Interactions In Pocket Research Articles

MMV 1804559 is a potential antistaphylococcal and antibiofilm agent targeting the clfA gene of Staphylococcus aureus.

Staphylococcus aureus, a high-priority pathogen proclaimed to cause infections ranging from mild to life-threatening, presents significant challenges in treatment. New therapies can be developed quicker using open drug discovery platforms offering a distinct approach to expedite the development of innovative antibacterial and anti-biofilm therapeutics. This study set out to address these issues by finding new uses for current medications to find compounds that are effective against S. aureus. In this study, we screened the global priority health box, launched by Medicines for Malaria Ventures containing 240 compounds, for their effectiveness against S. aureus. MMV1795508, MMV1542799, MMV027331, MMV1593278, and MMV1804559 showed potential antibacterial activity at 10µM concentration. These compounds underwent further evaluation for their ability to clear intracellular bacteria, disrupt biofilm formation and eradicate existing biofilms. MMV1804559 demonstrated strong efficacy across all tested parameters, achieving 94% inhibition of intracellular bacteria, 79.19% disruption of biofilm cells, and 66.18% inhibition of biofilm formation. Scanning electron microscopy revealed notable membrane perforations and blebbing in MMV1804559-treated cells, indicating its impact on bacterial membranes. Gene expression analysis of cells treated with MMV1804559 showed downregulation of clfA and clfB genes, critical for biofilm formation. Additionally, docking studies confirmed the binding affinity of MMV1804559 with clfA, supported by favorable docking scores, MM/GBSA binding energy, and increased hydrogen bond interactions in the binding pocket, suggesting clfA as a target for MMV1804559. MMV1804559 could serve as a potential therapy for S. aureus by targeting biofilm development and cell adhesion processes.

Targeted sulfur(VI) fluoride exchange-mediated covalent modification of a tyrosine residue in the catalytic pocket of tyrosyl-DNA phosphodiesterase 1

Developing effective inhibitors of the DNA repair enzyme tyrosyl-DNA phosphodiesterase 1 (TDP1) has been challenging because of the enzyme shallow catalytic pocket and non-specific substrate binding interactions. Recently, we discovered a quinolone-binding hot spot in TDP1’s active site proximal to the evolutionary conserved Y204 and F259 residues that position DNA. Sulfur (VI) fluoride exchange (SuFEx) is a biocompatible click chemistry reaction that enables acylation of protein residues, including tyrosine. Selective protein modifications can provide insights into the biological roles of proteins and inform ligand design. As we report herein, we used SuFEx chemistries to prepare covalent TDP1-bound binders showing site-specific covalent bonds with Y204. Our work presents the first application of SuFEx chemistries to TDP1 ligands. It validates the ability to covalently modify specific TDP1 residues by designed targeting and adds to the chemical biology resource toolbox for studying TDP1.

“In Silico” prediction of antibiotics biodegradation by Ganoderma lucidum GILCC 1 laccase

Antibiotics present a pressing environmental challenge as emerging pollutants due to their persistence and role in promoting antibiotic-resistant bacteria. To model the utilization of Ganoderma lucidum GlLCC1 laccase in degrading antibiotics, a 3D homology model of GILCC1, based on Lentinus tigrinus mushroom laccase, was utilized. Five broad-spectrum WHO-designated antibiotics with molecular weights between 100 and 500 Da were selected. Molecular dynamics simulations were conducted at pH 3.0 and 7.0 to evaluate the interactions between GILCC1 and antibiotics in a TIP3P water box, with system behaviour assessed at 300 °K using an NPT assembly. ABTS (2,2ʹ-Azino-bis (3-ethylbenzthiazoline-6-sulfonic Acid)) served as the comparison molecule. The binding free energy indicated a strong affinity between 3D GILCC1 and various ligands. At pH 3.0, GILCC1 exhibited significant Gibbs free energy (ΔG), indicating a high affinity for Levofloxacin (LVX; −8.2 kcal mol−1), Sulfisoxazole (SFX; −7.8 kcal mol−1), Cefuroxime (CXM; −7.5 kcal mol−1), Cephradine (CFD; −7. 5 kcal mol−1), ABTS (−7.6 kcal mol−1), and Tetracycline (TE; −7.5 kcal mol−1), attributed to pocket topology and interactions such as hydrogen bonds and van der Waals forces. Electron transfer in GILCC1 involved a chain of residues, including His395 and Phe239. Although the affinity decreased at pH 7.0, the potential of GILCC1 to degrade antibiotics remained plausible. This study accurately predicted the behaviour of the laccase-antibiotic system, providing atomic-level insights into molecular interactions and emphasizing the importance of experimental assays and assessments of antibiotic degradation in wastewater, considering various chemical compounds. The use of ABTS as a mediator was suggested to enhance molecule affinity.Graphical abstract

