Structure-based design, synthesis and structure-activity relationships of dibenzosuberyl- and benzoate-substituted tropines as ligands for acetylcholine-binding protein

Using structure-based optimization procedures on in silico hits, dibenzosuberyl- and benzoate substituted tropines were designed as ligands for acetylcholine-binding protein (AChBP). This protein is a homolog to the ligand binding domain of the nicotinic acetylcholine receptor (nAChR). Distinct SAR is observed between two AChBP species variants and between the α7 and α4β2 nAChR subtype. The AChBP species differences are indicative of a difference in accessibility of a ligand-inducible subpocket. Hereby, we have identified a region that can be scrutinized to achieve selectivity for nicotinic receptor subtypes.     

Selectivity of Imidacloprid for fruit fly versus rat nicotinic acetylcholine receptors by molecular modeling

For better understanding of the mechanisms of selective binding of the representative nicotinic acetylcholine receptor (nAChR) agonist neonicotinoid Imidacloprid (IMI), three-dimensional models of fruit fly α1β2 and rat α4β2 nAChRs were generated by homology modeling, using the crystal structure of the acetylcholine-binding protein (AChBP) of Lymnaea stagnalis and the nAChR of mus musculus as the templates, respectively. The conformational stability of the two models was studied by molecular dynamics (MD) and the quality of the models was confirmed. Especially, insecticide Imidacloprid was docked into the putative binding site of the fruit fly α1β2 and rat α4β2 nAChRs by Surflex-docking. The calculated docking energies were in agreement with the experimental data and the putative binding sites were also consistent with the results from labeling and mutagenesis experiments. Furthermore, the mechanisms of Imidacloprid selectively acting on fruit fly versus rat nAChRs were discussed.     

Crystal Structure of Lymnaea stagnalis AChBP Complexed with the Potent nAChR Antagonist DHβE Suggests a Unique Mode of Antagonism

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that belong to the Cys-loop receptor superfamily. These receptors are allosteric proteins that exist in different conformational states, including resting (closed), activated (open), and desensitized (closed) states. The acetylcholine binding protein (AChBP) is a structural homologue of the extracellular ligand-binding domain of nAChRs. In previous studies, the degree of the C-loop radial extension of AChBP has been assigned to different conformational states of nAChRs. It has been suggested that a closed C-loop is preferred for the active conformation of nAChRs in complex with agonists whereas an open C-loop reflects an antagonist-bound (closed) state. In this work, we have determined the crystal structure of AChBP from the water snail Lymnaea stagnalis (Ls) in complex with dihydro-β-erythroidine (DHβE), which is a potent competitive antagonist of nAChRs. The structure reveals that binding of DHβE to AChBP imposes closure of the C-loop as agonists, but also a shift perpendicular to previously observed C-loop movements. These observations suggest that DHβE may antagonize the receptor via a different mechanism compared to prototypical antagonists and toxins.     

Identification, characterization, and epitope mapping of human monoclonal antibody J19 that specifically recognizes activated integrin α4β7

Integrin α(4)β(7) is a lymphocyte homing receptor that mediates both rolling and firm adhesion of lymphocytes on vascular endothelium, two of the critical steps in lymphocyte migration and tissue-specific homing. The rolling and firm adhesions of lymphocytes rely on the dynamic shift between the inactive and active states of integrin α(4)β(7), which is associated with the conformational rearrangement of integrin molecules. Activation-specific antibodies, which specifically recognize the activated integrins, have been used as powerful tools in integrin studies, whereas there is no well characterized activation-specific antibody to integrin α(4)β(7). Here, we report the identification, characterization, and epitope mapping of an activation-specific human mAb J19 against integrin α(4)β(7). J19 was discovered by screening a human single-chain variable fragment phage library using an activated α(4)β(7) mutant as target. J19 IgG specifically bound to the high affinity α(4)β(7) induced by Mn(2+), DTT, ADP, or CXCL12, but not to the low affinity integrin. Moreover, J19 IgG did not interfere with α(4)β(7)-MAdCAM-1 interaction. The epitope of J19 IgG was mapped to Ser-331, Ala-332, and Ala-333 of β(7) I domain and a seven-residue segment from 184 to 190 of α(4) β-propeller domain, which are buried in low affinity integrin with bent conformation and only exposed in the high affinity extended conformation. Taken together, J19 is a potentially powerful tool for both studies on α(4)β(7) activation mechanism and development of novel therapeutics targeting the activated lymphocyte expressing high affinity α(4)β(7).     

