Search

[Image: see text] Allosteric coupling describes a reciprocal process whereby G-protein-coupled receptors (GPCRs) relay ligand-induced conformational changes from the extracellular binding pocket to the intracellular signaling surface. Therefore, GPCR activation is sensitive to both the type of extracellular ligand and intracellular signaling protein. We hypothesized that ligand-specific allosteric coupling may result in preferential (i.e., biased) engagement of downstream effectors. However, the structural basis underlying ligand-dependent control of this essential allosteric mechanism is poorly understood. Here, we show that two sets of extended muscarinic acetylcholine receptor M(1) agonists, which only differ in linker length, progressively constrain receptor signaling. We demonstrate that stepwise shortening of their chemical linker gradually hampers binding pocket closure, resulting in divergent coupling to distinct G-protein families. Our data provide an experimental strategy for the design of ligands with selective G-protein recognition and reveal a potentially general mechanism of ligand-specific allosteric coupling.

Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the

Background: Glioblastoma multiforme (GBM) is characterized by heterogeneous cell populations. Among these, the Glioblastoma Stem Cells (GSCs) fraction shares some similarities with Neural Stem Cells. GSCs exhibit enhanced resistance to conventional chemotherapy drugs. Our previous studies demonstrated that the activation of M2 muscarinic acetylcholine receptors (mAChRs) negatively modulates GSCs proliferation and survival. The aim of the present study was to analyze the ability of the M2 dualsteric agonist Iper-8-naphthalimide (N-8-Iper) to counteract GSCs drug resistance. Methods: Chemosensitivity to M2 dualsteric agonist N-8-Iper and chemotherapy drugs such as temozolomide, doxorubicin, or cisplatin was evaluated in vitro by MTT assay in two different GSC lines. Drug efflux pumps expression was evaluated by RT-PCR and qRT-PCR. Results: By using sub-toxic concentrations of N-8-Iper combined with the individual chemotherapeutic agents, we found that only low doses of the M2 agonist combined with doxorubicin or cisplatin or temozolomide were significantly able to counteract cell growth in both GSC lines. Moreover, we evaluated as the exposure to high and low doses of N-8-Iper downregulated the ATP-binding cassette (ABC) drug efflux pumps expression levels. Conclusions: Our results revealed the ability of the investigated M2 agonist to counteract drug resistance in two GSC lines, at least partially by downregulating the ABC drug efflux pumps expression. The combined effects of low doses of conventional chemotherapy and M2 agonists may thus represent a novel promising pharmacological approach to impair the GSC-drug resistance in the GBM therapy.

Marina Carini and Marco De Amici, Guest Editors of this Special Collection dedicated to NMMC 2019 and the 40th Anniversary of the DCF-SCI, look back at key events in the Italian medicinal chemistry community within the past year and introduce the collection.

α7 nicotinic acetylcholine receptors (nAChRs) are relevant therapeutic targets for a variety of disorders including neurodegeneration, cognitive impairment, and inflammation. Although traditionally identified as an ionotropic receptor, the α7 subtype showed metabotropic-like functions, mainly linked to the modulation of immune responses. In the present work, we investigated the structure-activity relationships in a set of novel α7 ligands incorporating the 5-(quinuclidin-3-ylmethyl)-1,2,4-oxadiazole scaffold, i.e. derivatives 21a-34a and 21b-34b, aiming to identify the structural requirements able to preferentially trigger one of the two activation modes of this receptor subtype. The new compounds were characterized as partial and silent α7 nAChR agonists in electrophysiological assays, which allowed to assess the contribution of the different groups towards the final pharmacological profile. Overall, modifications of the selected structural backbone mainly afforded partial agonists, among them tertiary bases 27a-33a, whereas additional hydrogen-bond acceptor groups in permanently charged ligands, such as 29b and 31b, favored a silent desensitizing profile at the α7 nAChR.

