Agonist Binding Properties Research Articles

New Glucosamine-Based TLR4 Agonists: Design, Synthesis, Mechanism of Action, and In Vivo Activity as Vaccine Adjuvants.

We disclose here a panel of small-molecule TLR4 agonists (the FP20 series) whose structure is derived from previously developed TLR4 ligands (FP18 series). The new molecules have increased chemical stability and a shorter, more efficient, and scalable synthesis. The FP20 series showed selective activity as TLR4 agonists with a potency similar to FP18. Interestingly, despite the chemical similarity with the FP18 series, FP20 showed a different mechanism of action and immunofluorescence microscopy showed no NF-κB nor p-IRF-3 nuclear translocation but rather MAPK and NLRP3-dependent inflammasome activation. The computational studies related a 3D shape of FP20 series with agonist binding properties inside the MD-2 pocket. FP20 displayed a CMC value lower than 5 μM in water, and small unilamellar vesicle (SUV) formation was observed in the biological activity concentration range. FP20 showed no toxicity in mouse vaccination experiments with OVA antigen and induced IgG production, thus indicating a promising adjuvant activity.

Small molecule somatostatin receptor subtype 4 (sst4) agonists are novel anti-inflammatory and analgesic drug candidates

We provided strong proof of concept evidence that somatostatin mediates potent analgesic and anti-inflammatory actions via its receptor subtype 4 (sst4) located both at the periphery and the central nervous system. Therefore, sst4 agonists are promising novel drug candidates for neuropathic pain and neurogenic inflammation, but rational drug design was not possible due to the lack of knowledge about its 3-dimensional structure.We modeled the sst4 receptor structure, described its agonist binding properties, and characterized the binding of our novel small molecule sst4 agonists (4-phenetylamino-7H-pyrrolo[2,3-d]pyrimidine derivatives) using an in silico platform. In addition to the in silico binding data, somatostatin displacement by Compound 1 was demonstrated in the competitive binding assay on sst4-expressing cells. In vivo effects were investigated in rat models of neurogenic inflammation and chronic traumatic neuropathic pain.We defined high- and low-affinity binding pockets of sst4 for our ligands, binding of the highest affinity compounds were similar to that of the reference ligand J-2156. We showed potent G-protein activation with the highest potency of 10 nM EC50 value and highest efficacy of 342%. Oral administration of 100 μg/kg of 5 compounds significantly inhibited acute neurogenic plasma protein extravasation in the paw skin by 40–60%, one candidate abolished and 3 others diminished sciatic nerve-ligation induced neuropathic hyperalgesia by 28–62%.The in silico predictions on sst4-ligands were tested in biological systems. Low oral dose of our novel agonists inhibit neurogenic inflammation and neuropathic pain, which opens promising drug developmental perspectives for these unmet medical need conditions.

Exploring the binding properties of agonists interacting with glucocorticoid receptor: an in silico approach.

The glucocorticoid receptors (GR) are members of the nuclear receptor superfamily that regulate growth, development, and many of the biological functions, including metabolism and inflammation, in a ligand dependent behavior. Thus, GRs are vital as therapeutic targets with steroid hormones and steroidal analogues, especially including the glucocorticoids. Studying the molecular mechanism of binding between GR and ligands is fundamentally important to develop applications in the pharmacological industry. The present study was carried out via molecular docking and molecular dynamic (MD) simulations of three GR-ligand complexes formed between the ligand binding domain (LBD) of GR with cortisol (a natural steroid), dexamethasone (a well-known synthetic steroid drug), and chonemorphine (a steroid virtually screened from the "Sri Lankan Flora" web-based information system). The investigation was mainly carried out in terms of macroscopic properties of the ligand-protein interactions and conformational fluctuations of the protein. The results indicated greater stability and a similar behavior of the GR protein in the chonemorphine-GR complex, compared to the other two complexes, GR-dexamethasone and GR-cortisol, in an aqueous medium. The integrity of the protein-substrate complexes was preserved by strong hydrogen bonds formed between the amino acid residues of the binding site of the proteins and ligands. The findings revealed that chonemorphine is a promising agonist to GR and may produce a pharmacological effect like that produced by glucocorticoids. Thus, the obtained knowledge could lead to further investigations of the pharmaceutical potential of chonemorphine and biological functions of GR in vivo.

