Identification of Novel Antagonists Targeting Cannabinoid Receptor 2 Using a Multi-Step Virtual Screening Strategy

The endocannabinoid system plays an essential role in the regulation of analgesia and human immunity, and Cannabinoid Receptor 2 (CB2) has been proved to be an ideal target for the treatment of liver diseases and some cancers. In this study, we identified CB2 antagonists using a three-step “deep learning–pharmacophore–molecular docking” virtual screening approach. From the ChemDiv database (1,178,506 compounds), 15 hits were selected and tested by radioligand binding assays and cAMP functional assays. A total of 7 out of the 15 hits were found to exhibit binding affinities in the radioligand binding assays against CB2 receptor, with a pK_i of 5.15–6.66, among which five compounds showed antagonistic activities with pIC₅₀ of 5.25–6.93 in the cAMP functional assays. Among these hits, Compound 8 with the 4H-pyrido[1,2-a]pyrimidin-4-one scaffold showed the best binding affinity and antagonistic activity with a pK_i of 6.66 and pIC₅₀ of 6.93, respectively. The new scaffold could serve as a lead for further development of CB2 drugs. Additionally, we hope that the model in this study could be further utilized to identify more novel CB2 receptor antagonists, and the developed approach could also be used to design potent ligands for other therapeutic targets.     

Novel Putative Positive Modulators of α4β2 nAChRs Potentiate Nicotine Reward-Related Behavior

The popular tobacco and e-cigarette chemical flavorant (−)-menthol acts as a nonselective, noncompetitive antagonist of nicotinic acetylcholine receptors (nAChRs), and contributes to multiple physiological effects that exacerbates nicotine addiction-related behavior. Menthol is classically known as a TRPM8 agonist; therefore, some have postulated that TRPM8 antagonists may be potential candidates for novel nicotine cessation pharmacotherapies. Here, we examine a novel class of TRPM8 antagonists for their ability to alter nicotine reward-related behavior in a mouse model of conditioned place preference. We found that these novel ligands enhanced nicotine reward-related behavior in a mouse model of conditioned place preference. To gain an understanding of the potential mechanism, we examined these ligands on mouse α4β2 nAChRs transiently transfected into neuroblastoma-2a cells. Using calcium flux assays, we determined that these ligands act as positive modulators (PMs) on α4β2 nAChRs. Due to α4β2 nAChRs’ important role in nicotine dependence, as well as various neurological disorders including Parkinson’s disease, the identification of these ligands as α4β2 nAChR PMs is an important finding, and they may serve as novel molecular tools for future nAChR-related investigations.     

Binding of Androgen- and Estrogen-Like Flavonoids to Their Cognate (Non)Nuclear Receptors: A Comparison by Computational Prediction

Flavonoids are plant bioactives that are recognized as hormone-like polyphenols because of their similarity to the endogenous sex steroids 17β-estradiol and testosterone, and to their estrogen- and androgen-like activity. Most efforts to verify flavonoid binding to nuclear receptors (NRs) and explain their action have been focused on ERα, while less attention has been paid to other nuclear and non-nuclear membrane androgen and estrogen receptors. Here, we investigate six flavonoids (apigenin, genistein, luteolin, naringenin, quercetin, and resveratrol) that are widely present in fruits and vegetables, and often used as replacement therapy in menopause. We performed comparative computational docking simulations to predict their capability of binding nuclear receptors ERα, ERβ, ERRβ, ERRγ, androgen receptor (AR), and its variant AR^T877A and membrane receptors for androgens, i.e., ZIP9, GPRC6A, OXER1, TRPM8, and estrogens, i.e., G Protein-Coupled Estrogen Receptor (GPER). In agreement with data reported in literature, our results suggest that these flavonoids show a relevant degree of complementarity with both estrogen and androgen NR binding sites, likely triggering genomic-mediated effects. It is noteworthy that reliable protein–ligand complexes and estimated interaction energies were also obtained for some suggested estrogen and androgen membrane receptors, indicating that flavonoids could also exert non-genomic actions. Further investigations are needed to clarify flavonoid multiple genomic and non-genomic effects. Caution in their administration could be necessary, until the safe assumption of these natural molecules that are largely present in food is assured.     