In previous studies, we found that the orthosteric muscarinic agonist arecaidine propargyl ester (APE) (100 μM) induced a decreased cell proliferation and severe apoptosis in glioblastoma cancer stem cells (GSCs). In this report, we have investigated the effects mediated by hybrid (orthosteric/allosteric) muscarinic agonists P-6-Iper and N-8-Iper on GSCs survival. At variance with APE, the agonist N-8-Iper inhibited cell growth in a dose dependent manner and also impaired cell survival at low doses. The inhibitory effects of the N-8-Iper action appear to be mediated by M2 receptor activation, since they were strongly reduced by co-administration of the selective M2 receptor antagonist methoctramine as well as upon M2 receptor silencing. Moreover, analysis of the expression of phosphorylated histone H2AX (γ-H2AX) indicated that the treatment with N-8-Iper produced a decreased cell survival by induction of DNA damage. The ability of N-8-Iper to produce a cytotoxic effect and apoptosis at low doses indicates that this muscarinic agonist is a suitable probe in a putative therapeutic intervention on glioblastoma through M2 receptor activation.

(±)-3-Methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene sesquifumarate (±)-1 was previously characterized as the most selective agonist at α7 neuronal nicotinic acetylcholine receptors in a series of spirocyclic quinuclidinyl-Δ2-isoxazoline derivatives. In this study, we performed different in vitro biological assays aimed at characterizing the ADMET properties of (±)-1. Then, we tested the compound in vivo in behavioral studies including classical novel object recognition and inhibitory avoidance tests in the rat, and a spatial memory assay in zebrafish involving a rapid T-maze task. The results indicated an overall favorable profile for (±)-1 in view of potential therapeutic applications targeting the central nervous system.

Due to the physiological properties of l -carnosine ( l -1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the 13C-labeled carnosine analogs and their application as internal standards for the quantitation of carnosine and anserine in a biological matrix.

Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor.

A set of hybrid compounds composed of the fragment of allosteric modulators of the muscarinic receptor, i.e. W84 and naphmethonium, and the well-known AChE inhibitor tacrine on the one hand, and the skeletons of the orthosteric muscarinic agonists, iperoxo and isox, on the other hand, were synthesized. The two molecular moieties were connected via a polymethylene linker of varying length. These bipharmacophoric compounds were investigated for inhibition of AChE (from electric eel) and BChE (from equine serum) as well as human ChEs in vitro and compared to previously synthesized dimeric inhibitors. Among the studied hybrids, compound 10-C10, characterized by a 10 carbon alkylene linker connecting tacrine and iperoxo, proved to be the most potent inhibitor with the highest pIC50 values of 9.81 (AChE from electric eel) and 8.75 (BChE from equine serum). Docking experiments with compounds 10-C10, 7b-C10, and 7a-C10 helped to interpret the experimental inhibitory power against AChE, which is affected by the nature of the allosteric molecular moiety, with the tacrine-containing hybrid being much more active than the naphthalimido- and phthalimido-containing analogs. Furthermore, the most active AChE inhibitors were found to have affinity to M1 and M2 muscarinic receptors. Since 10-C10 showed almost no cytotoxicity, it emerged as a promising lead structure for the development of an anti-Alzheimer drug.

We synthesized a set of new hybrid derivatives (7-C8, 7-C10, 7-C12 and 8-C8, 8-C10, 8-C12), in which a polymethylene spacer chain of variable length connected the pharmacophoric moiety of xanomeline, an M

In this paper, we designed, synthesized and tested a small set of three new derivatives potentially targeting the D3R-nAChR heteromer, a receptor complex recently identified and characterized as the molecular entity that, in dopaminergic neurons, mediates the neurotrophic effects of nicotine. By means of a partially rigidified spacer of variable length, we incorporated in the new compounds (1a-c) the pharmacophoric substructure of a known β2-subunit-containing nAChR agonist (A-84543) and that of the D2/D3R agonist drug ropinirole. All the compounds retained the ability to bind with high affinity both β2-subunit-containing nAChR and D3R. Compound 1a, renamed HyNDA-1, which is characterized by the shortest linker moiety, was the most interesting ligand. We found, in fact, that HyNDA-1 significantly modulated structural plasticity on both mice and human dopaminergic neurons, an effect strongly prevented by co-incubating this ligand with either nAChR or D3R antagonists. Moreover, the neurotrophic effects of HyNDA-1 were specifically lost by disrupting the complex with specific interfering peptides. Interestingly, by using the Bioluminescence Resonance Energy Transfer 2 (BRET2) assay in HEK-293 transfected cells, we also found that HyNDA-1 has the ability to increase the affinity of interaction between nAChR and D3R. Overall, our results indicate that the neurotrophic effects of HyNDA-1 are mediated by activation of the D3R-nAChR heteromeric complex specifically expressed on dopaminergic neurons.

