RÉSUMÉ
Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.
Sujet(s)
Maladie d'Alzheimer/traitement médicamenteux , Conception de médicament , Récepteur muscarinique de type M1/agonistes , Sujet âgé , Sujet âgé de 80 ans ou plus , Vieillissement/anatomopathologie , Maladie d'Alzheimer/complications , Maladie d'Alzheimer/imagerie diagnostique , Maladie d'Alzheimer/anatomopathologie , Séquence d'acides aminés , Animaux , Pression sanguine/effets des médicaments et des substances chimiques , Cellules CHO , Anticholinestérasiques/pharmacologie , Cricetulus , Cristallisation , Modèles animaux de maladie humaine , Chiens , Donépézil/pharmacologie , Électroencéphalographie , Femelle , Cellules HEK293 , Rythme cardiaque/effets des médicaments et des substances chimiques , Humains , Mâle , Souris de lignée C57BL , Modèles moléculaires , Simulation de dynamique moléculaire , Dégénérescence nerveuse/complications , Dégénérescence nerveuse/anatomopathologie , Primates , Rats , Récepteur muscarinique de type M1/composition chimique , Transduction du signal , Similitude structurale de protéinesRÉSUMÉ
Acetylcholine release in the hippocampus plays a central role in the formation of new memory representations. An influential but largely untested theory proposes that memory formation requires acetylcholine to enhance responses in CA1 to new sensory information from entorhinal cortex whilst depressing inputs from previously encoded representations in CA3. Here, we show that excitatory inputs from entorhinal cortex and CA3 are depressed equally by synaptic release of acetylcholine in CA1. However, feedforward inhibition from entorhinal cortex exhibits greater depression than CA3 resulting in a selective enhancement of excitatory-inhibitory balance and CA1 activation by entorhinal inputs. Entorhinal and CA3 pathways engage different feedforward interneuron subpopulations and cholinergic modulation of presynaptic function is mediated differentially by muscarinic M3 and M4 receptors, respectively. Thus, our data support a role and mechanisms for acetylcholine to prioritise novel information inputs to CA1 during memory formation.
Sujet(s)
Acétylcholine/métabolisme , Région CA1 de l'hippocampe/physiologie , Cortex entorhinal/physiologie , Potentiels post-synaptiques excitateurs/physiologie , Rétrocontrôle physiologique/physiologie , Transmission synaptique/physiologie , Animaux , Région CA1 de l'hippocampe/cytologie , Carbachol/pharmacologie , Agonistes cholinergiques/pharmacologie , Cortex entorhinal/cytologie , Potentiels post-synaptiques excitateurs/effets des médicaments et des substances chimiques , Rétrocontrôle physiologique/effets des médicaments et des substances chimiques , Interneurones/métabolisme , Interneurones/physiologie , Mâle , Souris de lignée C57BL , Souris knockout , Techniques de patch-clamp , Cellules pyramidales/métabolisme , Cellules pyramidales/physiologie , Récepteur muscarinique de type M3/génétique , Récepteur muscarinique de type M3/métabolisme , Transmission synaptique/effets des médicaments et des substances chimiquesRÉSUMÉ
The identification of cannabinoid ligands Cannabidiol and O-1918 as inverse agonists of the orphan receptor GPR52 is reported. Detailed characterisation of GPR52 pharmacology and modelling of the proposed receptor interaction is described. The identification of a novel and further CNS pharmacology for the polypharmacological agent and marketed drug Cannabidiol is noteworthy.
