30 research outputs found
G-protein coupled receptors activation mechanism: from ligand binding to the transmission of the signal inside the cell
G-protein coupled receptors (GPCRs) are the largest family of pharmaceutical drug targets in the human genome and are modulated by a large variety of en- dogenous and synthetic ligands. GPCRs activation usually depends on agonist binding (except for receptors with basal activity), which stabilizes receptor con- formations and allow the requirement and activation of intracellular transducers. GPCRs are unique receptors and very well studied, since they play an important role in a great number of diseases. They interact with different type of ligands (such as light, peptides, proteins) and different partners in the intracellular part (such as G-proteins or β-arrestins). Based on homology and function GPCRs are divided in five classes: Class A or Rhodopsin, Class B1 or Secretin, Class B2 or Adhesion, Class C or Glutamate, Class F or Frizzled. What is still missing in the state of the art of these receptor, and in particular in Class A, is a global study on different binding cavities with divergent properties, with the aim to discover common binding characteristics, preserved during years of evolution. Gaining more knowledge on common features for ligand recognition shared among all the recep- tors may become crucial to deeply understand the mechanism used to transmit the signal into the cell. In the first step of this thesis we have used all the solved Class A receptors structures to analyze and find, if exist, a common way to transmit the signal inside the cell. We identified and validated ten positions shared between all the binding cavities and always involved in the interaction with ligands. We demonstrated that residues in these positions are conserved and have co-evolved together. In a second step, we used these positions to understand how ligands could be positioned in the binding cavities of three study cases: Muscarinic receptors, Kisspeptin receptors and the GPR3 receptor. We did not have any experimental information a priori. We used homology modeling and docking techniques for the first two cases, adding molecular dynamics simulations in the third case. All the predictions and suggestions from the computational point of view, turned out to be very successful. In particular for the GPR3 receptor we were able to identify and validate by alanine-scanning mutagenesis the role of three functionally relevant residues. The latter were correlated with the constitutive and agonist-stimulated adenylate cyclase activity of GPR3 receptor. Taken together, these results suggest an important role of computational structural biology and pave the way of strong collaborations between computational and experimental researches
Novel Strategies for Drug Discovery Based on Intrinsically Disordered Proteins (IDPs)
Intrinsically disordered proteins (IDPs) are proteins that usually do not adopt well-defined native structures when isolated in solution under physiological conditions. Numerous IDPs have close relationships with human diseases such as tumor, Parkinson disease, Alzheimer disease, diabetes, and so on. These disease-associated IDPs commonly play principal roles in the disease-associated protein-protein interaction networks. Most of them in the disease datasets have more interactants and hence the size of the disease-associated IDPs interaction network is simultaneously increased. For example, the tumor suppressor protein p53 is an intrinsically disordered protein and also a hub protein in the p53 interaction network; Îą-synuclein, an intrinsically disordered protein involved in Parkinson diseases, is also a hub of the protein network. The disease-associated IDPs may provide potential targets for drugs modulating protein-protein interaction networks. Therefore, novel strategies for drug discovery based on IDPs are in the ascendant. It is dependent on the features of IDPs to develop the novel strategies. It is found out that IDPs have unique structural features such as high flexibility and random coil-like conformations which enable them to participate in both the âone to manyâ and âmany to oneâ interaction. Accordingly, in order to promote novel strategies for drug discovery, it is essential that more and more features of IDPs are revealed by experimental and computing methods
Selective inhibitors of the PSEN1-gamma-secretase complex
Clinical development of Y-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimerâs disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific Y-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1âAPH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different Y-secretase complexes might provide safer drugs in the future.The work was supported by an AIO-project (no. HBC.2016.0884). This project received funding from the European Research Council (ERC) under the European Unionâs Horizon 2020 Research and Innovation Programme (grant agreement no. ERC-834682 CELLPHASE_AD). This work was supported by the Flanders Institute for Biotechnology (VIB vzw), a Methusalem grant from KU Leuven and the Flemish Government, the Fonds voor Wetenschappelijk Onderzoek, KU Leuven, The Queen Elisabeth Medical Foundation for Neurosciences, the Opening the Future campaign of the Leuven Universitair Fonds, the Belgian Alzheimer Research Foundation (SAO-FRA), and the Alzheimerâs Association
