Accelerated Screen Identifies 25 Existing Drugs Capable of Disrupting SARS-CoV-2 Cell Entry

To date, the COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2 has infected > 39 million people worldwide and claimed > 1.1 million lives.* In an unprecedented effort to defeat this global health threat, researchers, industry experts and regulatory bodies have joined forces in attempts to better understand the virus and to develop effective and safe therapeutic strategies against it.

One group of researchers tasked with finding an effective COVID-19 treatment is that of the National Center for Advancing Translational Sciences (NCATS) – part of the National Institutes of Health (NIH).

“In general, with COVID-19 we are of seeing unprecedented levels of collaboration and agility across research, academic, corporate and government circles to drive towards quality therapeutics and vaccines solutions,” – Alan Fletcher, VP and GM Life Sciences, PerkinElmer.

The aim of the NCATS project is to rapidly screen existing drug compounds that have already passed through the entire drug development pipeline, to determine if any can disrupt the interaction between the SARS-CoV-2 spike protein and the host’s angiotensin converting enzyme 2 (ACE2) receptor.

What’s so special about SARS-CoV-2’s spike?

The single-stranded RNA-enveloped virus SARS-CoV-2 is a member of the coronavirus family. Coronaviruses get their name from the distinctive crown-like “spikes” presented on the surface of each virus particle – these spikes are actually 180–200 kDa glycoproteins, commonly referred to as “S proteins”. The polysaccharide molecules coating the S protein act as camouflage, shielding the virus from the host’s immune system.

“The first step in viral entry involves the interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein with the host ACE2 receptor. We reasoned that blocking this interaction could potentially stop the ability of the virus to infect cells,” explains Quinlin Hanson, Ph.D. postdoctoral fellow at NCATS, NIH.

SARS-CoV-2 cell entry and replication

SARS-CoV-2 entry into cells is mediated by the S protein. The S protein binds to the ACE2 receptor, located on the host cell surface, via its receptor-binding domain (Figure 1). Once inside, SARS-CoV-2 hijacks the cell, unloading its genetic information in the form of RNA. The RNA is then translated to produce three key viral structural components – S protein, envelope protein (E protein) and membrane protein (M protein). These proteins move to the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) of the host cell, where they associate with newly formed viral genomes enclosed in an N protein capsid. Once associated, the protein structure buds from the ERGIC membrane to produce new virions.

ACE2 is a receptor present on the surface of many cell types within the human body and can be found in the lungs, heart, kidneys, liver and GI tract. It is expressed in abundance on the surface of epithelial cells within the lungs – hence the adverse respiratory effects often seen in COVID-19 patients.

Figure 1: SARS-CoV-2 spike (S) protein is composed of three sections, the intracellular tail, transmembrane anchor and ectodomain. Binding to ACE2 is achieved via the receptor-binding domain at the top of the ectodomain. 

Kicking off the NCATS project

Although the drug discovery and development process can take 10–15 years, the team’s mandate was to work as quickly as possible – which is no easy task with pandemic protocols and social distancing in place, not to mention the challenge of securing the reagents and target analytes required to run the screening assay.

“Supply chain disruption is something everyone has dealt with in the COVID-19 pandemic and we were not spared from that,” – Quinlin Hanson.

“We are dedicated to putting safety first at NCATS, which meant enacting somewhat restrictive work policies limiting the number of personnel who could occupy a space at a given time. These practices (along with wearing masks, hand washing, and social distancing) are necessary and effective at combating the spread of the virus, but they do have the side effect of slowing down the speed with which certain things happen,” Hanson adds. 

“A lot of work has continued virtually to keep things moving ahead and our team has had to be very nimble and open to working together in new ways.”

Despite these challenges, the team set to work. Along with developing an assay to test the interaction between ACE2 and the SARS-CoV-2 RBD, the team also wanted to determine if any drug repurposing could be exploited to potentially streamline therapeutic development.

“Drug repurposing efforts help researchers get a potential jump-start on discovering therapies against novel or related indications be it around viruses such as COVID-19 or diseases,” – Alan Fletcher.

Fletcher continues: “With a repurposing approach, you are screening existing compounds that have been clinically studied and approved for other conditions or diseases. These compounds already have well-characterized pharmacokinetic, dosage, and safety data which could potentially be translated toward launching a clinical trial.”

The team selected 3,384 compounds which included many drugs approved for other treatments and other compounds with demonstrated activity in other anti-viral assays developed at NCATS. Twenty-five “hits” were identified from screening the compound library. These hits successfully disrupted the S protein–ACE2 binding interaction.

The biomolecular screening approach used by the team was carried out using PerkinElmer’s AlphaLISA – a no-wash bead-based technology. “The assay was designed to work in a 1536-well microtiter plate, making it a high-throughput assay. We can test hundreds of compounds in dose-response on a single plate, using rapid liquid handling technologies like acoustic dispensing, it is quick and simple to prepare large quantities of compounds for testing.” explains Hanson.

Binding of the analyte captured on the assay beads results in an energy transfer from one bead to the other at proximity – this produces a light emission signal.

The assay can be applied to a number of different modalities – including small molecules, proteins, polysaccharides and nucleic acid fragments. Fletcher highlights some of the assay’s key benefits: “The AlphaLISA offers greater sensitivity, a wider dynamic range and a simplified, streamlined workflow. It can detect and quantitate biomolecules in both simple and complex sample types. Altogether this leads to a faster time to results.”

“ELISA, western blot and other immunodetection technologies have commonly been used for quantitation of biomarkers and other analytes in a variety of sample types. However, these methods necessitate long, tedious protocols and often lack sensitivity due to limited detectable range,” continues Fletcher.

Next steps

The NCATS team is now working to evaluate the 25 hit compounds and will attempt to find analogs with superior potency which they can further optimize. The optimized compounds will then be rigorously tested in follow-up screening studies.

“Screening these compounds and identifying 25 hits is the first step on the long path to identifying and developing therapeutics. Each of these hits will have to be validated and tested in other assays before it can move forward for any sort of therapeutic indication,” explains Hanson. 

Hanson adds that there is a chance that none of the hits will move forward, but he explains that the possibility of being unsuccessful is to be anticipated when repurposing. If that is indeed the case, their efforts aren't wasted. "We can leverage our assay to test additional therapeutic types including other drug-like molecules, peptides, or neutralizing nanobodies,” says Hanson.

The NCATS researchers have published their data in an Open Data Portal to provide the wider scientific community with the ability to comment and collaborate.

“Information and best practices are being shared more openly and globally. Ideas and innovations are coming to the fore more rapidly. As part of this, and because labs are operating in a more virtual way due to social distancing guidelines, we are also seeing trends towards increased adoption of secure digitalization and automation of labs,” Fletcher concludes.

*Data obtained October 19, 2020 from Johns Hopkins coronavirus resource center.

Alan Fletcher and Quinlin Hanson were speaking with Laura Elizabeth Lansdowne, Senior Science Writer for Technology Networks.