A Small Molecule Mimetic of the Humanin Peptide as a Candidate for Modulating NMDA-Induced Neurotoxicity.

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aβ and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is reported to be primarily through its agonist binding properties to the gp130 receptor. However, the peptidic nature of HN presents challenges in its development as a therapeutic for AD. We report here for the first time the elucidation of the binding site of Humanin (HN) peptide to the gp130 receptor extracellular domain through modeling and the synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and provide protection against NMDA-induced neurotoxicity in primary hippocampal neurons. A brain permeable small molecule mimetic was identified through exploratory medicinal chemistry using microfluidic flow chemistry to facilitate the synthesis of new analogues for screening and SAR optimization.

An Unaltered Orthosteric Site and a Network of Long-Range Allosteric Interactions for PNU-120596 in α7 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptors (nAChRs) are vital toneuronal signaling, are implicated in important processes such as learning and memory, and are therapeutic targets for neural diseases. The α7 nAChR has been implicated in Alzheimer's disease and schizophrenia, and allosteric modulators have become one focus of drug development efforts. We investigate the mode of action of the α7-selective positive allosteric modulator, PNU-120596, and show that the higher potency of acetylcholine in the presence of PNU-120596 is not due to an altered agonist binding site. In addition, we propose several residues in the gating interface and transmembrane region that are functionally important to transduction of allosteric properties, and link PNU-120596, the acetylcholine binding region, and the receptor gate. These results suggest global protein stabilization from a communication network through several key residues that alter the gating equilibrium of the receptor while leaving the agonist binding properties unperturbed.

Exploring the binding properties of agonists interacting with human TGR5 using structural modeling, molecular docking and dynamics simulations

TGR5, act as a potential pharmacological target in the treatment of type II diabetes. In the computational study, structural modeling and binding site prediction of TGR5 receptor was performed. Two well-known agonists of TGR5 used to investigate the mode and mechanism of binding.

Molecular Simulations of Muscle AChR Agonist Binding Sites

We are attempting to design and control the agonist binding properties of muscle acetylcholine receptors (AChRs). These hetero-pentamers have 2 transmitter binding sites in the extracellular domain, at α-δ and either α-γ (fetal) or α-e (adult) subunit interfaces. We used computational modeling and MD simulation to investigate the structural dynamics and energetics of these 3 different binding interfaces. Starting from an Aplysia AChBP crystal structure (PDB id 2BYQ), we used homology modeling to build all-atom dimer and pentamer models of the extracellular domain, and docked acetylcholine (ACh) as the ligand. MD simulations (Charmm27) were run to obtain binding free energies at all 3 interfaces. The α-δ and α-e sites were found to be approximately equivalent, but α-γ provided ∼33% more favorable binding energy, which is in good agreement with experimental, single-channel electrophysiology results (Nayak and Auerbach, PNAS 2013). In the model, the packing of five aromatic residues is tighter, the tryptophan residues 149 and 55 are more orthogonal and loop C less is flexible at α-γ compared to the others. The dimer and pentamer models gave similar binding energies, supporting the experimental observation that the agonist sites behave independently. We are using the model as a tool, to identify residues in the e and δ subunits that are responsible for the lower ACh binding energy and, hence, to guide the wet-bench experiments. We targeted nine amino acids that are homologous in δ and e but different in γ, within 20 A of the quaternary ammonium of ACh. In the pentamer simulations, swapping all of these between α-δ and α-γ resulted in a full switch of the structures and energies. We are testing various subsets of these mutations in order to suggest a minimum construct that can be verified by using electrophysiology.

