Decoding the Molecular Effects of Atovaquone Linked Resistant Mutations on Plasmodium falciparum Cytb-ISP Complex in the Phospholipid Bilayer Membrane

Atovaquone (ATQ) is a drug used to prevent and treat malaria that functions by targeting the Plasmodium falciparum cytochrome b (PfCytb) protein. PfCytb catalyzes the transmembrane electron transfer (ET) pathway which maintains the mitochondrial membrane potential. The ubiquinol substrate binding site of the protein has heme bL, heme bH and iron-sulphur [2FE-2S] cluster cofactors that act as redox centers to aid in ET. Recent studies investigating ATQ resistance mechanisms have shown that point mutations of PfCytb confer resistance. Thus, understanding the resistance mechanisms at the molecular level via computational approaches incorporating phospholipid bilayer would help in the design of new efficacious drugs that are also capable of bypassing parasite resistance. With this knowledge gap, this article seeks to explore the effect of three drug resistant mutations Y268C, Y268N and Y268S on the PfCytb structure and function in the presence and absence of ATQ. To draw reliable conclusions, 350 ns all-atom membrane (POPC:POPE phospholipid bilayer) molecular dynamics (MD) simulations with derived metal parameters for the holo and ATQ-bound -proteins were performed. Thereafter, simulation outputs were analyzed using dynamic residue network (DRN) analysis. Across the triplicate MD runs, hydrophobic interactions, reported to be crucial in protein function were assessed. In both, the presence and absence of ATQ and a loss of key active site residue interactions were observed as a result of mutations. These active site residues included: Met 133, Trp136, Val140, Thr142, Ile258, Val259, Pro260 and Phe264. These changes to residue interactions are likely to destabilize the overall intra-protein residue communication network where the proteins’ function could be implicated. Protein dynamics of the ATQ-bound mutant complexes showed that they assumed a different pose to the wild-type, resulting in diminished residue interactions in the mutant proteins. In summary, this study presents insights on the possible effect of the mutations on ATQ drug activity causing resistance and describes accurate MD simulations in the presence of the lipid bilayer prior to conducting inhibitory drug discovery for the PfCytb-iron sulphur protein (Cytb-ISP) complex.     

Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro/3CLpro) and Human Cathepsin-L

After almost two years from its first evidence, the COVID-19 pandemic continues to afflict people worldwide, highlighting the need for multiple antiviral strategies. SARS-CoV-2 main protease (Mpro/3CLpro) is a recognized promising target for the development of effective drugs. Because single target inhibition might not be sufficient to block SARS-CoV-2 infection and replication, multi enzymatic-based therapies may provide a better strategy. Here we present a structural and biochemical characterization of the binding mode of MG-132 to both the main protease of SARS-CoV-2, and to the human Cathepsin-L, suggesting thus an interesting scaffold for the development of double-inhibitors. X-ray diffraction data show that MG-132 well fits into the Mpro active site, forming a covalent bond with Cys145 independently from reducing agents and crystallization conditions. Docking of MG-132 into Cathepsin-L well-matches with a covalent binding to the catalytic cysteine. Accordingly, MG-132 inhibits Cathepsin-L with nanomolar potency and reversibly inhibits Mpro with micromolar potency, but with a prolonged residency time. We compared the apo and MG-132-inhibited structures of Mpro solved in different space groups and we identified a new apo structure that features several similarities with the inhibited ones, offering interesting perspectives for future drug design and in silico efforts.     

Robustness of hen lysozyme monitored by random mutations

We investigated the robustness of hen lysozyme by using randommutant libraries. Six random mutant libraries containing 1,1.5, 2, 3, 5 and 14 amino acid mutations per hen lysozyme weresystematically constructed by varying the concentrations ofMg²⁺ and Mn²⁺ on polymerase chain reaction. The mutated genesfrom the six libraries were cloned to a yeast expression vectorand a total of 4000 clones were screened on the basis of lysisactivity and ELISA employing monoclonal antibody that recognizedonly lysozyme with native conformation. About 80% of the cloneswith an average of two amino acid mutations retained activestructure. Almost all clones with an average of five mutationslost active structure. On the other hand, 80% of the cloneswith an average of two amino acid mutations retained both grossconformation and active structure and 24% of the clones withan average of 14 amino acid mutations retained gross conformation.These results show that gross conformation is robust againstmutations and so is active structure to a lesser extent.     

