Allosteric communication between ACE2 active site and binding interface with SARS-CoV-2.

SARS-CoV-2, the virus causing COVID-19, initiates cell invasion by deploying a receptor binding domain (RBD) to recognize the host transmembrane peptidase angiotensin-converting enzyme 2 (ACE2). Numerous experimental and theoretical studies have adopted high-throughput and structure-guided approaches to (i) understand how the RBD recognizes ACE2, (ii) rationalize, and (iii) predict the effect of viral mutations on the binding affinity. Here, we investigate the allosteric signal triggered by the dissociation of the ACE2-RBD complex. To this end, we construct an Elastic Network Model (ENM), and we use the Structural Perturbation Method (SPM). Our key result is that complex dissociation opens the ACE2 substrate-binding cleft located away from the interface and that fluctuations of the ACE2 binding cleft are facilitated by RBD binding. These and other observations provide a structural and dynamical basis for the influence of SARS-CoV-2 on ACE2 enzymatic activity. In addition, we identify a conserved glycine (G502 in SARS-CoV-2) as a key participant in complex disassembly.

Protein-ligand interactions from a quantum fragmentation perspective: The case of the SARS-CoV-2 main protease interacting with α-ketoamide inhibitors.

We present a hybrid, multi-method, computational scheme for protein/ligand systems well suited to be used on modern and forthcoming massively parallel computing systems. The scheme relies on a multi-scale polarizable molecular modeling, approach to perform molecular dynamics simulations, and on an efficient Density Functional Theory (DFT) linear scaling method to post-process simulation snapshots. We use this scheme to investigate recent α-ketoamide inhibitors targeting the main protease of the SARS-CoV-2 virus. We assessed the reliability and the coherence of the hybrid scheme, in particular, by checking the ability of MM and DFT to reproduce results from high-end ab initio computations regarding such inhibitors. The DFT approach enables an a posteriori fragmentation of the system and an investigation into the strength of interaction among identified fragment pairs. We show the necessity of accounting for a large set of plausible protease/inhibitor conformations to generate reliable interaction data. Finally, we point out ways to further improve α-ketoamide inhibitors to more strongly interact with particular protease domains neighboring the active site.

Energetic vs. entropic stabilization between a Remdesivir analogue and cognate ATP upon binding and insertion into the active site of SARS-CoV-2 RNA dependent RNA polymerase.

SARS-CoV-2 RNA dependent RNA polymerase (RdRp) serves as a highly promising antiviral drug target such as for a Remdesivir nucleotide analogue (RDV-TP or RTP). In this work, we mainly used alchemical all-atom simulations to characterize relative binding free energetics between the nucleotide analogue RTP and natural cognate substrate ATP upon initial binding and pre-catalytic insertion into the active site of SARS-CoV-2 RdRp. Natural non-cognate substrate dATP and mismatched GTP were also examined for computation control. We first identified significant differences in dynamical responses between nucleotide initial binding and subsequent insertion configurations to the open and closed active sites of the RdRp, respectively, though the RdRp protein conformational changes between the active site's open and closed states are subtle. Our alchemical simulations indicated that upon initial binding (active site open), RTP and ATP show similar binding free energies to the active sites while in the insertion state (active site closed), ATP is more stabilized (∼-2.4 kcal mol-1) than RTP in free energetics. Additional analyses show, however, that RTP is more stabilized in binding energetics than ATP, in both the insertion and initial binding states, with RTP more stabilized due to the electrostatic energy in the insertion state and due to vdW energy in the initial binding state. Hence, it appears that natural cognate ATP still excels at association stability with the RdRp active site due to that ATP maintains sufficient flexibilities e.g., in base pairing with the template, which exemplifies an entropic contribution to the cognate substrate stabilization. These findings highlight the importance of substrate flexibilities in addition to energetic stabilization in antiviral nucleotide analogue design.

Unveiling the Potentiality of Shikonin Derivatives Inhibiting SARS-CoV-2 Main Protease by Molecular Dynamic Simulation Studies.

Shikonin, a phytochemical present in the roots of Lithospermum erythrorhizon, is well-known for its broad-spectrum activity against cancer, oxidative stress, inflammation, viruses, and anti-COVID-19 agents. A recent report based on a crystallographic study revealed a distinct conformation of shikonin binding to the SARS-CoV-2 main protease (Mpro), suggesting the possibility of designing potential inhibitors based on shikonin derivatives. The present study aimed to identify potential shikonin derivatives targeting the Mpro of COVID-19 by using molecular docking and molecular dynamics simulations. A total of 20 shikonin derivatives were screened, of which few derivatives showed higher binding affinity than shikonin. Following the MM-GBSA binding energy calculations using the docked structures, four derivatives were retained with the highest binding energy and subjected to molecular dynamics simulation. Molecular dynamics simulation studies suggested that alpha-methyl-n-butyl shikonin, beta-hydroxyisovaleryl shikonin, and lithospermidin-B interacted with two conserved residues, His41 and Cys145, through multiple bonding in the catalytic sites. This suggests that these residues may effectively suppress SARS-CoV-2 progression by inhibiting Mpro. Taken together, the present in silico study concluded that shikonin derivatives may play an influential role in Mpro inhibition.

