Purification and characterisation of heparin-like sulfated polysaccharides with potent anti-SARS-CoV-2 activity from snail mucus of Achatina fulica.

Heparin-like sulfated polysaccharide, acharan sulfate, was purified from the mucus of an African giant snail with unique sulfated glycosaminoglycans (GAGs). This study reported on finding novel and safe heparin resources from Achatina fulica for further use as well as easy isolation and purification of the active fraction from the initial raw material. Its structure was characterised by a strong-anion exchange combined with high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that the potential acharan sulfate fraction is a glycosaminoglycan composed of several repeating disaccharide units, namely, of →4)-α-IdoA(2S)(1→4)-α-GlcNAc/GlcNAc(6S)/GlcNSO3(6S)(1→, and hence, presents heterogeneity regarding negative net charge density. Furthermore, the heparinase digests inhibit the binding of SARS-CoV-2 spike protein to the ACE2 receptor. In summary, the acharan sulfate presented in this work has shown its great potential for application in the preparation of sulfated polysaccharides as an alternative to heparin with important biological activity.

Biological properties of reverse ankyrin engineered for dimer construction to enhance HIV-1 capsid interaction.

BACKGROUND: Assembly and budding in the late-stage of human immunodeficiency virus type 1 (HIV-1) production rely on Gag protein polymerization at the inner leaflet of the plasma membrane. We previously generated a monomeric ankyrin repeat protein (Ank1D4) that specifically interacts with capsid protein (CAp24) of HIV-1, however this protein had modest binding affinity. OBJECTIVE: This study aimed to improve the avidity of Ank1D4 by generating two Ank1D4 dimers: (Ank1D4NC-NC) and its inverted form (Ank1D4NC-CN), with each domain connected by a flexible (G4S)4 linker peptide. METHODS: Binding properties of monomeric and dimeric Ank1D4 was performed by capture enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA was used to examine bifunctional module of dimeric Ank1D4. Ank1D4NC-NC and Ank1D4NC-CN were evaluated using bio-layer interferometry (BLI), compared to monomeric Ank1D4. RESULTS: Similar binding surfaces were observed in both dimers which was comparable with monomeric Ank1D4. The interaction of Ank1D4NC-CN with CAp24 was significantly greater than that of Ank1D4NC-NC and Ank1D4 by capture ELISA. Ank1D4NC-CN also exhibited bifunctionality using a sandwich ELISA. The KD of Ank1D4NC-CN, Ank1D4NC-NC and monomeric Ank1D4 was 3.5 nM, 53.7 nM, and 126.2 nM, respectively using bio-layer interferometry analysis. CONCLUSIONS: This study provides a strategy for increasing Ank1D4 avidity through the construction of novel inverted dimers with a flexible linker. Ank1D4NC-CN may provide an alternative treatment strategy for inhibiting HIV-1 replication.

Mutational scanning of spike RBD protein for enhanced ACE2 affinity emerging Southeast Asia in the late transmission phase.

The COVID-19 pandemic has changed the quality of life and economic systems all over the world, as the virus can be transmitted from human to human via air-droplets. Since the SARS-CoV-2 virus was first identified in 2019, the virus has naturally mutated over time. Southeast Asia is one of the areas in the world that has implemented various procedures and measures to slow down the disease outbreaks. The first cluster of COVID-19 was identified from the tourist-travel history, and then the diversity of coronavirus victims has posed a serious issue of human security on a massive scale. To evaluate whether or not naturally occurring mutations have strengthened the infectivity of SARS-CoV-2, we computed in silico the structural dynamics of the RBD-spike protein mutation enhancing ACE2-binding. When considering emerging variations in Southeast Asia, 14 dominant mutations were analyzed by applying the structural and energetic characterization using MD simulations. The ones in the RBD region displayed higher affinity to ACE2 due to the improved interfacial stability of the RBD ß-strand surrounding the ACE2 across salt bridge hotspots. The binding hotspots and structurally conserved conformational-epitopes have been identified, which are deleterious for RBD mutation and ACE2 binding. We present an interactive visualization to facilitate the development of effective neutralizing agents for vaccination, prevention and treatment.