Potential Resistance of SARS-CoV-2 Main Protease (Mpro) against Protease Inhibitors: Lessons Learned from HIV-1 Protease

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome 2 (SARS-CoV-2), has been one of the most devastating pandemics of recent times. The lack of potent novel antivirals had led to global health crises; however, emergence and approval of potent inhibitors of the viral main protease (Mpro), such as Pfizer’s newly approved nirmatrelvir, offers hope not only in the therapeutic front but also in the context of prophylaxis against the infection. By their nature, RNA viruses including human immunodeficiency virus (HIV) have inherently high mutation rates, and lessons learnt from previous and currently ongoing pandemics have taught us that these viruses can easily escape selection pressure through mutation of vital target amino acid residues in monotherapeutic settings. In this paper, we review nirmatrelvir and its binding to SARS-CoV-2 Mpro and draw a comparison to inhibitors of HIV protease that were rendered obsolete by emergence of resistance mutations, emphasizing potential pitfalls in the design of inhibitors that may be of important relevance to the long-term use of novel inhibitors against SARS-CoV-2.     

In Silico Characterization of Masitinib Interaction with SARS-CoV-2 Main Protease

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a global health problem. Despite the current implementation of COVID-19 vaccination schedules, identifying effective antiviral drug treatments for this disease continues to be a priority. A recent study showed that masitinib (MST), a tyrosine kinase inhibitor, blocks the proteolytic activity of SARS-CoV-2 main protease (M^pro). Although MST is a potential candidate for COVID-19 treatment, a comprehensive analysis of its interaction with M^pro has not been done. In this work, we performed molecular dynamics simulations of the MST-M^pro complex crystal structure. The effect of the protonation states of M^pro H163 residue and MST titratable groups were studied. Furthermore, we identified the MST substituents and M^pro mutations that affect the stability of the complex. Our results provide valuable insights into the design of new MST analogs as potential treatments for COVID-19.     

State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2

Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a “bucket brigade.” This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication.     

The role of Glu259 in Escherichia coli elongation factor Tu in ternary complex formation

Determination of the crystal structure of the ternary complex formed between elongation factor Tu:GTP and aminoacylated tRNA revealed three regions of interaction between elongation factor Tu and tRNA. The structure indicates that the conserved glutamic acid at position 271 in Thermus aquaticus EF-Tu could be involved in the binding of the 3' CCA- Phe end of the aminoacylated tRNA. Therefore, the corresponding residue, Glu259, of Escherichia coli EF-Tu was mutated into alanine, aspartic acid, glutamine and tyrosine, in order to substantiate the crystallographic structural evidence and to obtain further knowledge of the importance of this residue. All of the mutated proteins showed nucleotide binding properties similar to the wild type. In addition the GTPase activities were similar to the wild type. The mutation of Glu259 to either alanine or aspartic acid resulted in a reduced strength of interaction with tRNA, while mutation to tyrosine abolished completely the interaction with tRNA. Finally, mutation to glutamine resulted in an elongation factor Tu variant behaving like the wild type. In conclusion, the environment around the site binding the CCA-Phe end of the tRNA is very restricted spatially and chemically so that only a residue with almost the same size and chemical properties as glutamic acid fulfils the requirements with regard to size, salt bridge- formation potential and maintenance of the backbone conformation at the 259 position.      