Identification and Investigation of a Cryptic Binding Pocket of the P37 Envelope Protein of Monkeypox Virus by Molecular Dynamics Simulations.

The spread of the monkeypox virus has surged during the unchecked COVID-19 epidemic. The most crucial target is the viral envelope protein, p37. However, lacking p37's crystal structure is a significant hurdle to rapid therapeutic discovery and mechanism elucidation. Structural modeling and molecular dynamics (MD) of the enzyme with inhibitors reveal a cryptic pocket occluded in the unbound structure. For the first time, the inhibitor's dynamic flip from the active to the cryptic site enlightens p37's allosteric site, which squeezes the active site, impairing its function. A large force is needed for inhibitor dissociation from the allosteric site, ushering in its biological importance. In addition, hot spot residues identified at both locations and discovered drugs more potent than tecovirimat may enable even more robust inhibitor designs against p37 and accelerate the development of monkeypox therapies.

pH profiles of 3-chymotrypsin-like protease (3CLpro) from SARS-CoV-2 elucidate its catalytic mechanism and a histidine residue critical for activity.

3-Chymotrypsin-like protease (3CLpro) is a promising drug target for coronavirus disease 2019 and related coronavirus diseases because of the essential role of this protease in processing viral polyproteins after infection. Understanding the detailed catalytic mechanism of 3CLpro is essential for designing effective inhibitors of infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Molecular dynamics studies have suggested pH-dependent conformational changes of 3CLpro, but experimental pH profiles of SARS-CoV-2 3CLpro and analyses of the conserved active-site histidine residues have not been reported. In this work, pH-dependence studies of the kinetic parameters of SARS-CoV-2 3CLpro revealed a bell-shaped pH profile with 2 pKa values (6.9 ± 0.1 and 9.4 ± 0.1) attributable to ionization of the catalytic dyad His41 and Cys145, respectively. Our investigation of the roles of conserved active-site histidines showed that different amino acid substitutions of His163 produced inactive enzymes, indicating a key role of His163 in maintaining catalytically active SARS-CoV-2 3CLpro. By contrast, the H164A and H172A mutants retained 75% and 26% of the activity of WT, respectively. The alternative amino acid substitutions H172K and H172R did not recover the enzymatic activity, whereas H172Y restored activity to a level similar to that of the WT enzyme. The pH profiles of H164A, H172A, and H172Y were similar to those of the WT enzyme, with comparable pKa values for the catalytic dyad. Taken together, the experimental data support a general base mechanism of SARS-CoV-2 3CLpro and indicate that the neutral states of the catalytic dyad and active-site histidine residues are required for maximum enzyme activity.

Green and efficient one-pot three-component synthesis of novel drug-like furo[2,3-d]pyrimidines as potential active site inhibitors and putative allosteric hotspots modulators of both SARS-CoV-2 MPro and PLPro.

In this paper, an environmentally benign, convenient, and efficient one-pot three-component reaction has been developed for the regioselective synthesis of novel 5-aroyl(or heteroaroyl)-6-(alkylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-diones (4a‒n) through the sequential condensation of aryl(or heteroaryl)glyoxal monohydrates (1a‒g), 1,3-dimethylbarbituric acid (2), and alkyl(viz. cyclohexyl or tert-butyl)isocyanides (3a or 3b) catalyzed by ultra-low loading ZrOCl2•8H2O (just 2 mol%) in water at 50 ˚C. After synthesis and characterization of the mentioned furo[2,3-d]pyrimidines (4a‒n), their multi-targeting inhibitory properties were investigated against the active site and putative allosteric hotspots of both SARS-CoV-2 main protease (MPro) and papain-like protease (PLPro) based on molecular docking studies and compare the attained results with various medicinal compounds which approximately in three past years were used, introduced, and or repurposed to fight against COVID-19. Furthermore, drug-likeness properties of the mentioned small heterocyclic frameworks (4a‒n) have been explored using in silico ADMET analyses. Interestingly, the molecular docking studies and ADMET-related data revealed that the novel series of furo[2,3-d]pyrimidines (4a‒n), especially 5-(3,4-methylendioxybenzoyl)-6-(cyclohexylamino)-1,3-dimethylfuro[2,3-d]pyrimidine-2,4(1H,3H)-dione (4g) as hit one is potential COVID-19 drug candidate, can subject to further in vitro and in vivo studies. It is worthwhile to note that the protein-ligand-type molecular docking studies on the human body temperature-dependent MPro protein that surprisingly contains zincII (ZnII) ion between His41/Cys145 catalytic dyad in the active site, which undoubtedly can make new plans for designing novel SARS-CoV-2 MPro inhibitors, is performed for the first time in this paper, to the best of our knowledge.

Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor.

We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (Tm) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [(-6)MProH41A and (-6*)MProH41A, respectively], and its corresponding mature MProH41A were systematically examined. While the H41A mutation exerts negligible effect on Tm and dimer dissociation constant (Kdimer) of MProH41A, relative to the wild type MPro, both miniprecursors show a 4-5 °C decrease in Tm and > 85-fold increase in Kdimer as compared to MProH41A. The Kd for the binding of the covalent inhibitor GC373 to (-6*)MProH41A increases ∼12-fold, relative to MProH41A, concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers' oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1-2 and 302-306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.

The Discovery of Small Allosteric and Active Site Inhibitors of the SARS-CoV-2 Main Protease via Structure-Based Virtual Screening and Biological Evaluation.

The main protease enzyme (Mpro) of SARS-CoV-2 is one of the most promising targets for COVID-19 treatment. Accordingly, in this work, a structure-based virtual screening of 3.8 million ligand libraries was carried out. After rigorous filtering, docking, and post screening assessments, 78 compounds were selected for biological evaluation, 3 of which showed promising inhibition of the Mpro enzyme. The obtained hits (CB03, GR04, and GR20) had reasonable potencies with Ki values in the medium to high micromolar range. Interestingly, while our most potent hit, GR20, was suggested to act via a reversible covalent mechanism, GR04 was confirmed as a noncompetitive inhibitor that seems to be one of a kind when compared to the other allosteric inhibitors discovered so far. Moreover, all three compounds have small sizes (~300 Da) with interesting fittings in their relevant binding sites, and they possess lead-like characteristics that can introduce them as very attractive candidates for the future development of COVID-19 treatments.

Autoprocessing and oxyanion loop reorganization upon GC373 and nirmatrelvir binding of monomeric SARS-CoV-2 main protease catalytic domain.

The monomeric catalytic domain (residues 1-199) of SARS-CoV-2 main protease (MPro1-199) fused to 25 amino acids of its flanking nsp4 region mediates its autoprocessing at the nsp4-MPro1-199 junction. We report the catalytic activity and the dissociation constants of MPro1-199 and its analogs with the covalent inhibitors GC373 and nirmatrelvir (NMV), and the estimated monomer-dimer equilibrium constants of these complexes. Mass spectrometry indicates the presence of the accumulated adduct of NMV bound to MProWT and MPro1-199 and not of GC373. A room temperature crystal structure reveals a native-like fold of the catalytic domain with an unwound oxyanion loop (E state). In contrast, the structure of a covalent complex of the catalytic domain-GC373 or NMV shows an oxyanion loop conformation (E* state) resembling the full-length mature dimer. These results suggest that the E-E* equilibrium modulates autoprocessing of the main protease when converting from a monomeric polyprotein precursor to the mature dimer.

Modeling the Enzymatic Mechanism of the SARS-CoV-2 RNA-Dependent RNA Polymerase by DFT/MM-MD: An Unusual Active Site Leading to High Replication Rates.

Viral infection relies on the hijacking of cellular machineries to enforce the reproduction of the infecting virus and its subsequent diffusion. In this context, the replication of the viral genome is a key step performed by specific enzymes, i.e., polymerases. The replication of SARS-CoV-2, the causative agent of the COVID-19 pandemics, is based on the duplication of its RNA genome, an action performed by the viral RNA-dependent RNA polymerase. In this contribution, by using highly demanding DFT/MM-MD computations coupled to 2D-umbrella sampling techniques, we have determined the chemical mechanisms leading to the inclusion of a nucleotide in the nascent viral RNA strand. These results highlight the high efficiency of the polymerase, which lowers the activation free energy to less than 10 kcal/mol. Furthermore, the SARS-CoV-2 polymerase active site is slightly different from those usually found in other similar enzymes, and in particular, it lacks the possibility to enforce a proton shuttle via a nearby histidine. Our simulations show that this absence is partially compensated by lysine whose proton assists the reaction, opening up an alternative, but highly efficient, reactive channel. Our results present the first mechanistic resolution of SARS-CoV-2 genome replication at the DFT/MM-MD level and shed light on its unusual enzymatic reactivity paving the way for the future rational design of antivirals targeting emerging RNA viruses.

