New SARS-CoV/ACE2 inhibitor assays & small molecules for drug discovery
Many initiatives led by worldwide scientific networks are currently being launched to decipher cellular mechanisms involved in SARS-CoV-2 cellular entry. For example, a recent study has identified 332 high confidence SARS-CoV-2-human protein-protein interactions (Gordon D.E. at al. “A SARS-CoV-2-Human Protein-Protein Interaction Map Reveals Drug Targets and Potential Drug-Repurposing” (March 2020) bioRxiv). All these efforts are aimed at rapidly discovering antiviral drugs with proven efficacy, and at developing vaccines to prevent COVID-19 outbreaks. This crisis also enables pharma & biotech companies to reposition their existing antiviral drugs (Harrison C. “Coronavirus puts drug repurposing on the fast track” (Feb. 2020), Nature). In this post, we’ll introduce new, recently released in vitro assays and SARS-CoV related chemical compounds and compound libraries to support COVID-19 early phase drug discovery projects.
SARS-CoV-2 uses the SARS-CoV receptor Angiotensin-Converting Enzyme 2 (ACE2) for host cell entry (Hoffmann M. et al. “SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor” (March 2020) Cell). This entry is indeed mediated by an interaction between the SARS-CoV Spike (S) glycoprotein-binding domain (RBD) and the ACE2 receptor present at the surface of numerous pulmonary cell types. Therefore, therapeutically active drugs targeting the interaction between the COVID-19 S protein and the ACE2 receptor may be a center of interest for drug discoverers. Human neutralizing antibodies targeting the Spike protein and blocking SARS-CoV-2 cellular entry are also promising therapeutical strategies; the 4D11 human mAB being the first neutralizing antibody being identified so far (Wang C. et al. “A human monoclonal antibody blocking SARS-Cov-2 infection” (May 2020) Nature).
Recently, a novel route involving CD147 (also known as Basigin (BSG) or extracellular matrix metalloproteinase inducer (EMMPRIN)) and SARS Spike (S) protein has been discovered (Wang K. et al. “SARS-CoV-2 invades host cells via a novel route: CD147-spike protein” (2020) , bioRxiv), opening new opportunities for the development of specific antiviral drugs targeted CD147 (e.g. Meplazumab for SARS-CoV-2 pneumonia) (Huijie B. et al. “Meplazumab treats COVID-19 pneumonia: an open-labelled, concurrent controlled add-on clinical trial” (2020) medRxiv).
COVID-19 inhibitor screening assay kits for drug discovery research
In this context, scientists at BPS Bioscience have released a series of functional in vitro assay kits to screen for inhibitors binding to the SARS-Cov Spike protein (cat. nr 79331) or the ACE2 receptor (cat. nr 79936).
These sensitive kits can detect inhibition of SARS-CoV S protein/ACE2 binding in a 96-well format using chemiluminescent detection.
First, ACE2 or SARS-CoV-2 Spike-Fc are attached to 96-well plates through nickel-His tag or anti-Fc:Fc tag interactions. Next, corresponding ligands are incubated with the coated plates. Finally, the plates are treated with HRP-labeled antibodies followed by the addition of an HRP substrate to produce chemiluminescence, which then can be measured using a chemiluminescence reader.
These two ready-to-use SARS:ACE2 inhibitors screening assay kits are available in Europe through tebu-bio. Interestingly, they are also available as laboratory services – tebu-bio’s scientists can perform the assay tests in our laboratories to evaluate the inhibitory effects of compounds of interest.
Interested in evaluating the possible SARS-ACE2 inhibitory effect of your compounds at tebu-bio labs?
Small molecules potentially modulating CoV-Human protein binding
In the work of Gordon et al., the authors have been able to identify 67 druggable human proteins or host factors targeted by 69 potential antiviral drugs located at various phases of the drug discovery workflow. This illustrates the possible therapeutic arsenal available for academic or private drug discoverers to slow down the progression of the epidemic (Li G. et al. “Therapeutic options for the 2019 novel coronavirus (2019-nCoV)” (2020) Nature Reviews Drug Discovery), without talking about the vaccine options under development.
Some of these small molecules are already available for research applications, and notably, for the early phases of the drug discovery process (e.g. assay development, lead generation, in vitro testing…). They are available either as predefined anti-COVID compound libraries or as individual compounds (Wang M. et al. “Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro (2020) Cell Research) that can be “cherry-picked”.
Several predefined libraries gathering compounds that have demonstrated antiviral activities are currently available:
- Library L1710 : a unique selection of compounds that have been demonstrated to have anti-coronavirus activity and some of them with broad-spectrum antiviral agents including Lopinavir/Ritonavir, Chloroquine diphosphate, Remdesivir, Polydatin…
- Anti-COVID-19 compound library (CADD) L1711 : The Swiss-Model homology modeling has been applied here to build all possible protein structures (e.g. viral papain like protease (PLpro), protease 3CLpro (a.k.a. 3-chymotrypsin-like protease), RNA-dependent RNA polymerase (RdRp), nsp16 (2′-O-methyltransferase, helicase), and RBD of Spike…). These proteins and human ACE2 as targets have been used to screen TargetMol’s compound libraries (7729 compounds) and Bioactive compound library (7647 compounds) by using Surflex-Dock in the Sybyl-X 2.0 package. To improve the virtual screening efficiency and reliability, three rounds of screening (2 rounds of molecular docking virtual screening plus 1 round of manual screening) have been performed. Finally, 238 compounds were selected into this library L1711 Anti-COVID-19 compound library (CADD). These compounds have been widely reported in the literature to have the potential of anti-cancer, anti-bacterial, anti-inflammation, or anti-oxidation activity; or have other potential targets.
