Ligand Binding Modes Research Articles

Structural basis for Y2 receptor-mediated neuropeptide Y and peptide YY signaling

Neuropeptide Y (NPY) and its receptors are expressed in various human tissues including the brain where they regulate appetite and emotion. Upon NPY stimulation, the neuropeptide Y1 and Y2 receptors (Y1R and Y2R, respectively) activate GI signaling, but their physiological responses to food intake are different. In addition, deletion of the two N-terminal amino acids of peptide YY (PYY(3-36)), the endogenous form found in circulation, can stimulate Y2R but not Y1R, suggesting that Y1R and Y2R may have distinct ligand-binding modes. Here, we report the cryo-electron microscopy structures of the PYY(3-36)‒Y2R‒Gi and NPY‒Y2R‒Gi complexes. Using cell-based assays, molecular dynamics simulations, and structural analysis, we revealed the molecular basis of the exclusive binding of PYY(3-36) to Y2R. Furthermore, we demonstrated that Y2R favors G protein signaling over β-arrestin signaling upon activation, whereas Y1R does not show a preference between these two pathways.

Molecular dynamics articulated multilevel virtual screening protocol to discover novel dual PPAR α/γ agonists for anti-diabetic and metabolic applications.

PPARα and PPARγ are isoforms of the nuclear receptor superfamily which regulate glucose and lipid metabolism. Activation of PPARα and PPARγ receptors by exogenous ligands could transactivate the expression of PPARα and PPARγ-dependent genes, and thereby, metabolic pathways get triggered, which are helpful to ameliorate treatment for the type 2 diabetes mellitus, and related metabolic complications. Herein, by understanding the structural requirements for ligands to activate PPARα and PPARγ proteins, we developed a multilevel in silico-based virtual screening protocol to identify novel chemical scaffolds and further design and synthesize two distinct series of glitazone derivatives with advantages over the classical PPARα and PPARγ agonists. Moreover, the synthesized compounds were biologically evaluated for PPARα and PPARγ transactivation potency from nuclear extracts of 3T3-L1 cell. Furthermore, glucose uptake assay on L6 cells confirmed the potency of the synthesized compounds toward glucose regulation. Percentage lipid-lowering potency was also assessed through triglyceride estimate from 3T3-L1 cell extracts. Results suggested the ligand binding mode was in orthosteric fashion as similar to classical agonists. Thus molecular docking and molecular dynamics (MD) simulation experiments were executed to validate our hypothesis on mode of ligands binding and protein complex stability. Altogether, the present study developed a newer protocol for virtual screening and enables to design of novel glitazones for activation of PPARα and PPARγ-mediated pathways. Accordingly, present approach will offer benefit as a therapeutic strategy against type 2 diabetes mellitus and associated metabolic complications.

The Efficacious Benefit of 25-Hydroxy Vitamin D to Prevent COVID-19: An In-Silico Study Targeting SARS-CoV-2 Spike Protein

The environment has rapidly looked at proven specialist task forces in the aftermath of the COVID-19 pandemic to build public health policies and measures to mitigate the effects of emerging coronaviruses. According to the researchers, taking 10 μg of 25-hydroxy vitamin D daily is recommended to keep us safe. There have been several studies recently indicating that there is a reduced risk of contracting Coronavirus by 25-hydroxy vitamin D consumption, even though there is no scientific data to prove that one would not affect the COVID-19 viral infection by 25-hydroxy vitamin D consumption. In this regard, the present study investigates the important literature and the role of 25-hydroxy vitamin D to prevent COVID-19 infection by conducting an in-silico study with SARS-CoV-2 spike protein as a target. Lopinavir, a previously reported drug candidate, served as a reference standard for the study. MD simulations were carried out to improve predictions of receptor-ligand complexes which offer novelty and strength to the current study. MD simulation protocols were explored and subjected to 25-hydroxy vitamin D and a known drug, Lopinavir. Comparison of ligands at refined models to the crystal structure led to promising results. Appropriate timescale simulations have been used to understand the activation mechanism, the role of water networks for receptor function, and the ligand binding process. Furthermore, MD simulations in combination with free energy calculations have also been carried out for lead optimization, evaluation of ligand binding modes, and assessment of ligand selectivity. From the results, 25-hydroxy vitamin D was discovered to have the vital interaction and highest potency in LBE, lower RMSD, and lower inhibition intensity similar to the standard. The findings from the current study suggested that 25-hydroxy vitamin D would be more effective in treating COVID-19. Compared with Lopinavir, 25-hydroxy vitamin D had the most potent interaction with the putative binding sites of the SARS-CoV-2 spike protein of COVID-19.

