GPCR Drug Discovery Research Articles

P.1.001 3-Nitropropionic acid induces autophagy by forming mitochondrial permeability transition pore, not by activating mitochondrial fission

and purified on an IMAC Nickel affinity column using a His-tag at the C terminus. Other expression systems that have been used for structural studies with GPCRs include yeast, E. coli and mammalian cells. Crystallisation screens are set up in a wide range of different detergent conditions. Two approaches are routinely used: vapour diffusion in short chain detergents and lipidic cubic phase (LCP) crystallisation. The lipidic cubic phase is a lipid matrix which produces a more native, membranelike environment. Extensive screening and optimisation of crystallisation conditions is then required to obtain the best diffracting crystals. Crystallisation in LCP is frequently facilitated by making fusion proteins with the GPCRwhich increase crystallisation contacts. For example T4 lysozyme may be fused into the third intracellular loop of the receptor. Antibodies may also be included as crystallisation chaperones. The resulting GPCR crystals are small and must be taken to specialised microfocussed X-ray beams at the synchrotron. Selection of the ligand for co-crystallisation studies is also important. In the absence of thermostabilising mutations a high affinity stabilising ligand is required to obtain crystals. The presence of thermostabilising mutations reduces this requirement and enables multiple co-crystal structures of weaker ligands to be obtained. This is important when using co-structures during the lead optimisation stage of drug discovery. Once the structure is solved a model of the receptor structure is obtained and this is used for virtual screening and structure based drug discovery. X-ray structures are highly enabling for GPCR drug discovery as they allow drug candidates to be designed which fit efficiently into the ligand binding pocket [3]. Compounds can be optimised for affinity, kinetics and selectivity using structure based approaches. Heptares have applied this approach to a range of CNS discovery projects including an adenosine A2A antagonist for the treatment of Parkinson’s disease, an orexin receptor antagonist for the treatment of sleep disorders and a highly selective muscarinic M1 agonist for the treatment of Alzheimer’s disease.

Circular plant peptides as templates for GPCR drug discovery?

Background Cyclotides are disulfide-rich miniproteins with the unique structural features of a circular backbone and knotted arrangement of three conserved disulfide bonds. These features make them exceptionally stable and they have applications as plant defense (insecticidal) agents and stable drug frameworks. So far they have been found mainly in two plant families, including in every species of the violet family (Violaceae) so far examined, and in a few species of the coffee family (Rubiaceae).

Thematic Analysis™ : A Chemogenomic Approach to GPCR Drug Discovery

Thematic Analysis™ is a chemogenomic tool which has been developed and used to aid the process of GPCR drug discovery. This review covers the scientific rationale behind the development of this tool and provides examples of the successful application of the chemogenomic method in both hit finding and hit to lead stages of the drug discovery process.

Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

Label-free biosensor technology based on dynamic mass redistribution (DMR) of cellular constituents promises to translate GPCR signaling into complex optical 'fingerprints' in real time in living cells. Here we present a strategy to map cellular mechanisms that define label-free responses, and we compare DMR technology with traditional second-messenger assays that are currently the state of the art in GPCR drug discovery. The holistic nature of DMR measurements enabled us to (i) probe GPCR functionality along all four G-protein signaling pathways, something presently beyond reach of most other assay platforms; (ii) dissect complex GPCR signaling patterns even in primary human cells with unprecedented accuracy; (iii) define heterotrimeric G proteins as triggers for the complex optical fingerprints; and (iv) disclose previously undetected features of GPCR behavior. Our results suggest that DMR technology will have a substantial impact on systems biology and systems pharmacology as well as for the discovery of drugs with novel mechanisms.

Platforms for the identification of GPCR targets, and of orthosteric and allosteric modulators

Areas covered in this review: The review provides a summary of old and new approaches for GPCR target identification and for the screening of molecules acting on GPCR targets. The new findings in the field are presented as well as an opinion about how these developments may help GPCR drug discovery.Importance in the field: GPCRs have been the most useful family of proteins in terms of targets for drug discovery. The expectations for GPCR target identification and discovery of new drugs acting on ‘old’ or ‘new’ GPCR targets are very high. Given the fact that the pace at which new ‘GPCR drugs’ appear in the market is decreasing and since the new developments in the field are not being translated into drug discovery there is a need to review the field from a critical perspective.Take home message: To overcome the limitation of the old approaches used in GPCR target identification and drugs discovery new approaches are required. In particular successful approaches in GPCR drug discovery should take into account that the real GPCR targets for a given disease are not GPCR monomers but GPCR heteromers.What the reader will gain: The reader will gain an overview of the strategies currently used and their pros and cons. The reader will also understand that new strategies may help in accelerating the access of GPCR into the market, and also notice that successful strategies should take advantage of the new findings in the field of GPCRs.

