Identification and characterization of alternative sites and molecular probes for SARS-CoV-2 target proteins

Abstract

Three protein targets from SARS-CoV-2, the viral pathogen that causes COVID-19, are studied: the main protease, the 2'-O-RNA methyltransferase, and the nucleocapsid (N) protein. For the main protease, the nucleophilicity of the catalytic cysteine C145 is enabled by coupling to three histidine residues, H163 and H164 and catalytic dyad partner H41. These electrostatic couplings enable significant population of the deprotonated state of C145. For the RNA methyltransferase, the catalytic lysine K6968 that serves as a Brønsted base has significant population of its deprotonated state via strong coupling with K6844 and Y6845. For the main protease, Partial Order Optimum Likelihood (POOL) predicts two clusters of biochemically active residues; one includes the catalytic H41 and C145 and neighboring residues. The other surrounds a second pocket adjacent to the catalytic site and includes S1 residues F140, L141, H163, E166, and H172 and also S2 residue D187. This secondary recognition site could serve as an alternative target for the design of molecular probes. From in silico screening of library compounds, ligands with predicted affinity for the secondary site are reported. For the NSP16-NSP10 complex that comprises the RNA methyltransferase, three different sites are predicted. One is the catalytic core at the conserved K-D-K-E motif that includes catalytic residues D6928, K6968, and E7001 plus K6844. The second site surrounds the catalytic core and consists of Y6845, C6849, I6866, H6867, F6868, V6894, D6895, D6897, I6926, S6927, Y6930, and K6935. The third is located at the heterodimer interface. Ligands predicted to have high affinity for the first or second sites are reported. Three sites are also predicted for the nucleocapsid protein. This work uncovers key interactions that contribute to the function of the three viral proteins and also suggests alternative sites for ligand design.

Keywords: Nucleocapsid; POOL; RNA methyltransferase; SARS-CoV-2; coupled amino acids; main protease; secondary sites.

FIGURE 1

(A) Homology model built on YASARA for N-terminal domain of the Nucleocapsid protein shown in cyan. (B) Homology model built on I-TASSER for the full length Nucleocapsid protein with domains colored as: N-terminal domain—cyan; C-terminal domain—blue; linker region—lavender; other regions—gray.

FIGURE 2

(A) A schematic representation of Partial Order Optimum Likelihood (POOL), our machine learning method to predict biochemically active sites. (B) Virtual screening workflow adopted in this project in conjunction with POOL.

FIGURE 3

(A) The different POOL-predicted pockets for the SARS-CoV-2 Main Protease (PDB ID: 6LU7). The POOL predicted residues in pocket one shown in yellow include the catalytic dyad H41-C145, shown in red. (B) The POOL-predicted secondary recognition pocket, shown in magenta, are the residues surrounding the catalytic dyad.

FIGURE 4

(A) Docking pose of the recognition peptide LQ↓SAG with two glycine caps (GG-LQSAG-GG) docked at the active site (in yellow) of the SARS-CoV-2 MPro with chain A in green and chain B in cyan. The peptide GG-LQSAG-GG is shown as red balls and sticks with the cleavage sequence Q↓S shown in blue. The H-bond interactions are shown in yellow and the interacting residues in teal. (B) The ligand interaction diagram showing the docked pose of GG-LQSAG-GG and the residues forming a pocket around it.

FIGURE 5

(A) Epigallocatechin gallate bound to the monomer of the SARS-CoV-2 MPro at the POOL predicted secondary site. The protein backbone is shown in green cartoon representation, ligand in red with the residues in gray. The hydrogen bonds are shown as yellow dashes and π- π stacking interactions as orange dashes. (B) Ligand interaction diagram of Epigallocatechin gallate at the secondary site. The hydrogen bonds are shown as pink arrows and π- π stacking interactions are shown as green lines.

FIGURE 6

The different POOL predicted sites for the SARS-CoV-2 RNA methyltransferase (NSP16/NSP10 complex) (A) The POOL-predicted residues in site 1, which contains the D-K-E part of the K-D-K-E conserved catalytic motif (D6928, K6968, and E7001) with an additional K6844, are shown in red (B) POOL-predicted site 2, containing residues surrounding the catalytic motif, is shown in magenta. (C) Site 3, and the POOL-predicted residues at the dimer interface, in blue.

FIGURE 7

(A) CAS#435297–57–7 bound to the SARS-CoV-2 NSP16/NSP10 complex (MTase) at the POOL-predicted site including the conserved catalytic motif. The protein backbone is shown in cartoon representation in green, ligand in red with the residue side chains in gray. The hydrogen bonds are shown as yellow dashes. (B) Ligand interaction diagram of CAS#435297–57–7 at the POOL-predicted site containing the conserved catalytic motif. The hydrogen bonds are shown as pink arrows and salt bridges in a bluish-red line.

FIGURE 8

(A) CAS# 926902–14–9 bound to SARS-CoV-2 NSP16/NSP10 complex (MTase) at the POOL-predicted residues surrounding the conserved catalytic motif, Site 2. The protein backbone is shown in cartoon representation in green, ligand in red with the residue side chains in gray. The hydrogen bonds are shown as yellow dashes. (B) Ligand interaction diagram of CAS# 926902–14–9 and the POOL-predicted residues surrounding the conserved catalytic motif, Site 2. The hydrogen bonds are shown as pink arrows and salt bridges in a bluish-red line.

FIGURE 9

The different POOL-predicted sites for the SARS-CoV-2 Nucleocapsid protein. POOL-predicted sites for the full-length SARS-CoV-2 Nucleocapsid protein built on I-TASSER. (A) Site 1 shown in blue, (B) Site 2 in green and (C) Site 3 in red.

FIGURE 10

(A) The POOL-predicted residues for the SARS-CoV-2 Nucleocapsid protein N-terminal model built in YASARA (B) The POOL-predicted residues for the SARS-CoV-2 Nucleocapsid protein C-terminal structure from the PDB (PDB ID: 7DE1).