The fast and constant development of drug resistant bacteria represents a serious medical emergency. To overcome this problem, the development of drugs with new structures and modes of action is urgently needed. In this work, we investigated, at the atomistic level, the mechanisms of hydrolysis of Meropenem by OXA-23, a class D β-lactamase, combining unbiased classical molecular dynamics and umbrella sampling simulations with classical force field-based and quantum mechanics/molecular mechanics potentials. Our calculations provide a detailed structural and dynamic picture of the molecular steps leading to the formation of the Meropenem-OXA-23 covalent adduct, the subsequent hydrolysis, and the final release of the inactive antibiotic. In this mechanistic framework, the predicted activation energy is in good agreement with experimental kinetic measurements, validating the expected reaction path.

The α7 nicotinic acetylcholine receptor (nAChR) silent agonists, able to induce receptor desensitization and promote the α7 metabotropic function, are emerging as new promising therapeutic anti-inflammatory agents. Herein, we report the structure-activity relationship investigation of the archetypal silent agonist NS6740 (1,4-diazabicyclo[3.2.2]nonan-4-yl(5-(3-(trifluoromethyl)-phenyl)-furan-2-yl)methanone) (1) to elucidate the ligand-receptor interactions responsible for the α7 silent activation. In this study, NS6740 fragments 11-16 and analogs 17-32 were designed, synthesized, and assayed on human α7 nAChRs expressed in Xenopus laevis oocytes with two-electrode voltage clamping experiments. All together the structural portions of NS6740 were critical to engender its peculiar activity profile. The diazabicyclic nucleus was essential but not sufficient for inducing α7 silent activation. The central hydrogen-bond acceptor core and the aromatic moiety were crucial for promoting prolonged α7 receptor binding and sustained desensitization. Compounds 13 and 17 were efficacious partial agonists. Compounds 12, 21, 23-26, and 30 strongly desensitized α7 nAChR and therefore may be of interest for additional investigation of inflammation responses. We gained key structural information useful for further silent agonist development.

We synthesized a set of new hybrid derivatives (7-C8, 7-C10, 7-C12 and 8-C8, 8-C10, 8-C12), in which a polymethylene spacer chain of variable length connected the pharmacophoric moiety of xanomeline, an M1/M4-preferring orthosteric muscarinic agonist, with that of tacrine, a well-known acetylcholinesterase (AChE) inhibitor able to allosterically modulate muscarinic acetylcholine receptors (mAChRs). When tested in vitro in a colorimetric assay for their ability to inhibit AChE, the new compounds showed higher or similar potency compared to that of tacrine. Docking analyses were performed on the most potent inhibitors in the series (8-C8, 8-C10, 8-C12) to rationalize their experimental inhibitory power against AChE. Next, we evaluated the signaling cascade at M1 mAChRs by exploring the interaction of Gαq-PLC-β3 proteins through split luciferase assays and the myo-Inositol 1 phosphate (IP1) accumulation in cells. The results were compared with those obtained on the known derivatives 6-C7 and 6-C10, two quite potent AChE inhibitors in which tacrine is linked to iperoxo, an exceptionally potent muscarinic orthosteric activator. Interestingly, we found that 6-C7 and 6-C10 behaved as partial agonists of the M1 mAChR, at variance with hybrids 7-Cn and 8-Cn containing xanomeline as the orthosteric molecular fragment, which were all unable to activate the receptor subtype response.

(±)-3-Methoxy-1-oxa-2,7-diaza-7,10-ethanospiro[4.5]dec-2-ene sesquifumarate (±)-1 was previously characterized as the most selective agonist at α7 neuronal nicotinic acetylcholine receptors in a series of spirocyclic quinuclidinyl-Δ

Hybrids of allosteric modulators of the muscarinic receptor and the AChE inhibitor tacrine and the orthosteric muscarinic agonists iperoxo and isox were synthesized.

This paper describes a practical approach to the preparation of 4-(2-hydroxyethyl)indolin-2-one, a key intermediate in the synthesis of dopaminergic agonists such as ropinirole—a drug used in the treatment of Parkinson’s disease and restless legs syndrome—and of two sets of protein kinase inhibitors. The sequence starts from commercially available 2-(2-methyl-3-nitrophenyl)acetic acid, which is converted in five steps into the desired target compound. This procedure offers a convenient alternative route to existing methodologies, given its milder reaction conditions, ease of implementation, and its overall yield (59 %).