High-throughput Virtual Screening- and Molecular Docking-based Prediction for Acetylcholinesterase Inhibitors and Exploring its Mechanisms against Alzheimer’s Disease based on Network Pharmacology

OBJECTIVE This study aimed to identify promising ligands for inhibiting acetylcholinesterase (AChE) activity using virtual screening (VS). METHODS VS was used to identify potential AChE inhibitors from the PubChem database. Ligands with favorable binding pocket interactions were selected. SwissADME and pkCSM tools were used to assess drug-likeness and pharmacokinetic properties. Molecular dynamic (MD) simulations provided insights into binding interactions. Network pharmacology was used to explore interactions between the target molecule and AD-related genes to determine its mechanism of action. RESULTS VS identified promising AChE inhibitor candidates with acridone, carbazole, and xanthone scaffolds. Docking simulations showed strong binding with AChE. These ligands displayed favorable drug-likeness and ADMET properties, with one (M5) lacking predicted hepatotoxicity. MD simulations suggested stable binding of M5 to AChE, potentially affecting both catalytic and peripheral sites, hinting at dual inhibition. M5’s interactions, especially near His440, appeared more favorable than donepezil. Network analysis implicated M5 in targeting multiple pathways in AD, with potential focus on neuroinflammation. CONCLUSIONS This study identified promising AChE inhibitor candidates through virtual screening. Ligand M5 emerged as particularly promising due to its favorable binding characteristics, lack of predicted hepatotoxicity, and potential for targeting multiple pathways in AD. However, further in vitro and in vivo validation is essential for clinical development. KEYWORDS virtual screening, acetylcholinesterase inhibitors, PubChem database, molecular dynamics simulation, gene ontology, KEGG pathways

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A2A Receptor.

The adenosine A2A receptor (A2AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A2AAR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A2AAR construct A2A-PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T883.36 was observed, an amino acid that was mutated to alanine in the most frequently used A2AAR crystallization constructs thus preventing the discovery of its interactions in most of the previous A2AAR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S913.39K mutation, although with an affinity lower than that at the native receptor. This could explain the compound's partial agonistic activity: while full A2AAR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs.

Identifying and characterising promising small molecule inhibitors of kinesin spindle protein using ligand-based virtual screening, molecular docking, molecular dynamics and MM‑GBSA calculations

The kinesin spindle protein (Eg5) is a mitotic protein that plays an essential role in the formation of the bipolar spindles during the mitotic phase. Eg5 protein controls the segregation of the chromosomes in mitosis which renders it a vital target for cancer treatment. In this study our approach to identifying novel scaffold for Eg5 inhibitors is based on targeting the novel allosteric pocket (α4/α6/L11). Extensive computational techniques were applied using ligand-based virtual screening and molecular docking by two approaches, MOE and AutoDock, to screen a library of commercial compounds. We identified compound 8-(3-(1H-imidazol-1-ylpropylamino)-3-methyl-7-((naphthalen-3-yl)methyl)-1H-purine-2, 6 (3H,7H)-dione (compound 5) as a novel scaffold for Eg5 inhibitors. This compound inhibited cancer cell Eg5 ATPase at 2.37 ± 0.15 µM. The molecular dynamics simulations revealed that the identified compound formed stable interactions in the allosteric pocket (α4/α6/L11) of the receptor, indicating its potential as a novel Eg5 inhibitor.Graphical

Molecular basis for differential recognition of an allosteric inhibitor by receptor tyrosine kinases.