Molecular crowders modulate ligand binding affinity to G-quadruplex DNA by decelerating ligand association

The study of kinetics of ligand binding to G-quadruplex DNA (GqDNA) structures is of paramount importance for comprehending (possible) ligand-induced anticancer activity involving GqDNA within cells. However, cellular environment is crowded with variety of small- and macro-molecules that occupy ∼30–40 % of cell-volume. While only few earlier studies dealt with deciphering the kinetics of ligands’ interactions with GqDNA in dilute solution, such studies in cell-like crowded milieu are absent. Here we investigate the effect of small and macro molecular crowders, glucose, sucrose and ficoll 70, on the kinetic steps of association and dissociation of a benzophenoxazine-ligand (Cresyl Violet: CV) with human telometic (hybrid) GqDNA structure using fluorescence correlation spectroscopy (FCS), aided by other methods. We find that the binding constants of the ligand to GqDNA change appreciably with nearly fivefold decrease in the presence of ficoll 70, compared to that in pure buffer solution. FCS measurements unfold that this decrease of binding constants is mainly modulated by the viscosity-induced deceleration of the association of the ligand to GqDNA in the crowded solution; however, the rate-determining dissociation rates remain nearly unchanged in the presence of all three crowders. These results have important implications in the context of ligand/GqDNA interactions within cellular environment, which indicate that even if the binding affinity of a ligand to GqDNA structures may be influenced by cellular crowders, they may not influence the unbinding rate of the ligand from a stable ligand/GqDNA complex formed by strong π-π stacking interactions.

Holo Protein Conformation Generation from Apo Structures by Ligand Binding Site Refinement.

An important part in structure-based drug design is the selection of an appropriate protein structure. It has been revealed that a holo protein structure that contains a well-defined binding site is a much better choice than an apo structure in structure-based drug discovery. Therefore, it is valuable to obtain a holo-like protein conformation from apo structures in the case where no holo structure is available. Meeting the need, we present a robust approach to generate reliable holo-like structures from apo structures by ligand binding site refinement with restraints derived from holo templates with low homology. Our method was tested on a test set of 32 proteins from the DUD-E data set and compared with other approaches. It was shown that our method successfully refined the apo structures toward the corresponding holo conformations for 23 of 32 proteins, reducing the average all-heavy-atom RMSD of binding site residues by 0.48 Å. In addition, when evaluated against all the holo structures in the protein data bank, our method can improve the binding site RMSD for 14 of 19 cases that experience significant conformational changes. Furthermore, our refined structures also demonstrate their advantages over the apo structures in ligand binding mode predictions by both rigid docking and flexible docking and in virtual screening on the database of active and decoy ligands from the DUD-E. These results indicate that our method is effective in recovering holo-like conformations and will be valuable in structure-based drug discovery.