The mechanism of RNA capping by SARS-CoV-2.

The RNA genome of SARS-CoV-2 contains a 5' cap that facilitates the translation of viral proteins, protection from exonucleases and evasion of the host immune response1-4. How this cap is made in SARS-CoV-2 is not completely understood. Here we reconstitute the N7- and 2'-O-methylated SARS-CoV-2 RNA cap (7MeGpppA2'-O-Me) using virally encoded non-structural proteins (nsps). We show that the kinase-like nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain5 of nsp12 transfers the RNA to the amino terminus of nsp9, forming a covalent RNA-protein intermediate (a process termed RNAylation). Subsequently, the NiRAN domain transfers the RNA to GDP, forming the core cap structure GpppA-RNA. The nsp146 and nsp167 methyltransferases then add methyl groups to form functional cap structures. Structural analyses of the replication-transcription complex bound to nsp9 identified key interactions that mediate the capping reaction. Furthermore, we demonstrate in a reverse genetics system8 that the N terminus of nsp9 and the kinase-like active-site residues in the NiRAN domain are required for successful SARS-CoV-2 replication. Collectively, our results reveal an unconventional mechanism by which SARS-CoV-2 caps its RNA genome, thus exposing a new target in the development of antivirals to treat COVID-19.

The SARS-CoV nsp12 Polymerase Active Site Is Tuned for Large-Genome Replication.

Positive-strand RNA viruses replicate their genomes using virally encoded RNA-dependent RNA polymerases (RdRP) with a common active-site structure and closure mechanism upon which replication speed and fidelity can evolve to optimize virus fitness. Coronaviruses (CoV) form large multicomponent RNA replication-transcription complexes containing a core RNA synthesis machine made of the nsp12 RdRP protein with one nsp7 and two nsp8 proteins as essential subunits required for activity. We show that assembly of this complex can be accelerated 5-fold by preincubation of nsp12 with nsp8 and further optimized with the use of a novel nsp8L7 heterodimer fusion protein construct. Using rapid kinetics methods, we measure elongation rates of up to 260 nucleotides (nt)/s for the core replicase, a rate that is unusually fast for a viral polymerase. To address the origin of this fast rate, we examined the roles of two CoV-specific residues in the RdRP active site: Ala547, which replaces a conserved glutamate above the bound NTP, and Ser759, which mutates the palm domain GDD sequence to SDD. Our data show that Ala547 allows for a doubling of replication rate, but this comes at a fidelity cost that is mitigated by using a SDD sequence in the palm domain. Our biochemical data suggest that fixation of mutations in polymerase motifs F and C played a key role in nidovirus evolution by tuning replication rate and fidelity to accommodate their large genomes. IMPORTANCE Replicating large genomes represents a challenge for RNA viruses because fast RNA synthesis is needed to escape innate immunity defenses, but faster polymerases are inherently low-fidelity enzymes. Nonetheless, the coronaviruses replicate their ≈30-kb genomes using the core polymerase structure and mechanism common to all positive-strand RNA viruses. The classic explanation for their success is that the large-genome nidoviruses have acquired an exonuclease-based repair system that compensates for the high polymerase mutation rate. In this work, we establish that the nidoviral polymerases themselves also play a key role in maintaining genome integrity via mutations at two key active-site residues that enable very fast replication rates while maintaining typical mutation rates. Our findings further demonstrate the evolutionary plasticity of the core polymerase platform by showing how it has adapted during the expansion from short-genome picornaviruses to long-genome nidoviruses.

Probing the Conformational States of Thimet Oligopeptidase in Solution.

Thimet oligopeptidase (TOP) is a metallopeptidase involved in the metabolism of oligopeptides inside and outside cells of various tissues. It has been proposed that substrate or inhibitor binding in the TOP active site induces a large hinge-bending movement leading to a closed structure, in which the bound ligand is enclosed. The main goal of the present work was to study this conformational change, and fluorescence techniques were used. Four active TOP mutants were created, each equipped with a single-Trp residue (fluorescence donor) and a p-nitro-phenylalanine (pNF) residue as fluorescence acceptor at opposite sides of the active site. pNF was biosynthetically incorporated with high efficiency using the amber codon suppression technology. Inhibitor binding induced shorter Donor-Acceptor (D-A) distances in all mutants, supporting the view that a hinge-like movement is operative in TOP. The activity of TOP is known to be dependent on the ionic strength of the assay buffer and D-A distances were measured at different ionic strengths. Interestingly, a correlation between the D-A distance and the catalytic activity of TOP was observed: the highest activities corresponded to the shortest D-A distances. In this study for the first time the hinge-bending motion of a metallopeptidase in solution could be studied, yielding insight about the position of the equilibrium between the open and closed conformation. This information will contribute to a more detailed understanding of the mode of action of these enzymes, including therapeutic targets like neurolysin and angiotensin-converting enzyme 2 (ACE2).

