Orphan G protein-coupled receptors: from DNA to drug targets

Although G protein-coupled receptors (GPCRs) are the most numerous therapeutic targets to date, ligands remain unknown for approximately two-thirds of these receptors. The challenge in the post-genomic era is to evaluate the role of these 'orphan' GPCRs in normal physiology and disease, and to develop new therapeutics based on this information. A prevalent strategy to determine the function of these orphan GPCRs is to identify specific ligands that conditionally modulate their function. These ligands can then be used to explore the role of the receptor-ligand pair in relevant models of disease. Some promise is emerging from this infant field of functional genomics as several recently deorphanized GPCRs may be potential drug targets for many diseases, including obesity, cardiovascular disease, inflammation and cancer.     

The potential use of PPARalpha agonists as immunosuppressive agents

Fibrates are peroxisome proliferator-activated receptor (PPAR)alpha ligands that have been used to treat hyperlipidemia and atherosclerosis for many years, and research has demonstrated that these agents have immunosuppressive effects. PPARalpha is expressed in multiple inflammatory cell types, and its ligands abrogate expression of inflammatory diseases. This review focuses on the use of fibrates in inflammatory disease models. It also describes proposed mechanisms of action of PPARalpha ligands and discusses the potential use of these medications as immunosuppressive agents.     

Pharmacological modulation of chemokine receptor function

G protein-coupled chemokine receptors and their peptidergic ligands are interesting therapeutic targets due to their involvement in various immune-related diseases, including rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, chronic obstructive pulmonary disease, HIV-1 infection and cancer. To tackle these diseases, a lot of effort has been focused on discovery and development of small-molecule chemokine receptor antagonists. This has been rewarded by the market approval of two novel chemokine receptor inhibitors, AMD3100 (CXCR4) and Maraviroc (CCR5) for stem cell mobilization and treatment of HIV-1 infection respectively. The recent GPCR crystal structures together with mutagenesis and pharmacological studies have aided in understanding how small-molecule ligands interact with chemokine receptors. Many of these ligands display behaviour deviating from simple competition and do not interact with the chemokine binding site, providing evidence for an allosteric mode of action. This review aims to give an overview of the evidence supporting modulation of this intriguing receptor family by a range of ligands, including small molecules, peptides and antibodies. Moreover, the computer-assisted modelling of chemokine receptor-ligand interactions is discussed in view of GPCR crystal structures. Finally, the implications of concepts such as functional selectivity and chemokine receptor dimerization are considered.     

PET imaging of norepinephrine transporters

The involvement of the norepinephrine transporter (NET) in the pathophysiology and treatment of attention deficit hyperactivity disorder (ADHD), substance abuse, neurodegenerative disorders (e.g., Alzheimer's disease (AD) and Parkinson's disease (PD)) and depression has long been recognized. However, many of these important findings have resulted from studies in vitro using postmortem tissues; as of now, these results have never been verified via in vivo methods because brain imaging of NET in living systems has been hampered due to the lack of suitable radioligands. The fact that all three monoamine (dopamine, norepinephrine, and serotonin) transporters (DAT, NET and SERT) are involved in various neurological and psychiatric diseases further emphasizes the need to develop suitable NET ligands so that researchers will be able to probe the contributions of each monoamine transporter system to specific CNS disorders. In this review article, the design and biological evaluation of several radioligands for imaging the brain NET system with PET are discussed. Based on these characterization studies, including C-11 labeled desipramine (DMI), 2-hydroxydesipramine (HDMI), talopram, talsupram, nisoxetine (Nis), oxaprotiline (Oxap), lortalamine (Lort) and C-11 and F-18 derivatives of reboxetine (RB), methylreboxetine (MRB) and their individual (R, R) and (S, S) enantiomers, in conjunction with studies with radiolabeled 4-iodo-tomoxetine and 2-iodo-nisoxetine, we have identified the superiority of (S, S)-[(11)C]MRB and the suitability of the MRB analogs as potential NET ligands for PET. In contrast, Nis, Oxap and Lort displayed high uptake in striatum (higher than thalamus). The use of these ligands is further limited by high non-specific binding and relatively low specific signal, as is characteristic of many earlier NET ligands. Thus, to our knowledge, (S, S)-[(11)C]MRB remains by far the most promising NET ligand for PET studies.     

