Biosensors for the Determination of SARS-CoV-2 Virus and Diagnosis of COVID-19 Infection

Monitoring and tracking infection is required in order to reduce the spread of the coronavirus disease 2019 (COVID-19), induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, the development and deployment of quick, accurate, and sensitive diagnostic methods are necessary. The determination of the SARS-CoV-2 virus is performed by biosensing devices, which vary according to detection methods and the biomarkers which are inducing/providing an analytical signal. RNA hybridisation, antigen-antibody affinity interaction, and a variety of other biological reactions are commonly used to generate analytical signals that can be precisely detected using electrochemical, electrochemiluminescence, optical, and other methodologies and transducers. Electrochemical biosensors, in particular, correspond to the current trend of bioanalytical process acceleration and simplification. Immunosensors are based on the determination of antigen-antibody interaction, which on some occasions can be determined in a label-free mode with sufficient sensitivity.

Grass Carp (Ctenopharyngodon idella) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

Post-translational modifications (PTMs), such as phosphorylation and ubiquitination, etc., have been reported to modulate the activities of IRF3 and IRF7. In this study, we found an acetyltransferase KAT8 in grass carp (CiKAT8, MW286472) that acetylated IRF3/IRF7 and then resulted in inhibition of IFN 1 response. CiKAT8 expression was up-regulated in the cells under poly I:C, B-DNA or Z-DNA stimulation as well as GCRV(strain 873) or SVCV infection. The acetyltransferase domain (MYST domain) of KAT8 promoted the acetylation of IRF3 and IRF7 through the direct interaction with them. So, the domain is essential for KAT8 function. Expectedly, KAT8 without MYST domain (KAT8-△264-487) was granularly aggregated in the nucleus and failed to down-regulate IFN 1 expression. Subcellular localization analysis showed that KAT8 protein was evenly distributed in the nucleus. In addition, we found that KAT8 inhibited the recruitment of IRF3 and IRF7 to ISRE response element. Taken together, our findings revealed that grass carp KAT8 blocked the activities of IRF3 and IRF7 by acetylating them, resulting in a low affinity interaction of ISRE response element with IRF3 and IRF7, and then inhibiting nucleic acids-induced innate immune response.

Molecular Basis for CPC-Sgo1 Interaction: Implications for Centromere Localisation and Function of the CPC

The Chromosomal Passenger Complex (CPC; consisting of Borealin, Survivin, INCENP and Aurora B kinase) and Shugoshin 1 (Sgo1) are key regulators of chromosome bi-orientation, a process essential for error-free chromosome segregation. Their functions rely on their ability to associate with centromeres. Two histone phosphorylations, histone H3 Thr3 (H3T3ph; directly recognised by Survivin) and histone H2A Thr120 (H2AT120ph; indirectly recognised via Sgo1), together with CPC’s intrinsic ability to bind nucleosome, facilitate CPC centromere recruitment. The molecular basis for CPC-Sgo1 binding and how their direct interaction influences CPC centromere localisation and function are lacking. Here, using an integrative structure-function approach, we show that the histone H3-like Sgo1 N-terminal tail interacts with Survivin acting as a hot-spot for CPC-Sgo1 assembly, while downstream Sgo1 residues, mainly with Borealin contributes for high affinity interaction. Disruption of the Sgo1 N-terminal tail-Survivin interaction abolished CPC-Sgo1 assembly in vitro and perturbed centromere localisation and function of CPC. Our findings provide evidence that CPC binding to Sgo1 and histone H3 N-terminal tail are mutually exclusive, suggesting that these interactions will likely take place in a spatially/temporally restricted manner and provide a rationale for the Sgo1-mediated ‘kinetochore proximal centromere’ pool of CPC.

Quantification of circadian interactions and protein abundance defines a mechanism for operational stability of the circadian clock

The mammalian circadian clock exerts substantial control of daily gene expression through cycles of DNA binding. Understanding of mechanisms driving the circadian clock is hampered by lack of quantitative data, without which predictive mathematical models cannot be developed. Here we develop a quantitative understanding of how a finite pool of BMAL1 protein can regulate thousands of target sites over daily time scales. We have used fluorescent correlation spectroscopy (FCS) to track dynamic changes in CRISPR-modified fluorophore-tagged proteins in time and space in single cells across SCN and peripheral tissues. We determine the contribution of multiple rhythmic processes in coordinating BMAL1 DNA binding, including the roles of cycling molecular abundance, binding affinities and two repressive modes of action. We find that nuclear BMAL1 protein numbers determine corresponding nuclear CLOCK concentrations through heterodimerization and define a DNA residence time of 2.6 seconds for this complex. Repression of CLOCK:BMAL1 is in part achieved through rhythmic changes to BMAL1:CRY1 affinity as well as a high affinity interaction between PER2:CRY1 which mediates CLOCK:BMAL1 displacement from DNA. Finally, stochastic modelling of these data reveals a dual role for PER:CRY complexes in which increasing concentrations of PER2:CRY1 promotes removal of BMAL1:CLOCK from genes consequently enhancing ability to move to new target sites.

