Receptor Binding Pocket Research Articles

Two Monoclonal Antibodies Targeting Distinct Subdomains of Human Norovirus P Protein.

The human norovirus (HuNov) major capsid VP1comprises an S (shell) and a P (protruding) domain; the latter is responsible for virus attachment and infection. The dimeric formation of P (containing P1 and P2 subdomains) is indispensable for forming a receptor-binding pocket, enabling HuNov to dock to attachment factor histo-blood group antigens (HBGAs) on the host cell. Thus, the P-specific antibody may hamper the engagement of P and HBGA, thereby inhibiting virus infection. In this study, we developed and characterized two HuNov P-specific murine monoclonal antibodies (MAbs), namely, 5C6 and 1H12. They can bind to P protein with high affinity, as evidenced by the results of indirect fluorescent assay, western blot, and Biolayer interferometry assay. Particularly, the MAb 1H12 recognizes the P2 subdomain, whereas the 5C6 targets the distal P1. These MAbs may contribute to the exploration of novel epitopes on HuNov VP1 and to the development of new antivirals.

Continuing evolution of H5N1 highly pathogenic avian influenza viruses of clade 2.3.2.1a G2 genotype in domestic poultry of Bangladesh during 2018–2021

ABSTRACT We characterized 15 H5N1 HPAI viruses from different small- and medium-scale poultry flocks across Bangladesh during 2018–2021 based on their complete genome sequences. The antigenic relatedness of H5N1 HPAI viruses from different timepoints was analysed. During 2020–2021, 42.11% of the flocks tested positive for at least one of the respiratory infections, with 15.79% showing influenza A virus, of which 8.77% tested positive for HPAIV H5N1. Co-infections with two to four pathogens were detected in 15.8% of flocks. Phylogeny and gene constellation analyses based on complete genome sequences of 15 HPAI viruses revealed the continuing circulation of H5 clade 2.3.2.1a genotype G2 viruses. In the HA protein of the study isolates, functionally meaningful mutations caused the loss of an N-linked glycosylation site (T156A), a modified antigenic site A (S141P), and a mutation in the receptor binding pocket (E193R/K). Consequently, antigenic analysis revealed a significant loss of cross-reactivity between viruses from different host species and periods. Most viruses displayed oseltamivir resistance markers at positions V96, I97, S227, and N275 (N1 numbering) of the NA protein. In addition, for the PB2, M1, and NS1 proteins, significant mutations were noticed that have been associated with polymerase activity and increased virulence for mammals in all study isolates. These results highlight the need for intensified genomic surveillance of HPAI circulating in poultry in Bangladesh and for establishing appropriate control measures to decrease the circulation of these viruses in poultry in the country.

Chemokine Receptor N-Terminus Charge Dictates Reliance on Post-Translational Modifications for Effective Ligand Capture and Following Boosting by Defense Peptides.

The chemokine receptors CCR1 and CCR5 display overlapping expression patterns and ligand dependency. Here we find that ligand activation of CCR5, not CCR1, is dependent on N-terminal receptor O-glycosylation. Release from O-glycosylation dependency is obtained by increasing CCR5 N-terminus acidity to the level of CCR1. Ligand activation of CCR5, not CCR1, drastically improves in the absence of glycosaminoglycans (GAGs). Ligand activity at both CCR1 and CCR5 is boosted by positively charged/basic peptides shown to interact with acidic chemokine receptor N-termini. We propose that receptors with an inherent low N-terminus acidity rely on post-translational modifications (PTMs) to efficiently compete with acidic entities in the local environment for ligand capture. Although crucial for initial ligand binding, strong electrostatic interactions between the ligand and the receptor N-terminus may counteract following insertion of the ligand into the receptor binding pocket and activation, a process that seems to be aided in the presence of basic peptides. Basic peptides bind to the naked CCR1 N-terminus, not the CCR5 N-terminus, explaining the loss of boosting of ligand-induced signaling via CCR5 in cells incapable of glycosylation.