One class classification for the detection of β2 adrenergic receptor agonists using single-ligand dynamic interaction data

G protein-coupled receptors are involved in many biological processes, relaying the extracellular signal inside the cell. Signaling is regulated by the interactions between receptors and their ligands, it can be stimulated by agonists, or inhibited by antagonists or inverse agonists. The development of a new drug targeting a member of this family requires to take into account the pharmacological profile of the designed ligands in order to elicit the desired response. The structure-based virtual screening of chemical libraries may prioritize a specific class of ligands by combining docking results and ligand binding information provided by crystallographic structures. The performance of the method depends on the relevance of the structural data, in particular the conformation of the targeted site, the binding mode of the reference ligand, and the approach used to compare the interactions formed by the docked ligand with those formed by the reference ligand in the crystallographic structure. Here, we propose a new method based on the conformational dynamics of a single protein–ligand reference complex to improve the biased selection of ligands with specific pharmacological properties in a structure-based virtual screening exercise. Interactions patterns between a reference agonist and the receptor, here exemplified on the β2 adrenergic receptor, were extracted from molecular dynamics simulations of the agonist/receptor complex and encoded in graphs used to train a one-class machine learning classifier. Different conditions were tested: low to high affinity agonists, varying simulation duration, considering or ignoring hydrophobic contacts, and tuning of the classifier parametrization. The best models applied to post-process raw data from retrospective virtual screening obtained by docking of test libraries effectively filtered out irrelevant poses, discarding inactive and non-agonist ligands while identifying agonists. Taken together, our results suggest that consistency of the binding mode during the simulation is a key to the success of the method.

Chemoproteomics-Enabled Identification of 4-Oxo-β-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-β-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

Promising SARS-CoV-2 main protease inhibitor ligand-binding modes evaluated using LB-PaCS-MD/FMO

Parallel cascade selection molecular dynamics-based ligand binding-path sampling (LB-PaCS-MD) was combined with fragment molecular orbital (FMO) calculations to reveal the ligand path from an aqueous solution to the SARS-CoV-2 main protease (Mpro) active site and to customise a ligand-binding pocket suitable for delivering a potent inhibitor. Rubraxanthone exhibited mixed-inhibition antiviral activity against SARS-CoV-2 Mpro, relatively low cytotoxicity, and high cellular inhibition. However, the atomic inhibition mechanism remains ambiguous. LB-PaCS-MD/FMO is a hybrid ligand-binding evaluation method elucidating how rubraxanthone interacts with SARS-CoV-2 Mpro. In the first step, LB-PaCS-MD, which is regarded as a flexible docking, efficiently samples a set of ligand-binding pathways. After that, a reasonable docking pose of LB-PaCS-MD is evaluated by the FMO calculation to elucidate a set of protein–ligand interactions, enabling one to know the binding affinity of a specified ligand with respect to a target protein. A possible conformation was proposed for rubraxanthone binding to the SARS-CoV-2 Mpro active site, and allosteric inhibition was elucidated by combining blind docking with k-means clustering. The interaction profile, key binding residues, and considerable interaction were elucidated for rubraxanthone binding to both Mpro sites. Integrated LB-PaCS-MD/FMO provided a more reasonable complex structure for ligand binding at the SARS-CoV-2 Mpro active site, which is vital for discovering and designing antiviral drugs.

MeDBA: the Metalloenzyme Data Bank and Analysis platform.

Metalloenzymes are attractive research targets in fields of chemistry, biology, and medicine. Given that metalloenzymes can manifest conservation of metal-coordination and ligand binding modes, the excavation and expansion of metalloenzyme-specific knowledge is of interest in bridging metalloenzyme-related fields. Building on our previous metalloenzyme-ligand association database, MeLAD, we have expanded the scope of metalloenzyme-specific knowledge and services, by forming a versatile platform, termed the Metalloenzyme Data Bank and Analysis (MeDBA). The MeDBA provides: (i) manual curation of metalloenzymes into different categories, that this M-I, M-II and M-III; (ii) comprehensive information on metalloenzyme activities, expression profiles, familyand disease links; (iii) structural information on metalloenzymes, in particular metal binding modes; (iv) metalloenzyme substrates and bioactive molecules acting on metalloenzymes; (v) excavated metal-binding pharmacophoresand (vi) analysis tools for structure/metal active site comparison and metalloenzyme profiling. The MeDBA is freely available at https://medba.ddtmlab.org.

