Probing <scp>3CL</scp> protease: Rationally designed chemical moieties for <scp>COVID</scp>‐19

Abstract

The outbreak of the COVID-19 pandemic has already pushed the worldwide morbidity and mortality rates to prodigious proportions, obligating deliberate efforts in identifying novel pharmacophores to manage the pathogenesis and, in addition, to target critical pathways that otherwise, sustain viral viability (Singh, Gupta, Satija, Negi, et al., 2020; Singh, Gupta, Satija, Pabreja et al., 2020). As per the latest WHO reports until June 2020, the COVID-19 pandemic claimed 0.3 million lives globally, with looming threat over 6.7 million confirmed cases (https://covid19.who.int/). Symptomized as severe acute respiratory syndrome (SARS), the COVID-19 infection, in addition to the Acute Respiratory Distress Syndrome (ARDS), manifests into moderate to acute pneumonia and aseptic shock (Gupta, Singh, Kumar Chellappan, & Dua, 2020; Xu et al., 2020). A multitude of regulatory protocols and lengthy clinical profile validations decelerated the de novo development of a novel drug molecule thereby creating an urgency to develop a swift chemotherapeutic regime to manage the present day health emergency. This encouraged a drug-repurposing approach to regulate and contain the disease eruption (Guy, DiPaola, Romanelli, & Dutch, 2020). However, the clinical limitations of several redeployed drugs apparently necessitated a paradigm shift in the strategy, which provided further impetus toward developing rationally designed pharmaceuticals. The prototypes for the development of such potentially active substances, that may inhibit the SARS-CoV-2 at the cellular level, then stemmed from the “lead” molecules that successfully countered MERS-CoV and SARS-CoV-1 (Anand, Ziebuhr, Wadhwani, Mesters, & Hilgenfeld, 2003). Coronaviruses constitute a group of closely related RNA viruses that have time and again generated considerable interest due to their unique pattern of emergence ever since their discovery in the early 20th century (Estola, 1970). Coronavirus membranes essentially carry four viral proteins found on the membrane of the coronavirus. These include glycoprotein Type I spike proteins, membrane proteins present over the membrane, strong hydrophobic proteins enveloping the entire structure of coronavirus, and nucleocapsid proteins (Thiel et al., 2003). Among the four viral proteins, the nucleocapsid protein and spike protein play critical role in virus development (Siu et al., 2008). The former promotes capsid development over the virus, whereas the latter prompts the binding of the virus with host cells (Walls et al., 2020). The genome of the SARS-CoV-1 consists of two open read frames that are connected by the ribosomal frame which encodes two large overlaps of polyprotein replicas, named as, pp1a and pp1ab (Ziebuhr, Heusipp, & Siddell, 1997). In the infected human cells, the SARS-CoV-1 generates functional proteins by breaking them from their overlapping “polyproteins,” pp1a, and pp1ab. Papain-like proteases 2 and chymotrypsin like cysteine protease known as 3C-like proteases (3CLpro) are the major proteases among them (Thiel et al., 2003). Papain like proteases cleave 3 sites, while 3CL proteases cleave 11 sites including its own N and C-terminal self-processing sites, by recognizing P4–P1 and P1' in the polyprotein to create unique functional proteins such as RNA-dependent polymerase, helicase, RNA-binding protein, exoribonuclease, endoribonuclease, and 2′-O-ribose (Yang et al., 2003). Proteases in SARS-CoV-2 bind to the angiotensin converting enzyme-2 (ACE-2) receptor present on respiratory epithelium to break the “S” protein into the S1 and S2 domains (Hoffmann et al., 2020). This cleavage initiates conformation transition thereby activating the S2 domain, followed by the introduction of the fusion protein into the membrane that further promotes the cellular internalization of the SARS-CoV-2 virus (Liu et al., 2020). The 3CL protease mediates a critical role in the viral replication and infection cycles, hence forming an ideal target for antiviral therapy for containing the SARS-CoV-2 (Li & De Clercq, 2020). Interestingly, rationally designed molecular mimics of the natural substrate of 3CL protease may serve as a potent inhibitor of the enzyme (Sisay, 2020). The carefully optimized chemical moieties and functional groups on the molecular mimics hold potential to alter the catalytic mechanism of the enzyme (Mengist, Fan, & Jin, 2020). The peptide or peptidomimetic