Development and Applications of Coronavirus Protease-Specific Covidyte™ and Covipyte™ Peptide Substrates

Abstract

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There is a high level of similarity in both amino acid sequence and structure between the SARS and SARS-CoV-2 virus, particularly in regards to the catalytic active sites of their proteases. Several coronavirus-specific amino acid sequences were discovered for use in investigating enzyme inhibitors for SARS, and have since been repurposed for SARS-CoV-2 research. By conjugating these sequences to FRET pairs, several fluorescent substrates have been developed to accurately measure protease activity. These substrates can be employed in sensitive assays for research into development of possible protease inhibitors for SARS-CoV-2 antiviral therapies.

Introduction

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Detected in late 2019 and subsequently spreading across the globe, the pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents the worst threat to human health in over a century. Also referred to as Covid-19, the virus has marshaled unprecedented efforts from the global scientific community in research into possible treatments and vaccines. There are several avenues of inquiry being pursued, of which one of the most promising is inhibiting essential enzymatic catalysis that the virus requires to function and replicate itself. Identifying and targeting proteases were helpful in identifying successful therapies during previous health crises, such as HIV, as well as the viral predecessor of the current pandemic, the related SARS-CoV pathogen that caused an epidemic in 2003.¹ SARS-CoV-2 has two cysteine proteases: the papain-like protease PLpro, and the chymotrypsin-like protease 3CLpro, (sometimes called the main protease or Mpro) both of which are high-priority therapeutic targets.² Via prior research with related coronaviruses, work on mapping the sequences and structure of these has led to the development of coronavirus-specific substrates which can be used to detect and measure enzymatic activity. These allow accurate evaluation of treatments, and pave the way to further exploration into possible inhibitors and treatments.

Sequence and Structure Conservation between Previous Coronaviruses and SARS-CoV-2

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Of particular interest to medical researchers is the knowledge that the catalytic sites for proteases are very highly conserved. There is only one amino acid difference (ser46) at the protease active site between SARS-CoV-2 and SARS.¹ This conservation of sequence strongly indicates that observed behavior and responses of these sites can be applied with confidence across types. Although the viruses aren't identical, comparative experiments demonstrate behavioral correlation. Unfortunately, the variations in viral structure that do exist lead to differences in binding affinities and presumed efficacy of experimental inhibiting compounds. Given the similarities, however, the research done during and in the wake of the SARS epidemic is able to be repurposed into promising investigations for the closely-related SARS-CoV-2 pandemic.



As seen in Figure 1, the overall structures of coronaviruses such as SARS, MERS, and SARS-CoV-2 overlap extensively. The SARS and SARS-CoV-2 genome sequences in particular have an 89.1% similarity.³ This is significant for researchers, since the structural similarity implies that similar treatments might be effective. The sequence overlap also means that there are many identical or near-identical stretches of amino acid sequences, including the functional sites for many enzymatic targets including both proteases. This permits prior knowledge of the functional sites for SARS to be immediately applicable to investigations into inhibiting the enzymatic targets of SARS-CoV-2.

Coronavirus 3CLpro Protease as a Treatment Target

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The PLpro and 3CLpro proteases are both essential for the function of SARS-CoV-2. PLpro has several roles in signaling and other processes, including replication, but 3CLpro is central to viral replication, making it the primary target of anti-infection research. 3CLpro is responsible for producing functional proteins during viral replication via hydrolysis of 2 larger polyproteins (pp1a and pp1ab).⁴ Without this enzyme, replication of the virus would be impossible. Additionally, the 11 known cleavage sites for this enzyme are unique and are not shared with human proteases, minimizing the risk of treatment toxicity.⁵

The 3CLpro protease is unique among the many possible therapeutic targets of SARS-CoV-2 in that the vast amounts of research done on the prior SARS coronavirus provides a generous foundation for research into treatments. Given that finding possible therapies aside from current symptomatic treatments is of a time-sensitive nature, having that background is promising. Some compounds used during the SARS epidemic are already seeing experimental clinical use during the current pandemic.³ As research continues worldwide, other potential drugs from these prior findings are likely to be improved or discovered that work as well or better against SARS-CoV-2.

Covidyte™ & Covipyte™ Fluorogenic Substrates: Research Applications

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A fundamental part of laboratory science is the development of the materials and methodology to accurately observe and measure a given process or compound. To measure the protease activity of SARS-CoV-2, several techniques have been created, with more constantly being proposed. The most sensitive and efficient method of measuring the SARS-CoV-2 enzyme activity uses a biochemical phenomenon known as Förster resonance energy transfer (FRET). In very simple terms, FRET is the energy exchange from a high-energy excited 'donor' molecule to an 'acceptor' molecule over a distance smaller than 10 nm. Within that distance, the excited donor molecule transfers energy to the acceptor via dipole-dipole interaction. When this energy transfer occurs between two fluorophores, the overall fluorescence intensity is quenched. There are many possible FRET pairs of donor/acceptors currently used in laboratories, with one of the most popular being EDANS/DABCYL. A variation on this energy exchange is known as 'dark' FRET, which is very similar, but instead of two fluorophores, consists of a pairing of an energy donor fluorophore and a 'dark' quencher chromophore. This is the interaction used for the Covidyte™ and Covipyte™ dark FRET substrates.
FRET is the basis for fluorescence-based activity assays using several amino acid peptides isolated for their efficacy in SARS enzymatic activity. For the investigation of 3CLpro protease, the first of these peptides is 12 amino acids in length, with the sequence VNSTLQSGLRKM, and the second is 14, with a sequence of KTSAVLQSGFRKME.⁶ Both of these sequences are specifically targeted by coronavirus proteases and not by human. By conjoining these amino acid sequences to FRET pairs, researchers can use the observed fluorescence as a sensitive measurement of enzymatic activity. When the protease cleaves the amino acid sequences, the conjoined fluorophore pair breaks and no longer is quenched by FRET. The more protease activity is present, the higher the increase in observed fluorescence. Using both classic FRET pairs and improved modern versions, several fluorescent peptide substrates are available for research use.

1. ? = Extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm^-1M^-1.

For the investigation of PLpro protease, the longest of these peptides is 9 amino acids long, with a sequence of RELNGGAPI. There are also 3 smaller tetra-peptide substrates that have been developed, each only 4 amino acids in length, with sequences of: KAGG, KKAG, and LRGG.

1. ? = Extinction coefficient at their maximum absorption wavelength. The units of extinction coefficient are cm^-1M^-1.

The development of fluorescent substrates such as these allow not only improved measurement of protease activity levels, but more sensitive detection of these enzymes overall. Protease activity assays are often used to assess the efficacy of potential enzyme inhibitors or of general viral activity. By using FRET pairs in particular, the assays can employ a fluorescent microplate format or similar, permitting conversion to a high-throughput workflow.

Discussion

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By using prior research on coronaviruses, and leveraging the structural similarities of SARS-CoV-2, the development of fluorescent substrates like Covidyte™ and Covipyte™ to investigate the behavior of both PLpro and 3CLpro coronavirus proteases is a promising tool for investigating possible antiviral treatments. Using modern computational power to help model, hundreds of thousands of compounds can be assessed for inhibitory interactions, and the best-performing simulations selected for further experiments. The continued combined efforts of researchers around the globe, and most particularly the highest levels of scientific cooperation ever seen, will be essential for preserving as many human lives as possible during the SARS-CoV-2 pandemic.

References

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