Chronic levels of inflammation lead to autoimmune diseases such as rheumatoid arthritis and atherosclerosis. A key molecular mediator responsible for the progression of these diseases is Chemokine C–C motif ligand 2 (CCL2), a homodimerized cytokine that dissociates into monomeric form and binds to the CCR2 receptor. CCL2, also known as monocyte chemoattractant protein‐1 (MCP‐1), attracts monocytes to migrate to areas of injury and mature into macrophages, leading to positive feedback inflammation with further release of proinflammatory molecules such as IL‐1β and TNF‐α. Sequestering CCL2 to prevent its binding to CCR2 may prevent this inflammatory activity. Prior work adapted an α‐helical CCL2‐binding peptide (WKNFQTI) from murine CCR2 through extracellular loop analysis. Here, higher‐affinity peptide binders are computationally designed through homology modeling and energy calculations, yielding an 11‐amino acid peptide with high binding affinity. In addition, Rosetta mutations improves binding affinity in silico with blockage of the CCL2 dimerization site. Future work in analyzing binding kinetics and in vivo inflammation abrogation will confirm the accuracy of computational modeling techniques in de novo rational design of CCL2 cytokine binders.

The human Betacoronavirus SARS-CoV-2 is a novel pathogen claiming millions of lives and causing a global pandemic that has disrupted international healthcare systems, economies, and communities. The virus is fast mutating and presenting more infectious but less lethal versions. Currently, some small-molecule therapeutics have received FDA emergency use authorization for the treatment of COVID-19, including Lagevrio (molnupiravir) and Paxlovid (nirmaltrevir/ritonavir), which target the RNA-dependent RNA polymerase and the 3CLpro main protease, respectively. Proteins downstream in the viral replication process, specifically the nonstructural proteins (Nsps1−16), are potential drug targets due to their crucial functions. Of these Nsps, Nsp4 is a particularly promising drug target due to its involvement in the SARS-CoV viral replication and double-membrane vesicle formation (mediated via interaction with Nsp3). Given the degree of sequence conservation of these two Nsps across the Betacoronavirus clade, their protein−protein interactions and functions are likely to be conserved as well in SARS-CoV-2. Through AlphaFold2 and its recent advancements, protein structures were generated of Nsp3 and 4 lumenal loops of interest. Then, using a combination of molecular docking suites and an existing library of lead-like compounds, we virtually screened 7 million ligands to identify five putative ligand inhibitors of Nsp4, which could present an alternative pharmaceutical approach against SARS-CoV-2. These ligands exhibit promising lead-like properties (ideal molecular weight and log P profiles), maintain fixed-Nsp4-ligand complexes in molecular dynamics (MD) simulations, and tightly associate with Nsp4 via hydrophobic interactions. Additionally, alternative peptide inhibitors based on Nsp3 were designed and shown in MD simulations to provide a highly stable binding to the Nsp4 protein. Finally, these therapeutics were attached to dendrimer structures to promote their multivalent binding with Nsp4, especially its large flexible luminal loop (Nsp4LLL). The therapeutics tested in this study represent many different approaches for targeting large flexible protein structures, especially those localized to the ER. This study is the first work targeting the membrane rearrangement system of viruses and will serve as a potential avenue for treating viruses with similar replicative function.

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