slate: A method for the superposition of flexible ligands

A novel program for the superposition of flexible molecules, slate, is presented. It uses simulated annealing to minimise the difference between the distance matrices calculated from the hydrogen-bonding and aromatic-ring properties of two ligands. A method for generating a molecular stack using multiple pairwise matches is illustrated. These stacks are used by the program doh to predict the relative positions of receptor atoms that could form hydrogen bonds to two or more ligands in the dataset. The methodology has been applied to ligands binding to dihydrofolate reductase, thermolysin, H₃ histamine receptors, ₂ adrenoceptors and 5-HT_1D receptors. When there are sufficient numbers and diversity of molecules in the dataset, the prediction of receptor-atom positions is applicable to compound design.     

Biophysical approaches to G protein-coupled receptors: Structure, function and dynamics

G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high-resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.     

Development and application of hybrid structure based method for efficient screening of ligands binding to G-protein coupled receptors

G-protein coupled receptors (GPCRs) comprise a large superfamily of proteins that are targets for nearly 50% of drugs in clinical use today. In the past, the use of structure-based drug design strategies to develop better drug candidates has been severely hampered due to the absence of the receptor’s three-dimensional structure. However, with recent advances in molecular modeling techniques and better computing power, atomic level details of these receptors can be derived from computationally derived molecular models. Using information from these models coupled with experimental evidence, it has become feasible to build receptor pharmacophores. In this study, we demonstrate the use of the Hybrid Structure Based (HSB) method that can be used effectively to screen and identify prospective ligands that bind to GPCRs. Essentially; this multi-step method combines ligand-based methods for building enriched libraries of small molecules and structure-based methods for screening molecules against the GPCR target. The HSB method was validated to identify retinal and its analogues from a random dataset of ∼300,000 molecules. The results from this study showed that the 9 top-ranking molecules are indeed analogues of retinal. The method was also tested to identify analogues of dopamine binding to the dopamine D2 receptor. Six of the ten top-ranking molecules are known analogues of dopamine including a prodrug, while the other thirty-four molecules are currently being tested for their activity against all dopamine receptors. The results from both these test cases have proved that the HSB method provides a realistic solution to bridge the gap between the ever-increasing demand for new drugs to treat psychiatric disorders and the lack of efficient screening methods for GPCRs. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Design of Drug Efficacy Guided by Free Energy Simulations of the β2-Adrenoceptor

G-protein-coupled receptors (GPCRs) play important roles in physiological processes and are modulated by drugs that either activate or block signaling. Rational design of the pharmacological efficacy profiles of GPCR ligands could enable the development of more efficient drugs, but is challenging even if high-resolution receptor structures are available. We performed molecular dynamics simulations of the β₂ adrenergic receptor in active and inactive conformations to assess if binding free energy calculations can predict differences in ligand efficacy for closely related compounds. Previously identified ligands were successfully classified into groups with comparable efficacy profiles based on the calculated shift in ligand affinity upon activation. A series of ligands were then predicted and synthesized, leading to the discovery of partial agonists with nanomolar potencies and novel scaffolds. Our results demonstrate that free energy simulations enable design of ligand efficacy and the same approach can be applied to other GPCR drug targets.     