USA.Peer ReviewedPostprint (published version
Selective inhibitors of the PSEN1âgamma-secretase complex
Clinical development of Îł-secretases, a family of intramembrane cleaving proteases, as therapeutic targets for a variety of disorders including cancer and Alzheimerâs disease was aborted because of serious mechanism-based side effects in the phase III trials of unselective inhibitors. Selective inhibition of specific Îł-secretase complexes, containing either PSEN1 or PSEN2 as the catalytic subunit and APH1A or APH1B as supporting subunits, does provide a feasible therapeutic window in preclinical models of these disorders. We explore here the pharmacophoric features required for PSEN1 versus PSEN2 selective inhibition. We synthesized a series of brain penetrant 2-azabicyclo[2,2,2]octane sulfonamides and identified a compound with low nanomolar potency and high selectivity (>250-fold) toward the PSEN1âAPH1B subcomplex versus PSEN2 subcomplexes. We used modeling and site-directed mutagenesis to identify critical amino acids along the entry part of this inhibitor into the catalytic site of PSEN1. Specific targeting one of the different Îł-secretase complexes might provide safer drugs in the future
Establishing Research Competitiveness in Biophysical Sciences in Maine
The Maine EPSCoR Research Infrastructure Improvement award is designed to enhance Maine\u27s competitiveness in molecular biophysical sciences through a partnership between the University of Maine and Maine\u27s non-profit research organizations. The proposed Biophysical Sciences Institute brings together University of Maine faculty in physics, chemistry, biology, mathematics, and spatial engineering, with biomedical researchers at the Jackson Laboratory and Maine Medical Center Research Institute. Maine EPSCoR proposes to hire additional tenure-track faculty in the fields of biophysics and advanced optics, biochemistry, structural biology, applied mathematics, computer science, image analysis and visualization, and material science. The new and existing investigators will form research teams to develop new measurement techniques, new sensors, and innovative approaches to data processing and interpretation in intracellular structures and dynamics, functional materials as a means to manipulate cellular reactions, and biocomputing. In addition to establishing the institute, Maine EPSCoR will integrate research and education through improvements to graduate training
Discovery of First-in-Class Small Molecule Agonists of the RXFP2 Receptor as Therapeutic Candidates for Osteoporosis
Osteoporosis is a chronic bone disease characterized by decreased bone mass and increased risk of developing fractures, predominantly observed in the elderly. The pathophysiological cause of the disease is a decrease in the activity of the bone-forming cells (osteoblasts) that alters bone remodeling in favor of bone resorption, leading to a decrease in bone mass. Recent studies identified the relaxin family peptide receptor 2 (RXFP2), the G protein-coupled receptor (GPCR) for insulin-like 3 peptide (INSL3), as an attractive target expressed in osteoblast cells to increase bone formation. The goal of this dissertation is to discover and characterize small molecule agonists of RXFP2 that are stable and can be delivered orally to promote bone growth. Several low molecular weight compounds were identified as agonists of the RXFP2 receptor using a cAMP high-throughput screen of the NCATS small molecule library. An extensive structure-activity relationship campaign resulted in highly potent and efficient full RXFP2 agonists. The selectivity and specificity of these compounds for human and mouse RXFP2 was shown in counter-screens against the related relaxin receptor RXFP1 and other GPCRs. Using a series of RXFP2/RXFP1 chimeric receptors, in silico modeling and RXFP2 point mutants, we established that the compounds are allosteric agonists of the RXFP2 receptor and identified the GPCR transmembrane domains as the specific region for compound interaction. We also showed that the candidate compounds promoted mineralization in primary human osteoblasts and had low cytotoxicity in various cell types. The compound with the highest activity in vitro was selected for pharmacokinetics profiling in mice, showing oral bioavailability and bone exposure. Moreover, an efficacy study in wild-type female mice treated orally with the lead compound showed a significant increase of the vertebral trabecular number and thickness compared to vehicle treated controls. Overall, our study has successfully identified and characterized the first-in-class small molecule series of RXFP2 agonists, which may lead to the development of a new class of orally bioavailable drugs for the treatment of diseases associated with bone loss
Charakterisierung der Agonistenspezifität am Nucleotidrezeptors P2Y6
In der vorliegenden Arbeit wurde die bisher kaum in der Literatur beschriebene Stoffklasse der Prostaglandin-Glycerolester (PG-G) in Hinblick ihrer biochemischen Wirkmechanismen genauer charakterisiert. Einige Studien beschreiben PG-G als relevante Mediatoren im Rahmen sowohl inflammatorischer als auch nozizeptiver Prozesse. Dieser Sachverhalt macht diese Stoffklasse zum interessanten Gegenstand aktueller pharmakologischer Forschung, dessen Grundlage die Identifizierung und Charakterisierung entsprechender Rezeptoren darstellt.
