An important stage in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) life cycle is the binding of the spike (S) protein to the angiotensin converting enzyme-2 (ACE2) host cell receptor. Therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (MD) simulation. The findings indicated that S2 subunit (heptad-repeat 1 (HR1), central helix (CH), and connector domain (CD) domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the receptor binding domain (RBD) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. Interpretations from MD simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an “up” and “down” state. In addition, the trimer cavity may function as a “bouncing spring” that may facilitate the homotrimer spike protein interactions with the ACE2 receptor. The feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. Several hits were identified that have already been validated or suggested to inhibit the SARS-CoV-2 virus in published cell models. In particular, the data suggest an action mechanism for molecules including Chitosan and macrolides such as the mTOR (mammalian target of Rapamycin) pathway inhibitor Rapamycin. These findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (CoV) family of viruses.

The detection of cancer antigens is a major aim of cancer research in order to develop better patient management through early disease detection. Many cancers including prostate, lung, and ovarian secrete a protein disulfide isomerase protein named AGR2 that has been previously detected in urine and plasma using mass spectrometry. Here we determine whether a previously developed monoclonal antibody targeting AGR2 can be adapted from an indirect two-site ELISA format into a direct detector using solid-phase printed gold electrodes. The screen-printed gold electrode was surface functionalized with the anti-AGR2 specific monoclonal antibody. The interaction of the recombinant AGR2 protein and the anti-AGR2 monoclonal antibody functionalized electrode changed its electrochemical impedance spectra. Nyquist diagrams were obtained after incubation in an increasing concentration of purified AGR2 protein with a range of concentrations from 0.01 fg/mL to 10 fg/mL. In addition, detection of the AGR2 antigen can be achieved from cell lysates in medium or artificial buffer. These data highlight the utility of an AGR2-specific monoclonal antibody that can be functionalized onto a gold printed electrode for a one-step capture and quantitation of the target antigen. These platforms have the potential for supporting methodologies using more complex bodily fluids including plasma and urine for improved cancer diagnostics.

An important stage in SARS-CoV-2 life cycle is the fusion of spike(S) protein with the ACE2 host-cell receptor. Therefore, to explore conserved features in S protein dynamics and to identify potentially novel regions for drugging, we measured variability derived from 791 viral genomes and studied its properties by MD simulation. The findings indicated that S2 subunit (HR1, CH, and CD domains) showed low variability, low fluctuations in MD, and displayed a trimer cavity. By contrast, the RBD domain, which is typically targeted in drug discovery programmes, exhibits more sequence variability and flexibility. Interpretations from MD suggest that the monomer is in constant motion showing transitions up-to-down state, and the trimer cavity may function as a 'bouncing spring' that may facilitates S protein interactions with ACE2. Feasibility of trimer cavity for potential drug target was examined by SBVS screening. Several hits that have already been validated or suggested to inhibit the SARS-CoV-2 virus in cell systems were identified; in particular, the data suggest an action mechanism for such molecules including Chitosan and macrolide types. These findings identify a novel binding-site formed by the S protein, that might assist in future drug discovery programmes aimed at targeting the CoV family of viruses.

Anti-idiotype monoclonal antibodies represent a class of reagents that are potentially optimal for analyzing the pharmacokinetics of fully human, anti-infective antibodies that have been developed as therapeutic candidates. This is particularly important where direct pathogen binding assays are complicated by requirements for biosafety level III or IV for pathogen handling. In this study, we describe the development of a recombinant, anti-idiotype monoclonal antibody termed E1 for the detection of a fully human, serotype-specific, therapeutic antibody candidate for the BSLIII pathogen Dengue virus termed 14c10 hG1. E1 was generated by naïve human Fab phage library panning technology and subsequently engineered as a monoclonal antibody. We show that E1 is highly specific for the fully-folded form of 14c10 hG1 and can be employed for the detection of this antibody in healthy human subjects’ serum by enzyme linked immunosorbent assay. In addition, we show that E1 is capable of blocking the binding of 14c10 hG1 to dengue virus serotype 1. Finally, we show that E1 can detect 14c10 hG1 in mouse serum after the administration of the therapeutic antibody in vivo. E1 represents an important new form of ancillary reagent that can be utilized in the clinical development of a therapeutic human antibody candidate.

Defining dynamic protein–protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.