Understanding kinase-inhibitor selectivity continues to be a major objective in kinase drug discovery. We probe the molecular basis of selectivity of an allosteric inhibitor (MSC1609119A-1) of the insulin-like growth factor-I receptor kinase (IGF1RK), which has been shown to be ineffective for the homologous insulin receptor kinase (IRK). Specifically, we investigated the structural and energetic basis of the allosteric binding of this inhibitor to each kinase by combining molecular modeling, molecular dynamics (MD) simulations, and thermodynamic calculations. We predict the inhibitor conformation in the binding pocket of IRK and highlight that the charged residues in the histidine-arginine-aspartic acid (HRD) and aspartic acid-phenylalanine-glycine (DFG) motifs and the nonpolar residues in the binding pocket govern inhibitor interactions in the allosteric pocket of each kinase. We suggest that the conformational changes in the IGF1RK residues M1054 and M1079, movement of the ⍺C-helix, and the conformational stabilization of the DFG motif favor the selectivity of the inhibitor toward IGF1RK. Our thermodynamic calculations reveal that the observed selectivity can be rationalized through differences observed in the electrostatic interaction energy of the inhibitor in each inhibitor/kinase complex and the hydrogen bonding interactions of the inhibitor with the residue V1063 in IGF1RK that are not attained with the corresponding residue V1060 in IRK. Overall, our study provides a rationale for the molecular basis of recognition of this allosteric inhibitor by IGF1RK and IRK, which is potentially useful in developing novel inhibitors with improved affinity and selectivity.

Proxy-approach in understanding the bisubstrate activity of strictosidine synthases

Besides tryptamine (1) and secologanin (2), non-cognate substrates also undergo a Pictet-Spengler reaction (PSR) catalyzed by strictosidine synthases (STR) with differing catalytic properties. We characterized the bisubstrate binding aspect of catalysis – order, affinity, and cooperativity – with STR orthologs from Rauvolfia serpentina (RsSTR) and Ophiorrhiza pumila (OpSTR) by an isothermal titration calorimetry (ITC) based ‘proxy approach’ that employed a non-reactive tryptamine analog (m1) to capture its inert ternary complexes with STRs and (2). ITC studies with OpSTR and (2) revealed ‘tryptamine-first’ cooperative binding with (1) and a simultaneous cooperative binding with (m1). Binding cooperativity among (m1) and (2) towards OpSTR was higher than RsSTR. Crystallographic study of RsSTR-(m1) complex helped to understand the unreactive binding of (m1) in terms of orientation and interactions in the RsSTR pocket. PSR with (m1) was revealed to be energetically unfeasible by the density functional theory (DFT) scans of the first hydrogen abstraction by RsSTR. The effect of pH on the bisubstrate binding to OpSTR was deciphered by molecular dynamics simulations (MDS), which also provided a molecular basis for the stability of complex of OpSTR with (m1) and (2). Therefore, we investigated STRs from a substrate binding perspective to inform drug-design and rational enzyme engineering efforts.

Inhibitory effects against SARSCoV-2 main protease (Mpro) of biflavonoids and benzophenones from the fruit of Platonia insignis

The SARS-CoV-2 mutation and the limitation of the approved drug against COVID-19 are still a challenge in many country healthcare systems and need to be affronted despite the set of vaccines to prevent this viral infection. To contribute to the identification of new antiviral agents, the present study focused on natural products from an edible fruit with potential inhibitory effects against the SARS-CoV-2 main protease (Mpro). First, LC-ESIMS analysis of Platonia insignis fruits was performed and showed the presence of biflavonoids and benzophenones in the seed and pulp, respectively. Then, maceration and chromatographic purification led to the identification of two triglycerides (1 and 2) alongside chamaejasmine (3) and volkensiflavone (4) from the seed and isogarcinol (5) and cycloxanthochymol (6), from the pulp. Compounds 1–6 after evaluating their inhibitory against Mpro, displayed from no to significant activity. Compound 5 was the most potent with an IC50 value of 0.72 μM and was more active than the positive control, Ebselen (IC50 of 3.4 μM). It displayed weak and no cytotoxicity against THP-1 (CC50 of 116.2 μM) and Vero cell lines, respectively. Other active compounds showed no cytotoxicity against THP-1. and Vero cell lines. Molecular docking studies revealed interactions in the catalytic pocket between compound 5 and amino acid residues that composed the catalytic dyads (His 41 and Cyst 145).

