Modeling DNA Oligonucleotide Binding to DNA-mimetic Stem-Loop 1 of the SARS-CoV-2 RNA

SARS-CoV-2 is a positive-sense RNA virus that contains open reading frame 1ab (ORF1ab) to produce 16 nonstructural proteins (nsps). Five stem-loops (SL) are found in the 5' UTR of the RNA that are involved in myriad viral functions and are labeled SL1 through SL5. SL1 is crucial to viral replication. Upon viral infection, nsp1 binds the ribosomal 40S subunit to inhibit all host mRNA translation. Upon SL1 binding to nsp1, viral mRNA can be processed by the ribosome, allowing viral proteins to be produced. In this study, we are examining small DNA oligonucleotides that bind to SL1-mimetic DNA in order to block SL1-nsp1 interactions. We designed a DNA analog of the SL1 hairpin and two small DNA oligonucleotides that are complementary to either the helical stem or the loop region of SL1. The binding of these oligonucleotides to the SL1 hairpin should allow the formation of either an alternate duplex or a triplex structure. Isothermal titration calorimetry (ITC) and circular dichroism (CD) techniques were performed in 1 MKCl and 10 mM MgCl2 at two different pH (5.5 and 7.0) to examine structural and thermodynamics of binding. ITC of the two oligonucleotides showed modest binding. Results from DNA binding experiments, thermal denaturation, and CD show the hairpin structure is thermodynamically more favored and mostly remains intact under the conditions examined.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Determining the Effect of KmtR Mutants, E101Q, H102Q, and H111Q on the Metal Binding Affinities

Mycobacterium tuberculosis is the second leading infectious killer after COVID-19. The bacteria utilizes several metal transport systems to help it survive in the host.With an increase in the number of multiresistant, extensively resistant and totally drug-resistant strains, the development of new therapeutic strategies that target other essential pathways in the bacteria is critical. The bacteria contain several metal transport systems which are necessary for its survival. Additionally, the bacteria has two metalloregulators that are associated with nickel and cobalt export, NmtR and KmtR. The focus of this research is on KmtR, which represses the expression of the genes, cdf (which encodes the export protein) and kmtR. The goal of our research is to identify the residues that are responsible for binding the cognate metals, nickel and cobalt, as well as the noncognate metal, zinc, to KmtR. Mutagenesis studies coupled with metal binding experiments will be used to determine how KmtR binds these metals. The E101Q, H102Q, and H111Q mutants, among others, have been made, expressed, and purified in our lab. Data obtained from Isothermal Titration Calorimetry determined that all three mutant proteins bind cobalt with nanomolar affinities and the H111Q mutant KmtR proteins binds cobalt an order of magnitude weaker than the other two mutant proteins. Research reported as supported fully by the RI Institutional Development Award (IDeA) Network for Biomedical Research Excellence (RI-INBRE) from the National Institute of General Medical Sciences of the National Institutes of Health under grant #P20GM103430.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Development of Aptasensor for the Diagnosis of Covid-19

Objectives: The objective is to develop a low-cost, practical, portable aptasensor platform for the diagnosis of COVID-19. Materials -Methods: Amino-terminated aptamers to be used for the design of an aptasensor were synthesized by SELEX method, and interaction of aptamers with SARS-CoV-2 S1 protein was investigated by isothermal titration calorimetry (ITC). Gold electrodes were used to design the biosensor platform. After the electrode surface was functionalized with cysteamine, the amino-terminated aptamer was conjugated to the surface via glutaraldehyde crosslinker. Then, the surface characterization and analytical parameters of the designed sensing platform were determined by adding commercial S1 proteins on the surface using differential pulse voltammetry (DPV), cyclic voltammetry (CV) and impedance spectroscopy (EIS). To evaluate the working performance of the system, S1 proteins were added to the synthetic serum samples using the standard addition method and the measurements were repeated. Result(s): Surface characterization of the platform designed with EIS and CV measurements was performed and it was found that the modification was successfully performed. In addition, DPV results and analytical parameters of the platform (calibration plot, limit of detection(LOD) , repeatability, coefficient of variation) were determined and the working performance of system was evaluated. Moreover, working performance of the biosensor in real samples and its specificity for COVID -19 were determined by experiments with synthetic serum and influenza A and B proteins. Conclusion(s): According the results, the system has potential to be used for the detection of COVID -19, and also it can be rapidly adapted in different pandemic situations that may occur in the future.