In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residueAsn14 that plays a crucial role in stabilizing the reactivesite loop conformation. This residue is found to be conservedin the Kunitz (STI) family of serine protease inhibitors. Tounderstand the contribution of this scaffolding residue on thestability of the reactive site loop, it was mutated in silicoto Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD)simulations were carried out on the mutants. The results ofMD simulations reveal the conformational variability and rangeof motions possible for the reactive site loop of differentmutants. The N-terminus side of the scissile bond, which isclose to a ß-barrel, is conformationally less variable,while the C-terminus side, which is relatively far from anysuch secondary structural element, is more variable and needsstability through hydrogen-bonding interactions. The simulatedstructures of WCI and the mutants were docked in the peptide-bindinggroove of the cognate enzyme chymotrypsin and the ability toform standard hydrogen-bonding interactions at P3, P1 and P2'residues were compared. The results of the MD simulations coupledwith docking studies indicate that hydrophobic residues likeLeu and Val at the 14th position are disruptive for the integrityof the reactive site loop, whereas a residue like Thr, whichcan stabilize the C-terminus side of the scissile bond, canbe predicted at this position. However, the size and chargeof the Asn residue made it most suitable for the best maintenanceof the integrity of the reactive site loop, explaining its conservednature in the family. Received October 17, 2002; revised June 6, 2003; accepted June 19, 2003.     

Mapping of residues involved in the interaction between the Bacillus subtilis xylanase A and proteinaceous wheat xylanase inhibitors

The Bacillus subtilis xylanase A was subjected to site-directed mutagenesis, aimed at changing the interaction with Triticum aestivum xylanase inhibitor, the only wheat endogenous proteinaceous xylanase inhibitor interacting with this xylanase. The published structure of Bacillus circulans XynA was used to target amino acids surrounding the active site cleft of B.subtilis XynA for mutation. Twenty-two residues were mutated, resulting in 62 different variants. The catalytic activity of active mutants ranged from 563 to 5635 XU/mg and the interaction with T.aestivum xylanase inhibitor showed a similar variation. The results indicate that T.aestivum xylanase inhibitor interacts with several amino acid residues surrounding the active site of the enzyme. Three different amino acid substitutions in one particular residue (D11) completely abolished the interaction between T.aestivum xylanase inhibitor and B.subtilis xylanase A.     

Computational Design of Miniproteins as SARS-CoV-2 Therapeutic Inhibitors

A rational therapeutic strategy is urgently needed for combating SARS-CoV-2 infection. Viral infection initiates when the SARS-CoV-2 receptor-binding domain (RBD) binds to the ACE2 receptor, and thus, inhibiting RBD is a promising therapeutic for blocking viral entry. In this study, the structure of lead antiviral candidate binder (LCB1), which has three alpha-helices (H1, H2, and H3), is used as a template to design and simulate several miniprotein RBD inhibitors. LCB1 undergoes two modifications: structural modification by truncation of the H3 to reduce its size, followed by single and double amino acid substitutions to enhance its binding with RBD. We use molecular dynamics (MD) simulations supported by ab initio density functional theory (DFT) calculations. Complete binding profiles of all miniproteins with RBD have been determined. The MD investigations reveal that the H3 truncation results in a small inhibitor with a −1.5 kcal/mol tighter binding to RBD than original LCB1, while the best miniprotein with higher binding affinity involves D17R or E11V + D17R mutation. DFT calculations provide atomic-scale details on the role of hydrogen bonding and partial charge distribution in stabilizing the minibinder:RBD complex. This study provides insights into general principles for designing potential therapeutics for SARS-CoV-2.     

Improved Binding Affinity of Omicron’s Spike Protein for the Human Angiotensin-Converting Enzyme 2 Receptor Is the Key behind Its Increased Virulence

The new variant of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), Omicron, has been quickly spreading in many countries worldwide. Compared to the original virus, Omicron is characterized by several mutations in its genomic region, including the spike protein’s receptor-binding domain (RBD). We have computationally investigated the interaction between the RBD of both the wild type and Omicron variant of SARS-CoV-2 with the human angiotensin-converting enzyme 2 (hACE2) receptor using molecular dynamics and molecular mechanics-generalized Born surface area (MM-GBSA)-based binding free energy calculations. The mode of the interaction between Omicron’s RBD with the hACE2 receptor is similar to the original SARS-CoV-2 RBD except for a few key differences. The binding free energy difference shows that the spike protein of Omicron has an increased affinity for the hACE2 receptor. The mutated residues in the RBD showed strong interactions with a few amino acid residues of hACE2. More specifically, strong electrostatic interactions (salt bridges) and hydrogen bonding were observed between R493 and R498 residues of the Omicron RBD with D30/E35 and D38 residues of the hACE2, respectively. Other mutated amino acids in the Omicron RBD, e.g., S496 and H505, also exhibited hydrogen bonding with the hACE2 receptor. A pi-stacking interaction was also observed between tyrosine residues (RBD-Tyr501: hACE2-Tyr41) in the complex, which contributes majorly to the binding free energies and suggests that this is one of the key interactions stabilizing the formation of the complex. The resulting structural insights into the RBD:hACE2 complex, the binding mode information within it, and residue-wise contributions to the free energy provide insight into the increased transmissibility of Omicron and pave the way to design and optimize novel antiviral agents.     