Discovery of a Novel Selective PPARγ Ligand with Partial Agonist Binding Properties by Integrated in Silico/in Vitro Work Flow

Full agonists to the peroxisome proliferator-activated receptor (PPAR)γ, such as Rosiglitazone, have been associated with a series of undesired side effects, such as weight gain, fluid retention, cardiac hypertrophy, and hepatotoxicity. Nevertheless, PPARγ is involved in the expression of genes that control glucose and lipid metabolism and is an important target for drugs against type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In an effort to identify novel PPARγ ligands with an improved pharmacological profile, emphasis has shifted to selective ligands with partial agonist binding properties. Toward this end we applied an integrated in silico/in vitro workflow, based on pharmacophore- and structure-based virtual screening of the ZINC library, coupled with competitive binding and transactivation assays, and adipocyte differentiation and gene expression studies. Hit compound 9 was identified as the most potent ligand (IC50 = 0.3 μM) and a relatively poor inducer of adipocyte differentiation. The binding mode of compound 9 was confirmed by molecular dynamics simulation, and the calculated free energy of binding was -8.4 kcal/mol. A novel functional group, the carbonitrile group, was identified to be a key substituent in the ligand-protein interactions. Further studies on the transcriptional regulation properties of compound 9 revealed a gene regulatory profile that was to a large extent unique, however functionally closer to that of a partial agonist.

Exploring Binding Properties of Agonists Interacting with a δ-Opioid Receptor

Ligand-receptor interactions are at the basis of the mediation of our physiological responses to a large variety of ligands, such as hormones, neurotransmitters and environmental stimulants, and their tuning represents the goal of a large variety of therapies. Several molecular details of these interactions are still largely unknown. In an effort to shed some light on this important issue, we performed a computational study on the interaction of two related compounds differing by a single methyl group (clozapine and desmethylclozapine) with a -opioid receptor. According to experiments, desmethylclozapine is more active than clozapine, providing a system well suited for a comparative study. We investigated stable configurations of the two drugs inside the receptor by simulating their escape routes by molecular dynamics simulations. Our results point out that the action of the compounds might be related to the spatial and temporal distribution of the affinity sites they visit during their permanency. Moreover, no particularly pronounced structural perturbations of the receptor were detected during the simulations, reinforcing the idea of a strong dynamical character of the interaction process, with an important role played by the solvent in addition.

Radiosynthesis and biological evaluation of the M1 muscarinic acetylcholine receptor agonist ligand [11C]AF150(S)

Purpose M1 muscarinic acetylcholine receptor (M1ACh-R) agonists are potential therapeutics for cognitive disorders. The aim of this study was to develop an orthosteric agonist positron emission tomography (PET) ligand for the M1ACh-R that would provide a tool to explore the active G-protein coupled receptor. To that extend, the radiosynthesis, receptor binding in vitro, and brain uptake of [11C]AF150(S), a selective M1ACh-R agonist, were investigated. Procedures [11C]AF150(S) was synthesized, its binding properties were investigated using autoradiography on rat brain sections, and brain uptake was determined in biodistribution studies ex vivo in rats. Metabolites in brain and blood were measured using high-performance liquid chromatography. Results Yield of the [11C]AF150(S) radiosynthesis was 69 ± 9% with radiochemical purity >99%. The logDoct,7.4 was 0.05. Autoradiography showed binding in M1ACh-R rich brain areas and selective inhibition by muscarinic agents in conditions promoting agonist binding. Biodistribution studies revealed rapid and high brain uptake, to levels exceeding five times the blood level. In M1ACh-R rich areas, specific uptake versus cerebellum was apparent, remaining constant over 60 min in spite of rapid decline of radioactivity from the brain and rapid peripheral metabolism. Brain uptake of [11C]AF150(S) may involve facilitated transport. Conclusion [11C]AF150(S) was successfully synthesized, showed M1ACh-R agonist binding properties, and high brain uptake. It provides an interesting lead for the development of an M1ACh-R agonist PET ligand meant to explore the active receptor state. Copyright © 2012 John Wiley & Sons, Ltd.

