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Concept: Allosteric regulation


BACKGROUND: Allostery is one of the most powerful and common ways of regulation of protein activity. However,for most allosteric proteins identified to date the mechanistic details of allosteric modulation are notyet well understood. Uncovering common mechanistic patterns underlying allostery would allow notonly a better academic understanding of the phenomena, but it would also streamline the design ofnovel therapeutic solutions. This relatively unexplored therapeutic potential and the putativeadvantages of allosteric drugs over classical active-site inhibitors fuel the attention allosteric-drugresearch is receiving at present. A first step to harness the regulatory potential and versatility ofallosteric sites, in the context of drug-discovery and design, would be to detect or predict theirpresence and location. In this article, we describe a simple computational approach, based on theeffect allosteric ligands exert on protein flexibility upon binding, to predict the existence and positionof allosteric sites on a given protein structure. RESULTS: By querying the literature and a recently available database of allosteric sites, we gathered 213allosteric proteins with structural information that we further filtered into a non-redundant set of 91proteins. We performed normal-mode analysis and observed significant changes in protein flexibilityupon allosteric-ligand binding in 70% of the cases. These results agree with the current view thatallosteric mechanisms are in many cases governed by changes in protein dynamics caused by ligandbinding. Furthermore, we implemented an approach that achieves 65% positive predictive value inidentifying allosteric sites within the set of predicted cavities of a protein (stricter parameters set,0.22 sensitivity), by combining the current analysis on dynamics with previous results on structuralconservation of allosteric sites. We also analyzed four biological examples in detail, revealing thatthis simple coarse-grained methodology is able to capture the effects triggered by allosteric ligandsalready described in the literature. CONCLUSIONS: We introduce a simple computational approach to predict the presence and position of allosteric sitesin a protein based on the analysis of changes in protein normal modes upon the binding of acoarse-grained ligand at predicted cavities. Its performance has been demonstrated using a newlycurated non-redundant set of 91 proteins with reported allosteric properties. The software developedin this work is available upon request from the authors.

Concepts: Enzyme kinetics, Hemoglobin, Allosteric regulation, Proteins, Scientific method, Protein, Protein structure


Serotonin (5-hydroxytryptamine; 5-HT) signaling through the 5-HT(2C) receptor (5-HT(2C)R) is essential in normal physiology, whereas aberrant 5-HT(2C)R function is thought to contribute to the pathogenesis of multiple neural disorders. The 5-HT(2C)R interacts with specific protein partners, but the impact of such interactions on 5-HT(2C)R function is poorly understood. Here, we report convergent cellular and behavioral data that the interaction between the 5-HT(2C)R and protein phosphatase and tensin homolog (PTEN) serves as a regulatory mechanism to control 5-HT(2C)R-mediated biology but not that of the closely homologous 5-HT(2A)R. A peptide derived from the third intracellular loop of the human 5-HT(2C)R [3L4F (third loop, fourth fragment)] disrupted the association, allosterically augmented 5-HT(2C)R-mediated signaling in live cells, and acted as a positive allosteric modulator in rats in vivo. We identified the critical residues within an 8 aa fragment of the 3L4F peptide that maintained efficacy (within the picomolar range) in live cells similar to that of the 3L4F peptide. Last, molecular modeling identified key structural features and potential interaction sites of the active 3L4F peptides against PTEN. These compelling data demonstrate the specificity and importance of this protein assembly in cellular events and behaviors mediated by 5-HT(2C)R signaling and provide a chemical guidepost to the future development of drug-like peptide or small-molecule inhibitors as neuroprobes to study 5-HT(2C)R allostery and therapeutics for 5-HT(2C)R-mediated disorders.

Concepts: Enzyme kinetics, Allosteric regulation, Protein, Enzyme, Cell biology, Receptor, Ligand, Serotonin


Their ubiquitous nature, wide cellular distribution and versatile molecular recognition and signalling help make G-protein binding receptors (GPCRs) the most important class of membrane proteins in clinical medicine, accounting for ∼40% of all current therapeutics. A large percentage of current drugs target the endogenous ligand binding (orthosteric) site, which are structurally and evolutionarily conserved, particularly among members of the same GPCR subfamily. With the recent advances in GPCR X-ray crystallography, new opportunities for developing novel subtype selective drugs have emerged. Given the increasing recognition that the extracellular surface conformation changes in response to ligand binding, it is likely that all GPCRs possess an allosteric site(s) capable of regulating GPCR signalling. Allosteric sites are less structurally conserved than their corresponding orthosteric site and thus provide new opportunities for the development of more selective drugs. Constitutive oligomerisation (dimerisation) identified in many of the GPCRs investigated, adds another dimension to the structural and functional complexity of GPCRs. In this review, we compare 60 crystal structures of nine GPCR subtypes (rhodopsin, ß(2)-AR, ß(1)-AR, A(2a)-AR, CXCR4, D(3)R, H(1)R, M(2)R, M(3)R) across four subfamilies of Class A GPCRs, and discuss mechanisms involved in receptor activation and potential allosteric binding sites across the highly variable extracellular surface of these GPCRs. This analysis has identified a new extracellular salt bridge (ESB-2) that might be exploited in the design of allosteric modulators.

