Scalable Inhibitors of the Nsp3–Nsp4 Coupling in SARS-CoV‑2

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.