Improved catalytic performance and molecular insight for lipoxygenase from Enterovibrio norvegicus via directed evolution.

Lipoxygenase (LOX) holds significant promise for food and pharmaceutical industries. However, albeit its application has been hampered by low catalytic activity and suboptimal thermostability. To address the drawbacks, a directed evolution strategy was explored to enhance the catalytic activity and thermostability of LOX from Enterovibrio norvegicus (EnLOX) for the first time. After two rounds of error-prone polymerase chain reaction (error-prone PCR) and one generations of sequential DNA shuffling, all of four different mutants showed a significant increase in the specific activity of EnLOX, ranging from 132.07 ± 9.34 to 330.17 ± 18.54U/mg. Among these mutants, D95E/T99A/A121H/S142N/N444W/S613G (EAHNWG) exhibited the highest specific activity, which was 8.25-fold higher than the wild-type enzyme (WT). Meanwhile, the catalytic efficiency (K cat /K m) of EAHNWG was also improved, which was 13.61 ± 1.67s-1μM-1, in comparison to that of WT (4.83 ± 0.38s-1μM-1). In addition, mutant EAHNWG had a satisfied thermostability with the t 1/2,50°C value of 6.44 ± 0.24h, which was 0.4h longer than that of the WT. Furthermore, the molecular dynamics simulation and structural analysis demonstrated that the reduction of hydrogen bonds number, the enhancement of hydrophobic interactions in the catalytic pocket, and the improvement of flexibility of the lid domain facilitated structural stability and the strength of substrate binding capacity for improved thermal stability and catalytic efficiency of mutant LOX after directed evolution. Overall, these results could provide the guidance for further enzymatic modification of LOX with high catalytic performance for industrial application.

A comprehension on structure guided alignment dependent 3D-QSAR modelling, and molecular dynamics simulation on 2,4-thiazolidinediones as aldose reductase inhibitors for the management of diabetic complications

Aldose reductase is an oxo-reductase enzyme belonging to the aldo-keto reductase class. Compounds having thiazolidine-2,4-dione scaffold are reported as potential aldose reductase inhibitors for diabetic complications. The present work uses structure-guided alignment-dependent Gaussian field- and atom-based 3D-QSAR on a dataset of 84 molecules. 3D-QSAR studies on two sets of dataset alignment have been carried out to understand the favourable and unfavourable structural features influencing the affinity of these inhibitors towards the enzyme. Using common pharmacophore hypotheses, the five-point pharmacophores for aldose reductase favourable features were generated. The molecular dynamics simulations (up to 100 ns) were performed for the potent molecule from each alignment set (compounds 24 and 65) compared to reference standard tolrestat and epalrestat to study target-ligand complexes’ binding energy and stability. Compound 65 was most stable with better interactions in the aldose reductase binding pocket than tolrestat. The MM-PBSA study suggests compound 65 possessed better binding energy than reference standard tolrestat, i.e. −87.437 ± 19.728 and −73.424 ± 12.502 kJ/mol, respectively. The generated 3D-QSAR models provide information about structure–activity relationships and ligand-target binding energy. Target-specific stability data from MD simulation would be helpful for rational compound design with better aldose reductase activity. Communicated by Ramaswamy H. Sarma

Riboflavin interactions with the chicken isolated carrier protein

Riboflavin, a member of the B vitamin family, is a water-soluble vitamin that participates in energy metabolism processes via two coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), in oxidized and reduced forms. Low levels of riboflavin have been associated with growth and developmental problems. In an effort to investigate the role of hydrogen bonding in the interactions between riboflavin and chicken riboflavin binding protein, the solid state geometry characteristics of a riboflavin derivative stripped of hydroxyl groups except the primary one, N-(6′-hydroxyhexyl)isoalloxazine, were investigated and found that π-stacking and hydrogen bonding involving the isoalloxazine rings are the primary intermolecular interactions. Subsequent comparative fluorescence studies showed that at neutral pH, in presence of the protein, quenching of N-(6′-hydroxyhexyl)isoalloxazine and riboflavin occurred similarly suggesting that the hydroxyl groups were not a key component of the vitamin protein interactions in the binding pocket.