Structure-Based Discovery of Novel Chemotypes for Adenosine A2A Receptor Antagonists

The recent progress in crystallography of G-protein coupled receptors opens an unprecedented venue for structure-based GPCR drug discovery. To test efficiency of the structure-based approach, we performed molecular docking and virtual ligand screening (VLS) of more than 4 million commercially available "drug-like" and ''lead-like'' compounds against the A(2A)AR 2.6 A resolution crystal structure. Out of 56 high ranking compounds tested in A(2A)AR binding assays, 23 showed affinities under 10 microM, 11 of those had sub-microM affinities and two compounds had affinities under 60 nM. The identified hits represent at least 9 different chemical scaffolds and are characterized by very high ligand efficiency (0.3-0.5 kcal/mol per heavy atom). Significant A(2A)AR antagonist activities were confirmed for 10 out of 13 ligands tested in functional assays. High success rate, novelty, and diversity of the chemical scaffolds and strong ligand efficiency of the A(2A)AR antagonists identified in this study suggest practical applicability of receptor-based VLS in GPCR drug discovery.

In this issue

AbstractCell‐based assays in GPCR drug discovery Siehler, S., Biotechnol. J. 2008, 3, 471‐483G protein‐coupled receptors (GPCRs) transmit extracellular signals into the intracellular space. They play key roles in the physiological regulation of virtually every cell and tissue and are a major class of drug targets. Sandra Siehler (Novartis Institutes for BioMedical Research, Basel, Switzerland) reviews cell‐based assays for screening GPCR drugs. In the past receptor binding assays comprised the main readout for receptor activity but these approaches are now complemented by measurement of the intracellular responses to receptor activation. This allows for better discrimination of agonism and antagonism and investigation of those receptors for which labeled ligands are not readily available. The sheer number of assay readouts for these receptors highlights how basic biological knowledge intertwines with drug discovery and characterization.Label‐free cell‐based assays for drug discoveryXi, B. et al.: Biotechnol. J. 2008, 3, 484‐495Cell‐based assays are an important part of the drug discovery process. Conventional label and reporter‐based cell assays may be more prone to artifacts due to considerable manipulation of the cell either by labeling or overexpression of targets or reporter proteins. Cell‐based label‐free technologies preclude the need for cellular labeling or over expression of reporter proteins, utilizing the inherent morphological and adhesive characteristics of the cell as a physiologically relevant and quantitative readout for various cellular assays. Furthermore, these technologies utilize non‐invasive measurements allowing for time resolution and kinetics in the assay. In this article, researchers from San Diego (CA, USA) review the various label‐free technologies that are being used in drug discovery settings.Computational chemistry approaches to drug discoveryFischer, P. M., Biotechnol. J. 2008, 3, 452‐470Whilst cell based functional screening constitutes an important approach in drug discovery, the availability of detailed structural information on many drugs targets has given impetus to approaches that use the structure of the target under investigation as a guiding principal. Computational techniques help predicting binding activity of chemical structures and they are widely used as components of the drug discovery screening cascade including discovery in the area of signal transduction. In this Review Peter Fischer (Nottingham, UK) introduces the enormous range of computational techniques currently available to aid the drug discovery process and describes their different rationales and underlying principles.

High Throughput Screening for Orphan and Liganded GPCRs

GPCRs had significant representation in the drug discovery portfolios of most major commercial drug discovery organizations for many years. This is due in part to the diverse biological roles mediated by GPCRs as a class, as well as the empirical discovery that they have proven relatively tractable to the development of small molecule therapeutics. Publication of the human genome sequence in 2001 confirmed GPCRs as the largest single gene superfamily with more than 700 members, furthering the already strong appeal of addressing this target class using efficient and highly parallelized platform approaches. The GPCR research platform implemented at Amgen is used as a case study to review the evolution and implementation of available assays and technologies applicable to GPCR drug discovery. The strengths, weaknesses, and applications of assay technologies applicable to G alpha s, G alpha i and G alpha q-coupled receptors are described and their relative merits evaluated. Particular consideration is made of the role and practice of "de-orphaning" and signaling pathway characterization as a pre-requisite to establishing effective screens. In silico and in vitro methodology developed for rapid, parallel high throughput hit characterization and prioritization is also discussed extensively.

G Proteins in Drug Screening: From Analysis of Receptor-G Protein Specificity to Manipulation of GPCR-Mediated Signalling Pathways

Seven transmembrane G protein coupled receptors (7TM GPCRs) represent one of the largest gene familes in the human genome. Because of the size of the GPCR family, their proven history of being valuable targets for small molecule drug design, the fact that the absolute number of GPCRs that are targets for current medicines represents only a small fraction of the total encoded by the human genome, and that ligands for GPCRs do not have to enter the cell to exert their function, it is very likely that GPCRs will remain major targets for the pharmaceutical industry in the foreseeable future. Despite recent evidence indicating that GPCRs can provide information to cells, that does not require activation of G proteins ("signaling at zero G"), most of the GPCRs known to date function via interaction with and activation of heterotrimeric (alphabetagamma) G proteins. Thus, assay systems translating ligand modulation of GPCRs into G protein-dependent intracellular responses are a key component of both basic research and the drug discovery process. This article will review the current knowledge and recent progress in understanding molecular aspects of specific receptor-G protein recognition. It will also highlight how the knowledge generated by such studies can be transformed into assay systems for GPCR drug discovery.

Lead the way in GPCR Drug Discovery

Lead the way in GPCR Drug Discovery