Identification of functional divergence sites in dopamine receptors of vertebrates

Dopamine is one of the major neurotransmitters in the brain and body, and regulates a wide variety of functions via its binding with dopamine receptors. Abnormalities in dopamine receptors have also been found to be related to various neurological disorders. For such reason, dopamine receptors are among the key components to understanding the molecular mechanisms of many diseases, they are also the potential drug targets for the treatment of many diseases. Till now, five different dopamine receptors (D1-D5) have been identified in mammals, which are assumed to be evolved from a common ancestor after multiple gene duplication events and functional divergence. Thus, identifying the specific features of each dopamine receptor, will not only provide clues for understanding the functional differences between the receptors, but also help us to design drugs specific for a certain subtype of receptor. In this study, we investigated the functional divergence in dopamine receptors in representative vertebrate species by analyzing their molecular evolution features. Our results showed that the coefficients for type I functional divergence (θ_I) were significantly greater than 0 for all the pairwise comparisons between the five dopamine receptors, suggesting that type I functional divergence, i.e., altered functional constraints or different evolutionary rates, may have taken place at some amino acids in the receptors. We further identified 84 potential type I functional divergence peptide sites for the pairwise comparisons between the D1-like and D2-like are identified in total. When these sites were mapped to the 3D structure of dopamine receptors, most of them were included in ICL3, M6 and M7 domains. Especially, sixteen of these sites may be the major sites associated with the changes of properties between D1-like and D2-like receptors. These sites provide molecular basis for further studies such as dopamine receptor function exploration and subtype specific drug design and screening.     

Chemoproteomic Approach to Explore the Target Profile of GPCR ligands: Application to 5‐HT1A and 5‐HT6 Receptors

Determination of the targets of a compound remains an essential aspect in drug discovery. A complete understanding of all binding interactions is critical to recognize in advance both therapeutic effects and undesired consequences. However, the complete polypharmacology of many drugs currently in clinical development is still unknown, especially in the case of G protein‐coupled receptor (GPCR) ligands. In this work we have developed a chemoproteomic platform based on the use of chemical probes to explore the target profile of a compound in biological systems. As proof of concept, this methodology has been applied to selected ligands of the therapeutically relevant serotonin 5‐HT_1A and 5‐HT₆ receptors, and we have identified and validated some of their off‐targets. This approach could be extended to other drugs of interest to study the targeted proteome in disease‐relevant systems.     

In silico analysis of the pyretic effect of drugs on antimalarial receptors

Malarial infection due to P. falciparum is prominent cause in worldwide fatality. PfCRT, PfDHPS, PfMDR1 and PfDHFR1 play vital role as targets in malarial infection. We chose PfCRT-specific ligands for our study and tested them against all P. falciparum targets. The study was carried out by performing MDS and MD analyses with the NAMD and AutoDock softwares, respectively. The study's goal is to find potential ligands that can act on all malarial targets at various temperatures.The MDS studies revealed structural conformational changes and protein stability from normal human body temperature, 98.6 ​°F, to maximum human body temperature, 107 ​°F. This MDS analysis of Plasmodium targets was carried out by creating a graph of RMSD vs time. Further MD analysis revealed that the ligands reported against PfCRT also had a high binding energy against PfDHPS and PfDHFR. As a result, those ligands can also be targeted for Plasmodium falciparum proteins other than the three mentioned above. These ligands can be subjected to SAR and QSAR studies in order to develop novel molecules for malaria treatment.     

Unsupervised guided docking of covalently bound ligands

Summary An approach for docking covalently bound ligands in protein enzymes or receptors was implemented in MacDOCK, a similarity-driven docking program based on DOCK 4.0. This approach was tested with a small number of covalent ligand–protein structures, using both native and non-native protein structures. In all cases, MacDOCK was able to generate orientations consistent with the known covalent binding mode of these complexes, with a performance similar to that of other docking programs. This method was also applied to search for known covalent thrombin inhibitors in a medium-sized molecular database (ca. 11,000 compounds). Detection of functional groups suitable for covalent docking was carried out automatically. A significant enrichment in known active molecules in the first 5% of the database was obtained, showing that MacDOCK can be used efficiently for the virtual screening of covalently bound ligands.     

Quantitative structure-affinity relationship of 5-HT1A receptor ligands by the classification tree method

The influence of molecular structure of 346 ligands on their affinity for 5-HT_1A receptors was investigated. It was shown that the effectiveness of the proposed novel approach for interpretation of decision tree models compared favourably with the PLS method. In the context of the proposed approach, molecular fragments and their values of the relative influence on the affinity for 5-HT_1A receptors were defined.     

