Each of the four aromatic -CH= of (S,R)-2-pyrrolidinyl-1,4-benzodioxane [(S,R)-6] and of its epimer at the dioxane stereocenter (S,S)-6, previously reported as α4β2 nAChR ligands, was replaced with nitrogen. The resulting four diastereoisomeric pairs of pyrrolidinyl-pyridodioxanes were studied for the nicotinic affinity and activity at α4β2, α3β4 and α7 nAChR subtypes and compared to their common carbaisostere. It turned out that such isosteric substitutions are highly detrimental, but with the important exception of the S,R stereoisomer of the pyrrolidinyl-pyridodioxane with the pyridine nitrogen adjacent to the dioxane and seven atoms distant from the pyrrolidine nitrogen. Indeed, this stereo/regioisomer not only maintained the α4β2 affinity of [(S,R)-6], but also greatly improved in selectivity over the α3β4 and α7 subtypes and, most importantly, exhibited a highly selective α4β2 partial agonism. The finding that [(S,R)-6] is, instead, an unselective α4β2 antagonist indicates that the benzodioxane substructure confers affinity for the α4β2 nAChR binding site, but activation of this receptor subtype needs benzodioxane functionalization under strict steric requirements, such as the previously reported 7-OH substitution or the present isosteric modification.

Some unichiral analogues of 2R,2'S-2-(1'-methyl-2'-pyrrolidinyl)-7-hydroxy-1,4-benzodioxane, a potent and selective α4β2-nAChR partial agonist, were designed by opening dioxane and replacing hydroxyl carbon with nitrogen. The resulting 3-pyridyl and m-hydroxyphenyl ethers have high α4β2 affinity and good subtype selectivity, which get lost if OH is removed from phenyl or the position of pyridine nitrogen is changed. High α4β2 affinity and selectivity are also attained by meta hydroxylating the 3-pyridyl and the phenyl ethers of (S)-N-methylprolinol and the phenyl ether of (S)-2-azetidinemethanol, known α4β2 agonists, although the interaction mode of the aryloxymethylene substructure cannot be assimilated to that of benzodioxane. Indeed, the α4β2 and α3β4 functional tests well differentiate behaviors that the binding tests homologize: both the 3-hydroxyphenyl and the 5-hydroxy-3-pyridyl ether of N-methylprolinol are α4β2 full agonists, but only the latter is highly α4β2/α3β4 selective, while potent and selective partial α4β2 agonism characterizes the hydroxybenzodioxane derivative and its two opened semirigid analogues.

A series of acetylcholine carbamoyl analogues, cyclised at the carbamate moiety or at the cationic head or at both, were tested for binding affinity at muscarinic and neuronal nicotinic receptors (nAChRs). While no muscarinic affinity was found, submicromolar Ki values, similar to that of carbachol, were measured at α4β2 nAChRs for the enantiomers of 5-dimethylaminomethyl- and 5-trimethylammoniomethyl-2-oxazolidinone, 2 and 2a, and for (S)-N-methylprolinol carbamate (S)-3. Methylation of oxazolidinone nitrogen of 2 and 2a and of N-methylprolinol nitrogen of (S)-3 and, even more, hybridization of cyclic carbamate substructure (oxazolidinone) with cyclic cationic head (N-methylpyrrolidine) markedly lower the nicotinic affinity. Docking results were consistent with SAR analysis highlighting the interaction capabilities of (R)-2a and (S)-3 and the negative effect of intracyclic nitrogen methylation and of double cyclisation.

Background Wounded leaves of Arabidopsis thaliana produce r eactive o xygen s pecies (ROS) within minutes after wounding and become resistant to the pathogenic fungus Botrytis cinerea at a local level. This fast response of the plants to the wound is called w ound- i nduced r esistance (WIR). However the molecular mechanisms of this response and the signal cascade between the wound and ROS production are still largely unknown. Calcium is a conserved signal and it is involved in many abiotic stress responses in plants, furthermore, calcium pathways act very fast. Results The results of this study show that leaves treated with calcium channels inhibitors (verapamil) or calcium chelators (oxalate and EGTA) are impaired in ROS production. Moreover, leaves treated with verapamil, EGTA or oxalate were more susceptible to B. cinerea after wounding. The intracellular measurements of calcium changes indicated quick but transient calcium dynamics taking place few seconds after wounding in cells neighbouring the wound site. This change in the cytosolic calcium was followed in the same region by a more stable ROS burst. Conclusions These data further extend our knowledge on the connection between wounding, calcium influx and ROS production. Moreover they provide for the first time the evidence that, following wounding, calcium changes precede a burst in ROS in the same location.

Background In a previous study we have shown that wounding of Arabidopsis thaliana leaves induces a strong and transient immunity to Botrytis cinerea , the causal agent of grey mould. Reactive oxygen species (ROS) are formed within minutes after wounding and are required for wound–induced resistance to B. cinerea . Results In this study, we have further explored ROS and resistance to B. cinerea in leaves of A. thaliana exposed to a soft form of mechanical stimulation without overt tissue damage. After gentle mechanical sweeping of leaf surfaces, a strong resistance to B. cinerea was observed. This was preceded by a rapid change in calcium concentration and a release of ROS, accompanied by changes in cuticle permeability, induction of the expression of genes typically associated with mechanical stress and release of biologically active diffusates from the surface. This reaction to soft mechanical stress (SMS) was fully independent of jasmonate (JA signaling). In addition, leaves exposed soft mechanical stress released a biologically active product capable of inducing resistance to B. cinerea in wild type control leaves. Conclusion Arabidopsis can detect and convert gentle forms of mechanical stimulation into a strong activation of defense against the virulent fungus B. cinerea .