Design, synthesis, and biological evaluation of novel imidazole derivatives as analgesic and anti-inflammatory agents: experimental and molecular docking insights

Imidazole moieties exhibit a broad range of biological activities, including analgesic, anti-depressant, anticancer, anti-fungal, anti-tubercular, anti-inflammatory, antimicrobial, antiviral, and antifungal properties. In this study, we explored the use of Schiff base for the synthesis of new imidazole derivatives as anti-inflammatory and pain-relieving agents. A series of eight novel imidazole analogues (2a–h) were prepared in three steps with excellent yields. All compounds were characterized using IR, NMR, and mass spectral data. Their analgesic and anti-inflammatory activities were evaluated using hot plate and paw oedema methods. Compound 2 g (1-(2,3-dichlorophenyl)-2-(3-nitrophenyl)-4,5-diphenyl-1H-imidazole) showed significant analgesic activity (89% at 100 mg/kg b.w.), while compounds 2a (2-(2,6-dichlorophenyl)-1-(4-ethoxyphenyl)-4,5-diphenyl-1H-imidazole) and 2b (2-(2,3-dichlorophenyl)-1-(2-chlorophenyl)-4,5-diphenyl-1H-imidazole) exhibited good anti-inflammatory activity (100% at 100 mg/kg b.w.), comparable to diclofenac salt (100% at 50 mg/kg b.w.). Molecular docking studies were conducted using Schrödinger software version 2021-2, employing the OPLS4 force field for both receptor and ligand preparation. The results were visualized using molecular visualization software such as PyMOL. These studies revealed that compound 2g exhibited the highest binding affinity with the COX-2 receptor (−5.516 kcal/mol). Compound 2g formed three conventional hydrogen bonds with residues GLN-242 (bond length: 2.3 Å) and ARG-343 (bond lengths: 2.2 Å & 2.4 Å). This binding affinity was comparable to that of Diclofenac salt, which showed the highest binding affinity of −5.627 kcal/mol with the COX-2 receptor. Diclofenac salt formed two conventional hydrogen bonds with the residues ARG-344 (bond length: 2.0 Å) and TRP-140 (bond length: 1.7 Å). Later, molecular dynamic simulations confirmed the stable binding affinity of compound 2g with the protein. Furthermore, other compounds also demonstrated potential binding to the receptor-binding pocket region. The anti-inflammatory potential of the synthesized compounds was evaluated using the carrageenan-induced rat hind paw oedema model, while the analgesic potential was assessed using the hot plate method. These evaluations were conducted in comparison with Diclofenac sodium, serving as the standard compound. However, compound 2g stood out for its superior analgesic activity, as confirmed by in-vivo examination. These findings suggest that these novel imidazole derivatives have potential as anti-inflammatory and analgesic agents.

Theoretical Investigations of a point mutation affecting H5 Hemagglutinin’s receptor binding preference

The avian influenza A H5N1 virus is a subtype of influenza A virus (IAV) that causes a highly infectious and severe respiratory illness in birds and poses significant economic losses in poultry farming. To infect host cell, the virus uses its surface glycoprotein named Hemagglutinin (HA) to recognize and to interact with the host cell receptor containing either α2,6- (SAα2,6 Gal) or α2,3-linked Sialic Acid (SAα2,3 Gal). The H5N1 virus has not yet acquired the capability for efficient human-to-human transmission. However, studies have demonstrated that even a single amino acid substitution in the HA can switch its glycan receptor preference from the avian-type SAα2,3 Gal to the human-type SAα2,6 Gal. The present study aims to explain the underlying mechanism of a mutation (D94N) on the H5 HA that causes the protein to change its glycan receptor-binding preference using molecular dynamics (MD) simulations. Our results reveal that the mutation alters the electrostatic interactions pattern near the HA receptor binding pocket, leading to a reduced stability for the HA-avian-type SAα2,3 Gal complex. On the other hand, the detrimental effect of the mutation D94N is not observed in the HA-human-type SAα2,6 Gal complex due to the glycan's capability to switch its topology.

DMXP: A De Novo Small-Molecule 3D Structure Predictor with Graph Attention Networks.

