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ABSTRACT The human angiotensin-converting enzyme 2 (hACE2) is the primary receptor for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Some human alleles of ACE2 exhibit an improved affinity for the SARS-CoV-2 Spike protein. However, the impact of ACE2 polymorphisms on SARS-CoV-2 infection remains unclear. Our previous study predicted that G431 and S514 in the receptor-binding domain (RBD) of SARS-CoV-2 S1 domain are important for S protein stability, and that S protein residues G496 and F497 and ACE2 residues D355 and Y41 are critical for the RBD-ACE2 interaction. In this study, we explored the potential of hACE2-derived neutralizing peptides as a therapeutic strategy against SARS-CoV-2 and investigated how ACE2 polymorphisms affect RBD-ACE2 binding affinity. We applied computational saturation mutagenesis to systematically screen the binding affinity changes among all possible ACE2 missense mutations within the ACE2-Wuhan-S1 complex. Mutations at ACE2 residues D355 and Y41 were predicted to weaken binding affinity, whereas those at N330 and D30 enhanced it. We identified six ACE2 regions (19–49, 65–102, 320–333, 348–359, 378–395, and 552–563) to be vital for ACE2-RBD interaction. We synthesized peptides corresponding to these six regions and tested them using a pseudotyped viral particle system and dot blot assay. Three peptides were confirmed to bind with the S protein, and four exhibited inhibitory effects. We aligned ACE2-Wuhan-S1 and ACE2-Omicron-S1 complexes, conducted correlation analysis, and observed similar binding patterns, suggesting that these peptides also have the potential to neutralize Omicron strains.IMPORTANCESARS-CoV-2 continues its global spread. In this research, we identified six regions within ACE2 that are vital for interaction with the viral S receptor-binding domain and have the potential to neutralize SARS-CoV-2 infection. Among the six peptides derived from ACE2, three were confirmed to bind with the S protein of the Wuhan strain, and four exhibited inhibitory effects on the Wuhan strain SARS-CoV-2. We also found ACE2 residues D355 and Y41 as weakening affinity, and N330 and D30 as enhancing it. We also aligned this complex with the ACE2-Omicron-S1 complex, performed correlation analyses, and compared their patterns of stability changes upon mutations and obtained similar results, indicating that these peptides may also be effective against Omicron variants. These results provide insight into the role of ACE2 polymorphism in viral entry and suggest that hACE2-derived peptides may offer a promising therapeutic strategy against SARS-CoV-2, demonstrating strong consistency between our computational predictions and experimental outcomes. 

The global effort to combat the COVID-19 pandemic faces ongoing uncertainty with the emergence of Variants of Concern featuring numerous mutations on the Spike (S) protein. In particular, the Omicron Variant is distinguished by 32 mutations, including 10 within its receptor-binding domain (RBD). These mutations significantly impact viral infectivity and the efficacy of vaccines and antibodies currently in use for therapeutic purposes. In our study, we employed structure-based computational saturation mutagenesis approaches to predict the effects of Omicron missense mutations on RBD stability and binding affinity, comparing them to the original Wuhan-Hu-1 strain. Our results predict that mutations such as G431W and P507W induce the most substantial destabilizations in the Wuhan-Hu-1-S/Omicron-S RBD. Notably, we postulate that mutations in the Omicron-S exhibit a higher percentage of enhancing binding affinity compared to Wuhan-S. We found that the mutations at residue positions G447, Y449, F456, F486, and S496 led to significant changes in binding affinity. In summary, our findings may shed light on the widespread prevalence of Omicron mutations in human populations. The Omicron mutations that potentially enhance their affinity for human receptors may facilitate increased viral binding and internalization in infected cells, thereby enhancing infectivity. This informs the development of new neutralizing antibodies capable of targeting Omicron’s immune-evading mutations, potentially aiding in the ongoing battle against the COVID-19 pandemic. 

PURPOSEThyroid peroxidase (TPO) is essential for the synthesis of thyroid hormones. However, specific mutations render TPO antigenic and prone to autoimmune attacks leading to thyroid cancer, TPO deficiency, and congenital hypothyroidism (CH). Despite technological advancement, most experimental procedures cannot quickly identify the genetic causes of CH nor detect thyroid cancer in the early stages. METHODSWe performed saturated computational mutagenesis to calculate the folding energy changes (∆∆G) caused by missense mutations and analyzed the mutations involved in post-translational modifications (PTMs). RESULTSOur results showed that the functional important missense mutations occurred in the heme peroxidase domain. Through computational saturation mutagenesis, we identified the TPO mutations in G393 and G348 affecting protein stability and PTMs. Our folding energy calculations revealed that seven of nine somatic thyroid cancer mutations destabilized TPO. CONCLUSIONThese findings highlight the impact of these specific mutations on TPO stability, linking them to thyroid cancer and other genetic thyroid-related disorders. Our results show that computational mutagenesis of proteins provides a quick insight into rare mutations causing Mendelian disorders and cancers in humans.