backbone serves as the vital constituent that plays a crucial role in the molecular inhibition of SARS-CoV-2 (Pillaiyar, Manickam, Namasivayam, Hayashi, & Jung, 2016). Considering the role of 3CL protease in virus propagation, the rationally designed molecular inhibitors of this enzyme present high perspectives for containing the further spread of COVID-19 pandemic (Macchiagodena, Pagliai, & Procacci, 2020). SARS-CoV-2 displays striking genetic similarity with the SARS-CoV-1 and MERS responsible for the earlier global outbreak by Betacoronavirus (Petrosillo, Viceconte, Ergonul, Ippolito, & Petersen, 2020). In addition, the structural similarity between the 3CL protease in SARS-CoV-1 and SARS-CoV-2 prompt the transferability of biological profile of rationally designed molecules mainly focused on SARS-CoV-1, to be effective in containing the SARS-CoV-2 infection (Walls et al., 2020). The present commentary considers the findings of Zhang, Lin, Sun, et al. (2020) and appraises the urgency of deliberated molecules as substrate mimics of 3CL protease enzyme of the SARS-CoV-2 virus, along with the identification of suitable pharmacophores with potential of capping the COVID-19 pandemic. The α-ketoamide group presents a potential pharmacophoric profile for targeting the 3CL protease enzyme of COVID-19, as validated by the inhibitory profiles of compounds 1–4 (Figure 1). Zhang, Lin, Sun, et al. (2020) identified the compound 1; intended as 3C protease inhibitor of enteroviruses, exhibited considerable submicromolar IC50 against SARS-CoV-1, and MERS-CoV as tested in Huh7 cells. Pharmacokinetic analysis of the test compound 1 in CD-1 mice revealed a lower half-life in blood plasma, and kinetic/ thermodynamic stability in systemic circulation (Zhang, Lin, Kusov, et al. 2020) Importantly the compounds did not manifest any toxicity symptoms in test animals. In addition to coronavirus, the test compound 1 displayed inhibitory potential against enterovirus by inhibiting the activity of 3CLpro and disrupting the virus replication, hence acting as a potential candidate for further screening against SARS-CoV-2. Replacement of the amide bond in compound 1 (Figure 1) with an intrinsic amide functionality in the pyridone ring improved the stability of the resulting compound 2 (Figure 1) against cellular proteases in systemic circulation. The pharmacokinetic parameters of the test compound 2; including plasma half-life, kinetic/thermodynamic stability improved remarkably with this structural modification. Preclinical analysis of the test compound 2 in CD-1 rats and human microsomes demonstrate a tolerable intrinsic clearance rate of 32 μl/min/mg protein and 21 μl/min/mg protein, respectively. Similarly, the replacement of the cinnamoyl group in compound 1; with a less hydrophobic tert-butyloxycarbonyl (Boc) moiety in compound 2 played a crucial role in improving the plasma solubility of the compound and in mitigating its binding to the plasma proteins due to the presence of sterically hindering bulkier substituent in the latter. The target 3CL protease in SARS-CoV-1 displays a striking 96% similarity to the same enzyme in SARS-CoV-2 (COVID-19) (Chen, Yiu, & Wong, 2020). The deliberated compound thus holds a huge potential in capping the morbidity caused by the latter. Further replacement of the cyclohexyl moiety in compound 2 (Figure 1) with a smaller cyclopropyl ring in compound 3 (Figure 1) resulted in the improvement in broad-spectrum activity by inhibiting the 3CLpro enzyme in SARS-CoV-1, MERS-CoV, and SARS-CoV-2 by promoting a significant reduction in viral RNA replication. While interacting with the active site residues of the target 3CLpro enzyme, the test compound 3 (Figure 1) reportedly undergoes a nucleophilic attack by the catalytic Cys145 residue at α-keto group to form thiohemiketal via a reversible reaction. Interestingly, the His41 residue, lining the active site of the target enzyme stabilizes the oxyanion of thiohemiketal via H-bonding, whereas, the “O” atom of amide functionality acts as H-bond acceptor from the main chain amide bond in the residues Gly143, Cys145, and Ser144 (Figure 2). Eventually, the removal of Boc group in compound 4 (Figure 1) lowered its inhibitory potency due to a marked decrease in interactions with the active site of 3CLpro enzyme. The test compound 1 displayed plasma half-life of 0.3 hr, with kinetic solubility of 6 μM and thermodynamic stability of 41 μM, respectively, in the blood plasma. These pharmacokinetic