Nucleoside-Driven Specificity of DNA Methyltransferase.

We have studied the adenosine binding specificities of two bacterial DNA methyltransferases, Taq methyltransferase (M.TaqI), and HhaI methyltransferase (M.HhaI). While they have similar cofactor binding pocket interactions, experimental data showed different specificity for novel S-nucleobase-l-methionine cofactors (SNMs; N=guanosyl, cytidyl, uridyl). Protein dynamics corroborate the experimental data on the cofactor specificities. For M.TaqI the specificity for S-adenosyl-l-methionine (SAM) is governed by the tight binding on the nucleoside part of the cofactor, while for M.HhaI the degree of freedom of the nucleoside chain allows the acceptance of other bases. The experimental data prove catalytically productive methylation by the M.HhaI binding pocket for all the SNMs. Our results suggest a new route for successful design of unnatural SNM analogues for methyltransferases as a tool for cofactor engineering.

A phenotypic screen with Trypanosoma brucei for discovering small molecules that target the SLiM-binding pocket of proliferating cell nuclear antigen orthologs.

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric protein complex that clamps around DNA to tether DNA polymerases to the template during replication and serves as a hub for many other interacting proteins. It regulates DNA metabolic processes and other vital cellar functions through the binding of proteins having short linear motifs (SLiMs) like the PIP-box (PCNA-interacting protein-box) or the APIM (AlkB homolog 2 PCNA-interacting motif) in the hydrophobic pocket where SLiMs bind. However, overproducing TbPCNA or human PCNA (hPCNA) in the pathogenic protist Trypanosoma brucei triggers a dominant-negative phenotype of arrested proliferation. The mechanism for arresting T. brucei proliferation requires the overproduced PCNA orthologs to have functional intact SLiM-binding pocket. Sight-directed mutagenesis studies showed that T. brucei overproducing PCNA variants with disrupted SLiM-binding pockets grew normally. We hypothesized that chemically disrupting the SLiM-binding pocket would restore proliferation in T. brucei, overproducing PCNA orthologs. Testing this hypothesis is the proof-of-concept for a T. brucei-based PCNA screening assay. The assay design is to discover bioactive small molecules that restore proliferation in T. brucei strains that overproduce PCNA orthologs, likely by disrupting interactions in the SLiM-binding pocket. The pilot screen for this assay discovered two hit compounds that linked to predetermined PCNA targets. Compound #1, a known hPCNA inhibitor, had selective bioactivity to hPCNA overproduced in T. brucei, validating the assay. Compound #6 had promiscuous bioactivity for hPCNA and TbPCNA but is the first compound discovered with bioactivity for inhibiting TbPCNA.

Biotransformation of isocostic acid from Dittrichia viscosa essential oil by Saccharomyces cerevisiae: in vitro anti-tyrosinase effect, insights from molecular docking and drug-likeness prediction

The purpose of this study was to investigate the biotransformation of the essential oil of Dittrichia viscosa leaves (LEO) by Saccharomyces cerevisiae (baker's yeast) and to evaluate its anti-tyrosinase property. The chemical analysis of the biotransformed essential oil (BEO) was determined by GC-FID and GC/MS. The results showed that isocostic acid, the predominant constituent of LEO (70.8%) was found to be totaly converted by S. cerevisiae into its isomer valerenic acid representing 78.4% of the total composition of BEO. The biotransformed essential oil (BEO) exhibited promising anti-tyrosinase activity (IC50 = 8.54 µg/mL) six times greater than that of LEO (IC50 = 53.44 µg/mL). The experimental results were reinforced by the molecular docking analysis and drug-likeness prediction carried out on the two major sesquiterpene acids detected in LEO and BEO (isocostic acid and valerenic acid, respectively). Valerenic acid showed lower binding energy (−6.3 kcal/mol) compared to isocostic acid (−5.9 kcal/mol), a correct mode and a large number of interactions in the active pocket of the enzyme which can explain the strong inhibitory effect of tyrosinase. Therefore, these results suggest that the bioconverted essential oil could be regarded as safe and effective source of skin whitening agent for cosmetic and medical applications.