Unique assignment of inter-subunit association in GABAA α1β3γ2 receptors determined by molecular modeling

Recent publications defined requirements for inter-subunit contacts in a benzodiazepine-sensitive GABA_A receptor (GABA_ARα1β3γ2). There is strong evidence that the heteropentameric receptor contains two α1, two β3, and one γ2 subunit. However, the available data do not distinguish two possibilities: When viewed clockwise from an extracellular viewpoint the subunits could be arranged in either γ2β3α1β3α1 or γ2α1β3α1β3 configurations. Here we use molecular modeling to thread the relevant GABA_AR subunit sequences onto a template of homopentameric subunits in the crystal structure of the acetylcholine binding protein (AChBP). The GABA_A sequences are known to have 15-18% identity with the acetylcholine binding protein and nearly all residues that are conserved within the nAChR family are present in AChBP. The correctly aligned GABA_A sequences were threaded onto the AChBP template in the γ2β3α1β3α1 or γ2α1β3α1β3  arrangements. Only the γ2α1β3α1β3 arrangement satisfied three known criteria: (1) α1 His¹⁰² binds at the γ2 subunit interface in proximity to γ2 residues Thr¹⁴², Phe⁷⁷, and Met¹³⁰; (2) α1 residues 80-100 bind near γ2 residues 91-104; and (3) α1 residues 58-67 bind near the β3 subunit interface. In addition to predicting the most likely inter-subunit arrangement, the model predicts which residues form the GABA and benzodiazepine binding sites.     

Nicotinic receptor agonists and antagonists increase sAPPα secretion and decrease Aβ levels in vitro

We have earlier reported that Aβ were significantly reduced in brains of smoking Alzheimer patients and control subjects compared with non-smokers, as well as in nicotine treated APPsw transgenic mice. To examine the mechanisms by which nicotine modulates APP processing we here measured levels of secreted amyloid precursor protein (sAPPα), total sAPP, Aβ40 and Aβ42 in different cell lines expressing different nicotinic receptor (nAChR) subtypes or no nAChRs. Treatment with nicotine increased release of sAPPα and at the same time lowered Aβ levels in both SH-SY5Y and SH-SY5Y/APPsw cells expressing α3 and α7 nAChR subtypes. These effects could also be evoked by co-treatment with the competitive α7 nAChR antagonists α-bungarotoxin and methyllycaconitine (MLA), and by these antagonists alone, suggesting that binding to the agonist binding site, rather than activation of the receptor, may be sufficient to trigger changes in APP processing. The nicotine-induced increase in sAPPα could only be blocked by co-treatment with the open channel blocker mecamylamine. In addition to nicotine, the agonists epibatidine and cytisine both significantly increased the release of sAPP in M10 cells expressing the α4/β2 nAChR subtype, and this effect was blocked by co-treatment with mecamylamine but not by the α4/β2 competitive antagonist dihydro-β-erythroidine. The lack of effect of nicotine on sAPPα and Aβ levels in HEK 293/APPsw cells, which do not express any nAChRs, demonstrates that the nicotine-induced attenuation of β-amyloidosis is mediated by nAChRs and not by a direct effect of nicotine. Our data show that nicotinic compounds stimulate the non-amyloidogenic pathway and that α4 and α7 nAChRs play a major role in modulating this process. Nicotinic drugs directed towards specific nAChR subtypes might therefore be beneficial for the treatment of AD not only by lowering Aβ production but also by enhance release of neuroprotective sAPPα.     