Discovery of Potent Pyrazoline-Based Covalent SARS-CoV-2 Main Protease Inhibitors**

While vaccines and antivirals are now being deployed for the current SARS-CoV-2 pandemic, we require additional antiviral therapeutics to not only effectively combat SARS-CoV-2 and its variants, but also future coronaviruses. All coronaviruses have relatively similar genomes that provide a potential exploitable opening to develop antiviral therapies that will be effective against all coronaviruses. Among the various genes and proteins encoded by all coronaviruses, one particularly “druggable” or relatively easy-to-drug target is the coronavirus Main Protease (3CL^pro or Mpro), an enzyme that is involved in cleaving a long peptide translated by the viral genome into its individual protein components that are then assembled into the virus to enable viral replication in the cell. Inhibiting Mpro with a small-molecule antiviral would effectively stop the ability of the virus to replicate, providing therapeutic benefit. In this study, we have utilized activity-based protein profiling (ABPP)-based chemoproteomic approaches to discover and further optimize cysteine-reactive pyrazoline-based covalent inhibitors for the SARS-CoV-2 Mpro. Structure-guided medicinal chemistry and modular synthesis of di- and tri-substituted pyrazolines bearing either chloroacetamide or vinyl sulfonamide cysteine-reactive warheads enabled the expedient exploration of structure-activity relationships (SAR), yielding nanomolar potency inhibitors against Mpro from not only SARS-CoV-2, but across many other coronaviruses. Our studies highlight promising chemical scaffolds that may contribute to future pan-coronavirus inhibitors.     

Development of Effective Therapeutic Molecule from Natural Sources against Coronavirus Protease

The SARS-CoV-2 main protease (M^pro) is one of the molecular targets for drug design. Effective vaccines have been identified as a long-term solution but the rate at which they are being administered is slow in several countries, and mutations of SARS-CoV-2 could render them less effective. Moreover, remdesivir seems to work only with some types of COVID-19 patients. Hence, the continuous investigation of new treatments for this disease is pivotal. This study investigated the inhibitory role of natural products against SARS-CoV-2 M^pro as repurposable agents in the treatment of coronavirus disease 2019 (COVID-19). Through in silico approach, selected flavonoids were docked into the active site of M^pro. The free energies of the ligands complexed with M^pro were computationally estimated using the molecular mechanics-generalized Born surface area (MM/GBSA) method. In addition, the inhibition process of SARS-CoV-2 Mpro with these ligands was simulated at 100 ns in order to uncover the dynamic behavior and complex stability. The docking results showed that the selected flavonoids exhibited good poses in the binding domain of M^pro. The amino acid residues involved in the binding of the selected ligands correlated well with the residues involved with the mechanism-based inhibitor (N3) and the docking score of Quercetin-3-O-Neohesperidoside (−16.8 Kcal/mol) ranked efficiently with this inhibitor (−16.5 Kcal/mol). In addition, single-structure MM/GBSA rescoring method showed that Quercetin-3-O-Neohesperidoside (−87.60 Kcal/mol) is more energetically favored than N3 (−80.88 Kcal/mol) and other ligands (Myricetin 3-Rutinoside (−87.50 Kcal/mol), Quercetin 3-Rhamnoside (−80.17 Kcal/mol), Rutin (−58.98 Kcal/mol), and Myricitrin (−49.22 Kcal/mol). The molecular dynamics simulation (MDs) pinpointed the stability of these complexes over the course of 100 ns with reduced RMSD and RMSF. Based on the docking results and energy calculation, together with the RMSD of 1.98 ± 0.19 Å and RMSF of 1.00 ± 0.51 Å, Quercetin-3-O-Neohesperidoside is a better inhibitor of M^pro compared to N3 and other selected ligands and can be repurposed as a drug candidate for the treatment of COVID-19. In addition, this study demonstrated that in silico docking, free energy calculations, and MDs, respectively, are applicable to estimating the interaction, energetics, and dynamic behavior of molecular targets by natural products and can be used to direct the development of novel target function modulators.     

