Structure-Guided Design of Potent Inhibitors of SARS-CoV-2 3CL Protease: Structural, Biochemical, and Cell-Based Studies

Abstract

The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe–Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro.

Structure-Guided Design of Potent Spirocyclic Inhibitors of Severe Acute Respiratory Syndrome Coronavirus-2 3C-like Protease

“…There is an array of advantages accrued through the judicious use of spirocycles in drug design, including improved physicochemical and PK characteristics, structural novelty, reduced conformational flexibility, and the capture of favorable binding interactions by probing and exploiting poorly explored regions of chemical space. − Importantly, the structural motifs embodied in spirocycles make possible the rigorous control of the spatial disposition of exit vectors; consequently, it was envisaged that the attachment of a suitably decorated spirocycle capable of engaging in favorable binding interactions with the S 4 subsite of SARS-CoV-2 3CL pro to a recognition element that is congruent with the known substrate specificity of the enzyme (a Leu-Gln surrogate fragment) can be leveraged to yield a molecule (Figure /general structure I) with high inhibitory prowess. The validity of the approach and the design of the inhibitors was further facilitated by the availability and use of high-resolution cocrystal structures. − ,− Finally, for comparative purposes, a series of azetidine-derived inhibitors (Figure /general structure II) were also synthesized and evaluated in biochemical and cell-based assays.…”

“…The inhibitory activity of compounds 1 – 18b/c toward SARS-CoV-2 3CL pro and MERS-CoV 3CLpro in biochemical assays − , as well as the cytotoxicity of the compounds were determined, and the results are listed in Tables and . For comparative purposes, the IC 50 and CC 50 values of GC376 are included in Table .…”

“…The SARS-CoV-2 genome (∼30 kb) encodes multiple structural (spike (S), envelope (E), membrane (M), and nucleocapsid (N)) and nonstructural proteins. , The homotrimeric spike protein plays a critical role in viral attachment, fusion, and entry by binding to the receptor-binding domain of the host receptor (ACE2), followed by the furin-, transmembrane serine protease 2-, and cathepsin L-mediated fusion of viral and endosomal membranes, and the release of viral RNA into the cytosol. − The replicase is expressed by two open reading frames that encode two large polyproteins (pp1a and pp1ab), which are processed by the 3C-like protease (3CL pro ) and papain-like protease (PL pro ), to generate mature structural and nonstructural proteins. The 3CL pro , also called main protease (M pro ), is an induced-fit enzyme with an extended binding cleft, a Cys-His catalytic dyad, and a primary substrate specificity for a P 1 Gln residue and a preference for a P 2 Leu. , The enzyme is essential for viral replication; consequently, it is an attractive validated target for the development of direct-acting antivirals. − SARS-CoV-2 3CL pro has been under intense investigation for the development of SARS-CoV-2 therapeutics by us − and others. ,− The rationale underlying the targeting of SARS-CoV-2 3CL pro is further buttressed by the first time demonstration of clinical efficacy by a feline coronavirus 3CL pro inhibitor. , We report herein the results of preliminary studies related to the structure-guided design of potent inhibitors of SARS-CoV-2 3CL pro (Figure /general structure I) that incorporate in their structure a spirocyclic component as a design element to optimally exploit new chemical space in the active site of the protease.…”

Computational molecular interaction between SARS-CoV-2 main protease and theaflavin digallate using free energy perturbation and molecular dynamics

“…We have also included a feline coronavirus inhibitor, GC376, with PDB ID 6WTJ and ligand ID K36 as GC376 [ 37 ]. We have included the PDB ID 7M01 complexed with inhibitor 14c [ 38 ]. The inhibitor 14c has two enantiomers, with ligand ID YKV and YKS.…”

“…The set includes PDB ID 7M03 with inhibitor 18c having R, S enantiomer with ligand ID YLJ, and YLD in a similar structure series. We have also included the reference set from PDB ID 7LZT with inhibitor 8b having enantiomer with ligand ID YMY and YN1 [ 39 ]. We have also included the reference set from covalent inhibitor 2k having enantiomer with ligand ID Y4V and Y7M [ 40 ].…”