Im Rahmen unserer Arbeit, identifizierten wir als ersten Schritt das Target des PGE2-G. Weder Prostaglandinrezeptoren noch bekannte orphane GPCR konnten durch die Stimulation mit PGE2-G aktiviert werden. Jedoch konnten in bestimmen Zelllinien wie RAW264.7 und H1819 Effekte durch PGE2-G erzielt werden, während andere Zelllinien keine Reaktionen zeigten. Dies machten wir uns zu Nutzen und sequenzierten das Transkriptom dieser Zelllinien, um so durch subtraktive Analysen Rezeptoren einzugrenzen, welche voraussichtlich als Target von PGE2-G fungieren. Als vielversprechender Kandidat exprimierten wir den UDP-Rezeptor P2Y6 heterolog in HEK293-Zellen und wiesen mittels Zellkultur-basierter Assays den Agonismus von PGE2-G am P2Y6 nach. Weitere Untersuchungen bestätigten diese hochspezifische Bindung zwischen beiden Interaktionspartnern. Auffällig war jedoch die ausordentlich hohe Potenz von PGE2-G, welche schon bei picomolaren Stoffkonzentrationen eine Aktivierung von P2Y6 initiiert. Insbesondere in Hinblick auf den deutlich hÜheren EC50-Wert von UDP stellt sich die Frage, inwiefern dies die Signaltransduktionsmechanismen und die damit einhergehenden physiologischen Effekte moduliert.
Es stellte sich weiterhin die Frage, wie der P2Y6 die Bindung zweier chemisch unterschiedlichen Substanzen realisieren kann. Ziel dieser Arbeit war es nun, die Struktur von P2Y6 auf molekularbiologischer Ebene zu untersuchen, um ein Verständnis ßber die Art und Weise der Bindung der unterschiedlichen Agonisten zu erhalten.:Entzßndungsreaktionen ................................................................................................................. 6
Prostaglandine ............................................................................................................................... 7
ProstaglandinâGlycerolester und die Identifizierung ihrer Rezeptoren ......................................... 9
ProstaglandinâGlycerolester ........................................................................................................9
GâProteinâgekoppelte Rezeptoren (GPCR) ............................................................................... 11
Orphane GPCR .......................................................................................................................... 12
Screening potentieller Targets von ProstaglandinâE2âGlycerolester ........................................ 13
Der Pyrimidinrezeptor P2Y6 .......................................................................................................... 14
Nucleotidrezeptoren ................................................................................................................ 14
Pharmakologische Bedeutung von Nucleotidrezeptoren ........................................................ 15
P2Y6 .......................................................................................................................................... 15
Fragestellung ................................................................................................................................ 18
Publikationen ................................................................................................................................... 20
Zusammenfassung der Ergebnisse und Diskussion .......................................................................... 50
ProstaglandinâE2âGlycerolester ist ein endogener Agonist am P2Y6 .......................................... 50
Die Bindungstasche des P2Y6 ....................................................................................................... 50
Evolutionäre Konservierung der AgonistenâPromiskuität........................................................ 50
Simulation der Ligandenbindung im Homologiemodell........................................................... 51
Experimentelle PrĂźfung der Interaktionspartner ......................................................................... 52
Interaktionspartner beider Agonisten: R103, Y107, R287 ....................................................... 53
Interaktionpartner mit UDP: Y262 ........................................................................................... 53
Interaktionspartner mit PGE2âG: Y75, N109, S291, N293 ........................................................ 53
Korrelation der experimentellen Daten mit einem Homologiemodell ........................................ 53
Ausblick ........................................................................................................................................ 54
Literatur ............................................................................................................................................ 56
Anhang ............................................................................................................................................. 62