Synthesis of N-glyoxyl prolyl and pipecolyl amides and thioesters and evaluation of their in vitro and in vivo nerve regenerative effects

The recent discovery that small molecule ligands for the peptidyl-prolyl isomerase (PPIase) FKBP12 possess powerful neuroprotective and neuroregenerative properties in vitro and in vivo suggests therapeutic utility for such compounds in neurodegenerative disease. The neurotrophic effects of these compounds are independent of the immunosuppressive pathways by which drugs such as FK506 and rapamycin operate. Previous work by ourselves and other groups exploring the structure-activity relationships (SAR) of small molecules that mimic only the FKBP binding domain portion of FK506 has focused on esters of proline and pipecolic acid. We have explored amide and thioester analogues of these earlier structures and found that they too are extremely potent in promoting recovery of lesioned dopaminergic pathways in a mouse model of Parkinson's disease. Several compounds were shown to be highly effective upon oral administration after lesioning of the dopaminergic pathway, providing further evidence of the potential clinical utility of a variety of structural classes of FKBP12 ligands.     

Adenosine receptor targeting in health and disease

INTRODUCTION: The adenosine receptors A(1), A(2A), A(2B) and A(3) are important and ubiquitous mediators of cellular signaling that play vital roles in protecting tissues and organs from damage. In particular, adenosine triggers tissue protection and repair by different receptor-mediated mechanisms, including increasing the oxygen supply:demand ratio, pre-conditioning, anti-inflammatory effects and the stimulation of angiogenesis. AREAS COVERED: The state of the art of the role of adenosine receptors which have been proposed as targets for drug design and discovery, in health and disease, and an overview of the ligands for these receptors in clinical development. EXPERT OPINION: Selective ligands of A(1), A(2A), A(2B) and A(3) adenosine receptors are likely to find applications in the treatment of pain, ischemic conditions, glaucoma, asthma, arthritis, cancer and other disorders in which inflammation is a feature. The aim of this review is to provide an overview of the present knowledge regarding the role of these adenosine receptors in health and disease.     

Computational elucidation of the structural basis of ligand binding to the dopamine 3 receptor through docking and homology modeling

The dopamine subtype 3 receptor (D3) is a promising therapeutic target for the treatment of cocaine addiction, schizophrenia, Parkinson's disease, and other disorders, but little is known about the binding of ligands to D3 at the atomic level. In the present study, binding of 29 known ligands to the D3 receptor was modeled computationally using four D3 receptor models which were obtained from homology modeling. The predicted binding models were validated with experimental data from site-directed mutagenesis, structure-activity relationship studies, and affinity labeling studies. Docking scores calculated for these 29 ligands correlate reasonably well with the experimentally determined binding affinities. A pharmacophore model is proposed that describes the binding of ligands at a single D3 receptor binding site and offers insights into the binding of structurally diverse D3 ligands to this receptor.     

Constitutively activated G protein-coupled receptors: a novel approach to CNS drug discovery

G protein-coupled receptors (GPCRs) represent a major class of signal transduction proteins that modulate various biological functions. GPCRs are one of the most common targets for drug development-currently, 39 of the top 100 marketed drugs in use act directly or indirectly through activation or blockade of GPCR-mediated receptors. Nearly 160 GPCRs have been identified based on their gene sequence and their ability to interact with known endogenous ligands. However, an estimated 500-800 additional GPCRs have been classified as "orphan" receptors (oGPCRs) because their endogenous ligands have not yet been identified. Given that known GPCRs have proven to be such clinically useful drug targets, these oGPCRs represent a rich group of receptor targets for the development of novel and improved medicines. To develop ligands for these potential drug targets requires the ability to identify groups or pools of GPCRs that are likely to be involved in a specific disease process (obesity, schizophrenia, depression, etc.) and to dissect out the pharmacological and signal transduction differences between these GPCR subtypes. It also requires the development of assays to detect ligands of GPCRs even when the endogenous ligands are unidentified. This paper will review novel strategies to identify clinically interesting oGPCRs and to screen for small molecules that act as ligands without prior knowledge of endogenous ligands. This involves the use of constitutively activated GPCRs, a technology that provides a unique opportunity to identify several classes of pharmacological agents, including agonists, inverse agonists and allosteric modulators.     

