A substrate for a cell free in vitro assay system to screen drugs targeting trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein and viral entry.

Host proteases trypsin and trypsin-like proteases have been reported to facilitate the entry of coronavirus SARS-CoV-2 in its host cells. These protease enzymes cleave the viral surface glycoprotein, spike, leading to successful cell surface receptor attachment, fusion and entry of the virus in its host cell. The spike protein has protease cleavage sites between the two domains S1 and S2. Since the cleavage site is recognized by the host proteases, it can be a potential antiviral therapeutic target. Trypsin-like proteases play an important role in virus infectivity and the property of spike protein cleavage by trypsin and trypsin-like proteases can be used to design assays for screening of antiviral candidates against spike protein cleavage. Here, we have documented the development of a proof-of-concept assay system for screening drugs against trypsin/trypsin-like proteases that cleave spike protein between its S1 and S2 domains. The assay system developed uses a fusion substrate protein containing a NanoLuc luciferase reporter protein, the protease cleavage site between S1 and S2 domains of SARS-CoV-2 spike protein and a cellulose binding domain. The substrate protein can be immobilized on cellulose via the cellulose binding domain of the substrate. When trypsin and trypsin-like proteases cleave the substrate, the cellulose binding domain remain bound to the cellulose and the reporter protein is dislodged. Reporter assay using the released reporter protein is the read out of the protease activity. We have demonstrated the proof-of-concept using multiple proteases like trypsin, TMPRSS2, furin, cathepsin B, human airway trypsin and cathepsin L. A significant increment in fold change was observed with increasing enzyme concentration and incubation time. Introduction of increasing amounts of enzyme inhibitors in the reaction reduced the luminescent signal, thus validating the assay. Furthermore, we used SDS-PAGE and immunoblot analyses to study the cleavage band pattern and re-confirm the cleavage for enzymes tested in the assay. Taken together, we have tested an in-vitro assay system using the proposed substrate for screening drugs against trypsin like protease-based cleavage of SARS-CoV-2 spike glycoprotein. The assay system can also be potentially used for antiviral drug screening against any other enzyme that might cleave the used cleavage site.

Plasmonic Micro-Channel Assisted Photonic Crystal Fiber Based Highly Sensitive Sensor for Multi-Analyte Detection.

A dual-channel propagation controlled photonic crystal fiber (PCF)-based plasmonic sensor was presented to detect multiple analytes simultaneously. Plasmonic micro-channels were placed on the outer surface of the PCF, which facilitates an easy sensing mechanism. The sensor was numerically investigated by the finite element method (FEM) with the perfectly matched layer (PML) boundary conditions. The proposed sensor performances were analyzed based on optimized sensor parameters, such as confinement loss, resonance coupling, resolution, sensitivity, and figure of merit (FOM). The proposed sensor showed a maximum wavelength sensitivity (WS) of 25,000 nm/refractive index unit (RIU) with a maximum sensor resolution (SR) of 4.0 × 10-6 RIU for channel 2 (Ch-2), and WS of 3000 nm/RIU with SR of 3.33 × 10-5 RIU for channel 1 (Ch-1). To the best of our knowledge, the proposed sensor exhibits the highest WS compared with the previously reported multi-analyte based PCF surface plasmon resonance (SPR) sensors. The proposed sensor could detect the unknown analytes within the refractive index (RI) range of 1.32 to 1.39 in the visible to near infrared region (550 to 1300 nm). In addition, the proposed sensor offers the maximum Figure of Merit (FOM) of 150 and 500 RIU-1 with the limit of detection (LOD) of 1.11 × 10-8 RIU2/nm and 1.6 × 10-10 RIU2/nm for Ch-1 and Ch-2, respectively. Due to its highly sensitive nature, the proposed multi-analyte PCF SPR sensor could be a prominent candidate in the field of biosensing to detect biomolecule interactions and chemical sensing.

Repurposing nonnucleoside antivirals against SARS-CoV2 NSP12 (RNA dependent RNA polymerase): In silico-molecular insight.

The pandemic of SARS-CoV-2 has necessitated expedited research efforts towards finding potential antiviral targets and drug development measures. While new drug discovery is time consuming, drug repurposing has been a promising area for elaborate virtual screening and identification of existing FDA approved drugs that could possibly be used for targeting against functions of various proteins of SARS-CoV-2 virus. RNA dependent RNA polymerase (RdRp) is an important enzyme for the virus that mediates replication of the viral RNA. Inhibition of RdRp could inhibit viral RNA replication and thus new virus particle production. Here, we screened non-nucleoside antivirals and found three out of them to be strongest in binding to RdRp out of which two retained binding even using molecular dynamic simulations. We propose these two drugs as potential RdRp inhibitors which need further in-depth testing.

Polymonoclonal (Not Polyclonal) Antibodies Derived from Convalescent Human B Cell Hybridomas Might Be a Better Therapeutic Option than Single Target Monoclonal Antibodies.

Both human B cell hybridoma technology and convalescent plasma therapy are promising immunological tools for therapeutic interventions. Here we propose using antibody producing B cells from convalescent SARS-CoV2 patients for developing human B cell hybridomas, and a combination of monoclonal antibodies against multiple immunogenic targets of SARS-CoV-2 spike protein might deliver an antibody cocktail for long-lasting therapeutic targeting.