APP and APLP2 Kunitz domains are identified as potent endogenous inhibitors of TMPRSS2-dependent respiratory virus infection and promising scaffolds for host-directed broad-spectrum antiviral strategies.
Abstract
Respiratory viruses depend on host proteases for activation of viral fusion proteins, making these enzymes attractive targets for broad-spectrum antiviral strategies. We previously identified Trypstatin, a human Bikunin-derived Kunitz domain, as a potent endogenous inhibitor of the airway serine protease TMPRSS2. Here, we investigated whether TMPRSS2 inhibition is shared by additional human Kunitz domains. Kunitz domains with high sequence similarity to Trypstatin were synthesized, refolded, and functionally characterized. Domains derived from amyloid precursor protein (APP) and amyloid precursor-like protein 2 (APLP2) potently inhibited TMPRSS2, with APP displaying subnanomolar activity comparable to camostat mesylate. APP and APLP2 selectively blocked SARS-CoV-2 Spike-mediated entry without affecting VSV-G-mediated entry or cell viability and inhibited infection by multiple coronaviruses and influenza viruses, but not TMPRSS2-independent rhinovirus. In primary human airway epithelial cultures, APP and Trypstatin reduced replication of SARS-CoV-2, endemic coronaviruses, and influenza A virus, and remained stable in airway mucus. These findings identify APP and APLP2 Kunitz domains as potent endogenous inhibitors of TMPRSS2-dependent respiratory virus infection and promising scaffolds for host-directed broad-spectrum antivirals.
The type II transmembrane serine protease TMPRSS2 plays a critical role in respiratory virus entry, including SARS-CoV-2. Using an mRNA display platform with genetic code reprogramming, we identified macrocyclic peptide (MCP) inhibitors of TMPRSS2 that bound to the TMPRSS2 active site. The most potent MCPs displayed picomolar binding affinities and excellent selectivity across a panel of 22 trypsin-fold serine proteases. Although the lead MCP showed strong in vitro potency, proteolytic instability reduced its cellular activity. Rational optimization yielded a stability-enhanced variant, T2-MCP-19, which bound TMPRSS2 with high affinity (KD = 80 pM) and inhibited the uptake of virus-like particles pseudotyped with SARS-CoV-2 spike protein in lung epithelial Calu-3 cells with nanomolar potency. Notably, several MCPs exhibited dual functionality by inhibiting both the enzymatic and receptor functions of TMPRSS2, as demonstrated by their blockade of the HKU1 coronavirus spike protein binding. These results highlight MCPs as promising, highly selective TMPRSS2-directed antiviral lead compounds.
Benjamin J. Tombling, Gabriel Lemieux, Alexandre Joushomme et al.· Journal of Medicinal Chemist...· 0 citations
HRBD demonstrated potent and broad-spectrum inhibition against Pangolin-CoV, SARS-CoV, SARS-CoV, SARS-CoV-2, and its variants, lowering the half-maximal inhibitory concentration (IC50) by approximately 1000-fold compared to the monomeric RBD.
Jintao Zou, Lingyu Su, Jiansheng Lu et al.· Antiviral Research· 0 citations
The receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein remains a central target for antiviral drug development. Recent in silico studies have revealed an expansion of amyloidogenic regions within the RBD of the Omicron variant, raising the possibility that amyloid-prone peptide fragments could modulate Spike function or host–virus interactions. In this study, we combined experimental assays with multiscale computational modeling to systematically characterise two short RBD-derived peptides: Pep-2 (YFPLQSYGFQ) from the ancestral Wuhan strain and Pep-3 (YFPLRSYSFR) from the Omicron BA.1 variant, the latter being predicted to have higher amyloidogenic potential. Cell-based assays demonstrated that neither peptide exhibited intrinsic cytotoxic or cytostatic effects on human lung fibroblasts or A549 lung adenocarcinoma cells at physiologically relevant concentrations, whereas significant cytotoxicity was observed in Vero E6 cells. In infection models with the B.1.1.1 (Wuhan) and BA.1 (Omicron) variants, the peptides unexpectedly enhanced virus-induced cytopathic effects at lower concentrations but inhibited viral infection at higher concentrations, indicating to a dose-dependent modulatory role for these short amyloidogenic RBD fragments. Fluorescence spectroscopy measurements did not detect the formation of stable thioflavin-T-positive amyloid fibrils. Computational analyses revealed that both peptides interact with the Spike RBD via multiple energetically favorable yet spatially heterogeneous modes, mostly outside the ACE2-binding site. Moreover, their predicted binding affinities for the ACE2 receptor were comparable, suggesting an additional route of interaction via the host receptor. Collectively, our findings demonstrate that these short amyloidogenic RBD-derived peptides exert a complex antiviral profile, with their interactions with both viral and host factors potentially shaping infection outcomes. This highlights the importance of spatially targeted and conformationally constrained peptide designs to effectively harness amyloidogenic features for antiviral therapy.