Identification of Potent Small Molecule Inhibitors of SARS-CoV-2 Entry

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, Calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals Calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

SARS-CoV-2 spike protein 13-mer subdomain corresponds to the drug-binding domain of glutamyl-propyl-tRNA synthetase 1 and is targetable by halofuginone

Since its emergence, SARS-CoV-2 has been the subject of intense investigation. Early sequence analysis identified a unique 13 amino acid region (13-mer) nested within the receptor-binding domain (RBD) of the spike protein that directly interacts with the ACE2 receptor. Blasting with the 13-mer identified a highly conserved segment in propyl-tRNA synthetase enzymes. Comparison with the human analogue, glutamyl-propyl-tRNA synthetase 1, showed a high level of identity with its drug binding domain, which is targeted by halofuginone, a drug recently shown to block SARS-CoV-2 infection in vitro. In silico experiments predicted a high affinity interaction between halofuginone and the 13-mer. In vitro addition of halofuginone effectively inhibited binding of recombinant S1 monomer to ACE2. Accordingly, it appears that halofuginone inhibits viral infection by preventing correct interactions between spike protein and ACE2. These findings indicate that viral entry can potentially be drug-targeted and support the application of halofuginone in mitigation of COVID-19.

SARS-CoV-2 spike protein 13-mer subdomain corresponds to the drug-binding domain of 3 glutamyl-propyl-tRNA synthetase 1 and is targetable by halofuginone

Since its emergence, SARS-CoV-2 has been the subject of intense investigation. Early sequence analysis identified a unique 13 amino acid region (13-mer) nested within the receptor-binding domain (RBD) of the spike protein that directly interacts with the ACE2 receptor. Blasting with the 13-mer identified a highly conserved segment in propyl-tRNA synthetase enzymes. Comparison with the human analogue, glutamyl-propyl-tRNA synthetase 1, showed a high level of identity with its drug binding domain, which is targeted by halofuginone, a drug recently shown to block SARS-CoV-2 infection in vitro. In silico experiments predicted a high affinity interaction between halofuginone and the 13-mer. In vitro addition of halofuginone effectively inhibited binding of recombinant S1 monomer to ACE2. Accordingly, it appears that halofuginone inhibits viral infection by preventing correct interactions between spike protein and ACE2. These findings indicate that viral entry can potentially be drug-targeted and support the application of halofuginone in mitigation of COVID-19.

Melanin, a potential new matrix material for recombinant His-tag protein purification

Nickel-Sepharose (Ni-Sepharose) has been being applied as the most common matrix in purifying His-tag proteins based on the affinity interaction between histidine residues and Ni2+ ion. However, Sepharose still comes at high cost for this purification purpose, especially in developing countries as Vietnam. Here, we show for the first time that melanin from ink sacs of squids which is considered as biowaste in the food industry, can be used as a new potential matrix material instead of Sepharose. We utilized either melanin or melanin charged with metal ions as the stationary phase of affinity purification of His-tag proteins. The results showed that a recombinant His-tag protein VP28 in a protein pool was captured by melanin and Ni2+/Fe3+/Zn2+ chelated melanin. Experiments for releasing VP28 were performed only on the melanin and Ni2+-melanin matrices. The result showed that VP28 was quite selectively eluted when applying elution buffer of 250 mM imidazole overnight. The relative efficiency in releasing VP28 of melanin and Ni-melanin matrices roughly compared to Ni-Sepharose were about 38 and 18% respectively. Further optimization of this process may allow higher efficiency in the purification of His-tag proteins.

The effect of protein mutations on drug binding suggests ensuing personalised drug selection

The advent of personalised medicine promises a deeper understanding of mechanisms and therefore therapies. However, the connection between genomic sequences and clinical treatments is often unclear. We studied 50 breast cancer patients belonging to a population-cohort in the state of Qatar. From Sanger sequencing, we identified several new deleterious mutations in the estrogen receptor 1 gene (ESR1). The effect of these mutations on drug treatment in the protein target encoded by ESR1, namely the estrogen receptor, was achieved via rapid and accurate protein–ligand binding affinity interaction studies which were performed for the selected drugs and the natural ligand estrogen. Four nonsynonymous mutations in the ligand-binding domain were subjected to molecular dynamics simulation using absolute and relative binding free energy methods, leading to the ranking of the efficacy of six selected drugs for patients with the mutations. Our study shows that a personalised clinical decision system can be created by integrating an individual patient’s genomic data at the molecular level within a computational pipeline which ranks the efficacy of binding of particular drugs to variant proteins.