parameters improved significantly on replacing of peptide linkage with pyridone ring mainly due to enhanced stability against cellular proteases. The pharmacokinetic analysis in CD-1 mice revealed that the subcutaneous administration of compound 2; at 20 mg/kg maintained its plasma sustainability for 4 hr with a prolonged urinary excretion for up to 24 hr. Even though the compound exhibited a rapid plasma clearance, it maintained a significant tissue distribution at concentration of 135 ng/g tissue in lungs and 52.7 ng/ml in the bronchioalveolar lavage fluid. The pharmacokinetic profile further improved for the compound 3; which demonstrated the mean resident time of 2.7 hr and plasma half-life of 1.8 hr with a less rapid clearance compared with the compound 2. Interestingly, the compounds 2, and 3 exhibit lung tropism due to the local lung concentration, which prove highly beneficial considering the effect of SARS-CoV-2 infection on lungs. The inhalation of test compound 3 by nebulizer at a dose of 3 mg/kg resulted in its localization in the lung tissues at 33 ng/g, with negligible adverse effect in the CD-1 mice, thereby validating a superior tolerability. These investigations also confirmed the possibility of a direct administration of the test compounds to the lungs (Zhang, Lin, Sun, et al. 2020). The drug discovery and development paradigms based on the synthesis of a library of diverse compounds for identifying the potential “hits” and “leads” face persistent challenges associated with the efficiency of synthesis and purification protocols of synthesized compounds (Akaji et al., 2011). The in silico drug discovery approaches provide an effective preliminary screening of the bioactive compounds from the large compound libraries, thereby saving the time and effort in generating these libraries in order to meet the urgency of therapeutic molecules as in COVID-19 (Cava, Bertoli, & Castiglioni, 2020). The binding site of the 3CL protease in SARS-CoV-2 constitutes of both hydrophilic and hydrophobic residues, including negatively charged Glu166 and Asp187 residues, and a positively charged residue, Arg188 (Figure 2). Importantly, the water molecules surrounding the residues such as, Hie41, His163, Glu166, and Gln192 constitute a salt bridge between the deliberated ligand molecule and the protein (PDB ID: 6Y2F). Similarly, the molecular docking simulations suggested the hydrogen bonding interactions between the test compounds 5–8 (Figure 3) in the active site of the target enzyme 3CLpro (PDB ID: 6 W63). The compound 5 (Figure 3) displayed the highest docking score of −11.02 kcal/mol while interacting with the active site residues Gln-189, Hie-164, Gly-143, Thr-26, Glu-166, and water bridges. The Gibbs free energy for ligand binding appeared in the range of −98.78 to −111.13 kcal/mol. The molecular dynamics simulations suggested that the compound 5 (Figure 3) adopted a stable conformation in the 3CLpro active site, whereas the compounds 6, 7, and 8 (Figure 3) showed slight deviations from an optimum orientation (Pant, Singh, Ravichandiran, Murty, & Srivastava, 2020). These investigations advocated the interactions of test compounds inside the binding loop of SARS-CoV-2 protease leading to its inhibition. Previous experiences with the anthelminthic drug “Niclosamide” [5-chloro-salicyl-(2-chloro-4-nitro) anilide] (9, Figure 4) have provided crucial information regarding the suitability of anilide derivatives in advancing the antiviral drug repurpose screening investigations (Pindiprolu & Pindiprolu, 2020). The anilide-based 3CL protease inhibitors (10–13), (Figure 4) were identified as potential pharmacophores, that effectively downregulated the signaling pathways in SARS-CoV-1 and MERS-CoV (Xu, Shi, Li, & Zhou, 2020). These peptide-appended anilides successfully inhibited SARS-CoV-1 3CL protease via competitive inhibition, while adopting a highly stabilized conformation within the active site loop of the target enzyme. The in silico investigations confirmed that the most active anilide interacts with –SH group of Cys145 residues via hydrogen-bonding interactions (4.95 Å), which prevents its nucleophilic proteolysis by the residue. The hydrolysis of N-nitrophenylamide requires a pertinent alignment of n, and π orbitals to facilitate the facile leaving of nitroaniline. However, the steric effect caused by “Cl” atoms at ortho-position disturbs the coplanarity of 2-chloro-4-nitrophenyl ring with the amido group, thereby, discouraging the process of hydrolysis. The