Ligand Binding Efficiency Research Articles

Structure‐Based Computational Approach in Search of the Potent Molecules Targeting Phosphoglycerate Dehydrogenase (PHGDH) Enzyme for Cancer Treatment

AbstractThis work aims to target phosphoglycerate dehydrogenase (PHGDH), a promising druggable target, that is overexpressed in various types of cancer. A structure‐based approach was employed to identify novel inhibitors against the enzyme. A common five‐feature pharmacophore model (RRHDA) was constructed using the active site co‐crystalized ligands. These chemical features were responsible for showing inhibition. The generated models were subsequently subjected to the validation method using a test set, receiver‐operator characteristic analysis, enrichment factor, and Güner–Henry studies. The validated models were subjected to the screening of a dataset of natural compounds. The screened unique natural compounds (1795) were further selected for the interaction analysis and study of ligand binding affinity considering the effect of the hydrogen bonding and desolvation and hydrophobic interactions contribution to the binding. The natural compounds which exhibited good ligand binding efficiency were selected and further subjected to pharmacokinetics and pharmacodynamic study. The natural compounds which exhibited good ligand binding efficiency were selected and further subjected to pharmacokinetics and pharmacodynamic study. The finalized complexes were selected for the simulation studies and MM/PBSA‐based binding free energy calculations. This study expands the possibilities for the development of shortlisted molecules as novel anti‐cancer compounds.

Arabidopsis METHYLENETETRAHYDROFOLATE REDUCTASE 2 functions independently of PENETRATION 2 during primary immunity against rice blast.

Nonhost resistance (NHR) is the most durable and robust form of innate immunity, with a surge of interest in crop improvement. Of the NHR genes identified against rice blast, a devastating disease caused by Magnaporthe oryzae, Arabidopsis PEN2 is indispensable for pre-penetration resistance against M. oryzae, while a consortium of genes orchestrates post-penetration resistance via lesser-known mechanisms. We identified M. oryzae-susceptible mosA (mthfr2 pen2-3) from a randomly mutagenized Arabidopsis pen2-3 population using forward genetics. Analysis of T-DNA inserted mthfr2 lines and pen2-3 complemented mosA lines enunciated that MTHFR2-dependent resistance to M. oryzae is independent of PEN2. MTHFR2-defective plants exhibited higher ROS accumulation and expression of SA-dependent defense markers. MTHFR2-ligand docking revealed that A55V nonsynonymous substitution in mosA altered ligand binding efficiency. This further affected the metabolomic profile of mosA, effectively allowing in vitro germination and development of M. oryzae conidia. Moreover, the loss of function mutation in mthfr2 (involved in 1C metabolic pathway) potentiated mosA immunity against Pst DC3000. In conclusion, our findings assert MTHFR2 as a positive modulator of NHR against M. oryzae. This work documents another layer of conserved yet divergent metabolomic defense in Arabidopsis regulated by folate-mediated 1C metabolism that has the potential to revolutionize crop improvement.

High-throughput virtual screening of potential inhibitors of GPR52 using docking and biased sampling method for Huntington's disease therapy.

