Novel Allosteric Modulators of G Protein-coupled Receptors

G protein-coupled receptors (GPCRs) are allosteric proteins, because their signal transduction relies on interactions between topographically distinct, yet conformationally linked, domains. Much of the focus on GPCR allostery in the new millennium, however, has been on modes of targeting GPCR allosteric sites with chemical probes due to the potential for novel therapeutics. It is now apparent that some GPCRs possess more than one targetable allosteric site, in addition to a growing list of putative endogenous modulators. Advances in structural biology are also shedding new insights into mechanisms of allostery, although the complexities of candidate allosteric drugs necessitate rigorous biological characterization.     

Chromatin Loops as Allosteric Modulators of Enhancer-Promoter Interactions

The classic model of eukaryotic gene expression requires direct spatial contact between a distal enhancer and a proximal promoter. Recent Chromosome Conformation Capture (3C) studies show that enhancers and promoters are embedded in a complex network of looping interactions. Here we use a polymer model of chromatin fiber to investigate whether, and to what extent, looping interactions between elements in the vicinity of an enhancer-promoter pair can influence their contact frequency. Our equilibrium polymer simulations show that a chromatin loop, formed by elements flanking either an enhancer or a promoter, suppresses enhancer-promoter interactions, working as an insulator. A loop formed by elements located in the region between an enhancer and a promoter, on the contrary, facilitates their interactions. We find that different mechanisms underlie insulation and facilitation; insulation occurs due to steric exclusion by the loop, and is a global effect, while facilitation occurs due to an effective shortening of the enhancer-promoter genomic distance, and is a local effect. Consistently, we find that these effects manifest quite differently for in silico 3C and microscopy. Our results show that looping interactions that do not directly involve an enhancer-promoter pair can nevertheless significantly modulate their interactions. This phenomenon is analogous to allosteric regulation in proteins, where a conformational change triggered by binding of a regulatory molecule to one site affects the state of another site.     

GABAB 受体变构剂药学研究进展

Y-氨基丁酸B受体(GABAB receptor,GABABR)是最具有药理学意义的药物靶点之一,具有复杂而精细的激活机制.传统的GABABR靶点药物开发集中于激动剂和拮抗剂,这类药物受到多种因素的制约,包括较强的副作用、药物代谢困难、机体耐药性明显等.变构剂结合于正构位点之外,能够调节GABABR异源二聚体亚基或结构域间的相互作用.正向变构剂(positive allosteric modulators,PAMs)和负向变构剂(negative allosteric modulators,NAMs)分别可以提高或降低GABABR的活性,并具有较高的特异性和药物安全性,同时还能够保持GABABR信号在时间和空间上的可控性.变构剂为GABABR靶点药物开发提供了新思路.     

GABAB受体变构剂药学研究进展

Y-氨基丁酸B受体(GABAB receptor,GABABR)是最具有药理学意义的药物靶点之一,具有复杂而精细的激活机制.传统的GABABR靶点药物开发集中于激动剂和拮抗剂,这类药物受到多种因素的制约,包括较强的副作用、药物代谢困难、机体耐药性明显等.变构剂结合于正构位点之外,能够调节GABABR异源二聚体亚基或结构域间的相互作用.正向变构剂(positive allosteric modulators,PAMs)和负向变构剂(negative allosteric modulators,NAMs)分别可以提高或降低GABABR的活性,并具有较高的特异性和药物安全性,同时还能够保持GABABR信号在时间和空间上的可控性.变构剂为GABABR靶点药物开发提供了新思路.     

