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Oct 15, 2008 (Vol. 28, No. 18)

Fine-Tuning the Design of GPCR Therapeutics

Understanding Allosteric Modulation and Functional Selectivity Are Key Priorities

  • Bryan Roth, M.D., Ph.D., director of the National Institute of Mental Health’s psychoactive drug screening program at the University of North Carolina, suggests that the information printed in textbooks of pharmacology is “far from reality.” He is referring to recent discoveries about the incredible complexity of GPCR, the theme of a conference organized by Dr. Roth and held last month in Wisconsin. Entitled “Emerging Themes in GPCR Signaling and Drug Discovery: Functional Selectivity and Allosteric Modulation,” the meeting was sponsored by Invitrogen.

    About half of all marketed drugs target GPCR, yet the mechanisms involved in regulating GPCR signaling are poorly understood. Data from GCPR studies repeatedly demonstrate that some drugs produce markedly different functional effects while acting through a single receptor. Such phenomena, once viewed as contradictory results, are now recognized as functional selectivity. Compounds with functional selectivity open doors to new avenues of novel drug targeting and therapeutics.

    Allosteric modulators are compounds that bind receptors at sites that are different from the orthostatic (primary) ligand-binding site. They enhance or reduce the action of orthostatic agents by fine-tuning signaling responses of GPCR systems. Allosteric modulators represent a new and safer mechanism for designing GPCR drugs. “Functional selectivity and allosteric modulation were theoretical ideas just five years ago,” said Dr. Roth, “but now they are the next wave of drug discovery.”

    Some researchers are addressing basic questions such as whether functional selectivity can be predicted by chemical structure. Bonnie Hanson, Ph.D., head of the GPCR program at Invitrogen’s Discovery Assays and Services, used dopamine D1 receptor agonists selected from three different structural classes. She tested compounds with a common ring structure that activates cyclic AMP.

    The compounds, however, performed differently in Invitrogen’s high-throughput Tango™ beta-arrestin assay, with some acting as agonists and others as antagonists. Therefore, compounds that are closely related structurally do not affect signaling pathways similarly. “Very subtle differences in structure can lead to profound differences in functional signaling,” Dr. Hanson said. The challenge, she added, “is to identify physiological consequences of functional selectivity receptors.”

  • Allosteric Modulation

    Allosteric modulators can increase the potency of other drugs. Researchers in the laboratory of Jeff Conn, Ph.D., at Vanderbilt University identified a compound called VU10010 as a potent allosteric modulator of M4 muscarinic acetylcholine receptors (mAChR). VU10010 increases the M4 response to acetylcholine 50-fold, yet shows no activity at other mAChRs.

    As testing progressed, VU10010 proved unfavorable for in vivo studies, but a screen of Vanderbilt’s drug library found two more favorable analogs, VU0152099 and VU0152100, which act similarly to VU10010. These new compounds do not interact with the orthostatic site and have no activity when used alone. “With further optimization, we can improve them,” said Dr. Conn.

    Dr. Conn also has identified allosteric modulators of M1 mAChR. Compounds that target M4 and M1 mAChRs are potentially novel therapeutics for schizophrenia. M1 and M4 receptors mediate the desired actions on the central nervous system, while avoiding undesirable side effects such as sweating and bradycardia activated through M2 and M3 receptors.

    Allosteric modulators represent a safer mechanism of action because they mimic the body’s own ligands, and they act only in cells where the natural ligand and receptor are present. In theory, allosteric modulators could lower the dosage of a main drug, reduce side effects, and prevent overdose, tolerance, abuse, or dependence. Whereas orthostatic binding sites are highly conserved with few options for subtype selectivity, allosteric binding sites are far less conserved with many subtypes available.

    Three main symptom clusters—positive, negative, and cognitive—characterize schizophrenia. Positive symptoms include paranoia and hallucinations; negative symptoms include loss of speech and emotions; and cognitive symptoms include loss of memory and attention. All currently prescribed drugs treat largely the positive symptoms, and no drugs treat the cognitive symptoms. “There’s a tremendous need for new medicines that control all three symptom clusters,” said Dr. Conn.

  • Functional Selectivity

    Functional selectivity can be exploited to learn more about marketed drugs and reveal unique actions that can guide the development of next-generation therapies. All clinically effective antipsychotics interact with the D2 class of dopamine receptors (D2R), and their physiological effects are mediated through GPCR or beta-arrestin signaling pathways.

    Bernard Masri, Ph.D., of Duke University Medical Center, tested several antipsychotics including halperidol, clozapine, and risperidone. He found that they share a common molecular mechanism involving inhibition of D2R/beta-arrestin signaling, yet they show highly diverse effects on D2R/G protein signaling. Selective targeting of D2R/beta-arrestin signaling pathways may lead to better antipsychotic drugs.

    Vince Setola, Ph.D., a research assistant professor in Dr. Roth’s laboratory, presented unpublished data revealed for the first time at the meeting. While screening drug libraries to find drugs that activate the serotonin 5-HT2B receptor, Dr. Setola stumbled on an example of functional selectivity with likely clinical relevance.

    Dr. Roth’s team previously showed that drugs known to cause valvular heart disease such as fenfluramine for weight loss and pergolide for Parkinson’s disease activate 5-HT2B receptors. These drugs also cause hyperproliferation of plaques in heart tissue that lead to valve dysfunction.

    Dr. Setola theorized that screening drug compounds for agonists of 5-HT2B might predict those at risk for causing valvular heart disease. He screened 2,500 compounds in libraries of commercial and investigational drugs and found several with clearcut 5-HT2B activity.

    Surprisingly, some are drugs prescribed today such as ropinirole for restless leg syndrome, guafacine for hypertension, oxymetazoline in over-the-counter decongestants, and quinidine for antirhythmia. None were previously known to act through 5-HT2B receptors, and none have yet to be associated with valvular heart disease. The greatest diversity in their functional selectivity was revealed in the heart-cell proliferation assays, rather than in standard tests of receptor activity such as calcium flux or inositol triphosphate accumulation. “When screening compounds to look for functional activity, it’s a good idea to use physiologically relevant screens,” Dr. Setola said.

    Other speakers agreed that assays must be optimized to detect allosteric modulation and functional selectivity, since drug efficacy and potency are assay dependent. In vitro assays must be designed to pick up subtleties and interactions that are cell-type dependent. In addition to in vitro assays, better preclinical animal models are needed to measure, for instance, the negative and cognitive symptoms associated with schizophrenia. “Assay design is the key,” said Roth.

    Invitrogen already supplies researchers with assays to measure GPCR receptor activity such as Tango cell-based assays and GeneBLAzer™. “Our assays meet some needs of these researchers, but we will be developing new ones based on what the researchers are describing here,” said Mark Keck, director of marketing with Invitrogen Discovery Sciences.

    “In 1991, one-third of drugs failed due to lack of efficacy, and it’s amazing that one-third of drugs still fail today,” said Arthur Christopoulos, co-director of the drug discovery biology laboratory at Monash University. A deeper understanding of allosteric modulation and functional selectivity could improve the design of new clinical compounds that act through GCPRs. Researchers are just starting to tackle this new frontier. Exactly how they will harness the emerging knowledge about allosteric modulation and functional selectivity to create better drugs remains to be seen.


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