Generating the three-dimensional (3D) structure of small molecules is crucial in both structure- and ligand-based drug design. Structure-based drug design needs bioactive conformations of compounds for lead identification and optimization. Ligand-based drug design techniques, such as 3D shape similarity search, 3D pharmacophore model, 3D-QSAR, etc., all require high-quality small-molecule ligand conformations to obtain reliable results. Although predicting a small molecular bioactive conformer requires information from the receptor, a crystal structure of the molecule is a proper approximation to its bioactive conformer in a specific receptor because the binding pose of a small molecule in its receptor's binding pockets should be energetically close to the crystal structures. This study presents a de novo small molecular structure predictor (dMXP) with graph attention networks based on crystal data derived from the Cambridge Structural Database (CSD) combined with molecular electrostatic information calculated by density-functional theory (DFT). Two featuring strategies (topological and atomic partial change features) were employed to explore the relation between these features and the 3D crystal structure of a small molecule. These features were then assembled to construct the holistic 3D crystal structure of a molecule. Molecular graphs were encoded using a graph attention mechanism to deal with the issues of the inconsistencies of local substructures contributing to the entire molecular structure. The root-mean-square deviation (RMSDs) of approximately 80% dMXP predicted structures and the native binding poses within receptors are less than 2.0 Å.

Design, Synthesis, and Biological Evaluation of Pyrido [1,2-α] Pyrimidinone Mesoionic Derivatives Bearing Propenylbenzene as the Vector Control Insecticide.

A series of novel pyrido [1,2-α] pyrimidinone mesoionic derivatives bearing a propenylbenzene group at the 1-position were synthesized on the basis of the structure of mesoionic insecticides triflumezopyrim and dicloromezotiaz via a rationally conceived pharmacophore model and evaluated for their insecticidal activities against three insect vectors. The bioassay results showed that some compounds exerted remarkable insecticidal activities against M. domestica, Ae. albopictus, and B. germanica. Particularly, compound 26l displayed outstanding insecticidal activity against Ae. Albopictus, with an LC50 value of 0.45 μg/mL, far superior to that of imidacloprid (LC50 = 1.82 μg/mL) and equivalent to that of triflumezopyrim (0.35 μg/mL). Meanwhile, compound 34l presented a broad insecticidal spectrum, with LC50 values of 1.51 μg/g sugar, 0.52 μg/mL and 0.14 μg/adult, which were about 2.88, 3.50, and 1.50 times better than that of imidacloprid (LC50 = 4.35 μg/g sugar, 1.82 μg/mL and 0.21 μg/adult against M. domestica, Ae. albopictus, and B. germanica, respectively) and equivalent to that of triflumezopyrim against M. domestica (1.13 μg/g sugar) and Ae. albopictus (0.35 μg/mL) but lower than the potency against B. germanica (0.06 μg/g sugar). The molecular docking study by energy minimizations revealed that introducing propenylbenzene at the 1-position of compounds 26l and 34l could embed into the binding pocket of nicotinic acetylcholine receptors and form pi-alkyl interaction with LEU306. These results demonstrated that compounds 26l and 34l could be promising candidates for vector control insecticides, which deserved further investigation.

Effects of tea catechin extracts from BB35 and purple (TRFK 306) tea clones on the gene expression of Egfr, App, Bcl2, Dnmt, Casp3, Hif1a, Gadd45b and Psmb5 genes involved in triple negative breast cancer diseases: In silico and in vitro study

Tea has been shown to contain metabolites that exhibit antioxidant, anti-inflammatory, anti-diabetic, anti-cancer and anti-cardio vascular diseases properties. This study aimed to carry out an in silico assessment of catechins on the genes previously shown to be involved in Triple Negative Breast Cancer (TNBC) disease and also to evaluate the effects of catechin extracts from purple (TRFK306) and BB35 tea clones on the expression patterns of genes in the 4T1 TNBC cell line as mentioned above. Identification and quantification of different catechin contents in these two tea clones were performed by using High-Performance Liquid Chromatography (HPLC). In silico assessment including Absorption, Distribution, Metabolism and Excretion (ADME) study for drug-likeness evaluation, Drug target prediction, Protein-protein interaction (PPI) network analysis and construction, Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment and analysis for targets and Molecular docking study was conducted. Simplified Molecular Input Line Entry System (SMILES) of 4 different tea catechins showed a target on 111 genes in TNBC and have high binding affinity to the receptor-binding pocket of chain A of Epidermal Growth Factor Receptor (EGFR) Extracellular Domains. Catechin extracts promoted the downregulation of expression of 6 genes (Egfr, App, Bcl2, Dnmt, Hif1a and Psmb5) and the upregulation of 2 genes (Casp3 and Gadd45b) in the 4T1 TNBC cell line. Catechin extracts from purple tea clones showed higher activity on gene expression levels of Egfr, Bcl2 and Casp3 than catechin extracts from BB35 tea clone.

