Discovery Of Novel Antagonists Research Articles

Discovery of novel antagonists targeting the DNA binding domain of androgen receptor by integrated docking-based virtual screening and bioassays.

Androgen receptor (AR), a ligand-activated transcription factor, is a master regulator in the development and progress of prostate cancer (PCa). A major challenge for the clinically used AR antagonists is the rapid emergence of resistance induced by the mutations at AR ligand binding domain (LBD), and therefore the discovery of novel anti-AR therapeutics that can combat mutation-induced resistance is quite demanding. Therein, blocking the interaction between AR and DNA represents an innovative strategy. However, the hits confirmed targeting on it so far are all structurally based on a sole chemical scaffold. In this study, an integrated docking-based virtual screening (VS) strategy based on the crystal structure of the DNA binding domain (DBD) of AR was conducted to search for novel AR antagonists with new scaffolds and 2-(2-butyl-1,3-dioxoisoindoline-5-carboxamido)-4,5-dimethoxybenzoicacid (Cpd39) was identified as a potential hit, which was competent to block the binding of AR DBD to DNA and showed decent potency against AR transcriptional activity. Furthermore, Cpd39 was safe and capable of effectively inhibiting the proliferation of PCa cell lines (i.e., LNCaP, PC3, DU145, and 22RV1) and reducing the expression of the genes regulated by not only the full-length AR but also the splice variant AR-V7. The novel AR DBD-ARE blocker Cpd39 could serve as a starting point for the development of new therapeutics for castration-resistant PCa.

The Schistosoma mansoni tegumental allergen protein, SmTAL1: Binding to an IQ-motif from a voltage-gated ion channel and effects of praziquantel

SmTAL1 is a calcium binding protein from the parasitic worm, Schistosoma mansoni. Structurally it is comprised of two domains – an N-terminal EF-hand domain and a C-terminal dynein light chain (DLC)-like domain. The protein has previously been shown to interact with the anti-schistosomal drug, praziquantel (PZQ). Here, we demonstrated that both EF-hands in the N-terminal domain are functional calcium ion binding sites. The second EF-hand appears to be more important in dictating affinity and mediating the conformational changes which occur on calcium ion binding. There is positive cooperativity between the four calcium ion binding sites in the dimeric form of SmTAL1. Both the EF-hand domain and the DLC-domain dimerise independently suggesting that both play a role in forming the SmTAL1 dimer. SmTAL1 binds non-cooperatively to PZQ and cooperatively to an IQ-motif from SmCav1B, a voltage-gated calcium channel. PZQ tends to strengthen this interaction, although the relationship is complex. These data suggest the hypothesis that SmTAL1 regulates at least one voltage-gated calcium channel and PZQ interferes with this process. This may be important in the molecular mechanism of this drug. It also suggests that compounds which bind SmTAL1, such as six from the Medicines for Malaria Box identified in this work, may represent possible leads for the discovery of novel antagonists.

Structure based virtual screening of novel noncompetitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor

The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype ionotropic glutamate receptors are attractive antiepileptic targets responsible for mediating the majority of excitatory neurotransmission and plasticity. The noncompetitive antagonists obtain more and more attention as drug candidates for treatment of the neurological diseases involving excessive activity of AMPARs, due to they regulate AMPA receptors (AMPARs) activity independently of endogenous glutamate levels unlike the competitive antagonists. Development of novel AMPAR noncompetitive antagonists, which are safer and more efficacious than competitive antagonists, is highly under demand. Here, we present the discovery of novel antagonists against AMPAR through Structure-Based Virtual Screening (SBVS). Three compounds were successfully distinguished by several different filtering strategies, namely STOCK6S-10902, STOCK1N-49134 and STOCK5S-68665. The interaction mode of these compounds was further explored through molecular dynamics simulation, binding free energy calculation and the binding free energy decomposition. It is demonstrated that some residues within the binding pocket, which have been proved their importance in antagonist binding and gating, form strong hydrogen bond interactions with these three molecules. In particular, H-bond interactions with high occupancies between Ser516, Ser788 and STOCK6S-10902 and Ser516, Asn791 and STOCK1N-49134 were observed. The three hit compounds with new scaffolds and the detailed binding modes could potentially serve as a starting point for further optimization and development.

Discovery of novel antagonists on β2-adrenoceptor from natural products using a label-free cell phenotypic assay.

