* = eUAA). See Fig. S1 for further information about the library design.

Fig. 3. Phage recovery after each round of panning. Percent phage recovery of individual eUAA-containing libraries through three rounds of affinity selection and amplification against the (a) DL and (b) FSS.

Fig. 4. Macrocyclic peptides identified from deconvoluted MOrPH-PhD libraries. (a) Top enriched cyclic peptides were identified from i/i ± 8 and i/i ± 10 libraries. Cyclic peptides identified from sequenced libraries panned against the FSS comprise the pCaaF i/i − 10 11-mer library. All other peptides were identified from NGS deconvoluted libraries panned against the DL RNA from SARS-CoV-2. (b) Cyclic peptides with the highest binding affinity for the DL RNA.

Scheme 1. SPPS methods for the generation of cyclic peptides analyzed in this study. (a) SPPS of peptides comprised by a Cys/pCaaF linkage. (b) SPPS of peptides comprised by an O4bbY/Cys linkage. (c) SPPS of peptides comprised by a Cys/O4bbY linkage.

Fig. 5. SHAPE-Seq data localizes peptide binding to U13518 within the dimerization loop. (a) Secondary structure of the RNA cassette containing the stable HIV-1 FSS hairpin at the 5′ end, followed by the SARS-CoV-2 FSS dimerization loop (DL) upon its stem, a strong 3′ linker hairpin, and the reverse transcriptase primer binding site. SARS-CoV-2 numbering corresponds to reference genome NC_045512.2. (b) Differential SHAPE reactivity (Δρ) profiles of the SARS-CoV-2 DL showing average differential acylation in the presence and absence of peptide (i.e., ρ^{+(pCaaF(i−8)-m1)} − ρ^{−(pCaaF(i−8)-m1)}) versus sequence position. (c) Differential SHAPE reactivity (Δρ) profiles of the SARS-CoV-2 DL showing average differential acylation in the presence and absence of peptide (i.e., ρ^{+(pCaaF(i−10)-m1)} − ρ^{−(pCaaF(i−10)-m1)}) versus sequence position. (d) Differential SHAPE reactivity (Δρ) profiles of the SARS-CoV-2 DL showing average differential acylation in the presence and absence of peptide (i.e., ρ^{+(O4bbY(i+10)-m1)} − ρ^{−(O4bbY(i+10)-m1)}) versus sequence position. Each bar represents the average of two replicates with standard deviations shown.