Huntington's disease (HD) is a rare and progressive neurodegenerative disorder caused by polyglutamine (poly-Q) mutations of the huntingtin (HTT) gene resulting in chorea, cognitive, and psychiatric dysfunctions. Being a monogenic condition, reducing the levels of the mutated huntingtin protein (mHTT) holds promise as an effective therapeutic approach. GPR52, an orphan G-protein coupled receptor (GPCR), enriched in the striatum, is a novel target for slowing down the progression of HD by lowering the mHTT levels. Therefore, the study focuses on identifying potent small-molecule inhibitors for GPR52 using a combination of robust high-throughput virtual screening (HTVS) and pharmacokinetics profiling followed by fast pulling of ligand (FPL) and umbrella sampling (US) simulations. Initially, screening a library of 2,36,545 compounds was done against the binding pocket of GPR52. Based on binding affinity, stereochemical and non-bonded interactions, and pharmacokinetic profiling, 50 compounds were shortlisted. Selected hit compounds 1, 2, and 3 were subjected to FPL simulations with applied external bias potential to investigate their unique dissociation pathways and intermolecular interactions over time. Subsequently, the US simulations were performed on the selected hit compounds to estimate their binding free energy (ΔG). The analysis of the trajectories obtained from simulations revealed that the residues TYR34, TYR185, GLY187, ASP188, ILE189, SER299, PHE300, and THR303 within the active site of GPR52 were significant for efficient ligand binding through the formation of various hydrogen bond interactions and hydrophobic contacts. Out of the three hit compounds, compound 3 had the lowest ΔG of - 20.82 ± 0.44kcal/mol. The study identified compounds 1, 2, and 3 as potential molecules that can be developed as GPR52 inhibitors holding promise for lowering mHTT levels.

Structural and mechanistic insights into ALS patient derived mutations in D-amino acid oxidase

The D-amino acid oxidase protein modulates neurotransmission by controlling the levels of D-serine, a co-agonist of N-methyl-D-aspartate receptors. Mutations in the DAO gene have been associated with ALS, with some studies reporting pathogenic mechanisms of the R199W mutation. We have characterized two novel mutations R38H and Q201R found in ALS patients and report certain novel findings related to the R199W mutation. We report the first instance of crystal structure analysis of a patient-derived mutant of DAO, R38H, solved at 2.10 Å. The structure revealed significant perturbations and altered binding with the cofactor (FAD) and the inhibitor benzoate, supported by biochemical assays. Q201R-DAO also exhibited significantly lower ligand binding efficiency. Furthermore, kinetic analysis across all variants revealed reduced oxidase activity and substrate binding. Notably, R38H-DAO exhibited near-WT activity only at high substrate concentrations, while R199W-DAO and Q201R-DAO displayed drastic activity reduction. Additionally, structural perturbations were inferred for R199W-DAO and Q201R-DAO, evident by the higher oligomeric state in the holoenzyme form. We also observed thermal instability in case of R199W-DAO mutant. We hypothesize that the mutant enzymes may be rendered non-functional in a cellular context, potentially leading to NMDAR-associated excitotoxicity. The study provides novel insights into structural and functional aspects of DAO mutations in ALS.

Molecular modelling reveals how abundance of α4 sub-type in synaptic GABARA receptor can lead to refractoriness toward GABA and BZ-type drugs

Epilepsy is a complex neurological disorder with genetic and acquired causes, and the drugs presently used to treat epilepsy are not effective in about 30% of the cases. Identification of the molecular mechanisms of resistance will help in the development of newer molecules for treatment. Recent clinical data indicate increased expression of α4- and γ2-containing synaptic GABARA receptors in patients of focal cortical dysplasia (FCD), which is associated with refractory epilepsy pathology. We have investigated, by molecular modelling and docking, the structure and ligand-binding efficiency of the α4-containing hetero-pentameric synaptic GABARA receptor. Though the overall conformation is similar to that of the α1-containing receptor, local conformational changes are seen due to differences between aligned α1 and α4 sub-type residues. The overlaps ALA209(α1)/PRO215(α4) and PHE73(α1)/TYR79(α4) have together caused conformational changes in ARG100(α4) (aligned with ARG94 in α1) thereby affecting key hydrogen bonding interactions with the inhibitory neurotransmitter GABA. This may influence the nature of seizures as strength of GABA-binding is known to affect the nature of Inhibitory Post-Synaptic Currents (IPSCs) from GABAergic neurons. The residue ARG135 (α4) aligns with the residue HIS129 (α1) in the benzodiazapine binding pocket. Molecular modelling also shows that a steric clash between benzodiazapine-type (BZ-type) drugs and ARG135 would reduce the binding of BZ-type drugs to α4-containing receptor. These two findings rationalize the observed association between over-expression of α4-containing synaptic GABARA receptors and refractory epilepsy pathology in FCD. The accurate three-dimensional geometry of the receptor-drug complex made available by these modelling studies will help in designing effective drugs. Communicated by Ramaswamy H. Sarma

