The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. 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 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD ∼ 7-300 μM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.
The virus life cycle depends on host-virus protein-protein interactions, which often involve a disordered protein region binding to a folded protein domain. 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 281 peptides from human proteins, and affinities of 31 interactions involving eight SARS-CoV-2 protein domains were determined (KD ∼ 7-300 μM). Key specificity residues of the peptides were established for six of the interactions. Two of the peptides, binding Nsp9 and Nsp16, respectively, inhibited viral replication. Our findings demonstrate how high-throughput peptide binding screens simultaneously identify potential host-virus interactions and peptides with antiviral properties. Furthermore, the high number of low-affinity interactions suggest that overexpression of viral proteins during infection may perturb multiple cellular pathways.
“…This strategy allows the design and identification of some Mpro peptide inhibitors, exemplified here by peptide KYLTWQNSQIN (IC50 = 3.11 µM). The obtained outcomes in this work could help in the Similarly, a recent work by G. Gallo and coworkers try to identify possible substrates and the primary specificity of the Mpro through the combination of terminal amine isobaric labeling of substrates (TAILS) and molecular mechanics Poisson-Boltzmann methodology [23] . These studies suggested high promiscuity of the enzyme, since only 6 out of 58 unique sequences have a Gln residue at P1 position, thus opening new opportunities for innovative inhibitor's design.…”
Section: Peptides As Sars-cov-mpro Inhibitorsmentioning
confidence: 91%
“…Similarly, a recent work by Gallo et al tries to identify possible substrates and the primary specificity of the Mpro through the combination of terminal amine isobaric labeling of substrates and molecular mechanics Poisson–Boltzmann methodology 23 . These studies suggested high promiscuity of the enzyme, because only 6 out of 58 unique sequences have a Gln residue at P1 position, thus opening new opportunities for innovative inhibitor's design.…”
Section: Peptides As Sars‐cov‐2 Mpro Inhibitorsmentioning
The COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still affecting people worldwide. Despite the good degree of immunological protection achieved through vaccination, there are still severe cases that require effective antivirals. In this sense, two specific pharmaceutical preparations have been marketed already, the RdRp polymerase inhibitor molnupiravir and the main viral protease (Mpro) inhibitor nirmaltrelvir (commercialized as Paxlovid, a combination with ritonavir). Nirmaltrelvir is a peptidomimetic acting as orally available, covalent and reversible inhibitor of SARS-CoV-2 Mpro. The success of this compound has revitalized the search for new peptide and peptidomimetic protease inhibitors. This Highlight collects some selected examples among those recently published in the field of SARS-CoV-2.
“…M pro selectively cleaves its substrates between the P1 and P1′ residues in the consensus sequence: [P4=Ala/Val/Pro/Thr]-[P3=X]-[P2=Leu/Phe/Val]-[P1=Gln]|[P1′=Ser/Ala/Asn], where "|" denotes the cleavage site and X any proteinogenic amino acid. 12,[14][15][16][17][18][19] The P4-P1′ residues bind in the corresponding S4-S1′ subsites on M pro . The presence of Gln at P1 is a conserved feature of M pro substrates of SARS-CoV-2 and other coronaviruses.…”
The main protease (Mpro) of the SARS-CoV-2 coronavirus employs a cysteine-histidine dyad in its active site to catalyse hydrolysis of the viral polyproteins. It is well established that binding of the substrate P1-Gln in the S1 subsite of Mproactive site is crucial for catalysis and this interaction has been employed to inform inhibitor design; however, how Mprodynamically recognises and responds to substrate binding remains difficult to probe by experimental methods. We thus employed the dynamical nonequilibrium molecular dynamics (D-NEMD) approach to probe the response of Mproto systematic substrate variations. The results emphasise the importance of P1-Gln for initiating a productive enzymatic reaction. Specifically, substituting P1-Gln with alanine disrupts the conformations of the Cys145 and His41 dyad, causing Cys145 to transition from the productivegaucheconformation to the non-productivetransconformation. Importantly, our findings indicate that Mproexhibits dynamic responses to substrate binding and likely to substrate-mimicking inhibitors within each of the S4-S2′ subsites. The results inform on the substrate selectivity requirements and shed light on the observed variations in hydrolytic efficiencies of Mprotowards different substrates. Some interactions between substrate residues and enzyme subsites involve more induced fit than others, implying that differences in functional group flexibility may optimise the binding of a substrate or inhibitor in a particular subsite.
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