Establishing an Analogue Based In Silico Pipeline in the Pursuit of Novel Inhibitory Scaffolds against the SARS Coronavirus 2 Papain-Like Protease

Abstract

The ongoing coronavirus pandemic has been a burden on the worldwide population, with mass fatalities and devastating socioeconomic consequences. It has particularly drawn attention to the lack of approved small-molecule drugs to inhibit SARS coronaviruses. Importantly, lessons learned from the SARS outbreak of 2002-2004, caused by severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), can be applied to current drug discovery ventures. SARS-CoV-1 and SARS-CoV-2 both possess two cysteine proteases, the main protease (M^pro) and the papain-like protease (PL^pro), which play a significant role in facilitating viral replication, and are important drug targets. The non-covalent inhibitor, GRL-0617, which was found to inhibit replication of SARS-CoV-1, and more recently SARS-CoV-2, is the only PL^pro inhibitor co-crystallised with the recently solved SARS-CoV-2 PL^pro crystal structure. Therefore, the GRL-0617 structural template and pharmacophore features are instrumental in the design and development of more potent PL^pro inhibitors. In this work, we conducted scaffold hopping using GRL-0617 as a reference to screen over 339,000 ligands in the chemical space using the ChemDiv, MayBridge, and Enamine screening libraries. Twenty-four distinct scaffolds with structural and electrostatic similarity to GRL-0617 were obtained. These proceeded to molecular docking against PL^pro using the AutoDock tools. Of two compounds that showed the most favourable predicted binding affinities to the target site, as well as comparable protein-ligand interactions to GRL-0617, one was chosen for further analogue-based work. Twenty-seven analogues of this compound were further docked against the PL^pro, which resulted in two additional hits with promising docking profiles. Our in silico pipeline consisted of an integrative four-step approach: (1) ligand-based virtual screening (scaffold-hopping), (2) molecular docking, (3) an analogue search, and, (4) evaluation of scaffold drug-likeness, to identify promising scaffolds and eliminate those with undesirable properties. Overall, we present four novel, and lipophilic, scaffolds obtained from an exhaustive search of diverse and uncharted regions of chemical space, which may be further explored in vitro through structure-activity relationship (SAR) studies in the search for more potent inhibitors. Furthermore, these scaffolds were predicted to have fewer off-target interactions than GRL-0617. Lastly, to our knowledge, this work contains the largest ligand-based virtual screen performed against GRL-0617.

Keywords: COVID-19; PLpro; SARS coronavirus 2; analogues; docking; drug discovery; in silico; papain-like protease; scaffold hopping; virtual screening.

Figure 1

The structure of PL^pro (PDB ID: 7JRN). The ubiquitin-binding domain is shown in dark blue. The catalytic triad is shown as silver sticks. Bound GRL-0617 is shown as magenta spheres, with BL2 (red) labelled nearby. The zinc-binding site is coordinated by residues shown as green sticks. The zinc ion is shown as a green sphere. In general, domain 1: dark blue, domain 2 (‘thumb’): green/cyan, domain 3 (‘fingers’): yellow/orange, domain 4 (‘palm’): red.

Figure 2

ROCS results (highest scoring molecules). (a) PubChem CID 121589399 had obtained the highest Shape Tanimoto; (b) PubChem CID 2732501 had obtained the highest Color Tanimoto.

Figure 3

Forge results of the compounds’ electrostatic similarity to GRL-0617 (hits scoring above 0.8 shown). (a) Query molecule GRL-0617 (shown as light green lines), which all compounds were aligned to; (b) Compound with PubChem CID: 5183914; (c) Compound with PubChem CID 53163980; (d) Compound with PubChem CID 121589399; (e) Compound with PubChem CID 2732501. Field scores for each of the compounds are shown in blue font. Regions of positive electrostatic potential are shown in red. Regions of negative electrostatic potential are shown in cyan. Regions of hydrophobicity are shown as gold spheres. The hits are shown as grey sticks.

Figure 4

Validation of the docking protocol using GRL-0617. (a)-i Reproducing the co-crystallised pose of GRL-0617 in AutoDock Vina (co-crystallised pose: pink sticks, docked pose: cyan sticks), with an RMSD of 0.430 Å between the two poses. The protein crystal structure is shown as white ribbons (PDB ID: 7JRN); (a)-ii The two-dimensional structure of GRL-0617 (drawn in MarvinSketch); (b) Co-crystallised pose: pink sticks, docked pose in AutoDock 4.2: cyan sticks, with an RMSD of 7.595 Å between the two poses; (c) Co-crystallised pose: pink sticks, docked pose in SwissDock: cyan sticks, with an RMSD of 0.545 Å between the two poses; (d) Co-crystallised pose: pink sticks, docked pose in GOLD: cyan sticks, with an RMSD of 0.720 Å between the two poses.