A new family of beta-hairpin mimetics based on a trypsin inhibitor from sunflower seeds

The ability of proteases to regulate many aspects of cell function and defense accounts for the considerable interest in the design of novel protease inhibitors. There are many naturally occurring proteinaceous serine protease inhibitors, one of which is a 14 amino acid cyclic peptide from sunflower seeds that shows both sequence and conformational similarity with the trypsin-reactive loop of the Bowman-Birk family of serine protease inhibitors. This inhibitor adopts a beta-hairpin conformation when bound at the active site of bovine beta-trypsin. We illustrate here an approach to inhibitor design in which the beta hairpin from the naturally occurring peptide is transplanted onto a hairpin-inducing template. Two mimetics with the sequences RC*TKSIPPIC*F (where C*C* is a disulfide) and TKSIPPI are studied, each mounted onto a D-Pro-L-Pro template. NMR studies revealed a well-defined beta-hairpin conformation for each mimetic in aqueous solution; this conformation is closely related to the trypsin-bound conformation of the natural inhibitor and includes a cis-Ile-Pro peptide bond. Both mimetics inhibit trypsin in the mid nanomolar range. An alanine scan revealed the importance for inhibitory activity of the specificity-determining Lys residue and of the first but not the second Pro residue in the IPPI motif. Since these hairpin mimetics can be prepared by parallel combinatorial synthesis, this family of molecules may be a useful starting point for the discovery of other biologically or medicinally useful serine protease inhibitors.     

Computational and experimental studies of the catalytic mechanism of Thermobifida fusca cellulase Cel6A (E2)

Mutagenesis experiments suggest that Asp79 in cellulase Cel6A(E2) from Thermobifida fusca has a catalytic role, in spiteof the fact that this residue is more than 13 Å from thescissile bond in models of the enzyme–substrate complexbuilt upon the crystal structure of the protein. This suggeststhat there is a substantial conformational shift in the proteinupon substrate binding. Molecular mechanics simulations wereused to investigate possible alternate conformations of theprotein bound to a tetrasaccharide substrate, primarily involvingshifts of the loop containing Asp79, and to model the role ofwater in the active site complex for both the native conformationand alternative low-energy conformations. Several alternativeconformations of reasonable energy have been identified, includingone in which the overall energy of the enzyme–substratecomplex in solution is lower than that of the conformation inthe crystal structure. This conformation was found to be stablein molecular dynamics simulations with a cellotetraose substrateand water. In simulations of the substrate complexed with thenative protein conformation, the sugar ring in the –1binding site was observed to make a spontaneous transition fromthe ⁴C₁ conformation to a twist-boat conformer, consistent withgenerally accepted glycosidase mechanisms. Also, from thesesimulations Tyr73 and Arg78 were found to have important rolesin the active site. Based on the results of these various MDsimulations, a new catalytic mechanism is proposed. Using thismechanism, predictions about the effects of changes in Arg78were made which were confirmed by site-directed mutagenesis.     