Mutational analysis of flavonol synthase of M. pinnata towards enhancement of binding affinity: a computational approach

Millettia pinnata is an important medicinal plant that has been used as a treatment of various diseases due to presence of wide range of pharmacological properties. The plant contains quercetin, kaempferol, karanjin, pongaglabrone, kanjone, kanugin, gammatin, pongaglabol, and other bioflavonoids. Kaempferol is a natural flavonol that shows many pharmacological properties including anti-inflammatory, antioxidant, anticancer, and antidiabetic activities etc. The enzyme flavonol synthase (FLS, EC 1.14.20.6) catalyses the conversion of dihydroflavonols to flavonols, i.e. biosynthesis of kaempferol from dihydrokaempferol. The current work examined the binding affinity-based approach to improve the enzyme catalytic activity using computational methods. Sequential site-directed mutagenesis was used to create four mutants with the goal to increase hydrogen bonds and further improving the ligand (dihydrokaempferol) binding efficiency. Simulations were done to monitor the stability of the mutants followed by molecular docking to confirm interactions with ligand. For structure validation, various dynamic analysis like RMSD, RMSF, ROG, SASA, H-bond, PCA, DCCM, and FEL were performed, which predicts the stability of wild-type (WT) proteins and mutants. The Mutant_2 and Mutant_3 showed maximum H-bonding and better stability than other mutants and WT that proved higher affinity suggesting improved catalysis. Mutant_2 and Mutant_3 exhibited binding affinities of −7.6 and −8.2 kcal/mol, respectively for the ligand. The outcome of present study will provide significant improvement in synthesis of kaempferol and other plant-based flavonoids. Communicated by Ramaswamy H. Sarma

Chemical probing profiling of 2'-deoxyguanosine sensing RNA reveals magnesium ion-dependent conformational switching.

Maintaining the homeostasis of cellular metabolites is essential for all living organisms. Noncoding mRNA in procaryotes strategically regulates the expression of downstream genes which are linked to the expression of proteins involved in specific metabolite salvage and biosynthesis, in response to binding to that specific metabolite. Such noncoding mRNAs are known as the Riboswitches. A class of riboswitches that recognizes purine analogs were identified. In this present contribution, we report the influence of magnesium and temperature on the conformation of the aptamer domain (the ligand binding domain) of 2’-deoxyguanosine riboswitch from Mesoplasma florum. Our comprehensive SHAPE (Selective 2′ Hydroxyl Acylation analyzed by Primer Extension) probing experiments have shown that the aptamer domain exhibited a strong dependence on the Mg2+ ion concentration for the efficient ligand binding and the concomitant structural change. Mg2+ ions reduce the conformational flexibility mainly by organizing P2 and P3 helices and thereby facilitate ligand binding by shifting the conformational ensemble of the ligand-free aptamer towards a pre-organized, ligand-binding competent conformation. The subtle conformational switching in the presence and absence of 2’-deoxyguanosine is observed at the (A) regulatory helix (P1) and (B) the interhelical junctions (which serve as the ligand binding pocket) of the aptamer. We have observed the formation of a regulatory helix, the folding of the binding pocket, and the subsequent encapsulation of the ligand is accomplished only in the simultaneous presence of Mg2+ and 2’-deoxyguanosine. We have also identified the existence of two conformations in equilibrium in a ligand-free state. Finally, we have shown that for the 2’-dG sensing riboswitch from Mesoplasma forum, the conformation of the full-length transcripts populates the functional OFF-state regardless of the presence or absence of ligand, consistent with a kinetic riboswitch mechanism.