RÉSUMÉ
The metabotropic glutamate (mGlu) receptors are a family of eight class C G protein-coupled receptors (GPCRs) which modulate cell signaling and synaptic transmission to the major excitatory neurotransmitter l-glutamate (l-glutamic acid). Due to their role in modulating glutamate response, their widespread distribution in the central nervous system (CNS) and some evidence of dysregulation in disease, the mGlu receptors have become attractive pharmacological targets. As the orthosteric (glutamate) binding site is highly conserved across the eight mGlu receptors, it is difficult not only to generate ligands with subtype selectivity but, due to the nature of the binding site, with suitable drug-like properties to allow oral bioavailability and CNS penetration. Selective pharmacological targeting of a single receptor subtype can be achieved by targeting alternative (allosteric) binding sites. The nature of the allosteric binding pockets allows ligands to be developed that have good physical chemical properties as evidenced by several allosteric modulators of mGlu receptors entering clinical trials. The first negative allosteric modulators of the metabotropic glutamate 5 (mGlu5) receptor were discovered from high throughput screening activities. An alternative approach to drug discovery is to use structural knowledge to enable structure-based drug design (SBDD), which allows the design of molecules in a more rational, rather than empirical, fashion. Here we will describe the process of SBDD in the discovery of the mGlu5 negative allosteric modulator HTL0014242 and describe how knowledge of receptor structure can also be used to gain insights into the receptor activation mechanisms.
Sujet(s)
Découverte de médicament , Récepteur-5 métabotropique du glutamate/composition chimique , Récepteur-5 métabotropique du glutamate/métabolisme , Régulation allostérique , Site allostérique , Animaux , Humains , Thérapie moléculaire ciblée , Récepteur-5 métabotropique du glutamate/génétique , Relation structure-activitéRÉSUMÉ
The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.
Sujet(s)
Antagonistes des récepteurs des orexines/métabolisme , Récepteurs des orexines/métabolisme , Sites de fixation , Simulation numérique , Cristallographie aux rayons X , Cellules HEK293 , Humains , Liaison hydrogène , Interactions hydrophobes et hydrophiles , Ligands , Antagonistes des récepteurs des orexines/composition chimique , Récepteurs des orexines/composition chimiqueRÉSUMÉ
The following chapter examines some of the current "state-of-the-art" tools for predicting, scoring, and examining explicit water molecules in proteins and protein/ligand complexes, highlighting some of the ways information can be readily examined in a manner that is useful in a drug discovery process.
Sujet(s)
Modèles moléculaires , Récepteurs couplés aux protéines G/composition chimique , Eau/composition chimique , Algorithmes , Découverte de médicament/méthodes , Simulation de docking moléculaire , Simulation de dynamique moléculaire , Liaison aux protéines , Relation quantitative structure-activité , LogicielRÉSUMÉ
Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.
Sujet(s)
Récepteur de type PAR-2/composition chimique , Récepteur de type PAR-2/métabolisme , Régulation allostérique/effets des médicaments et des substances chimiques , Site allostérique/effets des médicaments et des substances chimiques , Anticorps bloquants/composition chimique , Anticorps bloquants/pharmacologie , Benzimidazoles/composition chimique , Benzimidazoles/pharmacologie , Benzodioxoles/composition chimique , Benzodioxoles/pharmacologie , Alcools benzyliques/composition chimique , Alcools benzyliques/pharmacologie , Cristallographie aux rayons X , Humains , Imidazoles/composition chimique , Imidazoles/pharmacologie , Fragments Fab d'immunoglobuline/composition chimique , Fragments Fab d'immunoglobuline/pharmacologie , Cinétique , Ligands , Modèles moléculaires , Récepteur de type PAR-2/antagonistes et inhibiteurs , Transduction du signal/effets des médicaments et des substances chimiquesRÉSUMÉ
Class C G protein-coupled receptors encompass a range of promising therapeutic targets for a variety of diseases, yet to date only two members of this sub-family of GPCRs have been drugged. Recent advances in structural biology have revealed the X-ray crystallographic structures of allosteric ligands bound to two Class C metabotropic glutamate (mGlu) receptors, mGlu1 and mGlu5. Herein, we review how this information can be leveraged to help understand some of the historical challenges of mGlu receptor allosteric modulator drug discovery, and discuss how the structural enablement can be prospectively used for structurebased drug discovery approaches across Class C GPCR targets in general.
Sujet(s)
Conception de médicament , Récepteurs couplés aux protéines G/effets des médicaments et des substances chimiques , Régulation allostérique , Cristallographie aux rayons X , Conformation des protéines , Récepteurs couplés aux protéines G/composition chimique , Récepteurs couplés aux protéines G/métabolismeRÉSUMÉ
Recent developments in receptor stabilisation have facilitated major advances in G protein-coupled receptor (GPCR) research, notably structural biology, over the past eight years. Here we review the application of fragment, structure and biophysical techniques using stabilised GPCRs (StaR proteins), and their impact in the drug discovery process. These techniques have, most recently, been utilised in the discovery of the non-alkyne mGlu5 negative allosteric modulator HTL14242, in addition to the dual orexin receptor antagonist HTL6641, with differentiated residence time kinetics.