In silico characterization of cytisinoids docked into an acetylcholine binding protein

Homology models of nicotinic acetylcholine receptors (nAChRs) suggest that subtype specificity is due to non-conserved residues in the complementary subunit of the ligand-binding pocket. Cytisine and its derivatives generally show a strong preference for heteromeric α4β21 nAChRs over the homomeric α7 subtype, and the structural modifications studied do not cause large changes in their nAChR subtype selectivity. In the present work we docked cytisine, N-methylcytisine, and several pyridone ring-substituted cytisinoids into the crystallographic structure of the Lymnaea stagnalis acetylcholine binding protein (AChBP) co-crystallized with nicotine (1UW6). The graphical analysis of the best poses showed that cytisinoids have weak interactions with the side chains of the non-conserved amino acids in the complementary subunit justifying the use of PDB 1UWB as a surrogate for nAChR. Furthermore, we found a high correlation (R² = 0.96) between the experimental pIC₅₀ values at α4β21 nAChR and docking energy (S) of the best cytisinoid poses within the AChBP. Due to the quality of the correlation we suggest that this equation might be used as a predictive model to propose new cytisine-derived nAChRs ligands. Our docking results also suggest that further structural modifications of these cytisinoids will not greatly alter their α4β21/α7 selectivity.     

Identification of novel α7 nicotinic receptor ligands by in silico screening against the crystal structure of a chimeric α7 receptor ligand binding domain

A hierarchical in silico screening procedure using the crystal structure of an agonist bound chimeric α7/Ls-AChBP protein was successfully applied to both proprietary and commercial databases containing drug-like molecules. An overall hit rate of 26% (pK_i ?5.0) was obtained, with an even better hit rate of 35% for the commercial compound collection. Structurally novel and diverse ligands were identified. Binding studies with [³H]epibatidine on chimeric α7/5-HT₃ receptors yielded submicromolar inhibition constants for identified hits. Compared to a previous screening procedure that utilized the wild type Ls-AChBP crystal structure, the current study shows that the recently obtained α7/Ls-AChBP chimeric protein crystal structure is a better template for the identification of novel α7 receptor ligands.     

Potent and novel 11β-HSD1 inhibitors identified from shape and docking based virtual screening

Several potent and novel 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) inhibitors were discovered from in silico screening the commercially available Maybridge database. Among them, seven hit compounds showed good affinity, with IC(50) values lower than 100 nM and the best one 3.7 nM. To select the lead for further optimization, computational ADME/T prediction, the CYP3A4 inhibition and 11β-HSD1 over 11β-HSD2 selectivity test were also performed. Taking all of the above factors into consideration, two promising compounds were selected as lead structures for further development. The employed hierarchical virtual screening protocol not only demonstrates its efficiency, but also provides novel and selective compounds for developing 11β-HSD1 inhibitors to protect against metabolic syndrome.     

Structural determinants for interaction of partial agonists with acetylcholine binding protein and neuronal α7 nicotinic acetylcholine receptor

The pentameric acetylcholine‐binding protein (AChBP) is a soluble surrogate of the ligand binding domain of nicotinic acetylcholine receptors. Agonists bind within a nest of aromatic side chains contributed by loops C and F on opposing faces of each subunit interface. Crystal structures of Aplysia AChBP bound with the agonist anabaseine, two partial agonists selectively activating the α7 receptor, 3‐(2,4‐dimethoxybenzylidene)‐anabaseine and its 4‐hydroxy metabolite, and an indole‐containing partial agonist, tropisetron, were solved at 2.7–1.75 Å resolution. All structures identify the Trp 147 carbonyl oxygen as the hydrogen bond acceptor for the agonist‐protonated nitrogen. In the partial agonist complexes, the benzylidene and indole substituent positions, dictated by tight interactions with loop F, preclude loop C from adopting the closed conformation seen for full agonists. Fluctuation in loop C position and duality in ligand binding orientations suggest molecular bases for partial agonism at full‐length receptors. This study, while pointing to loop F as a major determinant of receptor subtype selectivity, also identifies a new template region for designing α7‐selective partial agonists to treat cognitive deficits in mental and neurodegenerative disorders.     