Free-energy simulations and experiments reveal long-range electrostatic interactions and substrate-assisted specificity in an aminoacyl-tRNA synthetase

Specific recognition of their cognate amino acid substrates by the aminoacyl-tRNA synthetase enzymes is essential for the correct translation of the genetic code. For aspartyl-tRNA synthetase (AspRS), electrostatic interactions are expected to play an important role, since its three substrates (aspartate, ATP, tRNA) are all electrically charged. We used molecular-dynamics free-energy simulations and experiments to compare the binding of the substrate Asp and its electrically neutral analogue Asn to AspRS. The preference for Asp is found to be very strong, with good agreement between simulations and experiment. The simulations reveal long-range interactions that electrostatically couple the amino acid ligand, ATP, and its associated Mg2+ cations, a histidine side chain (His448) next to the amino acid ligand and a flexible loop that closes over the active site in response to amino acid binding. Closing this loop brings a negatively charged glutamate into the active site; this causes His448 to recruit a labile proton, which interacts favorably with Asp and accounts for most of the Asp/Asn discrimination. Cobinding of the second substrate, ATP, increases specificity for Asp further and makes the system robust towards removal of His448, which is mutated to a neutral amino acid in many organisms. Thus, AspRS specificity is assisted by a labile proton and a cosubstrate, and ATP acts as a mobile discriminator for specific Asp binding to AspRS. In asparaginyl-tRNA synthetase, a close homologue of AspRS, a few binding-pocket differences modify the charge balance so that asparagine binding predominates.     

Design of Cyclic Peptides Featuring Proline Predominantly in the cis Conformation under Physiological Conditions

Turns are secondary‐structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four‐residue β‐turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI‐type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c‐(‐D Xaa‐Ser‐Pro‐D Xaa‐Lys‐Pro‐) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D ‐amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis‐Pro/cis‐Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.     

In silico allicin induced S-thioallylation of SARS-CoV-2 main protease

Coronavirus disease 2019 (COVID-19) is an ongoing pandemic caused due to new coronavirus infection with ³716075 deaths across the world as reported by the World Health Organization (WHO). SARS-CoV-2 main protease (M^pro) plays a vital role in the replication of coronavirus and thus an attractive target for the screening of inhibitors for the therapy of COVID-19. The preclinical drugs ebselen and PX-12 are potent inhibitors of SARS-CoV-2 M^pro and covalently modifies the active site Cys-145 residue of M^pro through selenosulfide/disulfide. In the current report, using virtual screening methods, reactive sulfur species allicin is subjecting for covalent docking at the active site of SARS-CoV-2 M^pro using PX-12 as a benchmark reference compound. The results indicate that allicin induces dual S-thioallylation of Cys-145 and Cys-85/ Cys-156 residues of SARS-CoV-2 M^pro. Using density functional theory (DFT), Gibbs free energy change (DG) is calculated for the putative reactions between N-acetylcysteine amide thiol and allicin/allyl sulfenic acid. The overall reaction is exergonic and allyl disulfide of Cys-145 residue of M^pro is involved in a sulfur mediated hydrogen bond. The results indicate that allicin causes dual S-thioallylation of SARS-CoV-2 M^pro which may be of interest for treatment and attenuation of ongoing coronavirus infection.     

Conformational stability of the N-terminal amino acid residues of mutated recombinant pigeon liver malic enzymes

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met- Lys-Lys-Gly-Tyr-Glu-Val-Leu-Arg-. Our previous results indicated that the N-terminus of the enzyme is located at or near the enzyme's active center involved in Mn(II)-L-malate binding and is also near to the subunits' interface. In the present study, the conformational stability of the various deletion (delta) and substitution mutants at Lys2/Lys3 of the enzyme was investigated with chemical and thermal sensitivities. The lysine residue at position 2 or 3 seems to be crucial for the correct active site conformation, probably through an ion-pairing with Glu6. Deletion at Lys2 or Lys3, delta(K2/K3), and the double mutant K(2,3)E were much less stable than the wild-type enzyme towards chemical denaturation. Kinetic analysis of the thermal inactivation at 58 degrees C of the recombinant enzymes indicated that mutation at position 3 to alanine (K3A) endows the protein with extra stability compared with the wild-type enzyme. K3A is also stable towards chemical denaturation. The concentration of urea that causes half unfolding, [urea]0.5, for K3A is 3.25 M compared with 2.54 M for the wild-type enzyme. The K3A mutant of malic enzyme might therefore have potential practical applications.