Selection of in silico drug screening results for G-protein-coupled receptors by using universal active probes

We developed a new protocol for in silico drug screening for G-protein-coupled receptors (GPCRs) using a set of "universal active probes" (UAPs) with an ensemble docking procedure. UAPs are drug-like compounds, which are actual active compounds of a variety of known proteins. The current targets were nine human GPCRs whose three-dimensional (3D) structures are unknown, plus three GPCRs, namely β(2)-adrenergic receptor (ADRB2), A(2A) adenosine receptor (A(2A)), and dopamine D3 receptor (D(3)), whose 3D structures are known. Homology-based models of the GPCRs were constructed based on the crystal structures with careful sequence inspection. After subsequent molecular dynamics (MD) simulation taking into account the explicit lipid membrane molecules with periodic boundary conditions, we obtained multiple model structures of the GPCRs. For each target structure, docking-screening calculations were carried out via the ensemble docking procedure, using both true active compounds of the target proteins and the UAPs with the multiple target screening (MTS) method. Consequently, the multiple model structures showed various screening results with both poor and high hit ratios, the latter of which could be identified as promising for use in in silico screening to find candidate compounds to interact with the proteins. We found that the hit ratio of true active compounds showed a positive correlation to that of the UAPs. Thus, we could retrieve appropriate target structures from the GPCR models by applying the UAPs, even if no active compound is known for the GPCRs. Namely, the screening result that showed a high hit ratio for the UAPs could be used to identify actual hit compounds for the target GPCRs.     

Snooker: a structure-based pharmacophore generation tool applied to class A GPCRs

G-protein coupled receptors (GPCRs) are important drug targets for various diseases and of major interest to pharmaceutical companies. The function of individual members of this protein family can be modulated by the binding of small molecules at the extracellular side of the structurally conserved transmembrane (TM) domain. Here, we present Snooker, a structure-based approach to generate pharmacophore hypotheses for compounds binding to this extracellular side of the TM domain. Snooker does not require knowledge of ligands, is therefore suitable for apo-proteins, and can be applied to all receptors of the GPCR protein family. The method comprises the construction of a homology model of the TM domains and prioritization of residues on the probability of being ligand binding. Subsequently, protein properties are converted to ligand space, and pharmacophore features are generated at positions where protein ligand interactions are likely. Using this semiautomated knowledge-driven bioinformatics approach we have created pharmacophore hypotheses for 15 different GPCRs from several different subfamilies. For the beta-2-adrenergic receptor we show that ligand poses predicted by Snooker pharmacophore hypotheses reproduce literature supported binding modes for ～75% of compounds fulfilling pharmacophore constraints. All 15 pharmacophore hypotheses represent interactions with essential residues for ligand binding as observed in mutagenesis experiments and compound selections based on these hypotheses are shown to be target specific. For 8 out of 15 targets enrichment factors above 10-fold are observed in the top 0.5% ranked compounds in a virtual screen. Additionally, prospectively predicted ligand binding poses in the human dopamine D3 receptor based on Snooker pharmacophores were ranked among the best models in the community wide GPCR dock 2010.     

New strategies in drug discovery for GPCRs: high throughput detection of cellular ERK phosphorylation

G-protein coupled receptors (GPCRs) are a large family of receptors for a wide range of stimulants, including hormones, neurotransmitters, and taste and olfactory chemicals. Due to their broad involvement in cellular responses, GPCRs affect many important body functions both in health and disease. Compared to other receptor families, the GPCRs have been a rich source of extracellularly-acting pharmaceuticals, due largely to the fact that many GPCR ligands are small molecules when compared with ligands for other receptors, such as the tyrosine kinase receptor family. This has allowed the development of small molecule modulators of receptor function that act on specific GPCRs, such as those involved in cardiovascular regulation. However, at several levels, current screening technologies of drug development for GPCRs are lacking. Firstly, responses from many GPCRs, such as the Gi-coupled GPCRs, are not easily measured in large screening programs by current techniques. Secondly, there are few options for detecting agonists of orphan GPCRs. Thirdly, it is now clear that the signaling from GPCRs is more complex than once thought, and the measurement of Ca(2+) and cAMP can account for only a fraction of the biological information emanating from an activated GPCR. Studies of the discrete and sometimes separable activation of the Ras/Raf/Mek/ERK cascade by many GPCRs is likely to offer development of new agonists and antagonists, contribute to new pharmacologies from receptors, and raise the potential for novel drug candidates in this important area of biology. Downstream activation of the ERK pathway, with or without transactivation of growth factor receptors, has not been measurable by high throughput methodologies. This article presents recent advances and associated applications for screening of GPCRs and other receptor species through the rapid measurement of protein phosphorylation events, such as ERK phosphorylation, as new readouts for drug discovery.     