Structural insights into translation regulation by the THF-II riboswitch.

In bacteria, expression of folate-related genes is controlled by the tetrahydrofolate (THF) riboswitch in response to specific binding of THF and its derivatives. Recently, a second class of THF riboswitches, named THF-II, was identified in Gram-negative bacteria, which exhibit distinct architecture from the previously characterized THF-I riboswitches found in Gram-positive bacteria. Here, we present the crystal structures of the ligand-bound THF-II riboswitch from Mesorhizobium loti. These structures exhibit a long rod-like fold stabilized by continuous base pair and base triplet stacking across two helices of P1 and P2 and their interconnecting ligand-bound binding pocket. The pterin moiety of the ligand docks into the binding pocket by forming hydrogen bonds with two highly conserved pyrimidines in J12 and J21, which resembles the hydrogen-bonding pattern at the ligand-binding site FAPK in the THF-I riboswitch. Using small-angle X-ray scattering and isothermal titration calorimetry, we further characterized the riboswitch in solution and reveal that Mg2+ is essential for pre-organization of the binding pocket for efficient ligand binding. RNase H cleavage assay indicates that ligand binding reduces accessibility of the ribosome binding site in the right arm of P1, thus down-regulating the expression of downstream genes. Together, these results provide mechanistic insights into translation regulation by the THF-II riboswitch.

Design and investigation of targeting agent orientation and density on nanoparticles for enhancing cellular uptake efficiency.

The design of targeting agent-conjugated systems is attracting much attention in cell targeted delivery and cancer therapy. However, quantitative study of the ligand density and binding efficiency is still limited due to the technical matters and tedious work involved. In this article, benzoboroxole-modified core-shell magnetic nanoparticles (MSP-AOPB NPs) as a drug carrier model were fabricated and transferrin (Tf) was immobilized on the nanoparticle surface in a site-oriented manner (Tf-MSP-AOPB NPs). The preparation conditions were investigated in detail to optimize the Tf binding efficiency. A suitable reaction temperature, time or initial feeding amount could significantly increase the Tf binding amount. The maximum Tf binding amount on the MSP-AOPB NPs was 184 mg g-1, and the targeting ligand density on the surface could be well controlled by simply adjusting the reaction conditions. In vitro studies demonstrated the excellent Tf-mediated targeting ability and enhanced cellular uptake efficacy by varying the ligand density. The optimal ligand binding amount for achieving the highest cellular uptake efficiency was 94 mg Tf/g, which corresponds to a ligand binding density of about 0.05 Tf/nm2, and the binding efficiency of conjugation was higher than 90%. Moreover, Tf-MSP-AOPB NPs prepared by a site-oriented conjugation strategy showed the best cell targeting ability, and their cellular uptake amount was 25 and 127 times higher than that of physical adsorption and EDC/NHS coupling reaction in HepG2 cells, respectively. This study provides a facile site-oriented bioconjugation technique for different kinds of antibodies, and a suitable ligand density can be easily attained to enhance the cellular uptake efficacy, which shows great significance for targeted delivery and cancer therapy.

Dose-dependent binding behavior of anthraquinone derivative purpurin interacting with tau-derived peptide protofibril.