G protein-coupled receptors constitute the largest family of membrane receptors and modulate almost every physiological process in humans. Binding of agonists to G protein-coupled receptors induces a shift from inactive to active receptor conformations. Biophysical studies of the dynamic equilibrium of receptors suggest that a portion of receptors can remain in inactive states even in the presence of saturating concentrations of agonist and G protein mimetic. However, the molecular details of agonist-bound inactive receptors are poorly understood. Here we use the model of bitopic orthosteric/allosteric (i.e. dualsteric) agonists for muscarinic M2 receptors to demonstrate the existence and function of such inactive agonist·receptor complexes on a molecular level. Using all-atom molecular dynamics simulations, dynophores (i.e. a combination of static three-dimensional pharmacophores and molecular dynamics-based conformational sampling), ligand design, and receptor mutagenesis, we show that inactive agonist·receptor complexes can result from agonist binding to the allosteric vestibule alone, whereas the dualsteric binding mode produces active receptors. Each agonist forms a distinct ligand binding ensemble, and different agonist efficacies depend on the fraction of purely allosteric (i.e. inactive) versus dualsteric (i.e. active) binding modes. We propose that this concept may explain why agonist·receptor complexes can be inactive and that adopting multiple binding modes may be generalized also to small agonists where binding modes will be only subtly different and confined to only one binding site.

We report the design, synthesis and pharmacological screening of a group of analogues of anabaseine 2, a naturally occurring unselective nicotinic agonist. The novel nAChR ligands 5-15 were planned following a molecular modeling analysis which suggested the replacement of the pyridine ring of 2 with a 3-substituted benzene ring as a means to gain selectivity for the α3β4 nAChR subtype. Overall, from binding experiments, the synthesized compounds showed high values of α3β4 affinity and α3β4 vs α4β2 selectivity, although they poorly discriminated the homomeric α7 subtype. The three analogues 6, 12 and 13 were also evaluated in electrophysiological assays, and 12 [6-(3-iodophenyl)-2,3,4,5-tetrahydropyridine] emerged as a rather interesting nicotinic ligand. Indeed, in addition to a noteworthy affinity (Ki = 4.7 nM) for the α3β4 subtype and to an excellent α3β4 vs α4β2 subtype selectivity (806-fold), compound 12 selectively activated the α3β4 nAChR (EC50 = 7.4 μM) while eliciting a negligible response at the α7 subtype and no effect at the α4β2 subtype.

A group of spirocyclic tropanyl-Δ2-isoxazolines was synthesized exploiting the 1,3-dipolar cycloaddition of nitrile oxides to olefins. Their interaction with the dopamine and serotonin transporters (DAT and SERT, respectively) was evaluated through binding experiments. The majority of the compounds had no inhibitory effects (IC50 >> 10 μM), while some had an IC50 value in the range 5–10 μM (8a–c, 10b and 11c on DAT, 12b on SERT). Unexpectedly, one of the tertiary amines under investigation, that is 3′-methoxy-8-methyl-spiro{8-azabicyclo[3.2.1]octane-3,5′(4′H)-isoxazole 7a, was able to enhance at a concentration of 10 μM both [3H]citalopram and [3H]paroxetine binding to SERT in rat brain homogenate (up to 25%, due to an increase of Bmax) and [3H]serotonin uptake (up to 30%) in cortical synaptosomes. This peculiar pharmacological profile of 7a suggests it binds to an allosteric site on SERT, and positions derivative 7a as a very useful tool to investigate SERT machinery.

Epiboxidine hydrochlorides (+)-2 and (−)-2, which are the structural analogs of the antipodes of epibatidine (±)-1, as well as the enantiomeric pairs (+)-3/(−)-3 and (+)-4/(−)-4 were synthesized and tested for binding affinity at α4β2 and α7 nicotinic acetylcholine receptor (nAChR) subtypes. Final derivatives were prepared through the condensation of racemic N-Boc-7-azabicyclo[2.2.1]heptane-2-one (±)-5 with the resolving agent (R)-(+)-2-methyl-2-propanesulfinamide. The pharmacological analysis carried out on the three new enantiomeric pairs evidenced an overall negligible degree of enantioselectivity at both nAChRs subtypes, a result similar to that reported for both natural and unnatural epibatidine enantiomers at the same investigated receptor subtypes. Chirality 24:543–551, 2012. © 2012 Wiley Periodicals, Inc.