G protein betagamma subunits as targets for small molecule therapeutic development

G proteins mediate the action of G protein coupled receptors (GPCRs), a major target of current pharmaceuticals and a major target of interest in future drug development. Most pharmaceutical interest has been in the development of selective GPCR agonists and antagonists that activate or inhibit specific GPCRs. Some recent thinking has focused on the idea that some pathologies are the result of the actions of an array of GPCRs suggesting that targeting single receptors may have limited efficacy. Thus, targeting pathways common to multiple GPCRs that control critical pathways involved in disease has potential therapeutic relevance. G protein betagamma subunits released from some GPCRs upon receptor activation regulate a variety of downstream pathways to control various aspects of mammalian physiology. There is evidence from cell- based and animal models that excess Gbetagamma signaling can be detrimental and blocking Gbetagamma signaling has salutary effects in a number of pathological models. Gbetagamma regulates downstream pathways through modulation of enzymes that produce cellular second messengers or through regulation of ion channels by direct protein-protein interactions. Thus, blocking Gbetagamma functions requires development of small molecule agents that disrupt Gbetagamma protein interactions with downstream partners. Here we discuss evidence that small molecule targeting Gbetagamma could be of therapeutic value. The concept of disruption of protein-protein interactions by targeting a "hot spot" on Gbetagamma is delineated and the biochemical and virtual screening strategies for identification of small molecules that selectively target Gbetagamma functions are outlined. Evaluation of the effectiveness of virtual screening indicates that computational screening enhanced identification of true Gbetagamma binding molecules. However, further refinement of the approach could significantly improve the yield of Gbetagamma binding molecules from this screen that could result in multiple candidate leads for future drug development.     

Latest development in drug discovery on G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the family of proteins with the highest impact from social, therapeutic and economic point of view. Today, more than 50% of drug targets are based on GPCRs and the annual worldwide sales exceeds 50 billion dollars. GPCRs are involved in all major disease areas such as cardiovascular, metabolic, neurodegenerative, psychiatric, cancer and infectious diseases. The classical drug discovery process has relied on screening compounds, which interact favorably with the GPCR of interest followed by further chemical engineering as a mean of improving efficacy and selectivity. In this review, methods for sophisticated chemical library screening procedures will be presented. Furthermore, development of cell-based assays for functional coupling of GPCRs to G proteins will be discussed. Finally, the possibility of applying structure-based drug design will be summarized. This includes the application of bioinformatics knowledge and molecular modeling approaches in drug development programs. The major efforts established through large networks of structural genomics on GPCRs, where recombinantly expressed GPCRs are subjected to purification and crystallization attempts with the intention of obtaining high-resolution structures, are presented as a promising future approach for tailor-made drug development.     

PyPLIF HIPPOS-Assisted Prediction of Molecular Determinants of Ligand Binding to Receptors

Identification of molecular determinants of receptor-ligand binding could significantly increase the quality of structure-based virtual screening protocols. In turn, drug design process, especially the fragment-based approaches, could benefit from the knowledge. Retrospective virtual screening campaigns by employing AutoDock Vina followed by protein-ligand interaction fingerprinting (PLIF) identification by using recently published PyPLIF HIPPOS were the main techniques used here. The ligands and decoys datasets from the enhanced version of the database of useful decoys (DUDE) targeting human G protein-coupled receptors (GPCRs) were employed in this research since the mutation data are available and could be used to retrospectively verify the prediction. The results show that the method presented in this article could pinpoint some retrospectively verified molecular determinants. The method is therefore suggested to be employed as a routine in drug design and discovery.     