Alzheimer's disease is hallmarked by microtubule-associated protein tau tangles and amyloid-β plaques. The β-structure propensity of tau inclusions is closely related to the hexapeptide motif VQIVYK (termed PHF6), and disruption of this motif prevents tau aggregation. Small-molecule inhibitors are considered a promising therapeutic strategy, but the molecular mechanisms underlying the correlation between dose and inhibitory effects are still unclear. In this work, we investigated the dose-induced influence of purpurin, an anthraquinone derivative, on the structural stability of the PHF6 fibrillar nucleus by performing microsecond all-atom molecular dynamics simulations in explicit water. The stability of PHF6 protofibrils of different sizes was first examined, and it was found that the structural stability of fibrillar oligomers increases with oligomer size, and that the octamer is the minimal stable nucleus for fibril formation. When purpurin molecules were added to the protofibril octamer at a low purpurin/peptide ratio, they bound to the octamer with different coupling states, and the different states may transition to each of the other states through an uncoupling state or directly through a short-time transition. With increasing purpurin/peptide ratio, purpurins tend to self-aggregate rather than bind to the protein surface. Interestingly, the contacts between individual purpurins and the octamer as a function of the purpurin number show a power-law behavior, which may serve as a useful indicator to reflect the binding efficiency of ligands to proteins in drug screening. The interaction analysis reveals that purpurin prefers to bind to the hydrophilic and aromatic Tyr and has the lowest probability with the hydrophobic Val located in the middle of PHF6. Aromatic stacking plays a key role in the octamer-purpurin interaction, in which the three aromatic rings of purpurin have different contributions. In addition, purpurin shows a remarkable disruptive effect on the protofibril octamer when the molar ratio of purpurin to peptide is 1 : 2; above this ratio, the binding mode and disruption effect of purpurin do not change significantly. Our work provides a detailed picture of the dynamics and interactions of purpurin binding to the PHF6 protofibril and expands the understanding of the dose-induced inhibitory mechanism.

Structure-based design of a novel inhibitor of the ZIKA virus NS2B/NS3 protease

Zika virus (ZIKV) has been a serious public health problem, and there is no vaccine or drug approved for the prevention or treatment of ZIKV yet. The ZIKV NS2B/NS3 protease plays an important role in processing the virus precursor polyprotein and is thus a promising target for antiviral drugs development. In order to discover novel inhibitors of this protease, we carried out a fragment-based hit screening and characterized protein-inhibitor interactions using the X-ray crystallography together with isothermal titration calorimetry. We reported two high-resolution crystal structures of the protease (bZiProC143S) in complex with an active fragment as well as a tetrapeptide, revealing that there is domain swapping in the protein structures and two ligands only occupy the substrate-binding pocket of one copy in a symmetric unit. Based on the detailed binding modes of two ligands revealed by crystal structures, we designed a novel inhibitor which inhibits the NS2B/NS3 protease with a higher potency than the fragment and possesses a higher ligand-binding efficiency and a comparable IC50 compared to the tetrapeptide. These results thus provide a structural basis and valuable hint for development of more potent inhibitors of the ZIKV NS2B/NS3 protease.

Interactions of selected cardiovascular active natural compounds with CXCR4 and CXCR7 receptors: a molecular docking, molecular dynamics, and pharmacokinetic/toxicity prediction study

BackgroundThe chemokine CXCL12 and its two receptors (CXCR4 and CXCR7) are involved in inflammation and hematopoietic cell trafficking. This study was designed to investigate molecular docking interactions of four popular cardiovascular-active natural compounds; curcumin, resveratrol, quercetin, and eucalyptol; with these receptors and to predict their drug-like properties. We hypothesize that these compounds can modify CXCL12/CXCR4/CXCR7 pathway offering benefits for coronary artery disease patients.MethodsDocking analyses were carried and characterized by Molecular Environment (MOE) software.Protein Data Bank (http://www.rcsb.org/) has been retrieved from protein structure generation and crystal structures of CXCR4 and CXCR7 receptors (PDB code = 3ODU and 6K3F). The active sites of these receptors were evaluated and extracted from full protein and molecular docking protocol was done for compounds against them. The presented parameters included docking scores, ligand binding efficiency, and hydrogen bonding. The pharmacokinetic/toxic properties (ADME/T) were calculated using SwissADME, ProTox-II, and Pred-hERG softwares to predict drug-like properties of the compounds. The thermochemical and molecular orbital analysis, and molecular dynamics simulations were also done.ResultsAll compounds showed efficient interactions with the CXCR4 and CXCR7 receptors. The docking scores toward proteins 3ODU of CXCR4 and 6K3F of CXCR7 were − 7.71 and − 7.17 for curcumin, − 5.97 and − 6.03 for quercetin, − 5.68 and − 5.49 for trans-resveratrol, and − 4.88 and − 4.70 for (1 s,4 s)-eucalyptol respectively indicating that all compounds, except quercetin, have more interactions with CXCR4 than with CXCR7. The structurally and functionally important residues in the interactive sites of docked CXCR4-complex and CXCR7-complex were identified. The ADME analysis showed that the compounds have drug-like properties. Only (1 s,4 s)-Eucalyptol has potential weak cardiotoxicity. The results of thermochemical and molecular orbital analysis and molecular dynamics simulation validated outcomes of molecular docking study.ConclusionsCurcumin showed the top binding interaction against active sites of CXCR4 and CXCR7 receptors, with the best safety profile, followed by quercetin, resveratrol, and eucalyptol. All compounds demonstrated drug-like properties. Eucalyptol has promising potential because it can be used by inhalation or skin massage. To our knowledge, this is the first attempt to find binding interactions of these natural agents with CXCR4 and CXCR7 receptors and to predict their druggability.