GABA_B受体变构剂药学研究进展

γ-氨基丁酸B受体（GABAB receptor,GABABR）是最具有药理学意义的药物靶点之一,具有复杂而精细的激活机制。传统的GABABR靶点药物开发集中于激动剂和拮抗剂,这类药物受到多种因素的制约,包括较强的副作用、药物代谢困难、机体耐药性明显等。变构剂结合于正构位点之外,能够调节GABABR异源二聚体亚基或结构域间的相互作用。正向变构剂（positiveallosteric modulators,PAMs）和负向变构剂（negative allosteric modulators,NAMs）分别可以提高或降低GABABR的活性,并具有较高的特异性和药物安全性,同时还能够保持GABABR信号在时间和空间上的可控性。变构剂为GABABR靶点药物开发提供了新思路。     

Introductory Lecture: Allosteric Modulation of Torpedo Nicotinic Acetylcholine Receptor Ion Channel Activity by Noncompetitive Agonists

Abstract

Similar to other neuroreceptors of the vertebrate central nervous system, the nicotinic acetylcholine receptor (nAChR) is subject to modulatory control by allosterically acting ligands. Of particular interest in this regard are allosteric ligands that enhance the sensitivity of the receptor to its natural agonist acetylcholine (ACh), as such ligands could be useful as drugs in diseases associated with impaired nicotinic neurotransmission. Here we discuss the action of a novel class of nAChR ligands which act as allosterically potentiating ligands (APL) on the nicotinic responses induced by ACh and competitive agonists. In addition, APLs also act as noncompetitive agonists of very low efficacy, and as direct blockers of ACh-activated channels. These actions are observed with nAChRs from brain, muscle and electric tissue, and they depend on the structure of the APL and the concentration range applied. We focus here on Torpedo nAChR because (i) the unusual pharmacology of these ligands was first discovered with this system, and (ii) large quantities of this receptor are readily available for biochemical studies.     

Allosteric Gating of a Large Conductance Ca-activated K+ Channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca²⁺. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca²⁺ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca²⁺ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca²⁺ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca²⁺ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.     

配体门控离子通道受体别构调节研究

配体门控离子通道(LGIC)在中枢神经系统信息处理的过程中起着极其重要的作用,与多种神经性疾病有着密切联系.与受体正位调节作用相比,别构调节效应具有类内源性生理作用、高选择性及不易过度调节的优点,从而避免了一系列不良反应发生.目前,各种LGIC受体超家族均有别构调节剂发现,部分已在临床上得到应用.在未来的研究中,通过建立及完善针对别构调节剂的筛选策略,别构调节剂的发现效率及生物活性将得到极大地提高,更多的药物将会不断涌现.     

Effects of Temperature and Allosteric Modulators on [3H]Nitrendipine Binding: Methods for Detecting Potential Ca2+ Channel Blockers

Abstract

The effects of incubation temperature and allosteric modulators were studied on [³H]nitrendipine binding to guinea-pig cardiac membranes. Incubation temperature only slightly affected the ability of nifedipine and verapamil derivatives to inhibit binding. By contrast, the Ca²⁺ channel blockers d-cis-diltiazem and fostedil (KB-944) stimulated [³H]nitrendipine binding in a temperature-dependent manner (37° > 25° > 4° C). The stimulatory effect of fostedil could be related to a decrease (2.3-fold at 37° C) in the rate of radioligand binding site dissociation, without significant effects on association kinetics. Both fostedil and d-cis-diltiazem caused a shift to the right of the concentration-inhibition curve of tiapamil, a negative allosteric modulator of [³H]nitrendipine binding. Neither compound affected the ability of nifedipine, a competitive antagonist, to inhibit radioligand binding. This selective effect of fostedil or d-cis-diltiazem may be useful for testing whether potential Ca²⁺ channel blockers interact in a competitive as opposed to allosteric manner with the dihydropyridine site. Varying the incubation temperature may also be useful in detecting compounds which act as positive allosteric modulators (stimulators) of dihydropyridine binding.     

Integrative Action of Receptor Mosaics: Relevance of Receptor Topology and Allosteric Modulators

After a definition of the receptor mosaic (RM, high order heteromer or homomer) concept, this study analyzes some relevant theoretical aspects related to receptor-receptor interactions (RRIs). In particular, the possible influence of the plasma membrane microdomain on RM integrative functions are discussed. Furthermore, a possible mathematical approach may identify the RM topologies [i.e., the spatial arrangements the receptors (tesserae of the mosaic) can assume within the RM assembly]. Finally, data are presented on homocysteine possible biasing action on the well-characterized heterodimer/receptor mosaic formed by adenosine A2A and dopamine D2 receptors. We discuss how these findings can lead to a new possible approach for developing drugs for the treatment of certain neuropsychiatric disorders.     

Zinc Potentiation of the Glycine Receptor Chloride Channel Is Mediated by Allosteric Pathways

Abstract: Molecular mechanisms of zinc potentiation were investigated in recombinant human α1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [³H]strychnine binding assays. In the wild-type (WT) GlyR, 1 µ M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1–M2 and M2–M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2–M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.     

Enhanced recognition of cystic fibrosis W1282X DNA point mutation by chiral peptide nucleic acid probes by a surface plasmon resonance biosensor

Peptide nucleic acids (PNAs) containing an insert of three chiral monomers based on D-lysine ('chiral box') were synthesized and used as probes in Biospecific Interaction Analysis (BIA) for the recognition of DNA containing the W1282X point mutation of the cystic fibrosis gene. Hybridization experiments carried out in solution showed enhanced mismatch recognition when compared with the analogous achiral PNAs and oligonucleotides. The signal intensity was lower, but the selectivity of the Biacore response was found to be much higher than that observed with achiral PNAs. The newly designed chiral PNA probes were also found to hybridize with a 1:1 mixture of normal (N-W1282X) and mutated (M-W1282X) DNA oligomers immobilized on the biosensor, thus allowing discrimination not only between a normal and a mutated sequence (healthy/homozygous), but also between homo- and heterozygous individuals. These results suggest that 'chiral box' PNAs are potential powerful tools for the analysis of single point mutations of biological/biomedical relevance.     

Stable Expression of Recombinant AMPA Receptor Subunits: Binding Affinities and Effects of Allosteric Modulators

Abstract: Homomeric AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors (GluRs) were stably expressed in kidney cells from cDNAs encoding GluR1 flop, GluR2 flip, GluR2 flop, and GluR3 flop subunits. The recombinant receptors were of the expected size and showed functional properties in whole-cell recording as previously reported. [³H]AMPA binding to all subunits was increased to a similar extent by the chaotropic ion thiocyanate (SCN^?). Significant differences were found in the Scatchard plots, however, which were linear and of high affinity for GluR1 and -3 receptors (K_D values of 33 and 52 nM, respectively) but showed curvature for GluR2 receptors, indicating the presence of two components with distinct affinities. As with brain AMPA receptors, solubilization of GluR2 receptors reduced the number of lower-affinity sites and correspondingly increased the number of higher-affinity sites. The sulfhydryl reagent p-chloromercuriphenylsulfonic acid, which increases binding to brain receptors, produced only minor changes except in the case of GluR2 flip. These results indicate that GluR2, among the subunits examined here, most closely resembles the native AMPA receptors in brain membranes. [³H]AMPA binding was inhibited in a noncompetitive manner by two drugs that change the desensitization kinetics of the AMPA receptor. In agreement with physiological observations, the apparent affinity of cyclothiazide for GluR2 flip (EC₅₀ = 7 µM) was higher than that for receptors made of flop subunits (49–130 µM). In contrast, BDP-37, a member of the benzamide family of drugs, exhibited a lower potency for GluR2 flip (58 µM) than for any of the flop isoforms (18–40 µM). These results predict that the action of centrally active AMPA-receptor modulators varies across brain regions depending on their flip/flop composition.     

Identification of Novel Specific Allosteric Modulators of the Glycine Receptor Using Phage Display

The glycine receptor (GlyR) is a member of the Cys-loop superfamily of ligand-gated ion channels and the major mediator of inhibitory neurotransmission in the spinal cord and brainstem. Many allosteric modulators affect the functioning of members of this superfamily, with some such as benzodiazepines showing great specificity and others such as zinc, alcohols, and volatile anesthetics acting on multiple members. To date, no potent and efficacious allosteric modulator acting specifically at the GlyR has been identified, hindering both experimental characterization of the receptor and development of GlyR-related therapeutics. We used phage display to identify novel peptides that specifically modulate GlyR function. Peptide D12-116 markedly enhanced GlyR currents at low micromolar concentrations but had no effects on the closely related γ-aminobutyric acid type A receptors. This approach can readily be adapted for use with other channels that currently lack specific allosteric modulators.     

Positive Allosteric Interaction of Structurally Diverse T-Type Calcium Channel Antagonists

Low-voltage-activated (T-type) calcium channels play a role in diverse physiological responses including neuronal burst firing, hormone secretion, and cell growth. To better understand the biological role and therapeutic potential of the target, a number of structurally diverse antagonists have been identified. Multiple drug interaction sites have been identified for L-type calcium channels, suggesting a similar possibility exists for the structurally related T-type channels. Here, we radiolabel a novel amide T-type calcium channel antagonist (TTA-A1) and show that several known antagonists, including mibefradil, flunarizine, and pimozide, displace binding in a concentration-dependent manner. Further, we identify a novel quinazolinone T-type antagonist (TTA-Q4) that enhanced amide radioligand binding, increased affinity in a saturable manner and slowed dissociation. Functional evaluation showed these compounds to be state-dependent antagonists which show a positive allosteric interaction. Consistent with slowing dissociation, the duration of efficacy was prolonged when compounds were co-administered to WAG/Rij rats, a genetic model of absence epilepsy. The development of a T-type calcium channel radioligand has been used to demonstrate structurally distinct TTAs interact at allosteric sites and to confirm the potential for synergistic inhibition of T-type calcium channels with structurally diverse antagonists.     

Insect Repellents: Modulators of Mosquito Odorant Receptor Activity

Background

DEET, 2-undecanone (2-U), IR3535 and Picaridin are widely used as insect repellents to prevent interactions between humans and many arthropods including mosquitoes. Their molecular action has only recently been studied, yielding seemingly contradictory theories including odorant-dependent inhibitory and odorant-independent excitatory activities on insect olfactory sensory neurons (OSNs) and odorant receptor proteins (ORs).

Methodology/Principal Findings

Here we characterize the action of these repellents on two Aedes aegypti ORs, AaOR2 and AaOR8, individually co-expressed with the common co-receptor AaOR7 in Xenopus oocytes; these ORs are respectively activated by the odors indole (AaOR2) and (R)-(−)-1-octen3-ol (AaOR8), odorants used to locate oviposition sites and host animals. In the absence of odorants, DEET activates AaOR2 but not AaOR8, while 2-U activates AaOR8 but not AaOR2; IR3535 and Picaridin do not activate these ORs. In the presence of odors, DEET strongly inhibits AaOR8 but not AaOR2, while 2-U strongly inhibits AaOR2 but not AaOR8; IR3535 and Picaridin strongly inhibit both ORs.

Conclusions/Significance

These data demonstrate that repellents can act as olfactory agonists or antagonists, thus modulating OR activity, bringing concordance to conflicting models.     

Bile Acids as Modulators of Enzyme Activity and Stability

Bile acids deactivate certain enzymes, such as prolyl endopeptidases (PEPs), which are investigated as candidates for protease-based therapy for celiac sprue. Deactivation by bile acids presents a problem for therapeutic enzymes targetted to function in the upper intestine. However, enzyme deactivation by bile acids is not a general phenomenon. Trypsin and chymotrypsin are not deactivated by bile acids. In fact, these pancreatic enzymes are more efficient at cleaving large dietary substrates in the presence of bile acids. We targeted the origin of the apparently different effect of bile acids on prolyl endopeptidases and pancreatic enzymes by examining the effect of bile acids on the kinetics of cleavage of small substrates, and by determining the effect of bile acids on the thermodynamic stabilities of these enzymes. Physiological amounts (5 mM) of cholic acid decrease the thermodynamic stability of Flavobacterium meningosepticum PEP from 18.5 ± 2 kcal/mol to 10.5 ± 1 kcal/mol, while thermostability of trypsin and chymotrypsin is unchanged. Trypsin and chymotrypsin activation by bile and PEP deactivation can both be explained in terms of a common mechanism: bile acid-mediated protein destabilization. Bile acids, usually considered non-denaturing surfactants, in this case act as a destabilizing agent on PEP thus deactivating the enzyme. However, this level of global thermodynamic destabilization does not account for a more than 50% decrease in enzyme activity, suggesting that bile acids most likely modulate enzyme activity through specific local interactions.