antiviral potential of these compounds, 10–13 (Figure 4) suggests the therapeutic potential of anilides, and amide functionality for the rational development of SARS-CoV-2 targeting drugs (Shie et al., 2005). The rationally designed compounds 14–15 (Figure 5) markedly inhibited 3CL protease enzyme in SARS-CoV-2, eventually delivering approximately 100% results at 1 μM concentration. The compounds 14 and 15 exhibited IC50 = 0.053 ± 0.005 μM and 0.040 ± 0.002 μM, respectively. Notably, the carbonyl carbon of the aldehyde group in the test compounds forms a C–S covalent bond of length 1.8 Å with the Cys145 residue present in the enzyme's active site, whereas, the “O” atom of the aldehyde group participates in conformational stabilization of the molecules by forming hydrogen bonds of length 1.8 Å with the Cys145 residue. The (S)-γ-lactam ring in the test compounds exhibited an ideal alignment in the active site of 3CLpro due to hydrogen bonding interactions with His163 residue through the “O” atom present on the lactam ring. Similarly, the “NH” group of lactam framework exhibits hydrogen bonding interactions with the enzyme's main chain residue Phe140, and the side chain residue Glu166 thereby providing a conformational stabilization to the lactam ring system in the active site of the enzyme. The –CONH2 functional groups present in the test compounds participate in hydrogen bonding interactions with His164 and Glu166 residues, whereas, the cyclohexyl ring embeds deeply into the active site cavity while stacking with the imidazole ring of the His41 residue. In addition, the side chains Tyr54, Met165, Arg188, Met49, and Asp187 residues surround the cyclohexyl ring via extensive hydrophobic interactions. Notably, the indole ring forms a 2.6 Å hydrogen bond with Glu166 residue, and displays hydrophobic interactions with the side chain residues Pro168, and Gln189. Similarly, the test compound with 3-fluorophenyl substituent experiences a marked downward rotation in the enzyme active site. The substituent displays hydrogen bonding interactions with Gln189 leading to its conformational stabilization, in addition to significant hydrophobic interactions with the side chains of residues Arg188, Val186, Met49, His41, Met165, and Asp187. The in vitro analysis of the test compounds 14–15 (Figure 5) further confirmed their inhibitory potential against the 3CL protease enzyme of SARS-CoV-2 (Figure 6). The in vivo analysis on Sprague Dawley rats (SD rats) and beagle dogs via intravenous and subcutaneous administration suggested favorable pharmacokinetics, good bioavailability, and metabolic stability of the test compounds, which further validated their robust candidature for clinical trials (Dai et al., 2020). The SARS-CoV-1 emerged as global pandemic during the early part of the year 2003. Soon after, the year 2015 saw another viral outbreak in the form of MERS-CoV. However, the timeframe between these two outbreaks produced fewer clinically proven pharmacophores from the pipeline, for countering an impending relapse of the viral pandemic. Hence, it resulted in the emergence of SARS-CoV-2, in 2020, which caused colossal proportions of strains in the global economy and healthcare systems. Subsequent failure of repurposed drugs further added to the urgency to contain the severity of the pandemic. One of the novel strategies is to employ chemical therapeutics that target the 3CL protease enzyme associated with viral propagation. The inhibition profile of 3CL protease previously investigated with SARS-CoV-1 and MERS-CoV outbreaks further validated its candidature for containing the novel SARS-CoV-2 transmission. The peptide backbone in most of the inhibitor classes enables the optimal accommodation of the designed molecule in the active site of the target 3CL protease enzyme thereby preventing the enzymatic metabolism of the natural substrate. Further appending of suitable moieties and functionalities including α-ketoamide, anilide, indole, and lactam on the peptide scaffold potentiate the interactions with key active site residues responsible for the catalytic action of the enzyme on the natural substrate. Eventually, the SAR analysis identifies the appropriate substituents and their position on the parent molecules for the exploration of effective molecules. Therefore, the rationally designed molecular inhibitors of 3CL protease enzyme provide the basis for the development of perspective anti COVID-19 chemotherapy. The authors declare no conflicts of interest.