Sujet(s)
Conception de médicament , Découverte de médicament/méthodes , Récepteurs couplés aux protéines G/composition chimique , Régulation allostérique/effets des médicaments et des substances chimiques , Humains , Antagonistes des récepteurs des orexines/pharmacologie , Conformation des protéines , Stabilité protéique , Pyridines/pharmacologie , Pyrimidines/pharmacologie , Récepteur-5 métabotropique du glutamate/effets des médicaments et des substances chimiques , Récepteur-5 métabotropique du glutamate/métabolisme , Récepteurs couplés aux protéines G/métabolisme , TempératureRÉSUMÉ
Virtual screening is routinely used to discover new ligands and in particular new ligand chemotypes for G protein-coupled receptors (GPCRs). To prepare for a virtual screen, we often tailor a docking protocol that will enable us to select the best candidates for further screening. To aid this, we created GPCR-Bench, a publically available docking benchmarking set in the spirit of the DUD and DUD-E reference data sets for validation studies, containing 25 nonredundant high-resolution GPCR costructures with an accompanying set of diverse ligands and computational decoy molecules for each target. Benchmarking sets are often used to compare docking protocols; however, it is important to evaluate docking methods not by "retrospective" hit rates but by the actual likelihood that they will produce novel prospective hits. Therefore, docking protocols must not only rank active molecules highly but also produce good poses that a chemist will select for purchase and screening. Currently, no simple objective machine-scriptable function exists that can do this; instead, docking hit lists must be subjectively examined in a consistent way to compare between docking methods. We present here a case study highlighting considerations we feel are of importance when evaluating a method, intended to be useful as a practitioners' guide.
Sujet(s)
Simulation de docking moléculaire , Récepteurs A2 à l'adénosine/composition chimique , Récepteurs A2 à l'adénosine/métabolisme , Référenciation , Bases de données de protéines , Évaluation préclinique de médicament , Ligands , Conformation des protéines , Interface utilisateur , Eau/métabolismeRÉSUMÉ
The metabotropic glutamate receptors have a wide range of modulatory functions in the central nervous system. They are among the most highly pursued drug targets, with relevance for several neurological diseases, and a number of allosteric modulators have entered clinical trials. However, so far this has not led to a marketed drug, largely because of the difficulties in achieving subtype-selective compounds with desired properties. Very recently the first crystal structures were published for the transmembrane domain of two metabotropic glutamate receptors in complex with negative allosteric modulators. In this analysis, we make the first comprehensive structural comparison of all metabotropic glutamate receptors, placing selective negative allosteric modulators and critical mutants into the detailed context of the receptor binding sites. A better understanding of how the different mGlu allosteric modulator binding modes relates to selective pharmacological actions will be very valuable for rational design of safer drugs.
Sujet(s)
Ligands , Mutation , Récepteurs métabotropes au glutamate/composition chimique , Récepteurs métabotropes au glutamate/génétique , Régulation allostérique , Site allostérique , Sites de fixation , Humains , Modèles moléculaires , Conformation moléculaire , Liaison aux protéines , Récepteurs métabotropes au glutamate/métabolisme , Relation structure-activitéRÉSUMÉ
Fragment screening of a thermostabilized mGlu5 receptor using a high-concentration radioligand binding assay enabled the identification of moderate affinity, high ligand efficiency (LE) pyrimidine hit 5. Subsequent optimization using structure-based drug discovery methods led to the selection of 25, HTL14242, as an advanced lead compound for further development. Structures of the stabilized mGlu5 receptor complexed with 25 and another molecule in the series, 14, were determined at resolutions of 2.6 and 3.1 Å, respectively.