Label-free cell phenotypic assays were performed to establish a β2-adrenoceptor (β2-AR) target model in A431 cells and a β1-AR target model in transfected HEK293-β1 cells, using known β2-AR and β1-AR agonists and antagonists. A list of natural compounds was screened on the target models, among which seven new compounds were found to be antagonistically active against β2-AR. After receptor specificity evaluations on hydroxyl carboxylic acid receptor-2 (ΗΧΑ-2), histamine receptor (H1R), and β1-adrenoceptor (β1-AR), six out of the seven compounds, including nuciferine, epiberberine, harmaline, harmine, palmatine, and columbamine, exhibited specific antagonistic activity against β2-AR. Epiberberine and palmatine showed the strongest antagonistic activities against β2-AR with IC50 values of 2.3 ± 0.2μM and 2.6 ± 0.3μM, respectively. Docking palmatine to the crystal structure of human β2-AR (PDB 5X7D) suggested that the ligand forms a hydrogen bond with N312 and hydrophobic interaction with several amino acid residues in the binding pocket, such as D113 and V114. The kinetic binding profile of palmatine was further investigated using co-stimulation assays. Results suggested that palmatine was a competitive antagonist for β2-AR. The six novel β2-AR antagonists provide a promising chemical starting point for identification and optimization of drugs used for treating hypertension, glaucoma, and infantile hemangiomas. This study also lays the foundation for the in-depth investigation of biochemical mechanisms and pharmacological properties of natural compounds.

E-Pharmacophore Model Assisted Discovery of Novel Antagonists of nNOS

The nitric oxide (NO) synthesized by neuronal nitric oxide synthase (nNOS) acts as a neurotransmitter and plays a crucial role in a series of neurobiological functions. In diseased condition, activated nNOS induces nitrosylation as well as phosphorylation of tau protein and glycogen synthase kinase 3 beta (GSK-3β) respectively. Hyper phosphorylation of tau accelerates tau oligomerization resulting in formation of neurofibrillary tangles (NFT), ensuring the neuronal cell death in hippocampus region; a hallmark of Alzheimer’s disease (AD). Thus, designing inhibitor towards nNOS may reduce the neuronal loss caused by nNOS. Hence nNOS has been one of the revitalizing targets for AD. In the present work, one energetically optimized structure-based pharmacophore (e-pharmacophore) was generated using nNOS co-crystal structure (4D1N) to map important pharmacophoric features of nNOS. Shape based similarity screening performed using e-pharmacophore against in-house library of more than one million compounds resulted 2701 library of compounds. Rigid receptor docking (RRD) was applied and followed by molecular mechanics and generalized Born and surface area (MM-GBSA) calculation which results 22 nNOS ligands. To define the leads, dock complexes were subjected to quantum-polarized ligand docking (QPLD) followed by free energy calculations revealed 3 leads. On comparison with 1 existing inhibitor,it concealed three best leads with lower binding energy and better binding affinity. The best lead was subjected to induced fit docking (IFD) with MM-GBSA calculation and further molecular dynamics (MD) simulations for 50 ns in solvated model system. Potential energy, root mean square deviation (RMSD) and root mean square fluctuations (RMSF) results disclosed constancy of lead 1 interactions throughout 50 ns MD simulations run. Thus proposed three leads are having favorable absorption distribution metabolism excretion toxicity (ADME/T) properties and provide a scaffold for designing nNOS antagonists.

Discovery of novel antagonists of human neurotensin receptor 1 on the basis of ligand and protein structure

Neurotensin receptor 1 (NTR1) is a cell surface receptor belonging to the G protein-coupled receptor (GPCR) A superfamily. NTR1 plays an important role in neuronal and non-neuronal systems. Using the previously identified crystal structure of rat NTR1 (rNTR1), we screened for potential candidates of human NTR1 (hNTR1) ligand. Approximately 10,000 compounds were selected using the docking score, followed by pharmacophore-based virtual screening and a two-dimensional (2D)-fingerprint structural similarity search. The identified molecules were tested by in vitro calcium flux assay. Four compounds showed micromolar level affinity, of which, one compound can inhibit hNTR1/CHO cells' proliferation by cell viability assays. To improve the affinity of these positive hit compounds, a homology model of hNTR1 was built on the basis of the crystal structure of rNTR1. The hit compounds will be further optimized on the basis of the structure of the hNTR1 receptor to be the targets for drugs directed against diseases associated with hNTR1. The results demonstrate that the method we used is valid, which will be treated as a useful tool to search for the agonists or antagonists of our interested target protein. Moreover, the compound we tested may provide a hopeful clue for treating the diseases related with hNTR1.

