Efficiency Improvements and Discovery of New Substrates for a SARS-CoV-2 Main Protease FRET Assay

ABSTRACTThe COVID-19 pandemic, caused by the SARS-CoV-2 virus, has a huge impact on the world. Although several vaccines have recently reached the market, the development of specific antiviral drugs against SARS-CoV-2 is an important additional strategy in fighting the pandemic. One of the most promising pharmacological targets is the viral main protease (Mpro). Here, we present an optimized biochemical assay procedure for SARS-CoV-2 Mpro. We have comprehensively investigated the influence of different buffer components and conditions on the assay performance, and characterized six FRET substrates with a 2-Abz/Tyr(3-NO2) FRET pair. The substrates 2-AbzSAVLQSGTyr(3-NO2)R-OH, a truncated version of the established DABCYL/EDANS FRET substrate, and a new substrate 2-AbzVVTLQSGTyr(3-NO2)R-OH are promising candidates for screening and inhibitor characterization. In the latter substrate, the incorporation of Val at the position P5 improved the catalytic efficacy. Based on the obtained results, we present here a reproducible, reliable assay protocol using highly affordable buffer components.

Perylene diimide-based nanoring architecture for exogenous and endogenous ATP detection: Biochemical assay for monitoring phosphorylation of glucose

The positively charged amphiphiles hold great significance in supramolecular chemistry due to their good solubility, physiochemical and molecular recognition properties. Herein, we report synthesis, characterization and molecular recognition properties of...

Tyramine Derivatives as Potent Therapeutics for Type 2 Diabetes: Synthesis and In Vitro Inhibition of α-Glucosidase Enzyme

Background: Tyramine derivatives 3-16 were prepared and tested first time for their α- glucosidase (Sources: Saccharomyces cerevisiae) inhibitory activity by using an in vitro mechanismbased biochemical assay. All the compounds were found to be new, except compounds 3, 10-12 and 16. Objective: In continuation of our research to synthesize and identify potent inhibitors of α-glucosidase enzyme, we intended to synthesize new inhibitors of α-glucosidase enzyme with enhanced efficacy in order to provide the basis for the better treatment of the type-II diabetic. Methods: Tyramine (1) was allowed to react with a variety of aryl chlorides (2) to yield the corresponding amides. Synthesized compounds were then purified through normal phase column chromatography. Compounds 3-16 were then assessed for their α-glucosidase inhibitory activity in an in vitro biochemical assay. The cytotoxicity of compounds 3-16 was determined by using 3T3 mouse fibroblast cell lines. Results: Compounds 3-5, 8, 13, and 15-16 were found to be more active (IC50 = 103.1±0.46, 37.3±4.51, 56.7±4.2, 23.9±2.31, 43.6±2.88, 55.8±1.73, and 38.2±0.86 μM, respectively) than the acarbose, the standard inhibitor of α-glucosidase enzyme, (IC50= 840.0±1.73 μM). To determine the dissociation constants and mode of inhibition, the kinetic studies were also performed for compounds 4 and 8 (the most potent inhibitors). It was observed that compounds 4 and 8 possess noncompetitive properties as the inhibitors of α-glucosidase. All the compounds were found to be noncytotoxic, except 5 and 12 (IC50= 14.7± 0.24 and 6.6± 0.38 μM, respectively). Conclusion: The current study gives the facile synthesis and identification of potent inhibitors of α- glucosidase. The new inhibitors reported here may be investigated further for the designing and development of novel anti-diabetic agents.

Oxidation of HDL in patients with coronary artery disease

The function of high-density lipoprotein (HDL) cholesterol may play a more important role in the prevention of cardiovascular disease compared to the concentration of the HDL. The aim of the present study is to assess a novel cell-free test to quantify oxidation of HDL and its association to coronary artery disease (CAD). We performed a prospective trial by including patients undergoing elective cardiac catheterization and healthy controls. A total of 895 subjects were included. In 483 patients CAD was confirmed (CAD group) and in 241 patients CAD was ruled-out (no CAD). Control patients (n=171) had no known CAD, had no diabetes and were not smokers. HDL function was measured in serum samples by determining its HDL-lipid peroxidation by a novel fluorometric cell-free biochemical assay. HDL lipid peroxide content (HDLox) is adjusted for the HDL cholesterol and has no units. Patients with confirmed CAD had higher levels of HDLox (0.92±0.58, no units) compared to patients with no CAD (0.8±0.46, no units) and controls (0.78±0.41, no units, p=0.003). HDL-Cholesterol was lower in the CAD group (50.7±17.7mg/dl) compared to no CAD (58.4±17.6mg/dl) and healthy subjects (59.1±15.9mg/dl, p<0.001). HDLox was a strong predictor of coronary artery disease status (odds ratio for coronary disease 1.69; 95% confidence interval [CI], 1.24 to 2.38; P=0.001). Oxidation of HDL, a metric of HDL function, measured by a cell-free biochemical assay, is increased in patients with CAD and is a predictor of the disease. Logistic Regression analysis Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Medical School of Brandenburg

Development of a cell-free split-luciferase biochemical assay as a tool for screening for inhibitors of challenging protein-protein interaction targets

Targeting the interaction of proteins with weak binding affinities or low solubility represents a particular challenge for drug screening. The NanoLuc ® Binary Technology (NanoBiT ®) was originally developed to detect protein-protein interactions in live mammalian cells. Here we report the successful translation of the NanoBit cellular assay into a biochemical, cell-free format using mammalian cell lysates. We show that the assay is suitable for the detection of both strong and weak protein interactions such as those involving the binding of RAS oncoproteins to either RAF or phosphoinositide 3-kinase (PI3K) effectors respectively, and that it is also effective for the study of poorly soluble protein domains such as the RAS binding domain of PI3K. Furthermore, the RAS interaction assay is sensitive and responds to both strong and weak RAS inhibitors. Our data show that the assay is robust, reproducible, cost-effective, and can be adapted for small and large-scale screening approaches. The NanoBit Biochemical Assay offers an attractive tool for drug screening against challenging protein-protein interaction targets, including the interaction of RAS with PI3K.