Sujet(s)
Pyridines/synthèse chimique , Pyridines/pharmacologie , Pyrimidines/synthèse chimique , Pyrimidines/pharmacologie , Récepteur-5 métabotropique du glutamate/effets des médicaments et des substances chimiques , Récepteurs couplés aux protéines G/effets des médicaments et des substances chimiques , Régulation allostérique , Animaux , Cellules Caco-2 , Chiens , Conception de médicament , Découverte de médicament , Cellules HEK293 , Humains , Ligands , Modèles moléculaires , Conformation moléculaire , Pyridines/pharmacocinétique , Pyrimidines/pharmacocinétique , Rats , Relation structure-activitéRÉSUMÉ
Class B GPCRs of the secretin family are important drug targets in many human diseases including diabetes, neurodegeneration, cardiovascular disease and psychiatric disorders. X-ray crystal structures for the glucagon receptor and corticotropin-releasing factor receptor 1 have now been published. In this review, we analyse the new structures and how they compare with each other and with Class A and F receptors. We also consider the differences in druggability and possible similarity in the activation mechanisms. Finally, we discuss the potential for the design of small-molecule modulators for these important targets in drug discovery. This new structural insight allows, for the first time, structure-based drug design methods to be applied to Class B GPCRs.
Sujet(s)
Conception de médicament , Récepteurs couplés aux protéines G/composition chimique , Récepteur hormone gastrointestinale/composition chimique , Animaux , Cristallographie aux rayons X , Découverte de médicament/méthodes , Humains , Thérapie moléculaire ciblée , Conformation des protéines , Récepteur CRH/composition chimique , Récepteurs couplés aux protéines G/effets des médicaments et des substances chimiques , Récepteurs couplés aux protéines G/métabolisme , Récepteur hormone gastrointestinale/effets des médicaments et des substances chimiques , Récepteur hormone gastrointestinale/métabolisme , Récepteurs au glucagon/composition chimiqueRÉSUMÉ
Several new pairs of active and inactive GPCR structures have recently been solved enabling detailed structural insight into the activation process, not only of rhodopsin but now also of the ß2 adrenergic, M2 muscarinic and adenosine A2A receptors. Combined with structural analyses they have enabled us to examine the different recent theories proposed for GPCR activation and show that they are all indeed parts of the same process, and are intrinsically related through their effect on the central hydrophobic core of GPCRs. This new unifying general process of activation is consistent with the identification of known constitutively active mutants and an in-depth conservational analysis of significant residues implicated in the process.
Sujet(s)
Récepteurs couplés aux protéines G/physiologie , Animaux , Humains , Interactions hydrophobes et hydrophiles , Récepteurs couplés aux protéines G/composition chimique , Relation structure-activitéRÉSUMÉ
Biophysical fragment screening of a thermostabilized ß1-adrenergic receptor (ß1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein-ligand crystal structures of the ß1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized ß1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively.
Sujet(s)
Phénomènes biophysiques , Conception de médicament , Pipérazines/composition chimique , Pipérazines/métabolisme , Récepteurs bêta-1 adrénergiques/métabolisme , Évaluation préclinique de médicament , Cellules HEK293 , Humains , Simulation de docking moléculaire , Pipérazine , Liaison aux protéines , Conformation des protéines , Récepteurs bêta-1 adrénergiques/composition chimique , Résonance plasmonique de surfaceRÉSUMÉ
Using isolated receptor conformations crystal structures of the adenosine A2A receptor have been solved in active and inactive states. Studying the change in affinity of ligands at these conformations allowed qualitative prediction of compound efficacy in vitro in a system-independent manner. Agonist 5'-N-ethylcarboxamidoadenosine displayed a clear preference to bind to the active state receptor; inverse agonists (xanthine amine congener, ZM241385, SCH58261, and preladenant) bound preferentially to the inactive state, whereas neutral antagonists (theophylline, caffeine, and istradefylline) demonstrated equal affinity for active and inactive states. Ligand docking into the known crystal structures of the A2A receptor rationalized the pharmacology observed; inverse agonists, unlike neutral antagonists, cannot be accommodated within the agonist-binding site of the receptor. The availability of isolated receptor conformations opens the door to the concept of "reverse pharmacology" whereby the functional pharmacology of ligands can be characterized in a system-independent manner by their affinity for a pair (or set) of G protein-coupled receptor conformations.