Concepts: DNA, Allosteric regulation, Protein, Signal transduction, Cell membrane, Receptor, Ligand, Membrane biology


A series of substrate analogues has been used to determine which chemical moieties of the substrate, phosphoenolpyruvate (PEP) contribute to the allosteric inhibition of rabbit muscle pyruvate kinase (M1-PYK) by phenylalanine. Replacing the carboxyl group of the substrate with a methyl alcohol, or removing the phosphate altogether, greatly reduces substrate affinity. However, removal of the carboxyl group is the only modification tested that removes the ability to allosterically reduce Phe binding. From this, it can be concluded that the carboxyl group of PEP is responsible for energetic coupling with Phe binding in the allosteric sites.

Concepts: Enzyme kinetics, Allosteric regulation, Alcohol, Adenosine triphosphate, Enzyme, Functional group, Ligand, Carboxylic acid


We previously reported the discovery of VU0364572 and VU0357017 as M(1)-selective agonists that appear to activate M(1) through actions at an allosteric site. Previous studies have revealed that chemical scaffolds for many allosteric modulators contain molecular switches that allow discovery of allosteric antagonists and allosteric agonists or positive allosteric modulators (PAMs) based on a single chemical scaffold. Based on this, we initiated a series of studies to develop selective M(1) allosteric antagonists based on the VU0364572 scaffold. Interestingly, two lead antagonists identified in this series, VU0409774 and VU0409775, inhibited ACh-induced Ca(2+) responses at rat M(1-5) receptor subtypes, suggesting they are nonselective muscarinic antagonists. VU0409774 and VU0409775 also completely displaced binding of the nonselective radioligand [(3)H]-NMS at M(1) and M(3) mAChRs with affinities similar to their functional IC(50) values. Finally, Schild analysis revealed that these compounds inhibit M(1) responses through a fully competitive interaction at the orthosteric binding site. This surprising finding prompted further studies to determine whether agonist activity of VU0364572 and VU0357017 may also engage in previously unappreciated actions at the orthosteric site on M(1). Surprisingly, both VU0364572 and VU0357017 completely displaced [(3)H]-NMS binding to the orthosteric site of M(1)-M(5) receptors at high concentrations. Furthermore, evaluation of agonist activity in systems with varying levels of receptor reserve and Furchgott analysis using a cell line expressing M(1) under control of an inducible promotor was consistent with an action of these compounds as weak orthosteric partial agonists of M(1). However, consistent with previous studies suggesting actions at a site that is distinct from the orthosteric binding site, VU0364572 or VU0357017 slowed the rate of [(3)H]-NMS dissociation from CHO-rM(1) membranes. Together, these results suggest that VU0364572 and VU0357017 act as bitopic ligands and that novel antagonists in this series act as competitive orthosteric site antagonists.

Concepts: Allosteric regulation, Pharmacology, Ligand, Receptor antagonist, Agonist, Inverse agonist, Muscarinic acetylcholine receptor, Functional selectivity


The operational model of agonism assumes that the maximum effect a particular receptor system can achieve (the E(m) parameter) is fixed. E(m) estimates are above but close to the asymptotic maximum effects of endogenous agonists. The concept of E(m) is contradicted by superagonists and those positive allosteric modulators that significantly increase the maximum effect of endogenous agonists. An extension of the operational model is proposed that assumes that the E(m) parameter does not necessarily have a single value for a receptor system but has multiple values associated to multiple active receptor conformations. The model provides a mechanistic link between active receptor conformation and agonist efficacy, which can be useful for the analysis of agonist response under different receptor scenarios.

Concepts: Allosteric regulation, Effectiveness, Agonist


Benzylquinolone carboxylic acid (BQCA) is a recently described cholinergic muscarinic M(1) receptor positive allosteric modulator having potential as cognitive enhancer in dementia. The present study focused on the characterisation of BQCA’s mode of action in relation to positive effects on memory and side-effects in an animal model. To get insight into this mode of action, in vitro receptor potency/left shift experiments in cells stably expressing the rat’s M(1) receptor were performed. They revealed an inflection point value of BQCA corresponding to 306nM, and potentiation of the agonist response up to 47-fold in presence of 10μM of BQCA. In vivo, brain microdialysis showed a maximal brain level of 270nM, 40min after i.p. administration at 10mg/kg. Based on in vitro data obtained with this dose, it can be concluded that BQCA reaches brain levels which should potentiate the agonist response about 4-fold. Behavioural data confirmed that BQCA used at 10mg/kg attenuated scopolamine-induced memory deficit in a spontaneous alternation task. Moreover, BQCA showed no side effect at 10mg/kg and above in spontaneous locomotion and salivation tests. The profile of BQCA observed in the present study displays a clear advantage over the M(1)-M(3) agonist cevimeline. The present data show the therapeutic potential of the M(1) receptor positive allosteric modulator BQCA for the treatment of memory deficits observed in Alzheimer’s disease.

Concepts: Alzheimer's disease, Allosteric regulation, Psychology, In vivo, Cognition, Ligand, In vitro, Positive allosteric modulator


Previous research has demonstrated that treatment with the positive allosteric modulator (PAM) of the GABA(B) receptor (GABA(B) PAM), rac-BHFF, suppressed lever-responding for alcohol and amount of self-administered alcohol in Sardinian alcohol-preferring (sP) rats. The present study was designed to extend the investigation on the anti-alcohol effects of rac-BHFF to alcohol drinking behavior. To this end, sP rats were exposed to the homecage, 2-bottle “alcohol (10%, v/v) vs water” choice regimen, with unlimited access for 24 h/day. rac-BHFF was administered once daily and for 7 consecutive days at the doses of 0, 50, 100, and 200 mg/kg (i.g.). Treatment with rac-BHFF resulted in an immediate, stable, and dose-related reduction in daily alcohol intake; the overall magnitude of reduction in alcohol intake averaged approximately 25%, 40%, and 65% in 50, 100, and 200 mg/kg rac-BHFF-treated rat groups, respectively. An increase in daily water intake fully compensated the reduction in alcohol intake, so that daily total fluid intake was unaffected by treatment with rac-BHFF. Daily food intake tended to be reduced only by the highest dose of rac-BHFF. These results complement closely with previous data indicating that (a) rac-BHFF suppressed operant, oral alcohol self-administration in sP rats and (b) the prototypic GABA(B) PAMs, CGP7930 and GS39783, reduced alcohol drinking in sP rats. However, while the reducing effect of CGP7930 and GS39783 on the daily alcohol intake tended to vanish after the first 2-3 days of treatment, the reducing effect of rac-BHFF on daily alcohol intake remained unchanged over the entire 7-day treatment period. These data strengthen the hypothesis that GABA(B) PAMs may represent a step forward in the search for GABA(B) receptor ligands with therapeutic potential for alcoholism.

Concepts: Allosteric regulation, Alcohol, Carbon dioxide, Redox, Ligand, Drinking culture


Introduction: There is substantial evidence from preclinical and early proof-of-concept studies suggesting that selective modulation of the M(1) muscarinic receptor is efficacious in cognitive models of Alzheimer’s disease (AD) and antipsychotic models of schizophrenia. For example, a number of nonselective M(1) muscarinic agonists have previously shown positive effects on cognitive function in AD patients, but were limited due to cholinergic adverse events thought to be mediated by pan activation of the M(2) to M(5) subtypes. Thus, there is a need to identify selective activators of the M(1) receptor to evaluate their potential in cognitive disorders. One strategy to confer selectivity for M(1) is the identification of allosteric agonists or positive allosteric modulators, which would target an allosteric site on the M(1) receptor rather than the highly conserved orthosteric acetylcholine binding site. Areas covered: This review discusses the M(1) muscarinic receptor and its potential therapeutic value in the treatment of CNS disorders such as AD and schizophrenia. Specifically, novel allosteric ligands that activate or positively modulate the M(1) receptor are examined and peer-reviewed articles associated with these patents publications are also described. Expert opinion: There is substantial evidence supporting activation of the M(1) receptor might be effective in treating symptoms of AD and schizophrenia, but therapeutic success has been elusive and is hypothesized to be due to the lack of selectivity among orthosteric agonists. During the past decade, allosteric modulation of GPCRs has evolved as a viable strategy toward generating subtype selective molecules. A number of novel, selective ligands in the form of allosteric agonists and positive allosteric modulators of the M(1) receptor have been identified offering the potential for clinical evaluation of M(1)-specific receptor activation.

Concepts: Allosteric regulation, Signal transduction, Ligand, Agonist, Acetylcholine, Modulation, Muscarinic acetylcholine receptor, Muscarinic agonist


Displacement of the αC helix in kinases by allosteric modulators is becoming a prominent approach in drug discovery, owing to its potential ability to provide inhibitor selectivity. According to recent evidence, this approach appears to be more generally applicable to a broader number of kinases of the human kinome than was previously expected. Owing to their crucial role in the modulation of cell pathways, protein kinases are important targets for a number of human diseases, including but not limited to cancer. The classic approach of targeting the ATP active site has recently come up against selectivity issues, which can be considerably reduced by following an allosteric modulation approach. Being closely related to protein kinase inactivation, allosteric targeting via displacement of the conserved structural αC helix enables a direct and specific modulation mechanism. A structure-based survey of the allosteric regulation of αC helix conformation in various kinase families is provided, highlighting key allosteric pockets and modulation mechanisms that appear to be more broadly conserved than was previously thought.

Concepts: Allosteric regulation, Signal transduction, Adenosine triphosphate, Enzyme, Kinase, Protein kinase, Enzyme inhibitor