Alpha7 nicotinic acetylcholine receptor is emerging as a central regulator in inflammatory processes, as documented by increasing studies reported in the literature. For instance, the activation of this nicotinic receptor subtype in resident macrophages inhibits the production of pro-inflammatory cytokines, thereby attenuating local inflammatory responses, and may open a new window in the treatment of chronic inflammatory disease, such as Crohn's disease, rheumatoid arthritis, psoriasis, and asthma. In continuation of our ongoing research for the development of new cholinergic drug candidates, we selected the nicotine derivative CAP55, which was previously shown to exert anti-inflammatory effects via nicotinic stimulation, as a suitable compound for lead optimization. Through the isosteric replacement of its 3,5-disubstituted 4,5-dihydroisoxazole core with a 1,4-disubstituted 1,2,3-triazole ring, we could rapidly generate a small library of CAP55-related analogs via a one-pot copper(I)-catalyzed azide-alkyne cycloaddition. Receptor binding assays at nAChRs led to the identification of two promising derivatives, compounds 4 and 10, worthy of further pharmacological studies.

A group of novel tricyclic Delta(2)-isoxazolines (4b, 5b, 7a-b, and 8a-b) and 3-oxo-isoxazolidines (6a-b and 9a-b), structurally related to cytisine or norferruginine, was prepared through 1,3-dipolar cycloadditions involving suitable olefins and bromonitrile oxide. The target compounds were assayed at alpha4beta2 and alpha7 neuronal acetylcholine receptors (nAChRs). The results of competition binding experiments indicated for the new derivatives a reduction of the affinity at the alpha4beta2 subtype in comparison with the reference molecules, coupled with an overall negligible affinity at the alpha7 subtype. The binding mode of the bromo-Delta(2)-isoxazolines 4b and 7b, which were the highest affinity ligands in the series (K(i)=0.92 and 0.75 microM, respectively), was analyzed by applying a recently developed model of the alpha4beta2 nAChRs.

Dualsteric ligands represent a novel mode of targeting G protein-coupled receptors (GPCRs). These compounds attach simultaneously to both, the orthosteric transmitter binding site and an additional allosteric binding area of a receptor protein. This approach allows the exploitation of favourable characteristics of the orthosteric and the allosteric site by a single ligand molecule. The orthosteric interaction provides high affinity binding and activation of receptors. The allosteric interaction yields receptor subtype-selectivity and, in addition, may modulate both, efficacy and intracellular signalling pathway activation. Insight into the spatial arrangement of the orthosteric and the allosteric site is far advanced in the muscarinic acetylcholine receptor, and the design of dualsteric muscarinic agonists has now been accomplished. Using the muscarinic receptor as a paradigm, this review summarizes the way from suggestive evidence for an orthosteric/allosteric overlap binding to the rational design and experimental validation of dualsteric ligands. As allosteric interactions are increasingly described for GPCRs and as insight into the spatial geometry of ligand/GPCR-complexes is growing impressively, the rational design of dualsteric drugs is a promising new approach to achieve fine-tuned GPCR-modulation. This article is part of a themed section on Molecular Pharmacology of GPCR. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00695.x

In the search for nicotinic acetylcholine receptor (nAChRs) agonists with a selective affinity for the homomeric alpha7 channels, we carried out the virtual screening of a test set of potential nicotinic ligands, and adopted a simplified MM-PBSA approach to estimate their relative binding free energy values. By means of this procedure, previously validated by a training set of compounds, we reached a realistic compromise between computational accuracy and calculation rate, and singled out a small group of novel structurally related derivatives characterized by a promising theoretical affinity for the alpha7 subtype. Among them, five new compounds were synthesized and assayed in binding experiments at neuronal alpha7 as well as alpha4beta2 nAChRs.