Towards New Antimicrobials - Targeting Bacterial Response Regulators

Purpose of Study: Antibiotic resistance remains one of the largest healthcare and public health challenges. Several studies have documented that the spread of antibiotic resistant bacteria in nosocomial settings has been exacerbated worldwide due to increased rates of hospitalization and intubation in the wake of the COVID-19 pandemic. One way to address antibiotic resistance is to identify novel compounds that inhibit essential microbial processes. Two-component regulatory systems are important mediators of signal transduction that allow bacteria to communicate with and respond to changes in their environment. The WalRK system is a two-component system that is conserved and essential for viability in many Gram-positive human pathogens. We hypothesize that a ligand that specifically binds with the DNA-interaction surface of the WalR protein can lead to cell death and can serve as a lead compound for future drug development efforts. Methods Used: We describe the development process of an assay to identify WalR binding compounds. In silico molecular dynamics docking approaches were utilized to identify potential WalR binding compounds from virtual compound libraries. To assess their WalR-binding capacity in vitro, overexpression strains for several WalR recombinant constructs were engineered and protein constructs were purified to homogenicity. Isothermal titration calorimetry (ITC) is a technique that measures heat release or absorption when two molecules interact. A MicroCal PEAQ ITC instrument was utilized to develop a WalR-binding assay. Summary of Results: WalR is a two-domain protein featuring a regulatory and a DNA-binding domain. Two constructs, a truncated DNA-binding domain and a full-length protein construct proved soluble, and pure quantities necessary to conduct ITC measurements could be successfully obtained (12 mg full-length protein and 23 mg truncated protein). These proteins were amenable to ITC experiments. We found that experiments were best run with at least a two-fold increase of ligand concentration to protein concentration supplied in identical buffer conditions over nineteen injections. We are currently assessing the binding affinities of our in silico hit compounds. Conclusion(s): Our results show that ITC enables the detailed, rapid, and reproducible characterization of the binding relationship between the DNA-binding domain of the WalR protein and any potential ligands. The protocol discussed herein will enable further drug discovery studies on the WalR response regulator protein to identify and characterize inhibitors, providing insight towards the development of novel antimicrobial compound.

Thermodynamic analysis of the interactions between human ACE2 and Spike RBD of Betacoronaviruses (SARS-CoV-1 and SARS-CoV-2).

There are many scientific reports on the interaction of the SARS-CoV-2 virus S protein (and its RBD) with the human ACE2 receptor protein. However, there are no reliable data on how this interaction differs from the interaction of the receptor binding domain of SARS-CoV-1 with ACE2, in terms of binding strength and changes in reaction enthalpy and entropy. Our studies have revealed these differences as well as the impact of zinc ions on this interaction. Intriguingly, the binding affinity of both RBDs (of SARS-CoV-1 and of SARS-CoV-2) to the ACE2 receptor protein is almost identical; however, there are some differences in the entropic and enthalpic contributions to these interactions.

Forces Driving a Magic Bullet to Its Target: Revisiting the Role of Thermodynamics in Drug Design, Development, and Optimization.

Drug discovery strategies have advanced significantly towards prioritizing target selectivity to achieve the longstanding goal of identifying "magic bullets" amongst thousands of chemical molecules screened for therapeutic efficacy. A myriad of emerging and existing health threats, including the SARS-CoV-2 pandemic, alarming increase in bacterial resistance, and potentially fatal chronic ailments, such as cancer, cardiovascular disease, and neurodegeneration, have incentivized the discovery of novel therapeutics in treatment regimens. The design, development, and optimization of lead compounds represent an arduous and time-consuming process that necessitates the assessment of specific criteria and metrics derived via multidisciplinary approaches incorporating functional, structural, and energetic properties. The present review focuses on specific methodologies and technologies aimed at advancing drug development with particular emphasis on the role of thermodynamics in elucidating the underlying forces governing ligand-target interaction selectivity and specificity. In the pursuit of novel therapeutics, isothermal titration calorimetry (ITC) has been utilized extensively over the past two decades to bolster drug discovery efforts, yielding information-rich thermodynamic binding signatures. A wealth of studies recognizes the need for mining thermodynamic databases to critically examine and evaluate prospective drug candidates on the basis of available metrics. The ultimate power and utility of thermodynamics within drug discovery strategies reside in the characterization and comparison of intrinsic binding signatures that facilitate the elucidation of structural-energetic correlations which assist in lead compound identification and optimization to improve overall therapeutic efficacy.