Substitutions of amino acids in the pore domain of homomeric α7 nicotinic receptors for analogous residues present in heteromeric receptors modify gating, rectification and binding properties

We have studied the role of different amino acids in the M2 transmembrane domain of the α7 neuronal nicotinic receptor by mutating residues that differ from the ones located at the same positions in other α (α2-α10) or β (β2-β4) subunits. Our aim was to investigate the contribution of these amino acids to the peculiar kinetic and inward rectification properties that differentiate the homomeric α7 receptor from other nicotinic receptors. Mutations of several residues strongly modified receptor function. We found that Thr245 had the most profound effect when mutated to serine, an amino acid present in all heteromeric receptors composed of α and β subunits, by dramatically increasing the maximal current, decreasing the decaying rate of the currents and decreasing receptor rectification. Some mutants also showed altered agonist-binding properties as revealed by shifts in the dose-response curves for acetylcholine. We conclude that residues in the M2 segment and flanking regions contribute to the unusual properties of the α7 receptor, especially to its characteristic fast kinetic behavior and strong inward rectification and furthermore to the potency of agonists.

Computational analysis of ligand recognition sites of homo- and heteropentameric 5-HT 3 receptors

Inhibition of the 5-hydroxytryptamine receptor (5-HT 3R), a member of the Cys-loop superfamily of Ligand-Gated Ion Channels (LGICs), has been recognized to have important antiemetic effects. With respect to the many other drugs already in use, such as the first generation 5-HT 3R antagonist granisetron, palonosetron, a second generation antagonist, clearly demonstrates superior inhibition potency towards the 5-HT 3Rs. Five different receptor monomers, the 5-HT 3R A–E, have been identified although the A and B subunits are the only known to build functional receptors, the homopentameric 5-HT 3AR and the heteropentameric 5-HT 3B–AR (with BBABA subunit arrangement). At present, however, no three-dimensional structure has been reported for any of the 5-HT 3R subunits. To understand the binding properties of agonists and antagonists, models of the extracellular portion of the 5-HT 3R A and B subunits are built and assembled into the receptor (homo- and hetero-) pentameric structure on the basis of the known three-dimensional structure of the nicotinic-acetylcholine receptor (nACh-R). The results of docking studies of the natural agonist serotonin and the antagonists palonosetron and granisetron into the modelled homomeric and heteromeric 5-HT 3R binding interfaces, provide a possible rationalization both of the higher potency of palonosetron with respect to other antagonists, and of its previously reported allosteric binding and positive cooperativity properties.

Roles of Tyrosine322 and Tyrosine324 in GnRH Receptor Function

GnRH receptor binding properties and cAMP signaling were analyzed in GT1‐7 cells expressing the endogenous GnRH‐R, and in COS‐7 cells transfected with the mouse GnRH‐R. Agonist stimulation of the wild type GnRH‐R expressed in COS‐7 cells using a pGFP2‐N vector caused a monotonic dose‐dependent increase in cAMP production. Deletion of Tyr322, or change of Tyr322 to Ser, significantly increased GnRH binding capacity of the mutant receptor (3‐5 fold). In such receptors, GnRH‐induced cAMP production was abolished, indicating the absence of Gs activation. GnRH‐activated phosphorylation of mitogen‐activated protein kinases (MAPKs) was also abolished in the mutant receptor, consistent with impairment of the protein kinase C (PKC)‐dependent signaling pathway. The GnRH‐R binding capacity was abolished when Tyr322 was substituted with Phe. Substitution of Tyr324 of the mouse GnRH‐R with non‐aromatic hydroxyl group‐containing Ser caused loss of agonist binding. In contrast, when Tyr324 was replaced with Phe the mutant receptor retained high binding capacity and cAMP signaling comparable to that of the wild type GnRH‐R. These findings demonstrate the importance of both Tyr322 and Tyr324 of the mouse GnRH receptor as crucial residues that determine its agonist binding and signaling properties.