Allosteric receptor ligands bind to a recognition site that is distinct from the binding site of the endogenous messenger molecule. As a consequence, allosteric agents may attach to receptors that are already transmitter-bound. Ternary complex formation opens an avenue to qualitatively new drug actions at G protein-coupled receptors (GPCRs), in particular receptor subtype selective potentiation of endogenous transmitter action. Consequently, suitable exploitation of allosteric recognition sites as alternative molecular targets could pave the way to a drug discovery paradigm different from those aimed at mimicking or blocking the effects of endogenous (orthosteric) receptor activators. The number of allosteric ligands reported to modulate GPCR function is steadily increasing and some have already reached routine clinical use. This review aims at introducing into this fascinating field of drug discovery and at providing an overview about the achievements that have already been made. Various case examples will be discussed in the framework of GPCR classification (family A, B, and C receptors). In addition, the behavior at muscarinic receptors of hybrid derivatives incorporating both an allosteric and an orthosteric fragment in a common molecular skeleton will be illustrated.

Racemic exo-epiboxidine 3, endo-epiboxidine 6, and the two unsaturated epiboxidine-related derivatives 7 and 8 were efficiently prepared taking advantage of a palladium-catalyzed Stille coupling as the key step in the reaction sequence. The target compounds were assayed for their binding affinity at neuronal alpha4beta2 and alpha7 nicotinic acetylcholine receptors. Epiboxidine 3 behaved as a high affinity alpha4beta2 ligand (K(i)=0.4 nM) and, interestingly, evidenced a relevant affinity also for the alpha7 subtype (K(i)=6 nM). Derivative 7, the closest analogue of 3 in this group, bound with lower affinity at both receptor subtypes (K(i)=50 nM for alpha4beta2 and K(i)=1.6 microM for alpha7) evidenced a gain in the alpha4beta2 versus alpha7 selectivity when compared with the model compound.

The present report deals with the synthesis of the two couples of enantiomers (+)-(3aS,4R,6aS)-/(−)-(3aR,4S,6aR)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-4-carboxylic acid [(+)-HIP-A and (−)-HIP-A] and (+)-(3aS,6S,6aS)-/(−)-(3aR,6R,6aR)-3-hydroxy-3a,4,6,6a-tetrahydro-pyrrolo[3,4-d]isoxazole-6-carboxylic acid [(+)-HIP-B and (−)-HIP-B], and the investigation of their inhibitory activity at EAATs. When tested in a [3H] d -aspartate uptake assay on native EAATs present in rat brain synaptosomal fractions, (−)-HIP-A and (+)-HIP-B turned out to be considerably more potent than their enantiomers; in particular, (−)-HIP-A showed an eudismic ratio (ER) higher than 100. Molecular modeling investigations gave clues with regard to the key amino acid residues involved in the binding with our inhibitors and provided explanations for the observed stereoselectivity.

Isoxazole derivative (±)- 4 and the three pairs of stereoisomeric 3-bromo-isoxazolyl amino alcohols ( S , R )-(−)- 7a /( R , R )-(+)- 7b , ( S , R )-(−)- 8a /( R , R )-(+)- 8b , and ( S , R )-(−)- 9a /( R , R )-(+)- 9b were synthesized and assayed for their affinity and efficacy at human β 1 -, β 2 -, and β 3 -adrenergic receptors (β-ARs) in membranes from Chinese hamster ovary (CHO) cells stably transfected with the respective receptor subtype. Whereas derivative (±)- 4 did not bind at all three β-ARs, stereoisomers ( S , R )- 7a -( S , R )- 9a behaved as high-affinity ligands at β 1 - and, particularly, at β 2 -ARs ( K i 2.82–66.7 nM). The K i values of isomers ( R , R )- 7b -( R , R )- 9b at β 1 - and β 2 -subtypes were about 30–100 times higher than those of their ( S , R )- 7a – 9a counterparts, indicating a sizable stereochemical effect. The affinity at β 3 -ARs was negligible for all the investigated compounds. When submitted to a functional assay, the three stereoisomeric pairs showed a comparable pattern of efficacy at all three β-AR subtypes. The highest value of efficacy (75–90%) was observed at β 2 -ARs, whereas all compounds behaved as partial agonists (30–60%) at the β 3 -subtype. The lowest degree of efficacy (15–35%) was found at β 1 -ARs. The affinity/efficacy profile of the derivatives under study has been compared with that of the two model compounds, Broxaterol [(±)- 1 ] and BRL 37344 [(±)- 6 ].