Structural comparison and finding similar ligands of target protein

There are some bacteria and protozoan that is a major public health problem. The protein which is responsible for disease as a suitable target for drug discovery for cholera, tuberculosis, kala azar and malaria. General conclusions and knowledge about ligand binding modes and protein flexibility are decisive for the drug design process, particularly in the structure-based part of it.In general, if two ligands are structurally similar, then the binding mode as defined by the relative orientation in the protein is conserved. That is, a majority of the ligand pairs occupy the same space in the binding sites. For understanding the various organisms’ protein ligand structure similarity with respect to all similar ligand of protein and comparative structural conformation study between bacteria and protozoan using structure model structure alignment and draw the ligand structure respectively by SWISS-MODEL, chimera software and chem-sketch.

A novel KPC-113 variant conferring carbapenem and ceftazidime-avibactam resistance in a multidrug-resistant Pseudomonas aeruginosa isolate

ObjectivesThis study aimed to characterize a novel KPC-113 variant from a clinical Pseudomonas aeruginosa isolate R20-14. MethodsGenomic DNA of R20-14 was subjected to Illumina and Oxford Nanopore sequencing. The horizontal transmission of plasmid was evaluated with conjugation experiments. Minimum inhibitory concentrations of bacterial strains were obtained using broth microdilution methods. KPC-113 detectability of different carbapenemase detection methods was tested. The kinetic parameters of KPC-113 were compared with those of KPC-2 by a spectrophotometer. Structure modelling and molecular docking of KPC-2 and KPC-113 were performed using Schrödinger. ResultsR20-14, a sequence type 3903 multidrug-resistant strain, was resistant to carbapenems and ceftazidime-avibactam (CZA) concurrently. S1-nuclease pulsed-field gel electrophoresis and genomic analysis revealed a blaKPC-113–carrying plasmid pR20-14, which resembled the previously reported type I KPC-encoding P. aeruginosa plasmids and exhibited a high conjugation frequency. KPC-113, with a glycine residue insertion between Ambler positions 266 and 267 in KPC-2, conferred both carbapenem and CZA resistance in DH5α and PAO1 transformants. Diagnostic tests showed that KPC-113 acted in a similar manner to KPC-2. Compared with KPC-2, KPC-113 presented reduced catalytic ability to carbapenems and ceftazidime, meanwhile responding poorly to avibactam inhibition. Modelling structure revealed that KPC-113 possibly had a more flattened binding pocket than KPC-2, leading to the change of ligand binding modes. ConclusionsKPC-113 is a novel KPC variant mediating both CZA resistance and carbapenem resistance. It is of great concern that blaKPC-113 could transfer horizontally with great efficiency and inactivate carbapenems and CZA simultaneously. Great efforts should be made to prevent its spread in clinical settings.

Structural basis for recognition of antihistamine drug by human histamine receptor

The histamine receptors belong to the G protein-coupled receptor (GPCR) superfamily, and play important roles in the regulation of histamine and other neurotransmitters in the central nervous system, as potential targets for the treatment of neurologic and psychiatric disorders. Here we report the crystal structure of human histamine receptor H3R bound to an antagonist PF-03654746 at 2.6 Å resolution. Combined with the computational and functional assays, our structure reveals binding modes of the antagonist and allosteric cholesterol. Molecular dynamic simulations and molecular docking of different antihistamines further elucidate the conserved ligand-binding modes. These findings are therefore expected to facilitate the structure-based design of novel antihistamines.