Identification of potent and selective retinoic acid receptor gamma (RARγ) antagonists for the treatment of osteoarthritis pain using structure based drug design

A series of triaryl pyrazoles were identified as potent pan antagonists for the retinoic acid receptors (RARs) α, β and γ. X-ray crystallography and structure-based drug design were used to improve selectivity for RARγ by targeting residue differences in the ligand binding pockets of these receptors. This resulted in the discovery of novel antagonists which maintained RARγ potency but were greater than 500-fold selective versus RARα and RARβ. The potent and selective RARγ antagonist LY2955303 demonstrated good pharmacokinetic properties and was efficacious in the MIA model of osteoarthritis-like joint pain. This compound demonstrated an improved margin to RARα-mediated adverse effects.

Discovery of novel antagonists of glycoprotein IIb/IIIa-mediated platelet aggregation through virtual screening.

The glycoprotein IIb/IIIa receptor is the final common pathway of platelet aggregation, regardless of the agonist, and thus represents an ideal therapeutic target for blocking thrombus formation. RUC-2 is a novel glycoprotein IIb/IIIa inhibitor of adenosine-5'-diphosphate (ADP)-induced platelet aggregation, importantly which exhibits a unique mode of binding with respect to classical Arg-Gly-Asp (RGD)-based glycoprotein IIb/IIIa antagonists. To identify new chemotypes that inhibit glycoprotein IIb/IIIa-mediated platelet aggregation like RUC-2, we performed a combination of structure-based pharmacophore screening and structure-based virtual screening approach to screen over 7.3 million small molecules based on the RUC-2-glycoprotein IIb/IIIa crystal structure. Three of the eleven hit compounds identified by virtual screening showed promising activity with IC50 values between 16.9 and 90.6μmolL(-1) in a human platelet aggregation assay induced by ADP and thrombin. The binding conformations of these three were analyzed to provide a rationalization of their activity profile. These compounds may serve as potential novel scaffolds for further development of glycoprotein IIb/IIIa antagonists.

Exploring Distinct Binding Site Regions of ß2 Adrenergic Receptor via Coarse-Grained Molecular Dynamics Simulations

\beta_2-Adrenergic receptor (\beta_2AR) is a G protein- coupled receptor that is highly flexible and able to recognize a wide range of ligands through its conformational variations. Active and inactive conformations revealed by recent crystallographic experiments do not provide a complete dynamic picture of the receptor, especially in the binding site. In this study, molecular dynamics (MD) simulation through a residue-based coarse-grained model is used as an alternative and efficient method to explore a wider conformational search space. The system was composed of \beta_2AR embedded into a 1-palmitoyl-2-oleoyl-phosphatidylcholine membrane bilayer with surrounding water. A total of 6 \mu s of simulation at constant NPT was performed for a system of 6868 coarse-grained beads. The system reached equilibrium at around 0.1 \mu s. The overall 3-dimensional structure was well preserved throughout the simulation. Local residue-based fluctuations were in good agreement with fully atomistic MD simulations. Four distinct snapshots were selected and reverse-mapped to all-atom representations with around 65,000 atoms. Each reverse-mapped system was later subjected to 100 ns of MD simulation for equilibration. Root mean square deviation clustering analysis yielded distinct receptor conformers for the binding site regions, which were suggested to be alternative representations of the binding pocket and thus were proposed as plausible targets in docking-based virtual screening experiments for the discovery of novel antagonists.

Direct selection of EGF mutants displayed on filamentous phage using cells overexpressing EGF receptor.

Understanding receptor-ligand interactions, and the signal transduction pathways they activate, is of great interest for the discovery of novel antagonists and agonists. In this report we describe a rapid and efficient procedure to evaluate the importance of several different epidermal growth factor (EGF) residues for the binding and activation of its receptor (EGFR). We constructed an EGF mutant library randomized at positions 13, 15 and 16 and expressed them on filamentous phages. Phage display is a powerful system, allowing rapid isolation of binding mutants. Since many of the most pharmacologically interesting receptors cannot be produced in a soluble form, we developed a technique to rapidly select receptor-binding molecules directly on cells. A luciferase assay, simple to perform, was then used to test their biological transduction activity and to rapidly detect mutants of interest. Analysis of the resulting sequences revealed that the wild-type amino acids at positions 13, 15 and 16 are optimized for binding and activity. EGF mutants with agonist properties were also isolated and tolerated substitutions were identified.