Structural specializations of α(4)β(7), an integrin that mediates rolling adhesion

The lymphocyte homing receptor integrin α(4)β(7) is unusual for its ability to mediate both rolling and firm adhesion. α(4)β(1) and α(4)β(7) are targeted by therapeutics approved for multiple sclerosis and Crohn's disease. Here, we show by electron microscopy and crystallography how two therapeutic Fabs, a small molecule (RO0505376), and mucosal adhesion molecule-1 (MAdCAM-1) bind α(4)β(7). A long binding groove at the α(4)-β(7) interface for immunoglobulin superfamily domains differs in shape from integrin pockets that bind Arg-Gly-Asp motifs. RO0505376 mimics an Ile/Leu-Asp motif in α(4) ligands, and orients differently from Arg-Gly-Asp mimics. A novel auxiliary residue at the metal ion-dependent adhesion site in α(4)β(7) is essential for binding to MAdCAM-1 in Mg(2+) yet swings away when RO0505376 binds. A novel intermediate conformation of the α(4)β(7) headpiece binds MAdCAM-1 and supports rolling adhesion. Lack of induction of the open headpiece conformation by ligand binding enables rolling adhesion to persist until integrin activation is signaled.     

Inhibition mechanism of rat α₃β₄ nicotinic acetylcholine receptor by the Alzheimer therapeutic tacrine

Nicotinic acetylcholine receptors (nAChRs) were studied in detail in the past regarding their interaction with therapeutic and drug addiction related compounds. Using fast kinetic whole-cell recording, we have now studied effects of tacrine, an agent used clinically to treat Alzheimer's disease, on currents elicited by activation of rat α(3)β(4) nAChR heterologously expressed in KXα3β4R2 cells. Characterization of receptor activation by nicotine used as agonist revealed a K(d) of 23 ± 0.2 μM and 4.3 ± 1.3 for the channel opening equilibrium constant, Φ(-1). Experiments were performed to investigate whether tacrine is able to activate the α(3)β(4) nAChR. Tacrine did not activate whole-cell currents in KXα3β4R2 cells but inhibited receptor activity at submicromolar concentration. Dose-response curves obtained with increasing agonist or inhibitor concentration revealed competitive inhibition of nAChRs by tacrine, with an apparent inhibition constant, K(I), of 0.8 μM. The increase of Φ(-1) in the presence of tacrine suggests that the drug stabilizes a nonconducting open channel form of the receptor. Binding studies with TCP and MK-801 ruled out tacrine binding to common allosteric sites of the receptor. Our study suggests a novel mechanism for action of tacrine on nAChRs besides inhibition of acetylcholine esterase.     

Insight to the binding mode of triazole RGD-peptidomimetics to integrin-rich cancer cells by NMR and molecular modeling

The binding features of a novel class of ‘click chemistry’-derived RGD mimics with integrin ligand capability were studied toward α_vβ₃ integrin using STD-NMR techniques on intact integrin-rich ECV340 bladder cancer cell line. STD is useful to identify which moieties of the ligand are closest to the receptor in the bound state. The NMR data were integrated with competitive binding assays to the purified α_vβ₃ receptor and were interpreted with the aid of docking calculations. The involvement of the triazole hydrogen atom in the interaction with the receptor was evinced for all compounds but 2, in agreement with docking studies showing a certain proximity between triazole and Tyr178. Moreover, the interaction of the hydroxylated ligands with the receptor was not as extended as in the compounds belonging to the corresponding series, with the exception of compound 4 having 2-aminobenzimidazole as the arginine bioisostere, in agreement with biological assay results showing reduced binding capability for the hydroxylated peptidomimetics.     

Alpha-conotoxin OmIA is a potent ligand for the acetylcholine-binding protein as well as alpha3beta2 and alpha7 nicotinic acetylcholine receptors

The molluskan acetylcholine-binding protein (AChBP) is a homolog of the extracellular binding domain of the pentameric ligand-gated ion channel family. AChBP most closely resembles the alpha-subunit of nicotinic acetylcholine receptors and in particular the homomeric alpha7 nicotinic receptor. We report the isolation and characterization of an alpha-conotoxin that has the highest known affinity for the Lymnaea AChBP and also potently blocks the alpha7 nAChR subtype when expressed in Xenopus oocytes. Remarkably, the peptide also has high affinity for the alpha3beta2 nAChR indicating that alpha-conotoxin OmIA in combination with the AChBP may serve as a model system for understanding the binding determinants of alpha3beta2 nAChRs. alpha-Conotoxin OmIA was purified from the venom of Conus omaria. It is a 17-amino-acid, two-disulfide bridge peptide. The ligand is the first alpha-conotoxin with higher affinity for the closely related receptor subtypes, alpha3beta2 versus alpha6beta2, and selectively blocks these two subtypes when compared with alpha2beta2, alpha4beta2, and alpha1beta1deltaepsilon nAChRs.     

