Chicoric acid, a potential candidate for the treatment of COVID-19

An article currently being reviewed in the journal Scientific Reports and currently available on the Research Square*preprint server reports on chicoric acid (CA) as a potential inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Study: Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor. Image credit: NIAID

Background

There continues to be a critical medical need for new targeted drugs capable of combating SARS-CoV-2 infections. The establishment of such antiviral agents, in most cases, requires knowledge of the function and structure of possible viral targets and the identification and profiling of small molecule modulatory binding sites in these proteins that allow for structure-based drug design (SBDD) techniques.

The SARS-CoV-2 nucleocapsid (N) protein is an important target for the development of new antivirals because it is essential for the transcription and encapsidation of the CoV genome. The N protein is most often expressed in infected cells. Yet, it has not been widely used as a target for anti-SARS-CoV-2 drug development, unlike structural envelope (E) and spike (S) proteins. Indeed, structural data on N-protein ligand binding are limited.

About the study

In the present study, researchers from the Brazilian Center for Energy and Materials Research and the State University of Campinas used a new high-throughput screening test (HTS) based on fluorescence polarization to discover small compounds that interfere with the ability of protein N to bind to a specific RNA, i.e. RNA1, obtained from the packaging signal of the SARS-CoV-2 (PS) genome.

To develop an assay to recognize possible inhibitors of the RNA-binding capacity of protein N, the team searched for an RNA target of protein N using the SARS-CoV-2 PS sequence as a candidate. A fluorescence polarization (FP) assay using different 5′-fluorescein isothiocyanate (FITC)-labeled RNAs as targets monitored the RNA-binding activities of protein N.

Scientists used an FP test to conduct an HTS experiment examining a library of approximately 3200 licensed drugs and bioactive compounds. Concentration-response studies were conducted using the preselected molecules to verify the efficacy of the hit candidates as inhibitors of N-RNA1 protein contact.

1H saturation transfer difference nuclear magnetic resonance (1H NMR-STD) and isothermal titration calorimetry (ITC) were used to further examine the characteristics of CA as a protein N ligand. The authors established the crystal structure of the C-terminal domain (CTD) of protein N coupled to AC to shed light on how AC binds to protein N. They sought to see if the AC could prevent SARS-CoV-2 infection in vitro after validation of AC as protein-ligand N with probable consequences on its RNA-binding activity.

Results

In the evaluation of bioactive small molecules, immensely polar compounds stood out, in particular polyphenols such as chebulinic acid (CI), ellagitannins, punicalin (PL) and punicalagin (PG), as well as diesters of tartaric acid, such as CA, and polysulfonated naphthyureas such as suramin (SUR). The latter substances interfered with the contact of an RNA probe generated from the SARS-CoV-2 PS sequence, RNA1, and the integral SARS-CoV-2 N protein at the submicromolar level.

The crystal structure of the SARS-CoV-2 N (CA) protein CTD-binding chicoric acid reveals a network of polar contacts and structural readjustments to accommodate the symmetric ligand in the protein CA-binding site. NOT.  A) Cartoon representation of the crystal structure of the N protein CTD dimer illustrating its secondary structure elements (in blue), including two 310 (η) helices, five α-helices and two antiparallel β-strands and the binding site CA (inset highlighted by the blue dotted square).  B) CA binding site detailed from the inset of panel A. The CA molecule is shown as sticks (orange) with its electron density map in blue.  CA binds to a shallow pocket formed between the α 1-2 helices and the η 2 helix, near the C-terminus (C-Ter).  C) CA atomic interactions with N protein residues. The CA carboxyl groups are at ideal distances to initiate electrostatic interactions and hydrogen bonds with the Arg276 side chain (NH1 atom), the main chain amine Arg277 and a structural water molecule (W288) stabilized by Arg276 NH2.  Thr271 and Gln289 can further position the hydrogen bond donors (Thr271O𝛾 and a structural water molecule, W478, stabilized by the Gln289 carbonyl) to engage in symmetric interaction with the carbonyl groups of the two caffeoyl units of CA.  One of the catechol motifs of CA is well lodged near the C-terminal Pro364, showing a well-defined electron density (open panel B).  D, E) Overlay of the CA-binding site in the CTD native N protein (blue rods, PDB ID 7UXZ) and the CA-N protein CTD complex (gray rods (PDB ID 7UXZ)) highlighting the induced structural readjustments by the CA bond (highlighted by red arrows).  Figures were generated with Pymol (Schroedinger Inc.).  Pole contacts are indicated by dotted lines with distances measured in Angstroms.  Oxygen atoms are shown in red, nitrogen atoms in blue.  Water molecules are represented by red spheres.