Unraveling the Role of π-Stacking Interactions in Ligand Binding to the Thiamine Pyrophosphate Riboswitch with High-level Quantum Chemical Calculations and Docking Study.

The thiamine pyrophosphate (TPP) riboswitch has emerged as the new target for designing new ligands for antibiotic purpose. Binding of the natural ligand TPP to the TPP riboswitch causes downregulation of the genes responsible for its biosynthesis. We have reported the role of π-stacking energy contributions to ligand binding with a TPP riboswitch. In conjunction with the docking study, the higher-level quantum chemical calculations performed with the wB97XD and Def2TZVPP basis set in the aqueous phase revealed that the optimum ring size is crucial to attain the effective binding efficiency of ligands with a TPP riboswitch. The π-stacking energy contributions observed for the ligands studied are largely similar; however, the cases studied with higher π-stacking energies with larger rings have a weaker ability to displace the radiolabeled thiamine from the riboswitch. The EDA and NCI analyses suggest the role of larger dispersive interactions in stabilizing the π-stacking rings. The contribution from hydrogen-bonding interactions of the hydrogen-bond donor groups on the A ring augments the binding affinity of the ligand. This study sheds light on various factors that contribute to the design of new ligands for efficient binding with a TPP riboswitch and inhibition of gene expression.

Critical APJ receptor residues in extracellular domains that influence effector selectivity.

Human APJ receptor/apelin receptor (APJR), activated by apelin peptide isoforms, regulates a wide range of physiological processes. The role of extracellular loop (ECL) domain residues of APJR in ligand binding and receptor activation has not been established yet. Based on multiple sequence alignment of APJ receptor from various organisms, we identified conserved residues in the extracellular domains. Alanine substitutions of specific residues were characterized to evaluate their ligand binding efficiency and Gq -, Gi -, and β-arrestin-mediated signaling. Mutation-dependent variation in ligand binding and signaling was observed. W197 A in ECL2 and L276 L277 W279 -AAA in ECL3 were deficient in Gi and β-arrestin signaling pathways with relatively preserved Gq -mediated signaling. T169 T170 -AA, Y182 A, and T190 A mutants in ECL2 showed impaired β-arrestin-dependent cell signaling while maintaining G protein- mediated signaling. Structural comparison with angiotensin II type I receptor revealed the importance of ECL2 and ECL3 residues in APJR ligand binding and signaling. Our results unequivocally confirm the specific role of these ECL residues in ligand binding and in orchestrating receptor conformations that are involved in preferential/biased signaling functions.