Figure 5

Poses of compounds from blind docking against PL^pro (PDB ID: 7JRN) in AutoDock Vina. (a) Compound 121589399 pose (cyan sticks) shown relative to reference GRL-0617 co-crystallised pose (pink sticks); (b) Compound 5384279 pose (cyan sticks) shown relative to reference GRL-0617 co-crystallised pose (pink sticks); (c) Compound 5183914 (cyan sticks) shown relative to reference co-crystallised GRL-0617 pose (pink sticks). The protein crystal structure is shown as white ribbons.

Figure 6

Three-dimensional protein-ligand interactions predicted by PLIP for the reference and three hits. (a) Reference ligand GRL-0617’s (cyan sticks) interactions with PL^pro residues (white/silver sticks); (b) Compound 121589399’s (cyan sticks) interactions with PL^pro residues (white/silver sticks); (c)) Compound 5384279’s (cyan sticks) interactions with PL^pro residues (white/silver sticks); (d) Compound 5183914’s (cyan sticks) interactions with PL^pro residues (white/silver sticks). 3-letter codes and sequence numbers for amino acids are given. Hydrogen bonds are shown as blue lines. π-π stacking interactions are shown as green dashes. Hydrophobic interactions are shown as grey dots. The PubChem CID is shown above each compound.

Figure 7

Two-dimensional protein-ligand interactions predicted by Maestro for the reference and three hits. (a) Reference ligand GRL-0617’s interactions with PL^pro residues; (b) Compound 121589399’s interactions with PL^pro residues; (c) Compound 5384279’s interactions with PL^pro residues; (d) Compound 5183914’s interactions with PL^pro residues. π-π stacking interactions are represented by green lines. π-cation interactions are represented by red lines. Hydrogen bonds are represented by purple dashed arrows (side chain) and purple solid arrow (backbone). Hydrophobic residues are shown as green circles. Positively charged residues are shown as purple circles. Negatively charged residues are shown as red circles. Polar residues are shown as cyan circles. Glycines are shown as light-yellow circles. The PubChem CID is shown above each compound.

Figure 8

Poses of analogues from blind docking against PL^pro in AutoDock Vina. (a) Compound 121558793’s pose (cyan sticks) relative to the reference ligand GRL-0617’s co-crystallised pose (pink sticks); (b) Compound 132344896’s pose (cyan sticks) relative to the reference ligand GRL-0617’s co-crystallised (pink sticks). The protein is shown as white ribbons.

Figure 9

Three-dimensional protein-ligand interactions predicted by PLIP for two best scoring analogues of compound 121589399. (a) Interactions of reference ligand GRL-0617 (cyan sticks) with PL^pro residues (white/silver sticks); (b) Interactions of analogue compound 121558793 (cyan sticks) with PL^pro residues (white/silver sticks); (c) Interactions of analogue compound 132344896 (cyan sticks) with PL^pro residues (white/silver sticks). 3-letter codes and sequence numbers for amino acids are given. Hydrogen bonds are shown as blue lines. π-π stacking interactions are shown as green dashes. Hydrophobic interactions are shown as grey dots. The PubChem CID is shown above each compound.

Figure 10

Two-dimensional protein-ligand interactions predicted for two best scoring analogues of compound 121589399 by Maestro. (a) Reference ligand GRL-0617’s interactions with PL^pro residues; (b) Compound 121558793’s interactions with PL^pro residues; (c) Compound 132344896’s interactions with PL^pro residues; π-π stacking interactions are represented by green lines. π-cation interactions are represented by red lines. Hydrogen bonds are represented by purple dashed arrows. Hydrophobic residues are shown as green circles. Positively charged residues are shown as purple circles. Negatively charged residues are shown as red circles. Polar residues are shown as cyan circles. Glycines are shown as light-yellow circles. A salt-bridge is shown as a multi-color (purple/red fusion) line. The PubChem CID is shown above each compound.

Figure 11

Comparison of poses from blind docking in Vina and focused docking (pose refinement) in AutoDock 4.2. The protein (PDB ID: 4JRN) is shown as white ribbons. (a) Reference ligand GRL-0617 docked pose from Vina (cyan sticks) and AutoDock 4.2 (gold sticks), relative to the co-crystallised pose (pink sticks); (b) Compound 121589399 docked pose from Vina (cyan sticks) and AutoDock 4.2 (gold sticks), relative to the co-crystallised pose of GRL-0617 (pink sticks); (c) Compound 5384279 docked pose from Vina (cyan sticks) and AutoDock 4.2 (gold sticks), relative to the co-crystallised pose of GRL-0617 (pink sticks); (d) Compound 5183914 docked pose from Vina (cyan sticks) and AutoDock 4.2 (gold sticks), relative to the co-crystallised pose of GRL-0617 (pink sticks); (e) Compound 121558793 docked pose from Vina (cyan sticks) and AutoDock 4.2 (gold sticks), relative to the co-crystallised pose of GRL-0617 (pink sticks); (f) Compound 132344896 docked pose from Vina (cyan sticks) and AutoDock 4.2 (gold sticks), relative to the co-crystallised pose of GRL-0617 (pink sticks).