Potential natural products that target the SARS-CoV-2 spike protein identified by structure-based virtual screening, isothermal titration calorimetry and lentivirus particles pseudotyped (Vpp) infection assay.

BACKGROUND AND AIM: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters cells through the binding of the viral spike protein with human angiotensin-converting enzyme 2 (ACE2), resulting in the development of coronavirus disease 2019 (COVID-19). To date, few antiviral drugs are available that can effectively block viral infection. This study aimed to identify potential natural products from Taiwan Database of Extracts and Compounds (TDEC) that may prevent the binding of viral spike proteins with human ACE2 proteins. METHODS: The structure-based virtual screening was performed using the AutoDock Vina program within PyRX software, the binding affinities of compounds were verified using isothermal titration calorimetry (ITC), the inhibitions of SARS-CoV-2 viral infection efficacy were examined by lentivirus particles pseudotyped (Vpp) infection assay, and the cell viability was tested by 293T cell in MTT assay. RESULTS AND CONCLUSION: We identified 39 natural products targeting the viral receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in silico. In ITC binding assay, dioscin, celastrol, saikosaponin C, epimedin C, torvoside K, and amentoflavone showed dissociation constant (K d) = 0.468 µM, 1.712 µM, 6.650 µM, 2.86 µM, 3.761 µM and 4.27 µM, respectively. In Vpp infection assay, the compounds have significantly and consistently inhibition with the 50-90% inhibition of viral infection efficacy. In cell viability, torvoside K, epimedin, amentoflavone, and saikosaponin C showed IC50 > 100 µM; dioscin and celastrol showed IC50 = 1.5625 µM and 0.9866 µM, respectively. These natural products may bind to the viral spike protein, preventing SARS-CoV-2 from entering cells. SECTION 1: Natural Products. TAXONOMY CLASSIFICATION BY EVISE: SARS-CoV-2, Structure-Based Virtual Screening, Isothermal Titration Calorimetry and Lentivirus Particles Pseudotyped (Vpp) Infection Assay, in silico and in vitro study.

Surfactants - Compounds for inactivation of SARS-CoV-2 and other enveloped viruses.

We provide here a general view on the interactions of surfactants with viruses, with a particular emphasis on how such interactions can be controlled and employed for inhibiting the infectivity of enveloped viruses, including coronaviruses. The aim is to provide to interested scientists from different fields, including chemistry, physics, biochemistry, and medicine, an overview of the basic properties of surfactants and (corona)viruses, which are relevant to understanding the interactions between the two. Various types of interactions between surfactant and virus are important, and they act on different components of a virus such as the lipid envelope, membrane (envelope) proteins and nucleocapsid proteins. Accordingly, this cannot be a detailed account of all relevant aspects but instead a summary that bridges between the different disciplines. We describe concepts and cover a selection of the relevant literature as an incentive for diving deeper into the relevant material. Our focus is on more recent developments around the COVID-19 pandemic caused by SARS-CoV-2, applications of surfactants against the virus, and on the potential future use of surfactants for pandemic relief. We also cover the most important aspects of the historical development of using surfactants in combatting virus infections. We conclude that surfactants are already playing very important roles in various directions of defence against viruses, either directly, as in disinfection, or as carrier components of drug delivery systems for prophylaxis or treatment. By designing tailor-made surfactants, and consequently, advanced formulations, one can expect more and more effective use of surfactants, either directly as antiviral compounds or as part of more complex formulations.