Dimerization Tendency of 3CLpros of Human Coronaviruses Based on the X-ray Crystal Structure of the Catalytic Domain of SARS-CoV-2 3CLpro.

3CLpro of SARS-CoV-2 is a promising target for developing anti-COVID19 agents. In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents.

A new inactive conformation of SARS-CoV-2 main protease.

The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a promising target for drugs against the COVID-19 pandemic. Here, a crystal structure is presented in which Mpro adopts an inactive state that has never been observed before, called new-inactive. It is shown that the oxyanion loop, which is involved in substrate recognition and enzymatic activity, adopts a new catalytically incompetent conformation and that many of the key interactions of the active conformation of the enzyme around the active site are lost. Solvation/desolvation energetic contributions play an important role in the transition from the inactive to the active state, with Phe140 moving from an exposed to a buried environment and Asn142 moving from a buried environment to an exposed environment. In new-inactive Mpro a new cavity is present near the S2' subsite, and the N-terminal and C-terminal tails, as well as the dimeric interface, are perturbed, with partial destabilization of the dimeric assembly. This novel conformation is relevant both for comprehension of the mechanism of action of Mpro within the catalytic cycle and for the successful structure-based drug design of antiviral drugs.

Fullerenes against COVID-19: Repurposing C60 and C70 to Clog the Active Site of SARS-CoV-2 Protease.

The persistency of COVID-19 in the world and the continuous rise of its variants demand new treatments to complement vaccines. Computational chemistry can assist in the identification of moieties able to lead to new drugs to fight the disease. Fullerenes and carbon nanomaterials can interact with proteins and are considered promising antiviral agents. Here, we propose the possibility to repurpose fullerenes to clog the active site of the SARS-CoV-2 protease, Mpro. Through the use of docking, molecular dynamics, and energy decomposition techniques, it is shown that C60 has a substantial binding energy to the main protease of the SARS-CoV-2 virus, Mpro, higher than masitinib, a known inhibitor of the protein. Furthermore, we suggest the use of C70 as an innovative scaffold for the inhibition of SARS-CoV-2 Mpro. At odds with masitinib, both C60 and C70 interact more strongly with SARS-CoV-2 Mpro when different protonation states of the catalytic dyad are considered. The binding of fullerenes to Mpro is due to shape complementarity, i.e., vdW interactions, and is aspecific. As such, it is not sensitive to mutations that can eliminate or invert the charges of the amino acids composing the binding pocket. Fullerenic cages should therefore be more effective against the SARS-CoV-2 virus than the available inhibitors such as masinitib, where the electrostatic term plays a crucial role in the binding.

Identification of Kukoamine A, Zeaxanthin, and Clexane as New Furin Inhibitors.

The endogenous protease furin is a key protein in many different diseases, such as cancer and infections. For this reason, a wide range of studies has focused on targeting furin from a therapeutic point of view. Our main objective consisted of identifying new compounds that could enlarge the furin inhibitor arsenal; secondarily, we assayed their adjuvant effect in combination with a known furin inhibitor, CMK, which avoids the SARS-CoV-2 S protein cleavage by means of that inhibition. Virtual screening was carried out to identify potential furin inhibitors. The inhibition of physiological and purified recombinant furin by screening selected compounds, Clexane, and these drugs in combination with CMK was assayed in fluorogenic tests by using a specific furin substrate. The effects of the selected inhibitors from virtual screening on cell viability (293T HEK cell line) were assayed by means of flow cytometry. Through virtual screening, Zeaxanthin and Kukoamine A were selected as the main potential furin inhibitors. In fluorogenic assays, these two compounds and Clexane inhibited both physiological and recombinant furin in a dose-dependent way. In addition, these compounds increased physiological furin inhibition by CMK, showing an adjuvant effect. In conclusion, we identified Kukoamine A, Zeaxanthin, and Clexane as new furin inhibitors. In addition, these drugs were able to increase furin inhibition by CMK, so they could also increase its efficiency when avoiding S protein proteolysis, which is essential for SARS-CoV-2 cell infection.

Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor.

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.