Novel approach to structure-based pharmacophore search using computational geometry and shape matching techniques

Computationally efficient structure-based virtual screening methods have recently been reported that seek to find effective means to utilize experimental structure information without employing detailed molecular docking calculations. These tools can be coupled with efficient experimental screening technologies to improve the probability of identifying hits and leads for drug discovery research. Commercial software ROCS (rapid overlay of chemical structures) from Open Eye Scientific is such an example, which is a shape-based virtual screening method using the 3D structure of a ligand, typically from a bound X-ray costructure, as the query. We report here the development of a new structure-based pharmacophore search method (called Shape4) for virtual screening. This method adopts a variant of the ROCS shape technology and expands its use to work with an empty crystal structure. It employs a rigorous computational geometry method and a deterministic geometric casting algorithm to derive the negative image (i.e., pseudoligand) of a target binding site. Once the negative image (or pseudoligand) is generated, an efficient shape comparison algorithm in the commercial OE SHAPE Toolkit is adopted to compare and match small organic molecules with the shape of the pseudoligand. We report the detailed computational protocol and its computational validation using known biologically active compounds extracted from the WOMBAT database. Models derived for five selected targets were used to perform the virtual screening experiments to obtain the enrichment data for various virtual screening methods. It was found that our approach afforded similar or better enrichment ratios than other related methods, often with better diversity among the top ranking computational hits.     

Ligand-steered modeling and docking: A benchmarking study in class A G-protein-coupled receptors

Class A G-protein-coupled receptors (GPCRs) are among the most important targets for drug discovery. However, a large set of experimental structures, essential for a structure-based approach, will likely remain unavailable in the near future. Thus, there is an actual need for modeling tools to characterize satisfactorily at least the binding site of these receptors. Using experimentally solved GPCRs, we have enhanced and validated the ligand-steered homology method through cross-modeling and investigated the performance of the thus generated models in docking-based screening. The ligand-steered modeling method uses information about existing ligands to optimize the binding site by accounting for protein flexibility. We found that our method is able to generate quality models of GPCRs by using one structural template. These models perform better than templates, crude homology models, and random selection in small-scale high-throughput docking. Better quality models typically exhibit higher enrichment in docking exercises. Moreover, they were found to be reliable for selectivity prediction. Our results support the fact that the ligand-steered homology modeling method can successfully characterize pharmacologically relevant sites through a full flexible ligand-flexible receptor procedure.     

Virtual screen for ligands of orphan G protein-coupled receptors

This paper describes a virtual screening methodology that generates a ranked list of high-binding small molecule ligands for orphan G protein-coupled receptors (oGPCRs), circumventing the requirement for receptor three-dimensional structure determination. Features representing the receptor are based only on physicochemical properties of primary amino acid sequence, and ligand features use the two-dimensional atomic connection topology and atomic properties. An experimental screen comprised nearly 2 million hypothetical oGPCR-ligand complexes, from which it was observed that the top 1.96% predicted affinity scores corresponded to "highly active" ligands against orphan receptors. Results representing predicted high-scoring novel ligands for many oGPCRs are presented here. Validation of the method was carried out in several ways: (1) A random permutation of the structure-activity relationship of the training data was carried out; by comparing test statistic values of the randomized and nonshuffled data, we conclude that the value obtained with nonshuffled data is unlikely to have been encountered by chance. (2) Biological activities linked to the compounds with high cross-target binding affinity were analyzed using computed log-odds from a structure-based program. This information was correlated with literature citations where GPCR-related pathways or processes were linked to the bioactivity in question. (3) Anecdotal, out-of-sample predictions for nicotinic targets and known ligands were performed, with good accuracy in the low-to-high "active" binding range. (4) An out-of-sample consistency check using the commercial antipsychotic drug olanzapine produced "active" to "highly-active" predicted affinities for all oGPCRs in our study, an observation that is consistent with documented findings of cross-target affinity of this compound for many different GPCRs. It is suggested that this virtual screening approach may be used in support of the functional characterization of oGPCRs by identifying potential cognate ligands. Ultimately, this approach may have implications for pharmaceutical therapies to modulate the activity of faulty or disease-related cellular signaling pathways. In addition to application to cell surface receptors, this approach is a generalized strategy for discovery of small molecules that may bind intracellular enzymes and involve protein-protein interactions.     