A newly synthesized small molecule: the evaluation against Alzheimer's Disease by in silico drug design and computational structure analysis methods

The submitted study reports the synthesis, crystal structure determination by the spectroscopic and non-spectroscopic methods, comparison of computational and experimental results, enclosure of the intermolecular interactions, comprehensive surface analysis by Hirshfeld method and electrostatic potential map, the examination of the electronic properties by Frontier Molecular Orbital (FMO) theory and Ultraviolet-Visible (UV-Vis) methods of an organic-structured molecule: (E)-1-(5-nitro-2-(piperidin-1-yl)phenyl)-N-(m-tolyl) methanimine. Drug-likeness properties such as Lipinski's rule of five, Adsorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) have been investigated by in silico web-based tools like SwissADME and ADMETlab. Besides the said, the determination studies of biological targets of the compound have also been carried out by in silico techniques. It has been found that the compound may be a substrate against both i) neurogenerative disease-related: microtubule-associated protein tau and ii) myotonic dystrophy-related: muscleblind like protein 1 targets. In this concept, the ligand's binding efficiency to the tau protein has been determined by in silico molecular docking tools such as AutoDock 4.2 and Molecular Graphics Laboratory (MGL) tools. The found results have shown that the compound is in harmony with Lipinski and others (Ghose, Veger, Egan, Muegge), which determine a compound's drug-likeness properties, with zero deviation. According to the results found by The Brain Or IntestinaL EstimateD (BOILED‐Egg) permeation method, the compound is active in both gastrointestinal absorption and Blood-Brain Barrier (BBB) crossing. We have found the lowest binding energy value determined by molecular docking is -5.28 kcal/mol. The title compound can be an inhibitor against tau aggregation to combat neurodegenerative diseases. But it needs further studies to verify the predictions said via both advanced in silico methods and in vitro and in vivo studies.

Discovery of Selective Pituitary Adenylate Cyclase 1 Receptor (PAC1R) Antagonist Peptides Potent in a Maxadilan/PACAP38-Induced Increase in Blood Flow Pharmacodynamic Model.

Inhibition of the pituitary adenylate cyclase 1 receptor (PAC1R) is a novel mechanism that could be used for abortive treatment of acute migraine. Our research began with comparative analysis of known PAC1R ligand scaffolds, PACAP38 and Maxadilan, which resulted in the selection of des(24-42) Maxadilan, 6, as a starting point. C-terminal modifications of 6 improved the peptide metabolic stability in vitro and in vivo. SAR investigations identified synergistic combinations of amino acid replacements that significantly increased the in vitro PAC1R inhibitory activity of the analogs to the pM IC90 range. Our modifications further enabled deletion of up to six residues without impacting potency, thus improving peptide ligand binding efficiency. Analogs 17 and 18 exhibited robust in vivo efficacy in the rat Maxadilan-induced increase in blood flow (MIIBF) pharmacodynamic model at 0.3 mg/kg subcutaneous dosing. The first cocrystal structure of a PAC1R antagonist peptide (18) with PAC1R extracellular domain is reported.

Mapping major SARS-CoV-2 drug targets and assessment of druggability using computational fragment screening: Identification of an allosteric small-molecule binding site on the Nsp13 helicase.

The 2019 emergence of, SARS-CoV-2 has tragically taken an immense toll on human life and far reaching impacts on society. There is a need to identify effective antivirals with diverse mechanisms of action in order to accelerate preclinical development. This study focused on five of the most established drug target proteins for direct acting small molecule antivirals: Nsp5 Main Protease, Nsp12 RNA-dependent RNA polymerase, Nsp13 Helicase, Nsp16 2’-O methyltransferase and the S2 subunit of the Spike protein. A workflow of solvent mapping and free energy calculations was used to identify and characterize favorable small-molecule binding sites for an aromatic pharmacophore (benzene). After identifying the most favorable sites, calculated ligand efficiencies were compared utilizing computational fragment screening. The most favorable sites overall were located on Nsp12 and Nsp16, whereas the most favorable sites for Nsp13 and S2 Spike had comparatively lower ligand efficiencies relative to Nsp12 and Nsp16. Utilizing fragment screening on numerous possible sites on Nsp13 helicase, we identified a favorable allosteric site on the N-terminal zinc binding domain (ZBD) that may be amenable to virtual or biophysical fragment screening efforts. Recent structural studies of the Nsp12:Nsp13 replication-transcription complex experimentally corroborates ligand binding at this site, which is revealed to be a functional Nsp8:Nsp13 protein-protein interaction site in the complex. Detailed structural analysis of Nsp13 ZBD conformations show the role of induced-fit flexibility in this ligand binding site and identify which conformational states are associated with efficient ligand binding. We hope that this map of over 200 possible small-molecule binding sites for these drug targets may be of use for ongoing discovery, design, and drug repurposing efforts. This information may be used to prioritize screening efforts or aid in the process of deciphering how a screening hit may bind to a specific target protein.