Other libraries available:
- 3CLpro-Targeted compound library (CADD): Based on the protein structure of 3CLpro, multiple research papers published identified several drugs having the potential to treat COVID-19 by virtual screening, such as drugs that target PLpro and 3CLpro in other viruses such as HIV drugs, Lopinavir and Ritonavir.
- ACE2-Targeted compound library (CADD): The prefusion SARS-CoV S1 subunit is structurally organized into four distinct domains: NTD, CTD1, CTD2 and CTD3. Among these, CTD1 is the receptor-binding domain, and one CTD1 in the trimer adopts an “up” conformation as a prerequisite for the binding of SARS-CoV to the cellular receptor angiotensin-converting enzyme 2 (ACE2). Similar observations of a protruding “up” CTD1 have also been reported for MERS-CoV S glycoproteins. Targeting the interaction of S protein RBD and ACE2 is an important therapeutic strategy to block coronavirus from entering host cells.
- RBD-Targeted compound library (CADD): 206 top-ranked docked molecules into RBD-Targeted compound library (CADD) by molecular docking virtual screening against 15,376 compound structures.
- nsp16-Targeted compound library (CADD): Nsp16 provides the viral mRNA with the ability to camouflage and obscure itself from the host cell by catalyzing methylation on m7GpppA-RNA, thus preventing recognition and activation of the host immune response which is essential for successful viral infection. Saquinavir and Lopinavir, two anti-HIV drugs, can inhibit the activity of NSP16 methyltransferase, thereby suppressing the function and replication of the virus.
- PLpro-Targeted compound library (CADD): Papain-like proteinase (PLpro), encoded in nsp3, is responsible for the cleavages of N-terminus of the replicase polyprotein to release Nsp1, Nsp2 and Nsp3, which is essential for correcting virus replication and antagonizing the host’s innate immunity.
- RdRP-Targeted compound library (CADD): RdRp is the core component of virus genome replication system, and has been used as a very important drug target in the research of SARS-CoV and MERS-CoV inhibitors. Remdesivir, an effective anti-SARS-CoV-2, is a nucleotide analog inhibitor of RdRp.
- X-Domain-Targeted compound library (CADD):X domain is a conserved structure of pp1a and becomes a part of nsp3 after pp1a cleaved by a virally encoded cysteine protease, the papain-like protease (PLpro). It is a catalytically active ADP-ribose-1″-phosphatase thought to play a role during synthesis of viral subgenomic RNAs thus can be used for drug screening or design.
Examples of anti COVID-19 compounds
- Nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps) Remdesivir (RDV – CAS:
- Autophagy Inhibitor Chloroquine phosphate (CAS: 50-63-5)
- Autophagy inhibitor Hydroxychloroquine sulfate (CAS:
- Marketed TMPRSS2 Inhibitor Camostat mesilate (CAS: 59721-29-8)
- FDA-approved anthelminthic drug STAT3 Inhibitor Niclosamide (CAS: 50-65-7)
- STAT3 signaling inhibitor Niclosamide (CAS: 50-65-7)
- New antiviral pro-drug clinical candidate NHC/EIDD-2801 (β-D-N4-hydroxycytidine – CAS: 3258-02-4)
Additional in vitro assays and reagents for drug discovery purposes
- Recombinant SARS-CoV S proteins and N proteins
- ACE2 Inhibitor Screening Assay Kit (cat. nr 79923): This in vitro assay is designed to measure the inhibitory activity of compounds against ACE2 for screening and profiling applications. ACE2, His-Tag (cat. nr 10003) is also available.
- ACE2 recombinant proteins and Human rec. CD147 / EMMPRIN / Basigin Proteins with various tags.
- PLP2, His-Tag (cat. nr 81090): The NL63 coronavirus genome encodes two papain-like proteases, PLP1 and PLP2; PLP2 being a deubiquitinase.
- PLPro, His-Tag (cat. nr 81091): The SARS genome encodes a number of proteases including papain-like protease (PLpro). Initially reported as a deubiquitylase, PLpro has been preferentially described as a deISGylase.
- 14-3-3 theta, His-Tag (cat. nr 79038): 14-3-3 theta has been observed to mediate nucleo-cytoplasmic shuttling of the coronavirus Nucleocapsid protein (N protein) of the SARS coronavirus. 14-3-3 theta phosphorylates N protein and localizes it to the cytoplasm.
- MnSOD plays an important role to prevent cellular oxydative stress and pulmonary inflammations induced by cytokine storms and ionizing radiation. Recently, some authors believe that MnSOD could be a promising therapeutically active target in Severe Acute Respiratory Syndromes. Dedicated MnSOD have been designed to cover these new emerging research areas:
I am very grateful to Suzan Oberle (BPS Bioscience) and Rob Zipkin (Focus Biomolecules) for helping me construct and edit this post related to SARS-CoV-2, by sharing with me their vision of the challenges in drug discovery represented by this COVID-19 epidemic.