Current Pharmaceutical Design - Volume 10, Issue 17, 2004

G protein-coupled receptors (GPCRs) comprise the largest class of cell surface receptor proteins and represent the major targets for currently used therapeutic agents. This issue of Current Pharmaceutical Design is the inaugural publication of a series devoted to GPCRs that will broadly deal with the utility of this receptor class in drug development. This issue contains invited review articles from preeminent researchers in the field. In their article, Graeme Milligan and colleagues [1] explore the utility of GPCR-fusion proteins in drug discovery. The paper discusses the wide range of potentially useful fusion partners, from fluorescent proteins and reporter genes through to specific G proteins, and how these can be used in assays of receptor activation and other aspects of receptor function including internalization and interaction with other proteins. The article also looks at the use of modified proteins that can engender increased constitutive receptor activity and how these may be useful for screening for inverse agonists. The article of Arthur Christopoulos and colleagues [2] looks at the potential of allosteric drugs acting at GPCRs. The paper discusses the potential therapeutic advantages of this class of drugs, some of the difficulties that can be encountered in drug screening and experimental and analytical methods that can be used to specifically identify drugs that act allosterically. In his article, Bill Messer [3] examines the development and utility of bivalent ligands as drugs for GPCRs. The paper discusses how these compounds can provide novel drug leads, both agonist and antagonist, that have improved receptor subtype specificity while maintaining high receptor affinity. The final two articles are comprehensive analyses of structural and functional aspects of small molecule compounds acting at either the adenosine A1 receptor or the CCR5 and CXCR4 chemokine receptors. Sally Hutchinson and Peter Scammells [4] explore the development and utility of agonist and partial agonist drugs that target the A1 receptor, including discussion of alternative pharmacophore models. The article of Christoph Seibert and Tom Sakmar [5] discusses antagonists of the CCR5 and CXCR4 receptors as promising new anti-HIV drugs and includes recent insights on how the small molecule compounds interact with each of these receptors. We are grateful to each of our contributors for their effort and also their patience in the preparation of this inaugural issue.

A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G proteincoupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein α subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist / inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.

G protein-coupled receptors (GPCRs) constitute the largest receptor superfamily in the human genome and represent the most common targets of drug action. Classic agonist and antagonist ligands that act at GPCRs tend to bind to the receptor's orthosteric site, that is, the site recognized by the endogenous agonist for that receptor. However, it is now evident that GPCRs possess additional, extracellular, allosteric binding sites that can be recognized by a variety of small molecule modulator ligands. Allosteric modulators offer many advantages over classic orthosteric ligands as therapeutic agents, including the potential for greater GPCR-subtype selectivity and safety. However, the manifestations of allosterism at GPCRs are many and varied and, in the past, traditional screening methods have generally failed to detect many allosteric modulators. More recently, there have been a number of major advances in high throughput screening, including the advent of cell-based functional assays, which have led to the discovery of more allosteric modulator ligands than previously appreciated. In addition, a number of powerful analytical techniques have also been developed exclusively for detecting and quantifying allosteric effects, based on an increased awareness of various mechanisms underlying allosteric modulator actions at GPCRs. Together, these advances promise to change the current paucity of GPCR allosteric modulators in the clinical setting and yield novel therapeutic entities for the treatment of numerous disorders.

Bivalent ligands have been developed for a variety of G protein-coupled receptor targets, including opioid, dopamine, serotonin and muscarinic receptors. The most successful application of the bivalent ligand approach has been in the development of selective opioid antagonists, such as norbinaltorphimine. Several important principles have emerged from the study of norbinaltorphimine and related compounds, including the utility of bivalent ligands for targeting particular receptor classes and serving as a scaffold for specific interactions with unique amino acid residues that render receptor subtype selectivity. In recent years, several novel bivalent compounds were synthesized and characterized for activity at muscarinic receptors. The compounds display an interesting profile of high binding affinity, strong agonist potency and receptor subtype selectivity. Bivalent ligands represent an important starting point for the development of selective muscarinic agonists with potential utility in treating a variety of neurological disorders, including Alzheimer's disease and schizophrenia. The bivalent ligand approach may be generally applicable to other G protein-coupled receptors.

Adenosine receptors are widely distributed in the body and modulate numerous physiological processes. Four receptor subtypes (termed A1, A2A, A2B and A3) have been identified based on their pharmacological profile and cloning. Activation of the A1 adenosine receptors produces a number of effects including a reduction in heart rate and atrial contractility, the attenuation of the stimulatory actions of catecholamines on the heart as well as a reduction of lipolysis in adipose tissue. As a result, A1AR agonists have been targeted as anti-arrhythmic and cardioprotective agents. This review discusses the synthesis, structure-activity relationships and therapeutic potential of A1AR agonists.