P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency.

Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7-0.9 µM) compared with wild-type (1.2-2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure-function relationship of human DAO.

In silico Molecular Docking Analysis Targeting SARS-CoV-2 Spike Protein and Selected Herbal Constituents

In modern drug discovery, molecular docking analysis is routinely used to understand and predict the interaction between a drug molecule and a target protein from a microbe. Drugs identified in this way may inhibit the entry and replication of pathogens in host cells. The SARS-CoV-2 associated coronavirus disease, COVID-19, has become the most contagious and deadly pandemic disease in the world today. In abeyance of any specific vaccine or therapeutic against SARS-CoV-2, the burgeoning situation urges a need for effective drugs to treat the virus-infected patients. Herbal medicines have been used as natural remedies for treating various infectious diseases since ancient times. The spike (S) protein of SARS-CoV-2 is important for the attachment and pathogenesis of the virus. Therefore, this study focused on the search of useful ligands for S protein among active constituents present in common herbs that could serve as efficient remedies for COVID-19. We analysed the binding efficiency of twelve compounds present in common herbs with the S protein of SARS-CoV-2 through molecular docking analysis and also results are validated with two different docking tools. The binding efficiency of ligands was scored based on their predicted pharmacological interactions coupled with binding energy estimates. In docking analysis, compound “I” (Epigallocatechin gallate (EGCG)) was found to have the highest binding affinity with the viral S protein, followed by compounds, “F” (Curcumin),“D” (Apigenin) and “E” (Chrysophanol). The present study corroborates that compound “I” (EGCG) mostly present in the integrants of green tea, shows the highest potentiality for acting as an inhibitor of SARS-CoV-2. Further, characterization of the amino acid residues comprising the viral binding site and the nature of the hydrogen bonding involved in the ligand-receptor interaction revealed significant findings with herbal compound “I” (EGCG) binding to the S protein at eight amino acid residues. The binding sites are situated near to the amino acids which are required for virus pathogenicity. The findings of the present study need in vivo experiments to prove the utility of “I”, “F”,“D” and “E” compounds and their further use in making herb-based anti-SARS-CoV-2 product in near future. This analysis may help to create a new ethno-drug formulation for preventing or curing the COVID-19.

FRET Assay for Ligands Targeting the Bacterial A-Site RNA.

A robust, fluorescence-based analysis and discovery platform is described for bacterial A-site binders. The assay relies on an incorporated isomorphic fluorescent uridine analog, which substitutes the A-site's U1406 and serves as a FRET donor to an A-site bound coumarin-labeled aminoglycoside that serves as the FRET acceptor. Binding efficiency of unlabeled A-site ligands can be determined by competition experiments, where the acceptor-labeled aminoglycoside is displaced. The replacement efficiency is gauged by the concentration-dependent loss of the sensitized FRET acceptor's signal with concomitant restoration of the donor's emission. Plotting the relative emission intensity of both the donor and acceptor as a function of ligand concentration followed by fitting of the data points to a dose-response curve yields IC50 values, one possible measure of the antibiotic potency of new A-site binders.