Embelin isolated from Embelia ribes derived silver nanoparticles and its application in breast cancer nanomedicine

Breast cancer is one of the most predominant cancers in female claiming millions of lives every year. Our current research work proposes that the synthesis of silver nanoparticles using phytochemical constituent Embelin from Embelia ribes fruits provides significant result as a potent anticancer agent along with its probable mechanism of action depicted by molecular docking. UV–Visible spectroscopy was used to characterise Embelin-AgNPs, and the maximum absorbance was recorded at 374.5 nm. Particle size determination indicated monodispersity of nanoparticles; particle size recorded as 25–30 nm and zeta potential analysis well explained the stability of the nanoparticles. In vitro cytotoxicity study was done by MTT assay on MCF-7 cell line. Embelin-AgNPs induced apoptosis in MCF-7 cells as measured by the Annexin-V PI apoptosis assay. To predict the binding mode of ligand and receptor and probable mechanism of action in-silico study was done against ER alpha and HER2 receptors of breast cancer cells by molecular docking and it has revealed Embelin could act as potential modulator against ER positive and HER2 positive breast cancers.

Molecular basis for the recognition of CIZ1 by ERH.

Enhancer of rudimentary homologue (ERH), a small protein conserved in eukaryotes, is involved in a wide spectrum of cellular events, including cell cycle progression, piRNA biogenesis, miRNA maturation and gene expression. Human ERH is recruited to replication foci by CDKN1A-interacting zinc finger protein 1 (CIZ1), and plays an important role in cell growth control. However, the molecular basis for CIZ1 recognition by ERH remains unknown. By using GST pull-down experiment, we found that a fragment within CIZ1, upstream of its first zinc finger, is sufficient for binding to ERH. We solved the structure of CIZ1-bound ERH, in which the ERH dimer binds to two CIZ1 fragments to form a 2 : 2 heterotetramer. CIZ1 forms intermolecular antiparallel β-strands with ERH, and its binding surface on ERH is distinct from those of other known ERH-binding ligands. The ERH-CIZ1 interface was further validated by mutagenesis and binding experiments. Our structural study complemented by biochemistry experiments not only provides insights into a previously unidentified ligand-binding mode for ERH but also sheds light on the understanding of evolutionarily conserved roles for ERH orthologs.

Probing ligand-binding modes by 'temperature-resolved' macromolecular crystallography

Probing ligand-binding modes by 'temperature-resolved' macromolecular crystallography

Protein-Based Virtual Screening Tools Applied for RNA-Ligand Docking Identify New Binders of the preQ1-Riboswitch.

Targeting RNA with small molecules is an emerging field. While several ligands for different RNA targets are reported, structure-based virtual screenings (VSs) against RNAs are still rare. Here, we elucidated the general capabilities of protein-based docking programs to reproduce native binding modes of small-molecule RNA ligands and to discriminate known binders from decoys by the scoring function. The programs were found to perform similar compared to the RNA-based docking tool rDOCK, and the challenges faced during docking, namely, protomer and tautomer selection, target dynamics, and explicit solvent, do not largely differ from challenges in conventional protein-ligand docking. A prospective VS with the Bacillus subtilis preQ1-riboswitch aptamer domain performed with FRED, HYBRID, and FlexX followed by microscale thermophoresis assays identified six active compounds out of 23 tested VS hits with potencies between 29.5 nM and 11.0 μM. The hits were selected not solely based on their docking score but for resembling key interactions of the native ligand. Therefore, this study demonstrates the general feasibility to perform structure-based VSs against RNA targets, while at the same time it highlights pitfalls and their potential solutions when executing RNA-ligand docking.

Synthesis, spectroscopic characterization, biological activities, X-ray diffraction and molecular docking studies of 2-methyl-3-(thiazol-2-ylcarbamoyl)phenylacetate