Darstellung des eigenen Beitrags ..................................................................................................... 85
Erklärung ßber die eigenständige Abfassung der Arbeit ................................................................. 87
Lebenslauf ........................................................................................................................................ 88
Publikationen und Vorträge ............................................................................................................. 90
Danksagung ...................................................................................................................................... 9
Pharmacokinetics/Pharmacodynamics and Analysis of the Effect of β-Amyloid Peptide on Acetylcholine Neurocycle and Alzheimerâs Disease Medications
The brain of Alzheimerâs disease (AD) is characterized by accumulations of β-amyloid peptide aggregates which promote neurodegentartive dysfunction. Comprehensive understanding of the interaction between β-amyloid aggregates and acetylcholine (ACh) neurocycle is required to uncover the physiological processes related to AD and might result in improving therapeutic approaches for AD. Pharmacokinetics (PK) and pharmacodynamics (PD) techniques were applied to allow predicting the extent of the interaction of certain doses of AD drugs and β-amyloid inhibitors and levels of ACh as well. Although many researchers focused on the β-amyloid interactions, the mechanisms by which β-amyloid affects cholinergic neurons and reduction of ACh are still unclear. The prediction of ACh and drug concentrations in the tissues and body needs an understanding of the physiology and mechanisms of β-amyloid aggregates processes and their compilation into a mechanistic model
In this work, two hypotheses are proposed to investigate the dynamic behavior of the interaction between β-amyloid peptide aggregates and cholinergic neurocycle and the possible therapeutic approaches through proposing pharmacokinetic/pharmacodynamics (PK/PD) models to represent the impact of β-amyloid aggregates in AD. The effect of β-amyloid peptide aggregates is formulated through incorporating β- amyloid aggregates into non-linear model for the neurocycle of ACh where the presynaptic neuron is considered as compartment 1 and both synaptic cleft and postsynaptic neurons are considered as compartment 2. In the first hypothesis which is choline leakage hypothesis, β-amyloid peptide aggregates are considered to be located in the membrane of the presynaptic neuron and create pathways inside the membrane to allow for the intracellular choline to leak outside the cholinergic system. It is observed that β-amyloid aggregates via the choline leakage hypothesis could cause significant reductions of ACh and choline levels in both compartments. Furthermore, the process rates of ACh synthesis and hydrolysis have been affected negatively by a wide range of β-amyloid aggregate concentrations. It is found that as the input rate of β-amyloid aggregates to compartment 1 increases, the loss of choline from compartment 1 increases leading to an increase in the intracellular concentration of β-amyloid.
In the second hypothesis, β-amyloid peptide aggregates are proposed to interact with the enzyme ChAT which is responsible for the synthesis of ACh in compartment 1; three different kinetic mechanisms are suggested to account for the interaction between β-amyloid aggregates and ChAT activity. In the first and second kinetic mechanisms, β-amyloid aggregate is supposed to attack different species in the enzyme. It is found that there is a significant decrease in the rate of ACh synthesis in compartment 1 and ACh concentrations in both compartments. However, it is observed that there is no effect on choline levels in both compartments, the rate of ACh hydrolysis in compartment 2, pH, and ACh levels in compartment 2. In the third kinetic mechanism, all species in ChAT are attacked by β-amyloid aggregates; it is observed that at very high input rates of β-amyloid aggregates, the oscillatory behavior dominates all components of the neurocycle of ACh. The disturbance observed in ACh levels in both compartments explains the harmful effect of the full attack of β-amyloid aggregates to all species of ChAT. It is found that to contribute significantly in ACh neurocycle, choline leakage hypothesis needs concentration of β-amyloid aggregates lower than that needed in ChAT activity hypothesis which is in agreement with experimental observations. The significant decrease in ACh levels observed in both choline leakage and loss of ChAT activity hypotheses leads to cognitive loss and memory impairment which were observed in individuals with AD.