Delineation of agonist binding to the human histamine H4 receptor using mutational analysis, homology modeling, and ab initio calculations

A three-dimensional homology model of the human histamine H 4 receptor was developed to investigate the binding mode of a series of structurally diverse H 4-agonists, i.e. histamine, clozapine, and the recently described selective, nonimidazole agonist VUF 8430. Mutagenesis studies and docking of these ligands in a rhodopsin-based homology model revealed two essential points of interactions in the binding pocket, i.e. Asp3.32 and Glu5.46 (Ballesteros-Weinstein numbering system). It is postulated that Asp3.32 interacts in its anionic state, whereas Glu5.46 interacts in its neutral form. The hypothesis was tested with the point mutations D3.32N and E5.46Q. For the D3.32N no binding affinity toward any of the ligands could be detected. This is in sharp contrast to the E5.46Q mutant, which discriminates between various ligands. The affinity of histamine-like ligands was decreased approximately a 1000-fold, whereas the affinity of all other ligands remained virtually unchanged. The proposed model for agonist binding as well as ab initio calculations for histamine and VUF 8430 explain the observed differences in binding to the H 4R mutants. These studies provide a molecular understanding for the action of a variety of H 4 receptor-ligands. The resulting H 4 receptor model will be the basis for the development of new H 4 receptor-ligands.     

Towards a rational spacer design for bivalent inhibition of estrogen receptor

Estrogen receptors are known drug targets that have been linked to several kinds of cancer. The structure of the estrogen receptor ligand binding domain is available and reveals a homodimeric layout. In order to improve the binding affinity of known estrogen receptor inhibitors, bivalent compounds have been developed that consist of two individual ligands linked by flexible tethers serving as spacers. So far, binding affinities of the bivalent compounds do not surpass their monovalent counterparts. In this article, we focus our attention on the molecular spacers that are used to connect the individual ligands to form bivalent compounds, and describe their thermodynamic contribution during the ligand binding process. We use computational methods to predict structural and entropic parameters of different spacer structures. We find that flexible spacers introduce a number of effects that may interfere with ligand binding and possibly can be connected to the low binding affinities that have been reported in binding assays. Based on these findings, we try to provide guidelines for the design of novel molecular spacers.     

Structural Insights into Ligand—Receptor Interactions Involved in Biased Agonism of G-Protein Coupled Receptors

G protein-coupled receptors (GPCRs) are versatile signaling proteins that mediate complex cellular responses to hormones and neurotransmitters. Ligand directed signaling is observed when agonists, upon binding to the same receptor, trigger significantly different configuration of intracellular events. The current work reviews the structurally defined ligand – receptor interactions that can be related to specific molecular mechanisms of ligand directed signaling across different receptors belonging to class A of GPCRs. Recent advances in GPCR structural biology allow for mapping receptors’ binding sites with residues particularly important in recognition of ligands’ structural features that are responsible for biased signaling. Various studies show particular role of specific residues lining the extended ligand binding domains, biased agonists may alternatively affect their interhelical interactions and flexibility what can be translated into intracellular loop rearrangements. Studies on opioid and angiotensin receptors indicate importance of residues located deeper within the binding cavity and direct interactions with receptor residues linking the ortosteric ligand binding site with the intracellular transducer binding domain. Collection of results across different receptors may suggest elements of common molecular mechanisms which are responsible for passing alternative signals from biased agonists.     

Metal-assembled anion receptors

This review describes the self-assembly of anion receptors from organic ligands and transition metal ions. These metal-assembled anion receptors can be synthesised from a number of different species; bidentate ligands with metals that prefer octahedral coordination geometries and monodentate ligands with metals that prefer square planar geometries are common. Anion binding transition metal helicates and systems where the coordination of metal ions results in the formation of an anion receptor by conformational locking are also reported. The effect of anion binding on the different properties of these complexes is discussed.