The erythro- and threo-amino-(3′-hydroxy-4′,5′-dihydro-isoxazol-5′-yl)-acetic acids, stereoisomers of tricholomic acid, were synthesized along with the corresponding higher homologues erythro- and threo-amino-(3′-carboxy-4′,5′-dihydro-isoxazol-5′-yl)-acetic acids. The target compounds were prepared via the 1,3-dipolar cycloaddition of a suitable nitrile oxide to (±)-2-tert-butoxycarbonylamino-3-buten-1-ol. Such a strategy allowed the synthesis of the two stereoisomeric amino acids in comparable amounts. The pharmacological activity of these compounds was investigated at ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs) by means of receptor binding assays to rat cortical membranes, electrophysiological tests and second messenger assays at cloned receptors expressed in CHO cells. Their pharmacological profiles were compared to those of l -glutamate and of the previously described selective NMDA receptor antagonists 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acids in order to highlight the effect of increasing/reducing the distance between the amino acid moiety and the distal acid group, which represent the two pharmacophoric entities.

Recent studies demonstrated that post-herpetical and inflammatory pain can be locally managed by morphine gels, empirically chosen. Aiming to rationalize the selection of the most suitable opioid for the cutaneous delivery, we studied the in vitro penetration through human epidermis of eight opioids, evidencing the critical modifications of the morphinan core. Log P, log D, solid-state features and solubility were determined. Docking simulations were performed using supramolecular assembly made of ceramide VI. The modifications on position 3 of the morphinan core resulted the most relevant in determining both physicochemical characteristics and diffusion pattern. The 3-methoxy group weakened the cohesiveness of the crystal lattice structure and increased the permeation flux (J). Computational studies emphasized that, while permeation is essentially controlled by molecule apolarity, skin retention depends on a fine balance of polar and apolar molecular features. Moreover, ChemPLP scoring the interactions between the opioids and ceramide, correlated with both the amount retained into the epidermis (Qret) and J. The balance of the skin penetration properties and the affinity potency for μ-receptors evidenced hydromorphone as the most suitable compound for the induction of local analgesia.

Increasing attention has recently been devoted to allosteric modulators, as they can provide inherent advantages over classic receptor agonists. In the field of nicotinic receptors (nAChRs), the main advantage is that allosteric modulators can trigger pharmacological responses, limiting receptor desensitization. Most of the known allosteric ligands are "positive allosteric modulators" (PAMs), which increase both sensitivity to receptor agonists and current amplitude. Intriguingly, some allosteric modulators are also able to activate the α7 receptor (α7-nAChR) even in the absence of orthosteric agonists. These compounds have been named "ago-allosteric modulators" and GAT107 has been studied in depth because of its unique mechanism of action. We here investigate by molecular dynamics simulations, metadynamics, and essential dynamics the activation mechanism of α7-nAChR, in the presence of different nicotinic modulators. We determine the free energy profiles associated with the closed-to-open motion of the loop C, and we highlight mechanistic differences observed in the presence of different modulators. In particular, we demonstrate that GAT107 triggers conformational motions and cross-talk similar to those observed when the α7-nACh receptor is in complex with both an agonist and an allosteric modulator.

Objectives: Acid-base dissociations constants of a series of poorly water-soluble nicotinic ligands designed as anti-inflammatory agents were determined in order to characterize the pharmacokinetic profile of this kind of ligands. Methods: pKas values were assessed by means of potentiometric and electrophoretic methods and investigated by computational protocols. Results: Both electrophoretic and potentiometric measurements produced reliable results. However, with the electrophoretic technique only an average value for close pKas was found, whereas the potentiometric method allowed determination of each pKa value in water-cosolvent mixtures. A theoretical treatment with various prediction programs - i.e. ADME Boxes v. 4.1, ACD/pKa DB and ACD/pKa GALAS - led in most cases to values which were not in accordance with the experimental ones. Conclusion: Electrophoretic and potentiometric techniques showed complementary features. Indeed, with capillary electrophoresis, the problem associated with the low water solubility of the studied samples could be easily overcome, although this technique did not allow to measuring all dissociation constants. In contrast, application of the potentiometric method afforded all the theoretical pKa values, although we had to perform the titrations in watercosolvent mixtures, a less precise, more laborious and time-consuming approach.

