A review article in the journal Scientific Reports and currently available on the Research Square * prepress server reports chicory acid (CA) as a potential inhibitor of coronavirus 2 (SARS-CoV-2) for severe acute respiratory syndrome.
Study: Discovery and structural characterization of chicory acid as a SARS-CoV-2 nucleocapsid protein ligand and RNA-binding disruptor. Image credit: NIAID
Fund
There is still a critical medical need for new, targeted drugs that can fight SARS-CoV-2 infections. The establishment of these antiviral agents, in most cases, requires knowledge of the function and structure of possible viral targets and the identification and profile of modulator binding sites of small molecules in these proteins that allow techniques. structure-based drug design (SBDD).
The nucleocapsid protein (N) SARS-CoV-2 is an important target for the development of new antivirals, as it is essential for the transcription and packaging of the CoV genome. Protein N is most frequently expressed in infected cells. However, it has not been widely used as a target for the development of anti-SARS-CoV-2 drugs, in contrast to the envelope (E) and ear (S) structural proteins. In fact, structural data on N protein ligand binding are limited.
About the study
In the present study, researchers from the Brazilian Center for Research in Energy and Materials and Campinas State University used a new high-performance screening (HTS) assay 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 N protein RNA binding capacity, the team looked for a target N protein RNA that employed the PS SARS-CoV-2 sequence as a candidate. A fluorescence polarization (FP) test using different 5′-fluorescein-labeled RNA isothiocyanate (FITC) as targets monitored RNA binding activities of the N protein.
The scientists used a FP trial to run an HTS experiment examining a library of about 3,200 authorized drugs and bioactive compounds. Concentration-response studies were performed using preselected molecules to verify the efficacy of successful candidates as N-RNA1 protein contact inhibitors.
1H saturation transfer difference nuclear magnetic resonance (1H-STD NMR) and isothermal titration calorimetry (ITC) were used to further examine the characteristics of CA as a protein N ligand. The authors established the crystalline structure of the C-terminal domain (CTD) of protein N together with CA to shed light on how CA binds to protein N. They tried to see if CA can prevent SARS-CoV-2 infection in vitro after validating CA as an N protein ligand with probable consequences on its RNA adhesion activity.
Results
In the evaluation of small bioactive molecules, immensely polar compounds stood out, especially polyphenols such as quebulinic acid (CI), elagitanins, punicalin (PL) and punicalagin (PG), as well as diesters of tartaric acid, such as CA, and naphthylureas. polysulfonates such as suramine (SUR). The latter substances interfered with the contact of an RNA probe generated from the PS SARS-CoV-2 PS sequence, RNA1, and the full-length SARS-CoV-2 N protein at the submicromolar level.
The crystal structure of the SARS-CoV-2 N CTD protein that binds chicory acid (CA) reveals a network of polar contacts and structural readjustments to accommodate the symmetrical ligand at the CA-N protein binding site. . A) Cartoon representation of the crystal structure of the CTD dimer of protein N representing its secondary structure elements (in blue), including two 310 helices (η), five α helices, and two antiparal β chains · Them and the AC junction site (insert highlighted by the dotted blue square). B) Detailed AC junction site of panel A insertion. The AC molecule is represented as poles (orange) with its electron density map in blue. CA binds to a shallow pocket formed between the α 1-2 helix and the η 2 helix, near the C-terminus (C-Ter). C) CA atomic interactions with N protein residues. CA carboxyl groups are at ideal distances to involve electrostatic interactions and hydrogen bonds with the Arg276 side chain (NH1 atom), the Arg277 main chain amine, and a structural water molecule (W288) stabilized by Arg276 NH2. Thr271 and Gln289 can further position hydrogen bond donors (Thr271O and a structural water molecule, W478, stabilized by the carbonyl Gln289) to interact symmetrically with the carbonyl groups of the two cafeoil units of CA. One of the reasons for AC catechol is well accommodated near the C-terminal Pro364, showing a well-defined electronic density (see panel B). D, E) Superposition of the CA binding site to the native CTD N protein (blue sticks, PDB ID 7UXX) and CA-N CTD protein complex (gray sticks (PDB ID 7UXZ)) highlighting the structural readjustments induced by the AC junction (highlighted). with red arrows). Figures were generated with Pymol (Schroedinger Inc.). Polar contacts are indicated by dashed lines with distances measured in Angstroms. Oxygen atoms are shown in red, nitrogen atoms in blue. Water molecules are represented as red spheres.
CI, PL, PG, and SUR were previously described to show different biological characteristics, including antiviral action. However, CA stood out as a new class of N protein modulators and one of the most effective successful compounds discovered in current HTS assays.
The findings showed that CA was an affinity ligand for N protein that binds to CTD and expels RNA from N protein 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 values of dissociation constant (KD) for N protein dissociation and the RNA1 complex.
Conclusions
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 modulating location of ligand binding to the CoV protein. N.
In the current study, the scientists described a new fluorescence-based HTS test that allows the discovery of small compounds that obstruct the ability of the SARS-CoV-2 N protein to bind RNA. They used a series of biophysical studies to characterize the best achievements. The researchers solved the crystal structure of the non-endogenous ligand that binds the CTD N protein for the first time, illuminating a new modulating region to the SARS-CoV-2 N protein. In particular, the CA-binding region was conserved in SARS-CoV and partially conserved in Middle East Respiratory Syndrome (MERS) N proteins.
Current data provide the structural basis for the rational design and establishment of new antiviral drugs that address the SARS-CoV-2 N protein, a relevant and as yet unstudied goal of CoVs, despite the need to further refine CA as antiviral agent.
* Important news
Preprints with Research Square publish preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guided by clinical practice or health-related behavior, or treated as established information.
Magazine reference:
- Discovery and structural characterization of chicory 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, Jose Luiz Proenca-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. Square research prepress. IT HURTS: