Identification of novel allosteric regulators of human-erythrocyte pyruvate kinase

Erythrocyte pyruvate kinase (PK) is an important glycolytic enzyme, and manipulation of its regulatory behavior by allosteric modifiers is of interest for medicinal purposes. Human-erythrocyte PK was expressed in Rosetta cells and purified on an Ni-NTA column. A search of the small-molecules database of the National Cancer Institute (NCI), using the UNITY software, led to the identification of several compounds with similar pharmacophores as fructose-1,6-bisphosphate (FBP), the natural allosteric activator of the human kinases. The compounds were subsequently docked into the FBP binding site using the programs FlexX and GOLD, and their interactions with the protein were analyzed with the energy-scoring function of HINT. Seven promising candidates, compounds 1-7, were obtained from the NCI, and subjected to kinetics analysis, which revealed both activators and inhibitors of the R-isozyme of PK (R-PK). The allosteric effectors discovered in this study could prove to be lead compounds for developing medications for the treatment of hemolytic anemia, sickle-cell anemia, hypoxia-related diseases, and other disorders arising from erythrocyte PK malfunction.     

The allosteric site regulates the voltage sensitivity of muscarinic receptors

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein–coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M₁-Rs and M₃-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the G_q protein cycle. In the presence of ACh, M₁-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M₃-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed “allosteric site” M₃/M₁-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M₃-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.     

Multiple allosteric sites on muscarinic receptors

Proteins and small molecules are capable of regulating the agonist binding and function of G-protein coupled receptors by multiple allosteric mechanisms. In the case of muscarinic receptors, there is the well-characterised allosteric site that binds, for example, gallamine and brucine. The protein kinase inhibitor, KT5720, has now been shown to bind to a second allosteric site and to regulate agonist and antagonist binding. The binding of brucine and gallamine does not affect KT5720 binding nor its effects on the dissociation of [3H]-N-methylscopolamine from M1 receptors. Therefore it is possible to have a muscarinic receptor with three small ligands bound simultaneously. A model of the M1 receptor, based on the recently determined structure of rhodopsin, has the residues that have been shown to be important for gallamine binding clustered within and to one side of a cleft in the extracellular face of the receptor. This cleft may represent the access route of acetylcholine to its binding site.     

G protein coupled receptors as allosteric proteins and the role of allosteric modulators

Seven transmembrane receptors (7TMRs) are proteins that convey signals through changes in conformation. These conformations are stabilized by external molecules (i.e. agonists, antagonists, modulators) and act upon other bodies (termed ‘guests’) which can be other molecules in the extracellular space, or proteins along the plane of the membrane (receptor oligomerization) or signaling proteins in the cytosol (i.e. G protein, β-arrestin). These elements comprise allosteric systems and a great deal of 7TMR pharmacology can be considered in terms of allosteric behavior. Allosteric ligands acting on 7TMRs possess four unique behaviors that can be valuable therapeutically; () the ability to alter the interaction of very large proteins, () probe dependence, () saturable effect, and () induction of separate changes in affinity and efficacy of other ligands. Two of these behaviors (namely probe dependence for CCR5-based HIV-1 entry inhibitors and functional selectivity for biased agonism) will be highlighted with examples.     

Nicotinic receptors, allosteric proteins and medicine

The nicotinic acetylcholine receptor (nAChR) was the first ion channel and membrane receptor of a neurotransmitter to be isolated and chemically identified and is one of the best known membrane proteins involved in signal transduction. Subsequently, nAChRs have been a target for drug discovery because of their potential to impact numerous brain diseases and disorders. Here, we consider recent developments in our understanding of nAChR structure and of the conformational transitions that link the acetylcholine (ACh)-binding site and the ion channel to mediate fast neurotransmission. The knowledge of such allosteric mechanisms is essential to understand pathologies such as congenital myasthenia, autosomal dominant nocturnal frontal lobe epilepsies, sudden infant death syndrome, attention deficit hyperactivity disorder and nicotine addiction and to design novel therapies.     

Dynamical probing of allosteric control in nuclear receptors

The dynamical behavior of the nuclear receptor LXR/RXR heterodimer was investigated with molecular dynamics simulations. The simulations reveal correlated motion between residues across the dimer interface that depends significantly on occupation of the ligand binding sites of the monomers. These results are broadly consistent with the observed experimental behavior of the dimers, where structural perturbation is thought to be a key element in signal transduction. Our results provide dynamical support for this model of allosteric control.     

Activation of synaptic receptors and its allosteric regulation

This review considers activation of synaptic receptors (SR) as a process of transduction of the energy of interaction between an agonist (A) and an agonist-recognizing module (R) toward an ion-conducting module (channel,Ch) orG protein within respective complexes (ARCh orARG). The agonist provides functional and, in the case of metabotropic SR, spatial association of protein components in these complexes. Conformational transformations of the latter, which are sufficient for dissociation ofG protein into subunits or opening of an ion-conducting channel, sometimes need additional energy expenditure. The source for this energy can be interaction with one more ligand: guanosine-5′-triphosphate (in the case of metabotropic SR) or glycine (in case of NMDA receptors), which act as co-agonists. The activation of SR is regulated by their allosteric modulators, which affect either the kinetics of interaction between the agonist and receptor by changing the rate of dissociation with the agonist or the kinetics of conformational transitions of the components into the activated or desensitized state.     

Fcgamma receptors as regulators of immune responses

In addition to their role in binding antigen, antibodies can regulate immune responses through interacting with Fc receptors (FcRs). In recent years, significant progress has been made in understanding the mechanisms that regulate the activity of IgG antibodies in vivo. In this Review, we discuss recent studies addressing the multifaceted roles of FcRs for IgG (FcgammaRs) in the immune system and how this knowledge could be translated into novel therapeutic strategies to treat human autoimmune, infectious or malignant diseases.     

The role of endogenous regulators in the compensatory process after salivary gland resection

The ways of penetration of submandibular salivary degeneration products into the blood after partial gland resection have been studied on 97 rats. Using isoenzyme LDH spectrum of the blood, as well as of gland and regional lymph node tissue, the acid-base balance values and morphological data, it has been shown that degeneration products of salivary glands penetrate into the general blood flow through regional lymph nodes, where they are partly modified. The true changes in LDH spectrum can be already registered half an hour after the injury of the gland. The influence of the degeneration products of gland tissue on the initiation of the compensation processes in cellular hyperplasia is discussed.     

The molecular and physiological aspects of endothelial dysfunction. The role of endogenous chemical regulators

Monolayer of endothelial cells that cover the vascular channels are the major regulator of haemo-vascular homeostasis. Endothelium secretes the chemical factors that affect contraction of the muscular vascular cells, permeability of tissue, blood fluidity, intercellular interaction in vascular structure of the channel as a whole and of different regions. In its turn, the secretory function of endothelial cells is stimulated by mechanical or hormonal factors under a feedback system principle. Special features of morphology and biochemistry of vascular endothelium cells determine the micro-organs heterogeneity of the vascular channel depending on phenotine, gene expression, size and growth of endothelium cells. On this basis the processing biochemical disintegration develop either selectively or in a generalised form, and results in development of endothelial dysfunctions, as the original factor of many cardiovascular pathologies. Endothelial disfunction is a systemic pathology related to pathology of microstructure and hormonal function of endothelial cells representing a major tissue system of the vascular channel. Formation of hypertension states, ischemic cardiopathology, haemostasis changes, metabolic pathology (hypercholesterinemia and hyperglycemia) that lead to pathogenesis of arteriosclerosis, diabetes (etc.) as result of modified function of endothelium, and above all, pathology of production by dilator and constrictor substances, and the factors regulating interaction of endothelium with blood cells. The basic mechanism for development of the endothelial dysfunction is related to modification of synthesis and releasing of nitrogen oxide, a key regulator of the endothelial-vasal system. Physiologically active peptides (angiotensin II, endothelin-I, bradykinin, adrenomedullin and ANP) contribute to development of the processes related to the endothelium function and dysfunction. An important role is played, apparently, by growth peptide factors and specific proteins of cellular adhesion and membrane interaction--to integrins and selectins.     

The impact of cryo-EM on determining allosteric modulator-bound structures of G protein-coupled receptors

G-protein coupled receptors (GPCRs) are important therapeutic targets for the treatment of human disease. Although GPCRs are highly successful drug targets, there are many challenges associated with the discovery and translation of small molecule ligands that target the endogenous ligand-binding site for GPCRs. Allosteric modulators are a class of ligands that target alternative binding sites known as allosteric sites and offer fresh opportunities for the development of new therapeutics. However, only a few allosteric modulators have been approved as drugs. Advances in GPCR structural biology enabled by the cryogenic electron microscopy (cryo-EM) revolution have provided new insights into the molecular mechanism and binding location of small molecule allosteric modulators. This review highlights the latest findings from allosteric modulator-bound structures of Class A, B, and C GPCRs with a focus on small molecule ligands. Emerging methods that will facilitate cryo-EM structures of more difficult ligand-bound GPCR complexes are also discussed. The results of these studies are anticipated to aid future structure-based drug discovery efforts across many different GPCRs.     

Astrocytes are endogenous regulators of basal transmission at central synapses

Basal synaptic transmission involves the release of neurotransmitters at individual synapses in response to a single action potential. Recent discoveries show that astrocytes modulate the activity of neuronal networks upon sustained and intense synaptic activity. However, their ability to regulate basal synaptic transmission remains ill defined and controversial. Here, we show that astrocytes in the hippocampal CA1 region detect synaptic activity induced by single-synaptic stimulation. Astrocyte activation occurs at functional compartments found along astrocytic processes and involves metabotropic glutamate subtype 5 receptors. In response, astrocytes increase basal synaptic transmission, as revealed by the blockade of their activity with a Ca(2+) chelator. Astrocytic modulation of basal synaptic transmission is mediated by the release of purines and the activation of presynaptic A(2A) receptors by adenosine. Our work uncovers an essential role for astrocytes in the regulation of elementary synaptic communication and provides insight into fundamental aspects of brain function.     

Low-molecular regulators of polypeptide hormone receptors containing LGR-repeats

During the last years the low-molecular non-peptidic regulators of the polypeptide hormone receptors containing LGR-repeats (LGR-receptors) were identified. The review summarizes and systematizes data on structure and molecular mechanisms realizing the effects of such regulators as agonists and antagonists of the luteinizing, follicle-stimulating and thyroid-stimulating hormones. The regulators interact with the serpentine domain of LGR-receptors and trigger the receptor-coupled signaling cascades. Low-molecular agonists and antagonists of the LGR-receptors are considered as a new generation of drugs that can demonstrate highly sensitive and selective regulation of the functional activity of signaling systems sensitive to pituitary glycoprotein hormones. These regulators are more available than these hormones and can be used orally.     

Changes in endogenous growth regulators in nasturtium leaves during senescence

Summary By use of lettuce-hypocotyl and wheat-coleoptile bioassay, the presence of both gibberellin (GA)-like and abscisic-acid(ABA)-like components in acidic ethyl-acetate extracts of fully expanded nasturtium (Tropaeolum majus) leaves has been shown. During senescence of detached leaves there was a progressive decline in GA-like components and an increase in ABA-like components. Pretreatment of detached leaves with GA₃ or kinetin prevented changes in the levels of endogenous growth regulators and delayed senescence. The observations provide experimental verification for the concept that senescence is associated with changes in endogenous growth regulators.     

Gibberellins as endogenous growth regulators in green and brown algae

Summary Gibberellin-like activity was detected in extracts from Enteromorpha prolifera (Chlorophyta) and Ecklonia radiata (Phaeophyta). Material eluted from chromatograms of extracts and active in dwarf maize also brought about growth responses in the species from which the extract had been made. The responses were similar to those brought about by gibberellic acid. It is concluded that gibberellins are normally involved in regulating growth in green and brown algae.     

Enhancer and competitive allosteric modulation model for G-protein-coupled receptors

A new mathematical model, referred to as Enhancer and Competitive Allosteric Modulator (ECAM) model, developed with the aim of quantitatively describing the interaction of an allosteric modulator with both enhancer and competitive properties towards G-protein-coupled receptors is described here. Model simulations for equilibrium (displacement-like and saturation-like), and kinetic (association and dissociation) binding experiments were performed. The results showed the ability of the model to interpret a number of possible ligand-receptor binding behaviors. In particular, the binding properties of PD81723, an enhancer and competitive allosteric modulator for the adenosine A₁ receptor, were experimentally evaluated by radioligand binding assays and interpreted by the ECAM model. The results also offer a theoretical background enabling the design and optimization of compounds endowed with allosteric enhancer, competitive, agonist, antagonist, and inverse agonist properties.