Discovery of Selective Tertiary Amide Inhibitors of Cyclin-Dependent Kinase 2 (CDK2).

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and are frequently altered in cancer cells, thereby leading to uncontrolled proliferation. In this context, CDK2 has emerged as an appealing target for anticancer drug development. Herein, we describe the discovery of a series of selective small molecule inhibitors of CDK2 beginning with historical compounds from our ERK2 program (e.g., compound 6). Structure-based drug design led to the potent and selective tool compound 32, where excellent selectivity against ERK2 and CDK4 was achieved by filling the lipophilic DFG-1 pocket and targeting interactions with CDK2-specific lower hinge binding residues, respectively. Compound 32 demonstrated 112% tumor growth inhibition in mice bearing OVCAR3 tumors with 50 mg/kg bis in die (BID) oral dosing.

Novel Insights on 6:6 Perfluoroalkyl Phosphonic Acid-Induced Melanin Synthesis Disorders Leading to Pigmentation in Tadpoles.

As alternatives to traditional per- and polyfluoroalkyl substances, perfluoroalkyl phosphonic acids (PFPiAs) are widely present in aquatic environments and can potentially harm aquatic organisms. Pigmentation affects the probability of aquatic organisms being preyed on and serves as an important toxic endpoint of development, but little is known about the impacts of PFPiAs on the development of aquatic organisms. In this study, Xenopus laevis embryos were exposed to 6:6 PFPiA (1, 10, and 100 nM) for 14 days. The developed tadpoles exhibited evident pigmentation with increased melanin particle size and density on the skin. Pathological and behavioral experiments revealed that the retinal layers became thinner, reducing the photosensitivity and disturbing the circadian rhythm of the tadpoles. Compared to the control group, the exposed tadpoles showed higher levels but less changes of melanin throughout the light/dark cycle, as well as distinct oxidative damage. Consequently, the expression level of microphthalmia-associated transcription factor (MITF), a key factor inducing melanin synthesis, increased significantly. Molecular docking analysis suggested that 6:6 PFPiA forms strong interactions in the binding pocket of MITF, implying that it could activate MITF directly. The activation of MITF ultimately promotes melanin synthesis, resulting in pigmentation on tadpoles.

Identification and evaluation of potential inhibitor molecules against TcyA from Candidatus Liberibacter asiaticus

Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.

In silico screening of potential inhibitors from Cordyceps species against SARS-CoV-2 main protease

Coronavirus disease 2019 (COVID-19) is a result of a retroviral infection of SARS-CoV-2. Due to its virulence and high infection rate, it is a matter of serious concern and a global health emergency. Currently available COVID-19 vaccines approved by regulatory bodies around the world have been shown to provide significant protection against COVID-19. But no vaccine is 100% effective at preventing infection, also they have varying efficacy rates and different side effects. However, the main protease (Mpro) of SARS-CoV-2 has been identified as a key drug target due to its essential role in viral infection and its minimal similarity with human proteases. Cordyceps mushrooms have been found to have various therapeutic properties that could effectively combat SARS-CoV-2, including improve lung functioning, anti-viral, immunomodulators, anti-infectious, and anti-inflammatory. The present study aims to screen and evaluate the inhibitory potential of the bioactive molecules from the Cordyceps species against the Mpro of SARS-CoV-2. The bioactive molecules were screened based on their docking score, molecular interactions in the binding pocket, ADME properties, toxicity, carcinogenicity, and mutagenicity. Among all the molecules that were tested, cordycepic acid was the most effective and promising candidate, with a binding affinity of −8.10 kcal/mol against Mpro. The molecular dynamics (MD) simulation and free binding energy calculations revealed that the cordycepic acid-Mpro complex was highly stable and showed fewer conformational fluctuations. These findings need to be investigated further through in-vitro and in-vivo studies for additional validation. Communicated by Ramaswamy H. Sarma

Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease.

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of Paxlovid, a drug approved by the U.S. Food and Drug Administration for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir's inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here, we investigated the H172Y Mpro's conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggest that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weaken the nirmatrelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intelligence approaches, and together with biochemical experiments, they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the optimization of antiviral drugs. The presented approach, in general, can be applied to characterize mutation effects on any protein drug targets.