Sujet(s)
Agonistes des récepteurs A2 à l'adénosine/pharmacologie , Antagonistes des récepteurs A2 à l'adénosine/pharmacologie , Récepteur A2A à l'adénosine/composition chimique , Récepteur A2A à l'adénosine/métabolisme , Animaux , Sites de fixation , Cellules CHO , Lignée cellulaire , Cricetinae , Ligands , Conformation moléculaire , Récepteurs couplés aux protéines G/métabolisme , Relation structure-activitéRÉSUMÉ
Potent, ligand efficient, selective, and orally efficacious 1,2,4-triazine derivatives have been identified using structure based drug design approaches as antagonists of the adenosine A(2A) receptor. The X-ray crystal structures of compounds 4e and 4g bound to the GPCR illustrate that the molecules bind deeply inside the orthosteric binding cavity. In vivo pharmacokinetic and efficacy data for compound 4k are presented, demonstrating the potential of this series of compounds for the treatment of Parkinson's disease.
Sujet(s)
Antagonistes des récepteurs A2 à l'adénosine/synthèse chimique , Antiparkinsoniens/synthèse chimique , Pyridines/synthèse chimique , Récepteur A2A à l'adénosine/métabolisme , Triazines/synthèse chimique , Antagonistes des récepteurs A2 à l'adénosine/pharmacocinétique , Antagonistes des récepteurs A2 à l'adénosine/pharmacologie , Administration par voie orale , Animaux , Antiparkinsoniens/pharmacocinétique , Antiparkinsoniens/pharmacologie , Cristallographie aux rayons X , Conception de médicament , Humains , Modèles moléculaires , Conformation des protéines , Pyridines/pharmacocinétique , Pyridines/pharmacologie , Dosage par compétition , Rats , Relation structure-activité , Résonance plasmonique de surface , Triazines/pharmacocinétique , Triazines/pharmacologieRÉSUMÉ
A new approach to generating information on ligand receptor interactions within the binding pocket of G protein-coupled receptors has been developed, called Biophysical Mapping (BPM). Starting from a stabilized receptor (StaR), minimally engineered for thermostability, additional single mutations are then added at positions that could be involved in small molecule interactions. The StaR and a panel of binding site mutants are captured onto Biacore chips to enable characterization of the binding of small molecule ligands using surface plasmon resonance (SPR) measurement. A matrix of binding data for a set of ligands versus each active site mutation is then generated, providing specific affinity and kinetic information (K(D), k(on), and k(off)) of receptor-ligand interactions. This data set, in combination with molecular modeling and docking, is used to map the small molecule binding site for each class of compounds. Taken together, the many constraints provided by these data identify key protein-ligand interactions and allow the shape of the site to be refined to produce a high quality three-dimensional picture of ligand binding, thereby facilitating structure based drug design. Results of biophysical mapping of the adenosine A(2A) receptor are presented.
Sujet(s)
Antagonistes des récepteurs A2 à l'adénosine/composition chimique , Conception de médicament , Modèles moléculaires , Récepteur A2A à l'adénosine/composition chimique , Sites de fixation/génétique , Ligands , Mutation , Pyrimidines/composition chimique , Récepteur A2A à l'adénosine/génétique , Bibliothèques de petites molécules , Triazines/composition chimique , Triazoles/composition chimique , Xanthines/composition chimiqueRÉSUMÉ
The antiviral treatment of chronic hepatitis B is limited by the selection of antiviral resistance mutations. Primary resistance to lamivudine occurs at rtM2041/V in the C Domain of the polymerase. Recently, resistance to adefovir has also been described in the D Domain at rtN236T. The treatment of patients with resistant virus without complete suppression can lead to the further selection of compensatory mutations. Thus, to gain an understanding of the hepatitis B virus (HBV) polymerase and also mutations associated with resistance, a three-dimensional model of the HBV reverse transcriptase core region based on homology with human immunodeficiency virus (HIV) was created. A comparative analysis of the HIV polymerase and the model of HBV polymerase was performed. In addition, the antiviral resistance mutations including potential compensatory mutations were mapped to determine their effect on the HBV polymerase model, especially in the nucleotide binding site.