A one-compartment drug PK/PD model is proposed to investigate a therapeutic approach for inhibiting β-amyloid aggregation via choline leakage hypothesis where the maximum feed rate of β-amyloid (KL2 = 1) is considered. The drug is assumed to interact with the tissues of the presynaptic neurons where β-amyloid aggregates are located. The PK/PD model is built based on the effect of β-amyloid aggregates via choline leakage hypothesis where the maximum feed rate of β-amyloid aggregates is considered. The dynamic behavior of all concentrations of β-amyloid aggregates, choline, ACh, acetate, and pH in both compartments in addition to the rate of ACh synthesis in compartment 1 and ACh hydrolysis are investigated by monitoring the impacts of the drug on β-amyloid aggregates and cholinergic neurocycle over a wide range of the input drug dosage. The PK/PD model is able to predict the reduction in levels of β-amyloid aggregates and the increase in choline and ACh, in both compartments as well as both rates of ACh synthesis and hydrolysis catalyzed. The parameters of the PK/PD model such as maximum concentration (Cmax), maximum time (Tmax), area under the curve (AUC), and maximum effect (Emax) were investigated. It was found that it takes a longer time (Tmax) (3-5 h) to reach Emax as the drug dose increases. Furthermore, AUC was found to increase with increasing drug dosage. The results of the current work show that drugs / therapeutic agents inhibiting β- amyloid aggregation in the brain represent a likely successful therapeutic approach to give systematic highlights to develop future trials, new diagnostic techniques, and medications for AD. This study is helpful in designing PK and PD and developing experimental animal models to support AD drug development and therapy in the future
Theoretical study of the interaction of agonists with the 5-HT2A receptor
The 5-HT2A receptor (5-HT2AR) is a biogenic amine receptor that belongs to the class A of G protein coupled receptors. It is characterized by a low affinity for serotonin (5-HT) and for other primary amines. Introduction of an ortho-methoxybenzyl substituent at the amine nitrogen increases the partial agonistic activity by a factor of 40 to 1400 compared with 5-HT.
The present study was to analyse the QSAR of a series of 51 5-HT2AR partial agonistic arylethylamines, tested in vascular in-vitro assays on rats, at a structure-based level and to suggest ligand binding sites. The compounds belong to three different structural classes, (1) indoles, (2) methoxybenzenes and (3) quinazolinediones. Following a hierarchical strategy, different methods have been applied which all contribute to the investigation of ligand-receptor interactions: fragment regression analysis (FRA), receptor modeling, docking studies and 3D QSAR approaches (comparative molecular field analysis, CoMFA, and comparative molecular similarity index analysis, CoMSIA).
An initial FRA indicated that methoxy substituents at indole and phenyl derivatives increase the activity and may be involved in polar interactions with the 5-HT2AR. The large contribution of lipophilic substituents in p position of phenethylamines suggests fit to a specific hydrophobic pocket. Secondary benzylamines are more than one order of magnitude more active than their NH2 analogs. An ortho-OH or -OMe substituent at the benzyl moiety further increases activity.
Homology models of the human and rat 5-HT2AR were generated using the crystal structure of bovine rhodopsin and of the beta2-adrenoceptor as templates. The derivation of the putative binding sites for the arylethylamines was based on the results from FRA and on mutagenesis data. Both templates led to 5-HT2AR models with similar topology of the binding pocket within the transmembrane domains TM3, TM5, TM6 and TM7. Docking studies with representative members of the three structural classes suggested that the aryl moieties and particularly para-substituents in phenyl derivatives fit into a hydrophobic pocket formed by Phe2435.47, Phe2445.48 and Phe3406.52. The 5-methoxy substituents in indole and phenyl compounds form H bonds with Ser2395.43. In each case, an additional H bond with Ser1593.36 may be assumed. The cationic amine interacts with the conserved Asp1553.32. The benzyl group of secondary arylethylamines is inserted into another hydrophobic pocket formed by Phe3396.51, Trp3677.40 and Tyr3707.43. In this region, the docking poses depend on the template used for model generation, leading to different interactions especially of ortho- substituents.
