Design of a bifunctional pan-sarbecovirus entry inhibitor targeting the cell receptor and viral fusion protein.

Development of highly effective antivirals that are robust to viral evolution is a practical strategy for combating the continuously evolved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Inspired by viral multistep entry process, we here focus on developing a bispecific SARS-CoV-2 entry inhibitor, which acts on the cell receptor angiotensin converting enzyme 2 (ACE2) and viral S2 fusion protein. First, we identified a panel of diverse spike (S) receptor-binding domains (RBDs) and found that the RBD derived from Guangdong pangolin coronavirus (PCoV-GD) possessed the most potent antiviral potency. Next, we created a bispecific inhibitor termed RBD-IPB01 by genetically linking a peptide fusion inhibitor IPB01 to the C-terminal of PCoV-GD RBD, which exhibited greatly increased antiviral potency via cell membrane ACE2 anchoring. Promisingly, RBD-IPB01 had a uniformly bifunctional inhibition on divergent pseudo- and authentic SARS-CoV-2 variants, including multiple Omicron subvariants. RBD-IPB01 also showed consistently cross-inhibition of other sarbecoviruses, including SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus (PCoV-GX). RBD-IPB01 displayed low cytotoxicity, high trypsin resistance, and favorable metabolic stability. Combined, our studies have provided a tantalizing insight into the design of broad-spectrum and potent antiviral agent. IMPORTANCE Ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution and spillover potential of a wide variety of sarbecovirus lineages indicate the importance of developing highly effective antivirals with broad capability. By directing host angiotensin converting enzyme 2 receptor and viral S2 fusion protein, we have created a dual-targeted virus entry inhibitor with high antiviral potency and breadth. The inhibitor receptor-binding domain (RBD)-IPB01 with the Guangdong pangolin coronavirus (PCoV-GD) spike RBD and a fusion inhibitor IPB01 displays bifunctional cross-inhibitions on pseudo- and authentic SARS-CoV-2 variants including Omicron, as well as on the sarbecoviruses SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus. RBD-IPB01 also efficiently inhibits diverse SARS-CoV-2 infection of human Calu-3 cells and blocks viral S-mediated cell-cell fusion with a dual function. Thus, the creation of such a bifunctional inhibitor with pan-sarbecovirus neutralizing capability has not only provided a potential weapon to combat future SARS-CoV-2 variants or yet-to-emerge zoonotic sarbecovirus, but also verified a viable strategy for the designing of antivirals against infection of other enveloped viruses.

Susceptibility and Resistance of SARS-CoV-2 Variants to LCB1 and Its Multivalent Derivatives.

LCB1 is a computationally designed three-helix miniprotein that precisely targets the spike (S) receptor-binding motif (RBM) of SARS-CoV-2, exhibiting remarkable antiviral efficacy; however, emerging SARS-CoV-2 variants could substantially compromise its neutralization effectiveness. In this study, we constructed two multivalent LCB1 fusion proteins termed LCB1T and LCB1T-Fc, and characterized their potency in inhibiting SARS-CoV-2 pseudovirus and authentic virus in vitro. In the inhibition of various SARS-CoV-2 variants, the two LCB1 fusion proteins exhibited markedly improved inhibitory activities compared to LCB1 as anticipated; however, it was observed that relative to the D614G mutation hosting variant, the variants Delta, Lambda, and Omicron BQ.1.1, XBB, XBB.1.5, and EG.5.1 caused various degrees of resistance to the two fusion proteins' inhibition, with XBB, XBB.1.5, and EG.5.1 variants showing high-level resistance. Moreover, we demonstrated that bat coronavirus RaTG13 and pangolin coronavirus PCoV-GD/PCoV-GX were highly sensitive to two LCB1 fusion proteins, but not LCB1, inhibition. Importantly, our findings revealed a notable decrease in the blocking capacity of the multivalent LCB1 inhibitor on the interaction between the virus's RBD/S and the cell receptor ACE2 when confronted with the XBB variant compared to WT and the Omicron BA.1 variant. In conclusion, our studies provide valuable insights into the antiviral profiling of multivalent LCB1 inhibitors and offer a promising avenue for the development of novel broad-spectrum antiviral therapeutics.

In situ manipulation of fluorescence resonance energy transfer between quantum dots and monolayer graphene oxide by laser irradiation.

The unique optical properties of solution-processable colloidal semiconductor quantum dots (QDs) highlight their promising applications in the next generation of optoelectronic and biomedical technologies. In order to optimize these applications, the tunability of QDs' optical properties is always highly desired. Although the tuning during synthesis stages has been intensively investigated, the in situ alteration after device fabrication is still limited. Here we report the tuning of the optical properties of CdSeTe/ZnS QDs through an in situ manipulation of fluorescence resonance energy transfer (FRET) between QDs and monolayer graphene oxide (GO). By increasing the acceptor's absorption ability of GO through laser irradiation, the efficiency of FRET between QDs and GO has been substantially improved from 29.7% to 70.0%. The corresponding energy transfer rate is enhanced by 5.5 times. These results can be well explored by a spectral overlap between the fluorescence emission of QDs and the absorption of original or reduced GO. Our scheme, with the features of in situ manipulation, high spatial resolution and wireless steering, enables the potential functionality of such hybrid structures in optoelectronic applications.

Co-adsorption of an anionic dye in the presence of a cationic dye and a heavy metal ion by graphene oxide and photoreduced graphene oxide.

To investigate the adsorption behavior of contaminants with different adsorbents and co-adsorbates under identical conditions, the adsorption capacities of anionic orange II (OII) dye onto graphene oxide (GO) and photoreduced GO (PRGO) in a single-component system and in the presence of cationic methylene blue (MB) dye as well as heavy metal ion Pb2+ were explored. In this work, PRGO was prepared by solar light irradiation of a GO dispersion. GO and PRGO were characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, Raman spectroscopy, atomic force microscopy, scanning electron microscopy, and transmission electron microscopy. The adsorption isotherms of OII, MB, and Pb2+ onto GO and PRGO in single and binary systems have been studied and analyzed by the Langmuir model. In the single system, the adsorption capacity of OII on GO can be promoted from 8.4 mg g-1 to 32.5 mg g-1 after solar light irradiation. While the adsorption capacities of MB and Pb2+ are not affected by the photoreduction process. In the binary system, a marked synergistic effect for the adsorption of OII has been determined in the presence of both MB and Pb2+, where the adsorption capacity of OII on PRGO has been improved from 8.4 mg g-1 to 295 mg g-1 and 105 mg g-1, enhancements of 35- and 12.5-fold, respectively. In contrast, the presence of OII leads to a mildly antagonistic effect on the adsorption of MB and Pb2+. These findings show that the adsorption of anionic dyes by graphene-based materials can be strongly improved in the presence of either cationic dyes or heavy metal ions, which will be of great value in practical applications.

Versatile and scalable micropatterns on graphene oxide films based on laser induced fluorescence quenching effect.

Here we report on the preparation of quasi-homogeneous fluorescence emission from graphene oxide (GO) film by modifying the local optical properties through the laser-induced fluorescence quenching effect, and the fabrication of single and multilayer micropatterns on quasi-homogeneous GO films. The modification is stemming from the photoreduction of GO, where the reduction degree and fluorescence intensity can be precisely tuned by changing the laser power and irradiation duration. This versatile approach with a mask-free feature can be readily used to fabricate various complex microstructures on quasi-homogeneous GO film from single layer to multilayer in vertical scale, as well as micrometers to centimeters in lateral scale. The micropatterns with varied optical properties are promising for applications in information storage, display technology, and optoelectronic devices.