乙酰胆碱受体α4β2亚型在非洲爪蟾卵母细胞中的表达

通过建立乙酰胆碱受体α4β2亚型(α4β2 nAChR)在非洲爪蟾卵母细胞中的表达模型,以便以α4β2 nAChR为作用靶点进行药物筛选。将乙酰胆碱受体亚基基因α4、β2体外转录获得的cRNA,通过显微注射的方法注入非洲爪蟾卵母细胞,并对该受体的表达情况进行检测。结果显示,乙酰胆碱受体α4β2亚型在蛙卵细胞中获得了有效表达,记录到典型的ACh配体门控的离子通道内向电流。     

Contribution of nicotinic receptors to the function of synapses in the central nervous system: The action of choline as a selective agonist of α7 receptors

The α7-nicotinic receptor (nAChR)-selective agonist choline and nAChR-subtype-selective antagonists led to the discovery that activation of both α7 and α4β2 nAChRs located in CA1 interneurons in slices taken from the rat hippocampus facilitates the tetrodotoxin (TTX)-sensitive release of γ-aminobutyric acid (GABA). Experiments carried out in cultured hippocampal neurons not only confirmed that preterminal α7 and α4β2 nAChRs modulate the TTX-sensitive release of GABA, but also demonstrated that evoked release of GABA is reduced by rapid exposure of the neurons to acetylcholine (ACh, 10 μM-1 mM) in the presence of the muscarinic receptor antagonist atropine (1 μM). This effect of ACh, which is fully reversible and concentration-dependent, is partially blocked by superfusion of the cultured neurons with external solution containing either the α7-nAChR-selective antagonist methyllycaconitine (MLA, 1 nM) or the α4β2-nAChR-selective antagonist dihydro-β-erythroidine (DHβE, 100 nM). A complete blockade of ACh-induced reduction of evoked release of GABA was achieved only when the neurons were perfused with external solution containing both MLA and DHβE, suggesting that activation of both α7 and α4β2 nAChRs modulates the evoked release of GABA from hippocampal neurons. Such mechanisms may account for the apparent involvement of nAChRs in the psychological effects of tobacco smoking, in brain disorders (e.g., schizophrenia and epilepsy), and in physiological processes, including cognition and nociception.     

Irisin supports integrin-mediated cell adhesion of lymphocytes

Irisin, a myokine released from skeletal muscle, has recently been found to act as a ligand for the integrins αVβ5, αVβ1, and α5β1 expressed on mesenchymal cells, thereby playing an important role in the metabolic remodeling of the bone, skeletal muscle and adipose tissues. Although the immune-modulatory effects of irisin in chronic inflammation have been documented, its interactions with lymphocytic integrins have yet to be elucidated. Here, we show that irisin supports the cell adhesion of human and mouse lymphocytes. Cell adhesion assays using a panel of inhibitory antibodies to integrins have shown that irisin-mediated lymphocyte adhesion involves multiple integrins including not only α4β1 and α5β1, but also leukocyte-specific αLβ2 and α4β7. Importantly, mouse lymphocytic TK-1 cells that lack the expression of β1 integrins have exhibited αLβ2- and α4β7-mediated cell adhesion to irisin. Irisin has also been demonstrated to bind to purified recombinant integrin αLβ2 and α4β7 proteins. Thus, irisin represents a novel ligand for integrin αLβ2 and α4β7, capable of supporting lymphocyte cell adhesion independently of β1 integrins. These results suggest that irisin may play an important role in regulating lymphocyte adhesion and migration in the inflamed vasculature.     

