A Small Molecule That In Vitro Neutralizes Infection of SARS-CoV-2 and Its Most Infectious Variants, Delta, and Omicron.

The COVID-19 pandemic has underscored the urgent need to develop highly potent and safe medications that are complementary to the role of vaccines. Specifically, it has exhibited the need for orally bioavailable broad-spectrum antivirals that are able to be quickly deployed against newly emerging viral pathogens. The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and its variants Delta and Omicron are still a major threat to patients of all ages. In this brief report, we describe that the small molecule CD04872SC was able to neutralize SARS-CoV2 infection with a half-maximal effective concentration (EC50) = 248 µM. Serendipitously, we also were able to observe that CD04872SC inhibited the infection of the SARS-CoV-2 variants; Delta (EC50 = 152 µM) and Omicron (EC50 = 308 µM). These properties may define CD04872SC as a potential broad-spectrum candidate lead for the development of treatments for COVID-19.

A NanoBiT assay to monitor membrane proteins trafficking for drug discovery and drug development.

Internalization of membrane proteins plays a key role in many physiological functions; however, highly sensitive and versatile technologies are lacking to study such processes in real-time living systems. Here we describe an assay based on bioluminescence able to quantify membrane receptor trafficking for a wide variety of internalization mechanisms such as GPCR internalization/recycling, antibody-mediated internalization, and SARS-CoV2 viral infection. This study represents an alternative drug discovery tool to accelerate the drug development for a wide range of physiological processes, such as cancer, neurological, cardiopulmonary, metabolic, and infectious diseases including COVID-19.

Investigating Real-Time Internalization of Membrane Proteins Using a Novel Technology Based on Bioluminescence

The trafficking of membrane proteins including G-protein coupled receptors (GPCRs) is an exciting area in pharmacology. Proteins, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. The objective of this work is the development of a universal method to study, under physiological conditions and in real-time, the different membrane protein internalization processes. This novel method to measure membrane protein internalization will accelerate the drug discovery process for a wide variety of diseases. Our hypothesis is that receptor trafficking can represent a universal feature of membrane proteins and can be exploited to study receptor pharmacology as well as drug development. Methods A short domain of the human endofin was synthesized and subcloned into the four NanoBiT vectors. Cells were seeded in 96-well plates at a density of 6?104 cells per well. The following day, cells were transfected using four different endofin-receptor plasmid combinations. The combination with the highest signal was chosen for further experimentation. At 24 hr post-transfection, the medium was aspirated and replaced with 100?l OPTIMEM (Life Technologies, Grand Island, NY, USA), which was then followed by the addition of 25?l substrate (furimazine). Luminescence measurements were taken once every minute for 10 min for signal stabilization. Finally, 10?l of ligand solution was added to each well, and luminescence measurements were immediately recorded. Three independent experiments were performed in triplicate, and each triplicate was averaged before calculating the s.e.m. Results In the quantitative pharmacological analysis of GPCRs, HEK293 cells were treated with increasing concentrations of ligand. The time course graph displays an increase in normalized luminescence over time with increasing ligand concentrations. The area under the curve analysis of this response demonstrates a clear concentration-dependent response to b2AR stimulation. We also explored the pharmacological analysis of a non-GPCR membrane protein, termed FAM19A5 Isoform II, and monitored its internalization by two monoclonal antibodies. Cells were treated with the antibodies A and B, with an EC50 value of 72.5±5nM for antibody A and 33.1±3nM for antibody B. Finally, we were also able to monitor virus entry in living cells via ACE2 internalization by binding with the SARS-CoV2 Spike protein in HEK293 cells treated with lentiviruses expressing the SARS-CoV2 Spike protein at their surface. Conclusions By using this novel approach we were able to characterize the trafficking of a wide variety of membrane proteins ranging from GPCRs to even antibody internalization and SARS-CoV2 virus entry in real-time living cells. We were also able to observe that internalization rates vary dramatically across different families of receptors, with the antibody-mediated internalization having the slowest kinetics. Compared to other technologies where specialized microscopes and fluorophores are required, our approach represents a simple low-cost universal assay to monitor the internalization of membrane proteins under physiological conditions.