Nevena Ilieva

Highlights: • NSP8-N binds NSP13, occupying residues required for TBK1 and NSP8-NSP13 association. • NSP8-N blocks NSP13-TBK1, occupies RTC, and inhibits immune evasion and replication. • NSP8-N restores the IFN-β expression in cells coexpressing NSP13.

The SARS-CoV-2 spike receptor-binding motif is crucial for viral entry via interaction with the human ACE2 receptor. Mutations N501Y and E484K, found in several variants of concern, impact viral transmissibility and immune escape, but experimental data on their binding effects remain inconsistent. Using isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations, we analyzed the thermodynamic and structural effects of these mutations. ITC confirmed that N501Y increases ACE2 affinity by 2.2-fold, while E484K enhances binding by 5.8-fold. The Beta/Gamma variant (carrying both mutations) showed the strongest affinity, with a 15-fold increase. E484K was enthalpy-driven, while N501Y introduced entropy-driven effects, suggesting hydrophobic interactions and conformational changes. MD simulations revealed distinct binding poses, with Beta/Gamma peptides interacting with a secondary ACE2 site. A strong correlation was found between entropy contributions and hydrophobic contacts. Additionally, a convolutional neural network was used to estimate the free binding energy of these complexes. Our findings confirm that N501Y and E484K enhance ACE2 binding, with the greatest effect when combined, providing insights into SARS-CoV-2 variant evolution and potential therapeutic strategies.

Human interferon γ (hIFNγ) is a crucial immunomodulatory cytokine that exerts its effects by binding to a high-affinity receptor complex on the cell surface. In addition to receptor binding, hIFNγ also displays strong affinity for heparin and heparan sulphate, primarily via its C-terminal domain. This interaction influences various physicochemical properties of the cytokine, including its receptor affinity, and could be harnessed for designing hIFNγ inhibitors. Current experimental methods cannot precisely determine sulphate group arrangements in heparin-like and HS-like octasaccharides, which influence charge distribution and potentially affect their binding affinity to the hIFNγ C-terminal peptide. Here, we propose an in silico approach to explore different sulphation patterns in octasaccharides containing one acetyl and seven sulphate groups, aiming to identify those that optimize binding to the hIFNγ C-terminal peptide.

Proteins are currently the fastest-growing class of new therapeutic compounds but smaller proteins and peptides are generally not suitable for use as drugs. Using cyclotides – special knotted proteins stabilized by three pairs of disulphide bonds – as a transport means by grafting onto them as a scaffolding the bioactive peptides can enhance their stability, cellular uptake, and overall efficacy. Experimental methods for creating peptide aptamers are highly time- and resources-consuming. In silico approaches may speed up this process by pre-selecting the drug candidates based on certain biodynamic criteria. In this study, we probe the hypothesis about a relation between the scaffolding conformational stability in conjunction with certain plasticity upon grafting of functionally important domains and the desired biological activity of the modified through the grafting process molecules.