Combination therapy with reverse transcriptase and protease inhibitors greatly reduces morbidity and mortality in HIV-1-infected individuals. However, current anti-retroviral treatment cannot eradicate the virus from infected individuals and is often limited by the emergence of drug-resistant HIV-1 strains and long-term toxicity. These problems emphasize the need to develop new anti-HIV-1 drugs targeting different steps in the viral replication cycle. HIV-1 entry into host cells represents a complex sequence of events involving several viral and cellular proteins that are potential drug targets. In particular, HIV-1 entry requires a sequential interaction of the viral envelope glycoprotein gp120 with CD4 and a co-receptor on the host cell plasma membrane. The CC-chemokine receptor 5 (CCR5) and the CXC-chemokine receptor 4 (CXCR4) are the primary HIV-1 co-receptors in vivo, and are attractive targets for the development of new anti-HIV-1 drugs. CCR5 and CXCR4 belong to the protein superfamily of G protein-coupled receptors (GPCRs). Many orally bioavailable small-molecules interact with specific GPCRs and many existing drugs are orally bioavailable smallmolecule agonists or antagonists of GPCRs. Several small-molecule antagonists of CCR5 and CXCR4 that block chemokine binding and HIV-1 entry have been identified in recent years and are now in pre-clinical or clinical development as drug candidates. This review discusses structural and functional aspects of these compounds and summarizes recent insights into how small-molecule antagonists interact with CCR5 and CXCR4, focusing on drug development programs that are well documented in the scientific literature.

The aim of antiviral therapy of chronic hepatitis B is to control Hepatitis B Virus (HBV) replication and to cure liver disease avoiding the progression of chronic hepatitis to cirrhosis and the end stage complications of cirrhosis. HBeAg / anti-HBe seroconversion is the hallmark of response in hepatitis B “e” antigen (HBeAg) positive patients. In the patients with antibody against HBeAg (anti-HBe positive) the combination of HBV DNA and anti-HBc IgM tests provides adequate diagnostic accuracy. Patients with biochemical and / or histological disease activity are eligible to therapy. The drug choice is based on age, disease severity, risk of complications, side effects and compliance, particularly in anti-HBe positive patients where prolonged treatment is needed. Interferon (5-6 MU daily or 9-10 MU thrice weekly for 4-6 months) is the first line therapy for HBeAg positive patients and (5-6 MU thrice weekly for 12-24 months) for anti-HBe positive patients. When IFN is contraindicated or ineffective, Lamivudine (100 mg) or Adefovir Dipivoxil (10 mg) are given as long as 4-6 months after HBeAg / anti-HBe seroconversion or for long-term treatments in HBeAg positive non-responders and anti-HBe positive patients. Patients with more advanced forms of cirrhosis and portal hypertension are to be treated within liver transplantation programs. Fifteen to 30% of treated patients achieve sustained response and more than 60% of them experience long-term disease remission during therapy. In perspectives, currently available molecular and immunologic tools and modelling of viral dynamics will help to address the therapy issue with more complex, efficacious and individually tailored treatment schedules.

Progression from acute to chronic HCV infection occurs in 50% to 84% of cases. In light of the risk of developing chronic disease and the response rate to treatment once the disease is established, it is important to consider early treatment of acute HCV infection before it progresses to the chronic state. Several studies evaluated the efficacy of either alpha or beta IFN monotherapy in patients with acute hepatitis C, but nearly all trials are small and present great variability regarding timing, schedule, response definition and patient characteristics. To overcome these limits, IFN efficacy has been assessed by meta-analyses demonstrating that antiviral therapy during the acute phase of HCV significantly reduces evolution to chronic hepatitis. Accordingly, treatment of persons with acute hepatitis C is warranted. However, several issues remain to be addressed, such as the optimal regimen and timing. Recent data would indicate that induction with daily IFN is needed to optimize response and pegylated IFN monotherapy could be the best option. Combination therapy with ribavirin does not seem to increase the response rate but could be proposed as a second choice to patients non responding to IFN monotherapy. Delaying treatment by 2-3 months might allow the identification of cases who would spontaneously resolve without compromising efficacy. However, additional data are required to improve the selection of those patients at great risk of progressing to chronic disease, and also to establish the optimal treatment in terms of risk / benefit and cost-effectiveness ratio.

Ribavirin is a very broad-spectrum anti-viral agent used clinically to treat infections by Lassa fever virus, respiratory syncytial virus (RSV) and, in combination with Interferon-α (IFN-α), hepatitis C virus (HCV). Although it was originally synthesized over 30 years ago, the precise mechanisms of its therapeutic activities are still not fully understood. Ribavirin was shown to possess both direct and indirect action mechanisms against several DNA and RNA viruses. These include direct inhibition of viral RNA-dependent RNA polymerases, inhibition of the host inosine monophosphate dehydrogenase, modulation of the host immune response and inhibition of viral capping enzymes. More recently, ribavirin was demonstrated to be able to act as an RNA virus mutagen, increasing mutations in the RNA virus genome and reducing their infectivity. Still the real challenge is to identify which of its biological properties is responsible for the observed clinical efficacy on specific infections. Under this aspect, renewed interest results from its synergistic enhancement of interferon-α (IFN-α) therapy, which could open the way to develop more powerful anti-HCV compounds. This work purpose is to provide a broad overview of all the recognized ribavirin action mechanisms against HCV, which can possibly also explain its synergistic behavior with IFN-α. An overview on the corresponding HCV treatment clinical observations is also provided in the second part of this work.