NMR structures of the human α7 nAChR transmembrane domain and associated anesthetic binding sites

The α7 nicotinic acetylcholine receptor (nAChR), assembled as homomeric pentameric ligand-gated ion channels, is one of the most abundant nAChR subtypes in the brain. Despite its importance in memory, learning and cognition, no structure has been determined for the α7 nAChR TM domain, a target for allosteric modulators. Using solution state NMR, we determined the structure of the human α7 nAChR TM domain (PDB ID: 2MAW) and demonstrated that the α7 TM domain formed functional channels in Xenopus oocytes. We identified the associated binding sites for the anesthetics halothane and ketamine; the former cannot sensitively inhibit α7 function, but the latter can. The α7 TM domain folds into the expected four-helical bundle motif, but the intra-subunit cavity at the extracellular end of the α7 TM domain is smaller than the equivalent cavity in the α4β2 nAChRs (PDB IDs: 2LLY; 2LM2). Neither drug binds to the extracellular end of the α7 TM domain, but two halothane molecules or one ketamine molecule binds to the intracellular end of the α7 TM domain. Halothane and ketamine binding sites are partially overlapped. Ketamine, but not halothane, perturbed the α7 channel-gate residue L9′. Furthermore, halothane did not induce profound dynamics changes in the α7 channel as observed in α4β2. The study offers a novel high-resolution structure for the human α7 nAChR TM domain that is invaluable for developing α7-specific therapeutics. It also provides evidence to support the hypothesis: only when anesthetic binding perturbs the channel pore or alters the channel motion, can binding generate functional consequences.     

Synthesis, in vitro and in vivo evaluation of 2-aryl-4H-chromene and 3-aryl-1H-benzo[f]chromene derivatives as novel α-glucosidase inhibitors

Herein we report a study of novel arylchromene derivatives as analogs of naturally occurring flavonoids with prominent α-glucosidase inhibitory properties. Novel inhibitors were identified via simple stepwise in silico screening, efficient synthesis, and biological evaluation. It is shown that 2-aryl-4H-chromene core retains pharmacophore properties while being readily available synthetically. A lead compound identified through screening inhibits yeast α-glucosidase with IC₅₀ of 62.26?µM and prevents postprandial hyperglycemia in rats at 2.2?mg/kg dose.     

Role for E-cadherin as an inhibitory receptor on epidermal gammadelta T cells

E-cadherin is a homophilic adhesion molecule that maintains homotypic intercellular adhesion between epithelial cells such as epidermal keratinocytes. E-cadherin is also expressed on resident murine epidermal γδ T cells, known as dendritic epidermal T cells (DETCs), but they express another receptor for E-cadherin, α(E)(CD103)β(7) integrin, as well. In this study, we analyzed functional differences between E-cadherin-mediated homophilic binding and heterophilic binding of α(E)β(7) integrin to E-cadherin in heterotypic intercellular adhesion of DETCs to keratinocytes. E-cadherin, but not α(E)β(7) integrin, was downregulated on activation of DETCs in vivo and in vitro. Short-term (1-h) adhesion of DETCs to keratinocytes in vitro was primarily mediated by α(E)β(7) integrin, and blocking of the binding of α(E)β(7) integrin to E-cadherin inhibited the lysis of keratinocytes by DETCs. Stable binding of E-cadherin on DETCs to plate-bound recombinant E-cadherin was observed only after 24-h culture in vitro. Cytokine production and degranulation by DETCs in response to suboptimal TCR cross-linking and mitogen stimulation were augmented by coligation of α(E)β(7) integrin. In contrast, engagement of E-cadherin on DETCs with immobilized anti-E-cadherin Ab, plate-bound recombinant E-cadherin, and E-cadherin on keratinocytes inhibited DETC activation. Therefore, E-cadherin acts as an inhibitory receptor on DETCs, whereas α(E)β(7) integrin acts as a costimulatory receptor. Differential expression of E-cadherin and α(E)β(7) integrin on resting and activated DETCs, as well as their opposite functions in DETC activation, suggests that E-cadherin and α(E)β(7) integrin on DETCs regulate their activation threshold through binding to E-cadherin on keratinocytes.