Inhibition of the Dopamine D1 Receptor Signaling by PSD-95

Dopamine D1 receptors play an important role in movement, reward, and learning and are implicated in a number of neurological and psychiatric disorders. These receptors are concentrated in dendritic spines of neurons, including the spine head and the postsynaptic density. D1 within spines is thought to modulate the local channels and receptors to control the excitability and synaptic properties of spines. The molecular mechanisms mediating D1 trafficking, anchorage, and function in spines remain elusive. Here we show that the synaptic scaffolding protein PSD-95 thought to play a role in stabilizing glutamate receptors in the postsynaptic density, interacts with D1 and regulates its trafficking and function. Interestingly, the D1-PSD-95 interaction does not require the well characterized domains of PSD-95 but is mediated by the carboxyl-terminal tail of D1 and the NH(2) terminus of PSD-95, a region that is recognized only recently to participate in protein-protein interaction. Co-expression of PSD-95 with D1 in mammalian cells inhibits the D1-mediated cAMP accumulation without altering the total expression level or the agonist binding properties of the receptor. The diminished D1 signaling is mediated by reduced D1 expression at the cell surface as a consequence of an enhanced constitutive, dynamin-dependent endocytosis. In addition, genetically engineered mice lacking PSD-95 show a heightened behavioral response to either a D1 agonist or the psychostimulant amphetamine. These studies demonstrate a role for a glutamatergic scaffold in dopamine receptor signaling and trafficking and identify a new potential target for the modulation of abnormal dopaminergic function.

On high- and low-affinity agonist sites in GABAA receptors.

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.

The noncompetitive inhibitor quinacrine modifies the desensitization kinetics of muscle acetylcholine receptors.

Quinacrine has been shown to act as a noncompetitive inhibitor of the nicotinic acetylcholine receptor (nAChR). However, its mechanism of action is still a matter of controversy. We analyzed in detail the action of quinacrine at both the single-channel and macroscopic current levels. The main effect of quinacrine is a profound concentration-dependent decrease in both the frequency of opening events and the duration of clusters elicited by high acetylcholine concentrations. Quinacrine also significantly increases (40-fold at 30 microM) the decay rate of macroscopic currents elicited by rapid perfusion of acetylcholine to outside-out patches. This decay is still well-described by a single exponential. Quinacrine has very little effect on the peak amplitude of the response, suggesting that it acts mainly on open channels. The recovery from desensitization after removal of acetylcholine is delayed in the presence of quinacrine. Results from both single-channel and macroscopic current recordings indicate that quinacrine increases the rate of nAChR desensitization and stabilizes the desensitized state. Interestingly, in equilibrium agonist-binding assays, quinacrine does not promote the typical high-affinity desensitized state. Thus, quinacrine seems to induce an intermediate state exhibiting the permeability but not the agonist binding properties of desensitization.

Prolonged effects of intraventricular galanin on a 5-hydroxytryptamine 1A receptor mediated function in the rat

Galanin (3 nmol/rat), 2 h after its intracerebroventricular (i.c.v.) administration to male rats, attenuated the passive avoidance (PA) retention deficit induced by the 5-hydroxytryptamine (HT) 1A receptor agonist 8-hydroxy-2-(di- N-propylamino)tetraline (8-OH-DPAT) (0.2 mg/kg) The reduction in the postjunctional 5-HT 1A receptor-mediated response after i.c.v. galanin was not associated with changes in the mRNA levels and agonist binding properties of cortical limbic 5-HT 1A receptors, believed to be the target receptors mediating the PA deficit caused by 8-OH-DPAT. These results suggest that acute increases of galanin transmission in vivo even after 2 h can counteract limbic 5-HT 1A receptor-mediated responses of relevance for affective disorders without significantly affecting gene expression and binding characteristics of cortical limbic 5-HT 1A receptors.