Engineering of Challenging G Protein-Coupled Receptors for Structure Determination and Biophysical Studies

Membrane proteins such as G protein-coupled receptors (GPCRs) exert fundamental biological functions and are involved in a multitude of physiological responses, making these receptors ideal drug targets. Drug discovery programs targeting GPCRs have been greatly facilitated by the emergence of high-resolution structures and the resulting opportunities to identify new chemical entities through structure-based drug design. To enable the determination of high-resolution structures of GPCRs, most receptors have to be engineered to overcome intrinsic hurdles such as their poor stability and low expression levels. In recent years, multiple engineering approaches have been developed to specifically address the technical difficulties of working with GPCRs, which are now beginning to make more challenging receptors accessible to detailed studies. Importantly, successfully engineered GPCRs are not only valuable in X-ray crystallography, but further enable biophysical studies with nuclear magnetic resonance spectroscopy, surface plasmon resonance, native mass spectrometry, and fluorescence anisotropy measurements, all of which are important for the detailed mechanistic understanding, which is the prerequisite for successful drug design. Here, we summarize engineering strategies based on directed evolution to reduce workload and enable biophysical experiments of particularly challenging GPCRs.     

High throughput screening of G-protein coupled receptors via flow cytometry

The molecular assemblies of signal transduction components, for example kinases and their target proteins or receptor-ligand complexes and intracellular signaling molecules, are critical for biological functions in cells. To better understand the interactions of these molecular assemblies and to screen for new pharmaceutics that could control and modulate these types of interactions, we have focused on developing high throughput approaches for the analysis of G-protein coupled receptors via flow cytometry. Flow cytometry offers a number of advantages including real-time collection of multicomponent data, and together with improvements in sample handling, the high throughput sampling rate is up to 100 samples per minute. For our targets, assemblies of solubilized GPCRs, a screening platform of a dextran bead has proven to be flexible, allowing different surface chemistries on the beads. The bead can be either ligand-labeled or have epitope-linked proteins attached to the bead surface, enabling several molecular assemblies to be constructed and analyzed. A major improvement with this system is that for screening ligands for GPCRs the underlying mechanism of action for these compounds can be investigated and incorporated into the definition of a 'hit'. Our current screening system is capable of simultaneously distinguishing GPCR agonists and antagonists.     

Structural genomics on membrane proteins: mini review

Structural genomics, structure-based analysis of gene products, has so far mainly concentrated on soluble proteins because of their less demanding requirements for overexpression, purification and crystallisation compared to membrane proteins. This so-called "low-hanging fruit" approach has generated more than 25,000 structures deposited in databases. In contrast, the substantially more complex membrane proteins, in relation to all steps from overexpression to high-resolution structure determination, represent less than 1% of available crystal structures. This is in sharp contrast to the importance of this type of proteins, particularly G protein-coupled receptors (GPCRs), as today 60-70% of the current drug targets are based on membrane proteins. The key to improved success with membrane protein structural elucidation is technology development. The most efficient approach constitutes parallel studies on a large number of targets and evaluation of various systems for expression. Next, high throughput format solubilisation and refolding screening methods for a wide range of detergents and additives in numerous concentrations should be established. Today, several networks dealing with structural genomics approaches of membrane proteins have been initiated, among them the Membrane Protein Network (MePNet) programme that deals with the pharmaceutically important mammalian GPCRs. In MePNet, three overexpression systems have been employed for the evaluation of 101 GPCRs, which has generated large quantities of numerous recombinant GPCRs, compatible for structural biology applications.     

A Photochromic Agonist for μ‐Opioid Receptors

Opioid receptors (ORs) are widely distributed in the brain, the spinal cord, and the digestive tract and play an important role in nociception. All known ORs are G‐protein‐coupled receptors (GPCRs) of family A. Another well‐known member of this family, rhodopsin, is activated by light through the cis/trans isomerization of a covalently bound chromophore, retinal. We now show how an OR can be combined with a synthetic azobenzene photoswitch to gain light sensitivity. Our work extends the reach of photopharmacology and outlines a general strategy for converting Family A GPCRs, which account for the majority of drug targets, into photoreceptors.     