Approaches to the rational design of selective melanocortin receptor antagonists

Introduction: When establishing the physiological roles of specific receptors in normal and disease states, it is critical to have selective antagonist ligands for each receptor in a receptor system with several subtypes. The melanocortin receptors have five subtypes referred to as the melanocortin 1 receptor, melanocortin 2 receptor, melanocortin 3 receptor, melanocortin 4 receptor and melanocortin 5 receptor, and they are of critical importance for many aspects of human health and disease. Areas covered: This article reviews the current efforts to design selective antagonistic ligands for the five human melanocortin receptors summarizing the currently published orthosteric and allosteric antagonists for each of these receptors. Expert opinion: Though there has been progress, there are still few drugs available that address the many significant biological activities and diseases that are associated with these receptors, which is possibly due to the lack of receptor selectivity that these designed ligands are currently showing. The authors believe that further studies into the antagonists' 3D conformational and topographical properties in addition to future mutagenesis studies will provide greater insight into these ligands which could play a role in the treatment of various diseases in the future.     

Nicotinic ligands as multifunctional agents for the treatment of neuropsychiatric disorders

The challenges associated with developing more effective treatments for neurologic and psychiatric illness such as Alzheimer’s disease and schizophrenia are considerable. Both the symptoms and the pathophysiology of these conditions are complex and poorly understood and the clinical presentations across different patients can be very heterogeneous. Moreover, it has become apparent that the reductionist approach to drug discovery for these illnesses that has dominated the field for decades (i.e., the development of highly selective compounds or other treatment modalities focused on a very specific pathophysiologic target) has not been widely successful. Accordingly, a variety of new strategies have emerged including the development of “multitarget-directed ligands” (MTDLs), the development and/or identification of compounds that exhibit “multifunctional” activity (e.g., pro-cognitive plus neuroprotective, pro-cognitive plus antipsychotic activity), “repurposing” strategies for existing compounds that have other clinical indications, and novel “adjunctive” treatment strategies that might enhance the efficacy of the currently available treatments. Interestingly, a variety of ligands at nicotinic acetylcholine receptors (nAChRs) appear to have the potential to fulfill one or more of these desirable properties (i.e., multifunctional, repurposing, or adjunctive treatment potential). The purpose of this review (while not all-inclusive) is to provide an overview of a variety of nAChR ligands that demonstrate potential in these categories, particularly, “multifunctional” properties. Due to their densities in the mammalian brain and the amount of literature available, the review will focus on ligands of the high affinity α4β2 nAChR and the low affinity α7 nAChR.     

Calculation of the binding affinity of beta-secretase inhibitors using the linear interaction energy method

It has been shown that the rate-limiting step in the production of beta-amyloid peptide (Abeta) is the proteolytric cleavage of the membrane-bound beta-amyloid precursor protein (APP) by beta-secretase (BACE). Since the accumulation of Abeta has been implicated as one of the key events in the progression of Alzheimer's disease, BACE has become an important therapeutic target. Recently, two crystal structures of BACE cocrystallized with the inhibitors OM99-2 and OM00-3 were published by Tang and co-workers. In addition, the Ghosh group has published binding data on a series of inhibitors based on their initial lead, OM99-2. Using this set as a basis, we have developed a model for the binding affinity of these ligands to BACE using the linear interaction energy method. The best binding affinity model for the full set of ligands had a RMSD of 1.10 kcal/mol. The best model excluding the two charged ligands had a RMSD of 0.87 kcal/mol.     

Estrogen receptor ligands in the control of pathogenic inflammation

Inflammation is recognized as a key component in a number of diseases, including rheumatoid arthritis, inflammatory bowel disease and atherosclerosis. Although well known for their classic effects on the reproductive tract and action by means of estrogen response elements in gene promoters, estrogens are also known to possess anti-inflammatory activity. This was originally highlighted with the observation that pregnancy ameliorates symptoms of rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease. Furthermore, the antagonistic cross talk between nuclear factor kappaB and estrogen receptor signaling pathways has been well documented. Recently, novel estrogen receptor ligands, pathway-selective ligands and estrogen receptor beta-selective ligands have been identified which demonstrate potent anti-inflammatory activity; these ligands are being analyzed for their therapeutic potential in pathogenic inflammation.     

Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology

A surface plasmon resonance biosensor assay was established for studying the interactions of 51 histaminergic and 15 GABAergic ligands with homo-oligomeric β3 GABA(A) receptors. Detergent solubilized receptors were successfully immobilized via affinity-capture on biosensor surfaces. The interaction kinetics of both histaminergic and GABAergic ligands were very rapid but affinities could be determined by steady-state analysis. Binding of several GABAergic ligands was observed, in agreement with previous data. Histamine and 16 histaminergic ligands were detected to directly bind to β3 GABA(A) receptors with micromolar affinity (K(D)<300 μM), thus extending previous evidence that β3 GABA(A) receptors can interact with histaminergic ligands. Histamine exhibited an affinity for these receptors comparable to that for human histamine type 1 (H1) or type 2 (H2) receptors. Furthermore, 13 of these histaminergic ligands appeared to compete with histamine. The discovery that H2, H3 and H4 receptor ligands interact with β3 receptors indicates a unique histaminergic pharmacology of these receptors. Due to their low affinity for the homo-pentameric β3 receptors these histaminergic drugs are not expected to modulate these receptors at clinically relevant concentrations. The results support the use of the new biosensor assay for the identification of drugs interacting with full length receptors and for fragment-based drug discovery of high affinity ligands for β3 receptors. Drugs with high affinity and selectivity for these receptors can be used to clarify the question whether β3 receptors do exist in the brain, and provide new avenues for the development of therapeutically active compounds targeting this novel histamine binding site.     

Novel ergopeptides as dual ligands for adenosine and dopamine receptors

Multivalent ligands are promising pharmacological tools that may be more efficacious for several diseases than highly selective single-target drugs. A combined therapy using dopaminergic agonists and adenosinergic antagonists is currently being evaluated for the treatment of Parkinson's disease. [(a) Kanda, T.; et al. Exp. Neurol. 2000, 162, 321-327. (b) Jenner, P. Expert Opin. Invest. Drugs 2005, 14, 729-738. (c) Kase, H.; et al. Neurology 2003, 61 (Suppl 6), S97-S100.] Here we prepared dual ligands acting on adenosine and dopamine receptors by applying a combinatorial approach based on the ergolene privileged structure. The potency and efficacy of these novel compounds were determined by radioligand binding studies and intracellular cAMP production assays in cells expressing adenosine and dopamine receptors. Selected compounds displayed dual dopamine agonist and adenosine antagonist activity. Molecules with this pharmacological profile are potentially useful for studying dopamine-adenosine cross-talk in the central nervous system and for testing the therapeutic potential of multivalent drugs for Parkinson's disease.     

Failure of GPI compounds to display neurotrophic activity in vitro and in vivo

The aim of this study was to evaluate the neurotrophic and neuroprotective properties of a series of immunophilin ligands and to assess the potential involvement of FK506 Binding Protein 12 kDa (FKBP12) rotamase inhibition in this activity. Both FK506 and rapamycin induced a potent inhibition of the FKBP12 rotamase activity (pIC(50) values of 7.3 and 7.4, respectively) but only a modest inhibition was observed with 1-(3,3-dimethyl-2-oxo-pentanoyl)-pyrrolidine-2-carboxylic acid S-3-pyridin-3-yl-propyl ester (GPI 1046) (5.8), its N-oxide (5.4) and thioester (6.3) analogues. Compared to nerve growth factor, all these immunophilin ligands only induced marginal increases in neurite outgrowth of rat dissociated newborn dorsal root ganglia cells. Furthermore, systemic administration of GPI 1046 and its N-oxide and thioester analogues failed to prevent striatal dopamine depletion induced by acute or chronic i.p. treatment with 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP). These results suggest that inhibition of FKBP12 rotamase activity is not predictive for neurotrophic and neuroprotective properties of immunophilin ligands and question their therapeutic utility in neurodegenerative diseases like Parkinson's disease.     

GABAC receptor ion channels

1. The present review gives an overview of studies conducted on GABAC receptors over the past 10 years since the author started at the University of Sydney. It concentrates on the structure-activity relationship profiles of the receptor and how these studies were used to: (i) develop selective GABAC receptor ligands; and (ii) understand the impact of amino acid changes on GABAC receptor pharmacology and function. 2. Structure-activity relationship studies involving variations of both ligands and their receptor targets are vital to the discovery of drugs that interact selectively with particular native and mutant receptor subtypes. Such agents may be useful for treating anxiety, depression, epilepsy and memory related disorders, such as Alzheimer's disease.