The docking studies with the beta2-adrenoceptor based rat 5-HT2AR model provided templates for a structure-based alignment of the whole series which was used in 3D QSAR analyses of the partial agonistic activity. Both approaches, CoMFA and CoMSIA, led to highly predictive models with low complexity (cross-validated q2 of 0.72 and 0.81 at 4 and 3 components, respectively). The results were largely compatible with the binding site and confirm the docking studies and the suggested ligand-receptor interactions. Steric and hydrophobic field effects on the potency indicate a hydrophobic pocket around the aryl moiety and near the para position of phenyl derivatives and account for the increased activity of secondary benzylamines. The effects of electrostatic and H-bond acceptor fields suggest a favourable influence of negative charges around the aryl moiety, corresponding to the increase in potency caused by methoxy substituents in 2-, 4-, 5- and 6-position of phenethylamines and by the quinazolinedione oxygens. This is in accord with the role of Ser1593.36 and Ser2395.43 as H bond donors. At the benzyl moiety, the negative charge and the acceptor potential of 2-hydroxy and -methoxy substituents is of advantage.
Agonists stabilize or induce active receptor states not reflected by the existing crystal structures. Based on models of different rhodopsin states, a homology modeling and ligand docking study on corresponding 5-HT2AR states suggested to be specific to agonist and partial agonist binding, respectively, was performed. The models indicate collective conformational changes of TM domains during activation. The different 5-HT2AR states are similar with respect to the amino acids interacting with the arylethylamines, but show individual topologies of the binding sites. The interconversion of states by TM movements may be accompanied by co-translations and rotations of the ligands. In the case of the secondary amines considered, the tight fit of the benzyl substituent into a hydrophobic pocket containing key residues in TM6 probably impedes the complete receptor activation due to inhibiting the rotation of this helix. High affinity of a partial agonist is therefore often at the expense of its ability to fully activate a receptor
Building and refinement of an in silico homology model of a novel G protein-coupled receptor: GPR35
Human GPR35 (hGPR35), a recently deorphanized Class A G-protein coupled receptor, has been shown to exhibit prominent expression in immune and gastrointestinal tissues, with additional expression in pancreatic islets, skeletal muscle, lung tissue, and the dorsal root ganglion. The rat GPR35 (rGPR35) analog, which has 72% sequence identity with human GPR35, has been shown to have expression in similar tissues as with human GPR35. GPR35 has been suggested to be involved in metabolism, heart failure, inflammation, asthma, a mental retardation syndrome associated with the deletion on 2q37.3, type II diabetes, as well as gastric cancer formation, making GPR35 a potential target for the treatment of multiple diseases. Both zaprinast, the well characterized cGMP-PDE inhibitor, and pamoic acid, a compound which the FDA has classified as an inactive compound, act as agonists at GPR35. However, interesting species differences have been found with these agonists and key mutations have also revealed differences between these two ligands. Pamoic acid is considerably lower in potency in rat GPR35, while zaprinast has increased efficacy in rat GPR35. Further, mutation studies suggest an increase in the potency of zaprinast in a human GPR35 R6.58A mutation. Pamoic acid, on the other hand shows similar potency to wild-type in this same mutant. To probe the molecular origins of these differences, three separate homology models, an active (R*) hGPR35, an R* hGPR35 R6.58A(240) mutant, and an R* rGPR35 model, were constructed and docking studies were performed with the aforementioned ligands. These studies revealed that the change in residue 5.43 (P5.43 in human; S5.43 in rat) alters the shape of the binding pocket for pamoic acid. In addition, arginines which contribute significantly to the interaction of pamoic acid in hGPR35 (R6.58 and R7.32) become uncharged residues (Q6.58 and S7.32) in rat GPR35. The increase of the potency of zaprinast in the hGPR35 R6.58A mutant receptor is due to the loss of bulk at position 6.58 (R6.58(240)¨ A6.58(240)), that allows for additional interactions with the ligand. The statistically equivalent potencies of pamoic acid for the wild-type and R6.58A(240) mutant hGPR35 receptors is due to the isoenergetic interchange of the direct interaction residue R6.58(240) with R7.32(255) in the R6.58(240)A mutant