An Integrated Approach toward NanoBRET Tracers for Analysis of GPCR Ligand Engagement

Gaining insight into the pharmacology of ligand engagement with G-protein coupled receptors (GPCRs) under biologically relevant conditions is vital to both drug discovery and basic research. NanoLuc-based bioluminescence resonance energy transfer (NanoBRET) monitoring competitive binding between fluorescent tracers and unmodified test compounds has emerged as a robust and sensitive method to quantify ligand engagement with specific GPCRs genetically fused to NanoLuc luciferase or the luminogenic HiBiT peptide. However, development of fluorescent tracers is often challenging and remains the principal bottleneck for this approach. One way to alleviate the burden of developing a specific tracer for each receptor is using promiscuous tracers, which is made possible by the intrinsic specificity of BRET. Here, we devised an integrated tracer discovery workflow that couples machine learning-guided in silico screening for scaffolds displaying promiscuous binding to GPCRs with a blend of synthetic strategies to rapidly generate multiple tracer candidates. Subsequently, these candidates were evaluated for binding in a NanoBRET ligand-engagement screen across a library of HiBiT-tagged GPCRs. Employing this workflow, we generated several promiscuous fluorescent tracers that can effectively engage multiple GPCRs, demonstrating the efficiency of this approach. We believe that this workflow has the potential to accelerate discovery of NanoBRET fluorescent tracers for GPCRs and other target classes.     

High throughput screening (HTS) in identification new ligands and drugable targets of G protein-coupled receptors (GPCRs)

G protein-coupled receptors (GPCRs) which constitute one of the largest and most versatile families of cell surface receptors are involved in a wide spectrum of physiological functions, such as, neuronal transmission, chemotaxis, pacemaker activity and embryonic development. Therefore, in the past a few years GPCR families have become very important targets in pharmaceutical design. However, according to the human genome project, there are approximately 1000 genes encoding GPCRs, only about 200 of GPCRs have known ligands and functions. Searching for ligands of the unknown GPCRs and better modulators of known GPCRs are currently attracting lots of interest. High throughput screening (HTS), which is commonly defined as an automatic process of testing potential drug candidates efficiently, is widely used in drug discovery. In this review, the use of high throughput screening (HTS) in studying GPCRs and the choice of screening technology in different G-protein signaling pathways were summarized.     

The complexity of G-protein coupled receptor-ligand interactions

The G-protein coupled receptors (GPCRs) play fundamental roles in the human biololgy and drug discovery. GPCRs function as signalling molecules that transduce extracellular signals into cells. The signalling transduction is generally triggered by interacting with ligands, including photons, ions, small organic compounds, peptides, proteins and lipids. In this review, we focus on interactions with diffusible ligands such as hormones and neurotransmitters. We discuss three aspects of the complexity of the GPCR-ligand interactions: functional selectivity of ligands, receptor subtype selectivity of ligands and orphan GPCRs.     

A virtual screen for diverse ligands: discovery of selective G protein-coupled receptor antagonists

Virtual screening has become a major focus of bioactive small molecule lead identification, and reports of agonists and antagonists discovered via virtual methods are becoming more frequent. G protein-coupled receptors (GPCRs) are the one class of protein targets for which success with this approach has been limited. This is likely due to the paucity of detailed experimental information describing GPCR structure and the intrinsic function-associated structural flexibility of GPCRs which present major challenges in the application of receptor-based virtual screening. Here we describe an in silico methodology that diminishes the effects of structural uncertainty, allowing for more inclusive representation of a potential docking interaction with exogenous ligands. Using this approach, we screened one million compounds from a virtual database, and a diverse subgroup of 100 compounds was selected, leading to experimental identification of five structurally diverse antagonists of the thyrotropin-releasing hormone receptors (TRH-R1 and TRH-R2). The chirality of the most potent chemotype was demonstrated to be important in its binding affinity to TRH receptors; the most potent stereoisomer was noted to have a 13-fold selectivity for TRH-R1 over TRH-R2. A comprehensive mutational analysis of key amino acid residues that form the putative binding pocket of TRH receptors further verified the binding modality of these small molecule antagonists. The described virtual screening approach may prove applicable in the search for novel small molecule agonists and antagonists of other GPCRs.