Molecular Dynamics Simulation of Serotonin Transport Protein Complex with 6-Hydroxy-1-Methyl-1,2,3,4-Tetrahydro-β-Carboline Ligand from Chocolate (Theobroma cacao L.) Isolate

6-hydroxy-1-methyl1,2,3,4-tetrahydro-β-carboline (6OHMTHβC) is a chocolate derivate that has antidepressant potency. It can increase dopamine and serotonin secretion, which leads to mood improvement. This method was carried out using computational molecular docking simulations (in silico). The research design was computational-based exploratory descriptive. The results of molecular docking showed the lowest energy score and the backbone RMSD value ≤2Å. The procedure performed was 6AWP receptor docking without ligand, with native ligand (6OHMTHβC), and with reference ligand (fluvoxamine). This study also performed molecular docking simulations of 6OHMTHβC towards 6AWP to find compounds in the receptor binding pocket. This study also performed dynamics simulations and identified the molecular determinants using PyPLIF-HIPPOS and YASARA Structure software 20.1.24.10 with the Windows 10 operating system. This study succeeded in determining the stability of the dynamics simulation of the serotonin transport protein complex with the reference ligand 6OHMTHβC for 50 ns, and this result corresponds to the RMSD value and binding energy. The determination of binding energy (BE) was calculated from the BE calculation available at YASARA and Ubuntu. The binding energy value of the original ligand was -11.6590 kJ/mol, and the reference ligand was -83880 kJ/mol. The highest RMSD value of the original ligand was 1.39292Å, while the RMSD value of the reference ligand was 1.71072Å. The essential amino acid carried out was 438Ser with hydrogen bond interactions, so 6OHMTHβC was considered a competent antidepressant candidate.

Development of Photoswitchable Tethered Ligands that Target the μ-Opioid Receptor.

Converting known ligands into photoswitchable derivatives offers the opportunity to modulate compound structure with light and hence, biological activity. In doing so, these probes provide unique control when evaluating G-protein-coupled receptor (GPCR) mechanism and function. Further conversion of such compounds into covalent probes, known as photoswitchable tethered ligands (PTLs), offers additional advantages. These include localization of the PTLs to the receptor binding pocket. Covalent localization increases local ligand concentration, improves site selectivity and may improve the biological differences between the respective isomers. This work describes chemical, photophysical and biochemical characterizations of a variety of PTLs designed to target the μ-opioid receptor (μOR). These PTLs were modeled on fentanyl, with the lead disulfide-containing agonist found to covalently interact with a cysteine-enriched mutant of this medically-relevant receptor.

Bridelia ferruginea phytocompounds interact with SARS-COV-2 drug targets: Experimental validation of corilagin contribution

The high mutation rate of SARS-CoV-2 genomic RNA has made COVID-19 more difficult to eradicate using currently available interventions. Hence, newer pharmaceutical strategies must be developed, especially those generally complementary and alternative medicines. In the current study, stem bark of Bridelia ferruginea Benth is presented as plausible source of ethno-pharmaceuticals actionable against key SARS-CoV-2 life cycle-dependent enzymes based on in vitro inhibition studies, LC-ESI-MS characterization and molecular docking studies. Bridelia ferruginea stem bark extracted with 1 % HCL-acidified water, water, butanol, chloroform, ethyl-acetate, and petroleum ether and assayed for in vitro inhibition of SARS-COV-2-gp/human ACE2 interaction. The lowest and the highest IC50 value were recorded for ethyl-acetate (3.550 mg/L) and chloroform (413.4 mg/L) extracts respectively. When the ethyl-acetate extract was tested for SARS-COV-2 protease inhibition in vitro, papain-like protease (PL-pro, IC50=1.981 mg/L) presented as the better target in comparison to the main protease (3CL-pro, IC50=10.13 mg/L). LC/MS analysis identified corilagin and Gallocatechin-[4-O-7]-epigallocatechin as the active principles whilst molecular docking revealed the plausible poses of the compounds within the binding pockets of SARS-COV-2-glycoprotien receptor binding pocket, 3CL-pro and PL-pro. Taken together, these findings identified Bridelia ferruginea stem bark as a plausible source of anti-SARS-COV-2 phytochemicals and propose that corilagin may play important role in this activity.