M. Nikiforova, S. Grishin, A. Aksenova et al.· International Journal of Mol...· 0 citations
The rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and associated complement overactivation challenge current antiviral strategies that mainly target viral entry. This study aimed to develop dual targeting engineered binding proteins capable of simultaneously blocking viral infection and complement activation. Two proteins, SBP10 and SBP16, were engineered by integrating an ACE2-mimicking peptide with a mannose-binding lectin (MBL) domain. Binding affinity and antiviral activity were evaluated using biochemical and functional assays, including inhibition of S protein-ACE2 interaction, neutralization of multiple SARS-CoV-2 variants, and assessment of lectin pathway-mediated complement activation. Both SBP10 and SBP16 bound the spike protein with low-nanomolar affinity and effectively blocked its interaction with ACE2. The proteins exhibited broad-spectrum neutralizing activity against several variants, including Alpha, Beta, Delta, and Omicron. Moreover, they significantly suppressed spike-induced activation of the lectin complement pathway. In vivo experiments further demonstrated that treatment with SBP10 or SBP16 markedly reduced spike protein-induced lung injury. In conclusion, SBP10 and SBP16 function as dual targeting engineered binding proteins that inhibit viral entry while attenuating complement-mediated inflammation, highlighting a promising therapeutic strategy for controlling SARS-CoV-2 infection and its associated immune dysregulation.
Fan Pu, Yiwan Guo, Xinni Pan et al.· Antiviral Research· 0 citations
PDZ domains are one of the largest families of short linear motif (or peptide) binding domains in the human proteome. These scaffolding domains are important in signaling and trafficking pathways, such as in the formation of tight junctions or in the postsynaptic density of neurons. PDZ domains are also targeted by several pathogenic viral proteins, including human papillomavirus (HPV), influenza, hepatitis, rabies, coronaviruses, among others. Previously, we investigated the specificity determinants of modulator, or non‐motif, residues for two PDZ binding motifs (or PBMs) from the cystic fibrosis transmembrane conductance regulator (CFTR) and HPV16 E6 proteins to better understand differences in relative promiscuity between these similar sequences. To test whether viral PDZ‐binding motifs are inherently more promiscuous than endogenous targets, we measured binding affinities for 7 endogenous and 7 viral PBMs across 8 well‐studied Class I PDZ domains. Fluorescence anisotropy assays revealed no significant difference in relative binding affinities between viral and endogenous PBMs, indicating that viral versus endogenous origin alone does not explain PBM promiscuity. Our results were consistent with available data from high throughput holdup assays. Taken together, our data support previously reported sequence hotspots, whereby certain PBMs are recognized by large numbers of PDZ domains, likely due to a combination of widely favorable modulator residues.
Cole D. Masuga, Caroline Ceravolo, Elise F. Tahti et al.· Protein Science· 0 citations
It is demonstrated that nanobodies targeting the PLpro/ISG15 interface can achieve synergistic antiviral and immunomodulatory effects, providing a proof-of-concept for a novel therapeutic approach to combat SARS-CoV-2 and potentially other emerging coronaviruses.
Guo-Long Liu, Jiantao Chen, Fang Wu et al.· Journal of Virology· 0 citations