The crystal structure of the SARS-CoV-2 N (CA) protein CTD-binding chicoric acid reveals a network of polar contacts and structural readjustments to accommodate the symmetric ligand in the protein CA-binding site. NOT. A) Cartoon representation of the crystal structure of the N protein CTD dimer illustrating its secondary structure elements (in blue), including two 310 (η) helices, five α-helices and two antiparallel β-strands and the binding site CA (inset highlighted by the blue dotted square). B) CA binding site detailed from the inset of panel A. The CA molecule is shown as sticks (orange) with its electron density map in blue. CA binds to a shallow pocket formed between the α 1-2 helices and the η 2 helix, near the C-terminus (C-Ter). C) CA atomic interactions with N protein residues. The CA carboxyl groups are at ideal distances to initiate electrostatic interactions and hydrogen bonds with the Arg276 side chain (NH1 atom), the main chain amine Arg277 and a structural water molecule (W288) stabilized by Arg276 NH2. Thr271 and Gln289 can further position the hydrogen bond donors (Thr271O and a structural water molecule, W478, carbonyl-stabilized Gln289) to engage in symmetrical interaction with the carbonyl groups of the two caffeoyl units of CA. One of the catechol motifs of CA is well lodged near the C-terminal Pro364, showing a well-defined electron density (open panel B). D, E) Overlay of the CA-binding site in the CTD native N protein (blue rods, PDB ID 7UXZ) and the CA-N protein CTD complex (gray rods (PDB ID 7UXZ)) highlighting the induced structural readjustments by the CA bond (highlighted by red arrows). Figures were generated with Pymol (Schroedinger Inc.). Pole contacts are indicated by dotted lines with distances measured in Angstroms. Oxygen atoms are shown in red, nitrogen atoms in blue. Water molecules are represented by red spheres.

CI, PL, PG, and SUR have been described previously to display different biological characteristics, including antiviral action. However, AC has been highlighted as a new class of N-protein modulators and one of the most effective compounds discovered in current HTS assays.

The results demonstrated that CA was an affinity ligand for protein N that binds to CTD and expels protein N RNA at micromolar levels. The team found that CA suppresses the multiplication of SARS-CoV-2 in cell culture at micromolar concentrations, which was consistent with the dissociation constant (KD) values ​​for the dissociation of the N protein and the RNA1 complex.

conclusion

According to the study authors, the present research was the first characterization of a non-endogenous ligand for the SARS-CoV-2 N protein and the initial account of this modulator binding location of the ligand on the CoV N protein.

In the current study, the scientists described a novel fluorescence-based HTS assay that enables the discovery of small compounds that obstruct the ability of the SARS-CoV-2 N protein to bind to RNA. They used a series of biophysical studies to characterize the best results. Researchers solved the crystal structure of the non-endogenous ligand binding N CTD protein for the first time, illuminating a novel modulator region in the SARS-CoV-2 N protein. Notably, the CA-binding region was conserved in SARS -CoV and partly conserved in Middle East Respiratory Syndrome (MERS) N proteins.

The current data provide the structural basis for the rational design and establishment of new antiviral drugs targeting the SARS-CoV-2 N protein, a relevant and as yet unstudied target of CoVs, despite the need to further refine the AC in as an antiviral agent.

*Important Notice

Preprints with Research Square publish preliminary scientific reports that are not peer-reviewed and, therefore, should not be considered conclusive, guide clinical practice/health-related behaviors, or treated as established information.

Journal reference:

  • Discovery and structural characterization of chicoric acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA binding disruptor; Gustavo Mercaldi, Eduardo Bezerra, Fernanda Aparecida Batista, Celisa Tonoli, Adriana Soprano, Jacqueline Shimizu, Alice Nagai, Jaqueline da Silva, Helder Ribeiro-Filho, Jessica Faria, Marcos da Cunha, Ana Zeri, Andrey Fabricio Nascimento, José Luiz Proença-Modena , Marcio Bajgelman, Silvana Rocco, Paulo Lopes-de-Oliveira, Artur Cordeiro, Marjorie Bruder, Rafael Elias Marques, Mauricio Sforca, Kleber Franchini, Celso Benedetti, Ana Carolina Figueira, Daniela Trivella. Quest square preprint. DOI: https://doi.org/10.21203/rs.3.rs-1720953/v1, https://www.researchsquare.com/article/rs-1720953/v1

Valerie J. Wallis