Comparison of SARS-CoV-2 entry inhibitors based on ACE2 receptor or engineered Spike-binding peptides.

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. IMPORTANCE The increasing resistance of SARS-CoV-2 variants to therapeutic antibodies has highlighted the need for new treatment options, especially in individuals who do not respond to vaccination. Receptor decoys that block viral entry are an attractive approach because of the presumed high bar to developing viral resistance. Here, we compare two entry inhibitors based on derivatives of the ACE2 receptor, or engineered peptides that bind to the receptor-binding pocket of the SARS-CoV-2 Spike protein. In each case, the inhibitors were fused to immunoglobulin Fc domains, which can further enhance therapeutic properties, and compared for activity against different SARS-CoV-2 variants. Potent inhibition against multiple SARS-CoV-2 variants was demonstrated in vitro, and even relatively low single doses of optimized reagents provided some protection in a mouse model, confirming their potential as an alternative to antibody therapies.

Design, Synthesis, and Computational Studies with ADMET of Novel Cardiovascular Hybrid Drugs

Recently the drug discovery trend has been to design hybrid drug molecules consisting of different pharmacophore groups linked together via spacers. Calcium channel blockers have an important role in the treatment of several cardiovascular diseases. The objective of the present research work is to encompass the strategic design, synthesis, and computational assessment of novel 1,4- dihydropyridine containing calcium channel blocker hybrid containing beta blocker side chain along with NO group as promoting cardiovascular agents as a viable alternative to well-established drugs like Amlodipine, Nifedipine, Felodipine, etc. In this research, we synthesized new 18 cardiovascular hybrid compounds based on structure-activity relationship properties. The 3D crystallographic structure of the calcium channel receptor (6M7H) was obtained from PDB. RCSB and used for docking study. The molecular docking studies were carried out by using the standard option Glide 5.5 in Maestro. In silico molecular docking analysis of all the synthesized compounds, AE1 to AE18, showed good docking scores and remarkable interactions with the essential amino acid residues located within the receptor's binding pocket of 6M7H. In comparison to amlodipine, AE12, AE6, AE8, AE11, AE5, AE16, and AE14 exhibit good scores as well as good binding patterns. AE12 exhibits the highest docking score of -8.455 kcal mol?1. Extensive ADMET profiling and structure–activity relationship (SAR) elucidated favorable pharmacokinetic properties and essential structural modifications influencing antihypertensive effectiveness.

Design and synthesis of thiadiazole-oxadiazole-acetamide derivatives: Elastase inhibition, cytotoxicity, kinetic mechanism, and computational studies

Considering the biological significance of 1,3,4-thiadiazole/oxadiazole heterocyclic scaffolds, a novel series of 1,3,4-thiadiazole-1,3,4-oxadiazole-acetamide derivatives (7a–j) was designed and synthesized using molecular hybridization. The inhibitory effects of the target compounds on elastase were evaluated, and all of these molecules were found to be potent inhibitors compared to the standard reference oleanolic acid. Compound 7f exhibited the excellent inhibitory activity (IC50 = 0.06 ± 0.02 μM), which is 214-fold more active than oleanolic acid (IC50 = 12.84 ± 0.45 μM). Kinetic analysis was also performed on the most potent compound (7f) to determine the mode of binding with the target enzyme, and it was discovered that 7f inhibits the enzyme in a competitive manner. Furthermore, the MTT assay method was used to assess their toxicity on the viability of B16F10 melanoma cell lines, and all compounds did not display any toxic effect on the cells even at high concentrations. The molecular docking studies of all compounds also justified with their good docking score and among them, compound 7f had a good conformational state with hydrogen bond interactions within the receptor binding pocket, which is consistent with the experimental inhibition studies.

Stimulation of platelet P2Y1 receptors by different endogenous nucleotides leads to functional selectivity via biased signalling.

