The assembly of two monomeric constructs spanning segments 1-199 (MPro^1-199) and 10-306 (MPro^10-306) of SARS-CoV-2 main protease (MPro) was examined to assess the existence of a transient heterodimer intermediate in the N-terminal autoprocessing pathway of MPro model precursor. Together, they form a heterodimer population accompanied by a 13-fold increase in catalytic activity. Addition of inhibitor GC373 to the proteins increases the activity further by ∼7-fold with a 1:1 complex and higher order assemblies approaching 1:2 and 2:2 molecules of MPro^1-199 and MPro^10-306 detectable by analytical ultracentrifugation and native mass estimation by light scattering. Assemblies larger than a heterodimer (1:1) are discussed in terms of alternate pathways of domain III association, either through switching the location of helix 201 to 214 onto a second helical domain of MPro^10-306 and vice versa or direct interdomain III contacts like that of the native dimer, based on known structures and AlphaFold 3 prediction, respectively. At a constant concentration of MPro^1-199 with molar excess of GC373, the rate of substrate hydrolysis displays first order dependency on the MPro^10-306 concentration and vice versa. An equimolar composition of the two proteins with excess GC373 exhibits half-maximal activity at ∼6 μM MPro^1-199. Catalytic activity arises primarily from MPro^1-199 and is dependent on the interface interactions involving the N-finger residues 1 to 9 of MPro^1-199 and E290 of MPro^10-306. Importantly, our results confirm that a single N-finger region with its associated intersubunit contacts is sufficient to form a heterodimeric MPro intermediate with enhanced catalytic activity.

01 Jul 2024·Journal of Molecular Biology

Visualizing the Active Site Oxyanion Loop Transition Upon Ensitrelvir Binding and Transient Dimerization of SARS-CoV-2 Main Protease

Article

Author: Nashed, Nashaat T ; Louis, John M ; Ghirlando, Rodolfo ; Aniana, Annie ; Kovalevsky, Andrey ; Coates, Leighton

N-terminal autoprocessing from its polyprotein precursor enables creating the mature-like stable dimer interface of SARS-CoV-2 main protease (MPro), concomitant with the active site oxyanion loop equilibrium transitioning to the active conformation (E*) and onset of catalytic activity. Through mutagenesis of critical interface residues and evaluating noncovalent inhibitor (ensitrelvir, ESV) facilitated dimerization through its binding to MPro, we demonstrate that residues extending from Ser1 through Glu14 are critical for dimerization. Combined mutations G11A, E290A and R298A (MPro™) restrict dimerization even upon binding of ESV to monomeric MPro™ with an inhibitor dissociation constant of 7.4 ± 1.6 µM. Contrasting the covalent inhibitor NMV or GC373 binding to monomeric MPro, ESV binding enabled capturing the transition of the oxyanion loop conformations in the absence of a reactive warhead and independent of dimerization. Characterization of complexes by room-temperature X-ray crystallography reveals ESV bound to the E* state of monomeric MPro as well as an intermediate approaching the inactive state (E). It appears that the E* to E equilibrium shift occurs initially from G138-F140 residues, leading to the unwinding of the loop and formation of the 3₁₀-helix. Finally, we describe a transient dimer structure of the MPro precursor held together through interactions of residues A5-G11 with distinct states of the active sites, E and E*, likely representing an intermediate in the autoprocessing pathway.

01 Apr 2024·Bioorganic & Medicinal Chemistry Letters

Synthesis, SARS-CoV-2 main protease inhibition, molecular docking and in silico ADME studies of furanochromene-quinoline hydrazone derivatives

Article

Author: Cassel, Joel ; Henry, Geneive E ; Montaner, Luis J ; Salvino, Joseph M ; Tietjen, Ian ; Basile, Olivia N ; Shellenberger, Blake M ; Olsen, Morgan R

Seven furanochromene-quinoline derivatives containing a hydrazone linker were synthesized by condensing a furanochromene hydrazide with quinoline 2-, 3-, 4-, 5-, 6-, and 8-carbaldehydes, including 8-hydroxyquinoline-2-carbaldehye. Structure-activity correlations were investigated to determine the influence of the location of the hydrazone linker on the quinoline unit on SARS-CoV-2 M^pro enzyme inhibition. The 3-, 5-, 6- and 8-substituted derivatives showed moderate inhibition of SARS-CoV-2 M^pro with IC₅₀ values ranging from 16 to 44 μM. Additionally, all of the derivatives showed strong interaction with the SARS-CoV-2 M^pro substrate binding pocket, with docking energy scores ranging from -8.0 to -8.5 kcal/mol. These values are comparable to that of N3 peptide (-8.1 kcal/mol) and more favorable than GC-373 (-7.6 kcal/mol) and ML-188 (-7.5 kcal/mol), all of which are known SARS-CoV-2 M^pro inhibitors. Furthermore, in silico absorption, distribution, metabolism, and excretion (ADME) profiles indicate that the derivatives have good drug-likeness properties. Overall, this study highlights the potential of the furanochromene-quinoline hydrazone scaffold as a SARS-CoV-2 M^pro inhibitor.

100 Deals associated with GC-373

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Indication	Highest Phase	Country/Location	Organization	Date
Virus Diseases	Phase 1	US	University of Kansas	12 Aug 2012
Norovirus Infections	Preclinical	US	Wichita State University	01 Sep 2011

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