Previous data demonstrated that an elevated percentage of hepatitis C virus (HCV) infected patients are endotoxemic. Endotoxemic patients are poor responders to the interferon (IFN)- α / ribavirin (RIB) treatment and exhibit lower serum levels of IFN-γ and interleukin (IL)-10 than the responder counterpart. Here we provide evidence that in endotoxemic HCV+ patients absolute numbers of CD19+ cells (B cells) are higher than those observed in the non-endotoxemic counterpart at the end of the combined treatment. Moreover, anti lactoferrin (LF) antibodies are more elevated in non-responder HCV+ patients than in the responders. In turn, these autoantibodies may affect the antiviral activity of LF, on the one hand , and, on the other hand abrogate the LF binding to lipopolysaccharides (LPS). Such an interaction hampers the binding of LPS to LPS binding protein, thus inhibiting LPS fixation to CD14+ cells and, ultimately, leading to a decreased release of proinflammatory cytokines.

Combined therapy using Interferon alfa (IFN) and Ribavirin (RIB) represents the standard treatment in patients with chronic hepatitis C. However, the percentage of responders to this regimen is still low, while its cost and side effects are elevated. Therefore, the possibility to predict patient's response to the above treatment is of paramount importance. The progress in the field of informatics and its large use for decision making has led to the development of novel techniques related to the so-called Artificial Intelligence, even including artificial neural networks (ANNs). In chronic viral hepatitis data are lacking. By means of an artificial neural network (ANN), 300 patients treated with IFN plus RIB were retrospectively analyzed with the aim to predict the response to the treatment. One hundred patients resulted responders and 200 non-responders at the end of treatment and during the follow up. For evaluating the prediction of treatment response, six ANNs with 16 neurons of input, an hidden layer with 7 neurons and an output layer with one neuron were utilized. The ANN model generated a positive predictive value (i.e. posterior probability of treatment response) ranging from 57% to 75% while the negative one (i.e. posterior probability of no response to treatment) was comprised between 52% and 71%. The highest level of diagnostic accuracy was 70%. In conclusion, ANNs appear to be a promising tool in the prediction of treatment response in patients with chronic hepatitis C. However, additional prospective studies are necessary to ultimately validate this predictive method.

The introduction of highly active anti-retroviral therapy (HAART) for Human Immunodeficiency Virus (HIV) infection has significantly improved the life expectancy of HIV positive patients. Hepatitis C virus (HCV) co-infection is common in HIV infected patients and is now a significant cause of morbidity and mortality. Optimal management and treatment of HCV in HIV infected patients is therefore essential. Interferon-alpha (IFN-α) and ribavirin is the mainstay of treatment for HCV infection in HIV infected people. The sustained virological response rate (SVR) with combination therapy is lower than that commonly observed in HCV monoinfected patients. This is, at least in part, due to the very high treatment drop out rates. Ribavirin in combination with HAART is associated with particular side effects such as mitochondrial toxicity. Therefore, vigilant monitoring of patients during therapy, in specialist centers is essential. Pegylated interferon (PEG-IFN) plus ribavirin is particularly promising as it is easier to administer and will probably become the treatment of choice for co-infected patients. A SVR is associated with genotype 2 and 3, in addition to a high CD4+ cell count and a low HCV load prior to therapy. The progression of HCV related liver disease in HIV positive patients is faster than in subjects with HCV infection alone. As a result, there is an increasing incidence of cirrhosis and end-stage liver disease in co-infected patients. Liver transplantation is being evaluated in many centers. To date the experiences are very limited but encouraging in term of survival rate.

Hepatitis C virus chronic infection is currently the most common cause of end-stage liver disease. The benefit of antiviral therapy on liver histology and its impact on the long-term course of the disease has been extensively studied. However, the results are still equivocal and the overall assessment of treatment effect remains difficult to evaluate. Although the conclusions of the last National Institute of Health Consensus Development Conferences on Hepatitis C have recently been published, several important issues still remain unanswered. We review the available data by an evidence-based approach and conclude that: 1) peginterferon alfa is more effective than conventional interferon in improving liver histology; 2) monotherapy with PEG-interferon induces a marked reduction in staging in virological sustained responders and to a lesser degree in relapsers, but provides no benefit to nonresponders after 24-48 weeks of treatment; 3) maintenance therapy aiming to improve histology in virological nonresponders should be considered experimental and of unproven benefit; 4) although the reduction in the number of events in sustained responders suggests a long-term benefit of IFN therapy, available evidence is still insufficient to confirm that IFN prolongs life in HCV infected patients. Data of the long-term benefit of subjects treated with IFN plus ribavirin are still not available; 5) pooling of published data suggests a slight preventive effect of IFN on HCC development in patients with HCV-related cirrhosis. The magnitude of this effect is low and the observed benefit might be due to spurious associations. The preventive effect is more evident among sustained responders to interferon.