Platelet function during inflammation is dependent on activation by endogenous nucleotides. Non-canonical signalling via the P2Y1 receptor is important for these non-thrombotic functions of platelets. However, apart from ADP, the role of other endogenous nucleotides acting as agonists at P2Y1 receptors is unknown. This study compared the effects of ADP, Ap3A, NAD+ , ADP-ribose, and Up4A on platelet functions contributing to inflammation or haemostasis. Platelets obtained from healthy human volunteers were incubated with ADP, Ap3A, NAD+ , ADP-ribose, or Up4A, with aggregation and fibrinogen binding measured (examples of function during haemostasis) or before exposure to fMLP to measure platelet chemotaxis (an inflammatory function). In silico molecular docking of these nucleotides to the binding pocket of P2Y1 receptors was then assessed. Platelet aggregation and binding to fibrinogen induced by ADP was not mimicked by NAD+ , ADP-ribose, and Up4A. However, these endogenous nucleotides induced P2Y1 -dependent platelet chemotaxis, an effect that required RhoA and Rac-1 activity, but not canonical PLC activity. Analysis of molecular docking of the P2Y1 receptor revealed distinct differences of amino acid interactions and depth of fit within the binding pocket for Ap3A, NAD+ , ADP-ribose, or Up4A compared with ADP. Platelet function (aggregation vs motility) can be differentially modulated by biased-agonist activation of P2Y1 receptors. This may be due to the character of the ligand-binding pocket interaction. This has implications for future therapeutic strategies aimed to suppress platelet activation during inflammation without affecting haemostasis as is the requirement of current ant-platelet drugs. This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.

Latent Biases in Machine Learning Models for Predicting Binding Affinities Using Popular Data Sets.

Drug design involves the process of identifying and designing molecules that bind well to a given receptor. A vital computational component of this process is the protein-ligand interaction scoring functions that evaluate the binding ability of various molecules or ligands with a given protein receptor binding pocket reasonably accurately. With the publicly available protein-ligand binding affinity data sets in both sequential and structural forms, machine learning methods have gained traction as a top choice for developing such scoring functions. While the performance shown by these models is optimistic, there are several hidden biases present in these data sets themselves that affect the utility of such models for practical purposes such as virtual screening. In this work, we use published methods to systematically investigate several such factors or biases present in these data sets. In our analysis, we highlight the importance of considering sequence, protein-ligand interaction, and pocket structure similarity while constructing data splits and provide an explanation for good protein-only and ligand-only performances in some data sets. Through this study, we provide to the community several pointers for the design of binding affinity predictors and data sets for reliable applicability.

Structural Elucidation of Relevant Gibberellic Acid Impurities and In Silico Investigation of Their Interaction with Soluble Gibberellin Receptor GID1.

Gibberellin derivatives are a family of tetracyclic diterpenoid plant hormones used in agriculture as plant growth regulators included in the European Directive 91/414. In the pesticide peer review process and to assess their toxicological relevance and product chemical equivalence, the European Food Safety Authority (EFSA) highlighted data gaps such as the identification of hydrolysis products and unknown impurities. The aspect of impurity characterization and quantitation is challenging and requires the use of hyphenated analytical techniques. In this regard, we used an LC-QTOF/MS and NMR analysis for the characterization of gibberellic acid impurities found in technical products. Gibberellic acid impurities such as gibberellin A1 (GA 1 ), 3-isolactone gibberellic acid (iso-GA 3 ), gibberellenic acid, 1α,2α-epoxygibberellin A3 (2-epoxy- GA 3 ), and (1α,2β,3α,4bβ,10β)-2,3,7-trihydroxy-1-methyl-8-methylenegibb-4-ene-1,10-dicarboxylic acid were identified and successfully characterized. Moreover, an in silico investigation on selected gibberellic acid impurities and derivatives and their interactions with a gibberellin insensitive dwarf1 (GID1) receptor has been carried out by means of induced fit docking (IFD), generalized-Born surface area (MM-GBSA), and metadynamics (MTD) experiments. A direct HPLC method with DAD and MS for the detection of gibberellic acid and its impurities in a technical sample has been developed. Moreover, by means of the in silico characterization of the GID1 receptor-binding pocket, we investigated the receptor affinity of the selected gibberellins, identifying compounds (2) and (4) as the most promising hit to lead compounds.