We designed, prepared and tested a set of structural analogs 1–4 as new hybrid compounds by incorporating, through a common alkyl chain of variable length, the pharmacophoric elements of N-n-alkyl nicotinium salts (non-α7 nicotinic acetylcholine receptors antagonists) and of 7-hydroxy-2-(aminomethyl)chromanes (dopaminergic D2 receptor agonists). The target compounds, which were assayed in binding experiments and electrophysiological, functional and Erk1/2 activation tests, essentially combined the pharmacological profiles of their individual receptor ligands. Among the studied derivatives, hybrid 2, one of the shortest homologs, in addition to the antagonist nicotinic profile similar to the other three congeners, behaved as a high affinity ligand at the investigated heteromeric nAChRs and as a low efficacy agonist at D2Rs. These bifunctional derivatives represent novel pharmacological tools in the study of nicotine addiction.

The group of Δ2-isoxazoline derivatives 5a–c and 6a–c, structurally related to epibatidine, and the simplified analogues 7a–c were synthesized by means of a 1,3-dipolar cycloaddition-based strategy and tested at α4β2 and α7 neuronal acetylcholine receptor (nAChR) subtypes. Competition binding experiments at α4β2 nAChR subtypes showed an overall significant reduction in affinity for the compounds under study in comparison to the reference radioligand [3H]-epibatidine. These outcomes have been rationalized by taking into account the ligand-based pharmacophore models reported in the literature and the recently proposed molecular model of the α4β2 receptor subtype. Conversely, compounds 5b, 5c, and 6b exhibited a noticeable affinity for the α7 receptors and, in the case of 5c, also some subtype selectivity. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

The four stereoisomers of 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid(+)-4, (-)-4, (+)-5, and (-)-5 were prepared by stereoselective synthesis of two pairs of enantiomers, which were subsequently resolved by enzymatic procedures. These four stereoisomers and the four stereoisomers of the bicyclic analogue 5-amino-4,5,6,6a-tetrahydro-3aH-cyclopenta[d]isoxazole-3,5-dicarboxylic acid (+)-2, (-)-2, (+)-3, and (-)-3 were tested at ionotropic and metabotropic glutamate receptor subtypes. The most potent NMDA receptor antagonists [(+)-2, (-)-4, and (+)-5] showed a significant neuroprotective effect when tested in an oxygen glucose deprivation (OGD) cell culture test. The same compounds were preliminarily assayed using Xenopus oocytes expressing cloned rat NMDA receptors containing the NR1 subunit in combination with either NR2A, NR2B, NR2C, or NR2D subunit. In this assay, all three derivatives showed high antagonist potency with preference for the NR2A and NR2B subtypes, with derivative (-)-4 behaving as the most potent antagonist. The biological data are discussed on the basis of homology models reported in the literature for NMDA receptors and mGluRs.

The two diastereomeric pairs of acidic amino acids 5-(2-amino-2-carboxyethyl)-4,5-dihydroisoxazole-3-carboxylic acid (8A/8B) and 4-(2-amino-2-carboxyethyl)-5,5-dimethyl-4,5-dihydroisoxazole-3-carboxylic acid (10A/10B) were prepared via a strategy based on a 1,3-dipolar cycloaddition. The four amino acids were tested at ionotropic and metabotropic glutamate receptors. None of the compounds was active, neither as agonists nor as antagonists, at 1 mM on metabotropic receptors (mGluR1, -2, -4, and -5 expressed in CHO cell lines). Conversely, the pair of stereoisomers 8A/8B showed a remarkable affinity, antagonist potency, and selectivity for NMDA receptors, when tested on ionotropic glutamate receptors. The affinity of 8A proved to be 5 times higher than that of diastereomer 8B (K(i) values 0.21 and 0.96 microM, respectively). Furthermore, compounds 8A and 8B exhibited a noteworthy anticonvulsant activity in in vivo tests on DBA/2 mice. Derivative 10A was inactive at all ionotropic glutamate receptors, whereas its stereoisomer 10B displayed a seizable binding to both NMDA and AMPA receptors.

The two enantiomeric pairs of bicyclic isoxazoline acidic amino acids, designed as conformationally constrained homologues of glutamic acid, have been prepared by taking advantage of a nitrile oxide cycloaddition coupled to highly selective biocatalyzed transformations. All four isomeric target amino acids were obtained with enantiomeric excesses higher than 99%. The absolute configurations were attributed to the compounds under study by comparing the experimental and theoretical vibrational circular dichroism features of two selected reaction intermediates. These absolute configurations were further confirmed using experimental and theoretical specific rotations.