We performed a different methodology for amide bond formation, the 2-methyl-3-(thiazol-2-ylcarbamoyl)phenylacetate (MTP) compound, which was prepared from the reaction of 3-acetoxy-2-methylbenzoic anhydride with thiazol-2-amine. The MTP compound was characterized with the assistance of various spectral techniques including IR, 1H NMR, 13C NMR, XRD and elemental analysis. The MTP has been crystallized in the monoclinic space group P21/n. The ground state molecular structure (GSMS) of the optimized MTP was obtained using the DFT/B3LYP/6–31G(d,p) method. Then, intermolecular interactions for the MTP crystal were conducted by the 2D and 3D Hirshfeld analyses. Next, the DFT-optimized structure of MTP compound was used to perform molecular docking studies with the proteins of bacterial and fungal organisms in order to find the most preferred binding mode of ligand within the protein cavity. Druglikeness assay, ADME and Toxicology studies have been carried out to predict whether the MTP has an effective drug characteristics or not. The antimicrobial activity of the MTP was tested in terms of antibacterial and antifungal activities. The results revealed that this MTP showed the best activity against B. licheniformis among four bacterial species and A. flavus and C. utilis among five fungal species. These findings indicate that this and similar compounds with thiazole ring can be used as antibacterial agents in the future.

Structural basis for receptor selectivity and inverse agonism in S1P5 receptors

The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family.

Biochemical Evolution of a Potent Target of Mosquito Larvicide, 3-Hydroxykynurenine Transaminase.

A specific mosquito enzyme, 3-hydroxykynurenine transaminase (HKT), is involved in the processing of toxic metabolic intermediates of the tryptophan metabolic pathway. The HKT enzymatic product, xanthurenic acid, is required for Plasmodium spp. development in the mosquito vectors. Therefore, an inhibitor of HKT may not only be a mosquitocide but also a malaria-transmission blocker. In this work, we present a study investigating the evolution of HKT, which is a lineage-specific duplication of an alanine glyoxylate aminotransferases (AGT) in mosquitoes. Synteny analyses, together with the phylogenetic history of the AGT family, suggests that HKT and the mosquito AGTs are paralogous that were formed via a duplication event in their common ancestor. Furthermore, 41 amino acid sites with significant evidence of positive selection were identified, which could be responsible for biochemical and functional evolution and the stability of conformational stabilization. To get a deeper understanding of the evolution of ligands’ capacity and the ligand-binding mechanism of HKT, the sequence and the 3D homology model of the common ancestor of HKT and AGT in mosquitoes, ancestral mosquito AGT (AncMosqAGT), were inferred and built. The homology model along with 3-hydroxykynurenine, kynurenine, and alanine were used in docking experiments to predict the binding capacity and ligand-binding mode of the new substrates related to toxic metabolites detoxification. Our study provides evidence for the dramatic biochemical evolution of the key detoxifying enzyme and provides potential sites that could hinder the detoxification function, which may be used in mosquito larvicide and design.

Towards an Enrichment Optimization Algorithm (EOA)-based Target Specific Docking Functions for Virtual Screening.

Docking-based virtual screening (VS) is a common starting point in many drug discovery projects. While ligand-based approaches may sometimes provide better results, the advantage of docking lies in its ability to provide reliable ligand binding modes and approximated binding free energies, two factors that are important for hit selection and optimization. Most docking programs were developed to be as general as possible and consequently their performances on specific targets may be sub-optimal. With this in mind, in this work we present a method for the development of target-specific scoring functions using our recently reported Enrichment Optimization Algorithm (EOA). EOA derives QSAR models in the form of multiple linear regression (MLR) equations by optimizing an enrichment-like metric. Since EOA requires target-specific active and inactive (or decoy) compounds, we retrieved such data for six targets from the DUD-E database, and used them to re-derive the weights associated with the components that make up GOLD's ChemPLP scoring function yielding target-specific, modified functions. We then used the original ChemPLP function in small-scale VS experiments on the six targets and subsequently rescored the resulting poses with the modified functions. In addition, we used the modified functions for compounds re-docking. We found that in many although not all cases, either rescoring the original ChemPLP poses or repeating the entire docking process with the modified functions, yielded better results in terms of AUC and EF1% , two metrics, common for the evaluation of VS performances. While work on additional datasets and docking tools is clearly required, we propose that the results obtained thus far hint to the potential benefits in using EOA-based optimization for the derivation of target-specific functions in the context of virtual screening. To this end, we discuss the downsides of the methods and how it could be improved.