Binding Properties of Agonists and Antagonists to Distinct Allosteric States of the Nicotinic Acetylcholine Receptor Are Incompatible with a Concerted Model

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde, which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low affinity to a conformation with high affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model, including the postulate of a preformed equilibrium between the different states of the nAChR in the absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.

Neuronal Nicotinic Receptor β2 and β4 Subunits Confer Large Differences in Agonist Binding Affinity

We used equilibrium binding analysis to characterize the agonist binding properties of six different rat neuronal nicotinic receptor subunit combinations expressed in Xenopus laevis oocytes. The alpha4beta2 receptor bound [3H]cytisine with a Kdapp of 0.74 +/- 0. 14 nM. The rank order of Kiapp values of additional nicotinic ligands, determined in competition assays, was cytisine < nicotine < acetylcholine < carbachol < curare. These pharmacological properties of alpha4beta2 expressed in oocytes are comparable to published values for the high affinity cytisine binding site in rat brain (alpha4beta2), demonstrating that rat neuronal nicotinic receptors expressed in X. laevis oocytes display appropriate pharmacological properties. Use of [3H]epibatidine allowed detailed characterization of multiple neuronal nicotinic receptor subunit combinations. Kdapp values for [3H]epibatidine binding were 10 pM for alpha2beta2, 87 pM for alpha2beta4, 14 pM for alpha3beta2, 300 pM for alpha3beta4, 30 pM for alpha4beta2, and 85 pM for alpha4beta4. Affinities for six additional agonists (acetylcholine, anabasine, cytisine, 1, 1-dimethyl-4-phenylpiperazinium, lobeline, and nicotine) were determined in competition assays. The beta2-containing receptors had consistently higher affinities for these agonists than did beta4-containing receptors. Particularly striking examples are the affinities displayed by alpha2beta2 and alpha2beta4, which differ in 1,1-dimethyl-4-phenylpiperazinium, nicotine, lobeline, and acetylcholine affinity by 120-, 86-, 85-, and 61-fold, respectively. Although smaller differences in affinity could be ascribed to different alpha subunits, the major factor in determining agonist affinity was the nature of the beta subunit.

Prostacyclin receptor desensitization is a reversible phenomenon in human platelets.

Long-term exposure of platelets to endogenous or exogenous prostacyclin or its analogues might result in desensitization of the platelet prostacyclin receptor in vitro and in vivo accompanied by a loss in receptor density on the platelet surface and a reduced sensitivity toward the inhibitory effects of prostacyclins. However, the reversibility of this process in platelets has not yet been investigated. Human platelets desensitized by the chemically stable prostacyclin analogue iloprost showed a significant reduction in [3H]-iloprost binding sites that was reversed by saponin permeabilization. This indicates functionally active internalized prostacyclin receptors. To assess whether the internalized prostacyclin receptors recycle to the cell surface after withdrawal of the agonist, iloprost sensitivity and prostacyclin receptor binding properties of iloprost (30 nmol/L, 2 hours) desensitized platelets incubated in iloprost-free autologous plasma were investigated. While desensitized platelets showed a significant increase in IC50 for iloprost inhibition of thrombin-induced platelet aggregation, serotonin release, and p-selectin expression and a reduced iloprost-stimulated cAMP formation, platelet iloprost sensitivity was restored 3 hours after iloprost withdrawal. In addition, the significant reduction in Bmax and the increase in K(D) of prostacyclin receptors in desensitized platelets as revealed by [3H]-iloprost binding studies also returned to the initial values. These results indicate that prostacyclin receptors internalized during short-term desensitization are not degraded but can be recycled rapidly to the platelet surface in a functionally active form after withdrawal of the agonist.