Molecular Basis for Non-Covalent, Non-Competitive FAAH Inhibition

Fatty acid amide hydrolase (FAAH) plays a key role in the control of cannabinoid signaling and it represents a promising therapeutic strategy for the treatment of a wide range of diseases, including neuropathic pain and chronic inflammation. Starting from kinetics experiments carried out in our previous work for the most potent inhibitor 2-amino-3-chloropyridine amide (TPA14), we have investigated its non-competitive mechanism of action using molecular dynamics, thermodynamic integration and QM-MM/GBSA calculations. The computational studies highlighted the impact of mutations on the receptor binding pockets and elucidated the molecular basis of the non-competitive inhibition mechanism of TPA14, which prevents the endocannabinoid anandamide (AEA) from reaching its pro-active conformation. Our study provides a rationale for the design of non-competitive potent FAAH inhibitors for the treatment of neuropathic pain and chronic inflammation.

Pea eggplant (Solanum torvum Swartz) is a source of plant food polyphenols with SARS-CoV inhibiting potential.

Pea eggplant (Solanum torvum Swartz) commonly known as turkey berry or 'terung pipit' in Malay is a vegetable plant widely consumed by the local community in Malaysia. The shrub bears pea-like turkey berry fruits (TBFs), rich in phytochemicals of medicinal interest. The TBF phytochemicals hold a wide spectrum of pharmacological properties. In this study, the TBF phytochemicals' potential inhibitory properties were evaluated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of the Coronavirus disease 2019 (COVID-19). The TBF polyphenols were screened against SARS-CoV receptors via molecular docking and the best receptor-ligand complex was validated further by molecular dynamics (MD) simulation. The SARS-CoV receptor structure files (viral structural components) were retrieved from the Protein Data Bank (PDB) database: membrane protein (PDB ID: 3I6G), main protease (PDB ID: 5RE4), and spike glycoproteins (PDB ID: 6VXX and 6VYB). The receptor binding pocket regions were identified by Discovery Studio (BIOVIA) for targeted docking with TBF polyphenols (genistin, kaempferol, mellein, rhoifolin and scutellarein). The ligand and SARS-CoV family receptor structure files were pre-processed using the AutoDock tools. Molecular docking was performed with the Lamarckian genetic algorithm using AutoDock Vina 4.2 software. The best pose (ligand-receptor complex) from the molecular docking analysis was selected based on the minimum binding energy (MBE) and extent of structural interactions, as indicated by BIOVIA visualization tool. The selected complex was validated by a 100 ns MD simulation run using the GROMACS software. The dynamic behaviour and stability of the receptor-ligand complex were evaluated by the root mean square displacement (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), solvent accessible surface volume (SASV) and number of hydrogen bonds. At RMSD = 0, the TBF polyphenols showed fairly strong physical interactions with SARS-CoV receptors under all possible combinations. The MBE of TBF polyphenol-bound SARS CoV complexes ranged from -4.6 to -8.3 kcal/mol. Analysis of the structural interactions showed the presence of hydrogen bonds, electrostatic and hydrophobic interactions between the receptor residues (RR) and ligands atoms. Based on the MBE values, the 3I6G-rhoifolin (MBE = -8.3 kcal/mol) and 5RE4-genistin (MBE = -7.6 kcal/mol) complexes were ranked with the least value. However, the latter showed a greater extent of interactions between the RRs and the ligand atoms and thus was further validated by MD simulation. The MD simulation parameters of the 5RE4-genistin complex over a 100 ns run indicated good structural stability with minimal flexibility within genistin binding pocket region. The findings suggest that S. torvum polyphenols hold good therapeutics potential in COVID-19 management.

2-Arylmelatonin analogues: Probing the 2-phenyl binding pocket of melatonin MT1 and MT2 receptors

In crystal structures of melatonin MT1 and MT2 receptors, a lipophilic subpocket has been characterized which accommodates the phenyl ring of the potent agonist 2-phenylmelatonin. This subpocket appears a key structural element to achieve high binding affinity and selectivity for the MT2 receptor. A series of 2-arylindole ligands was synthesized to probe the requirements for the optimal occupation and interaction with the 2-phenyl binding pocket. Thermodynamic integration simulations applied to MT1 and MT2 receptors in complex with the α-naphthyl derivative provided a rationale for the MT2-selectivity and investigation on the binding mode of a couple of atropisomers allowed to define the available space and arrangement of substituents inside the subpocket. Interestingly, more hydrophilic 2-aza-substituted compounds displayed high binding affinity and molecular dynamics simulations highlighted polar interaction with residues from the subpocket that could be responsible for their potency.