Carnitinamide chloride, an immediate synthetic precursor of carnitine, was chlorinated at the amide nitrogen. The resultant carnitinechloramide chloride, when treated with a base, revealed that the first-formed isolable and characterisable carnitinechloramide inner salt undergoes solid state conversion into 5-trimethylammoniomethyl-2-oxazolidinone chloride via Hoffmann rearrangement and intramolecular cyclization of the β-hydroxyisocyanate. The trimethylaminomethyl substituent at C-5 of the 2-oxazolidinone was converted into a dimethylaminomethyl group by microwave-assisted demethylation in DMF and then into a methylaminomethyl group by decomposition of the α-chloroethyl carbamate obtained by treatment with α-chloroethyl chloroformate. This sequence of reactions was then applied to both (S)- and (R)-carnitinamide chloride without any racemization to yield both enantiomers of 5-aminomethyl-2-oxazolidinones that are mono-, di- and tri-methylated at the exocyclic nitrogen.

(S)-(+)-3-Aminomethyl-5-methylhexanoic acid (pregabalin) was converted in one-pot to (S)-(−)-3-cyano-5-methylhexanoic acid (pregabalin nitrile) by N-dichlorination and double dehydrochlorination. The (S) β-cyanoacid was racemized under mild conditions by treatment with a base. This very simple and efficient procedure, applied to (R)-(−)-3-aminomethyl-5-methylhexanoic acid, would enable the recycling of the undesired enantiomer of pregabalin, an anticonvulsant drug manufactured by the synthesis of rac-3-aminomethyl-5-methylhexanoic acid and subsequent classical resolution.

A series of unichiral 7-substituted 2-(1'-methyl-2'-pyrrolidinyl)-1,4-benzodioxanes were synthesized and tested for the affinity for the α4β2 and α7 central nicotinic receptors; the 2R,2'S diastereomer of the 7-OH analogue [(R,S)-7], unique in the series, has a high α4β2 affinity (12nM K(i)). N-Demethylation and configuration inversion of the stereocenters greatly weaken its α4β2 affinity, confirming that such a rigid molecule can be considered a new template for α4β2 ligands. Docking analysis showed how (R,S)-7 is capable of strongly and specifically interacting with the amino acidic counterpart of the α4β2 receptor binding site. Further pharmacological characterization demonstrated that (R,S)-7 also has a high affinity for the α6β2 receptor, and in vitro functional tests indicated that it is a potent α4β2 and α6β2 partial agonist, with modest affinity and potency for the α3β4 receptor. Comparison with varenicline, a well-known nicotinic partial agonist used as a smoking cessation aid, interestingly reveals similar nicotinoid profiles.

Wounded leaves of Arabidopsis thaliana show transient immunity to Botrytis cinerea, the causal agent of grey mould. Using a fluorescent probe, histological staining and a luminol assay, we now show that reactive oxygen species (ROS), including H(2)O(2) and O(2) (-), are produced within minutes after wounding. ROS are formed in the absence of the enzymes Atrboh D and F and can be prevented by diphenylene iodonium (DPI) or catalase. H(2)O(2) was shown to protect plants upon exogenous application. ROS accumulation and resistance to B. cinerea were abolished when wounded leaves were incubated under dry conditions, an effect that was found to depend on abscisic acid (ABA). Accordingly, ABA biosynthesis mutants (aba2 and aba3) were still fully resistant under dry conditions even without wounding. Under dry conditions, wounded plants contained higher ABA levels and displayed enhanced expression of ABA-dependent and ABA-reporter genes. Mutants impaired in cutin synthesis such as bdg and lacs2.3 are already known to display a high level of resistance to B. cinerea and were found to produce ROS even when leaves were not wounded. An increased permeability of the cuticle and enhanced ROS production were detected in aba2 and aba3 mutants as described for bdg and lacs2.3. Moreover, leaf surfaces treated with cutinase produced ROS and became more protected to B. cinerea. Thus, increased permeability of the cuticle is strongly linked with ROS formation and resistance to B. cinerea. The amount of oxalic acid, an inhibitor of ROS secreted by B. cinerea could be reduced using plants over expressing a fungal oxalate decarboxylase of Trametes versicolor. Infection of such plants resulted in a faster ROS accumulation and resistance to B. cinerea than that observed in untransformed controls, demonstrating the importance of fungal suppression of ROS formation by oxalic acid. Thus, changes in the diffusive properties of the cuticle are linked with the induction ROS and attending innate defenses.

Cells of all living organisms contain complex signal transduction networks to ensure that a wide range of physiological properties are properly adapted to the environmental conditions. The fundamental concepts and individual building blocks of these signalling networks are generally well-conserved from yeast to man; yet, the central role that growth factors and hormones play in the regulation of signalling cascades in higher eukaryotes is executed by nutrients in yeast. Several nutrient-controlled pathways, which regulate cell growth and proliferation, metabolism and stress resistance, have been defined in yeast. These pathways are integrated into a signalling network, which ensures that yeast cells enter a quiescent, resting phase (G0) to survive periods of nutrient scarceness and that they rapidly resume growth and cell proliferation when nutrient conditions become favourable again. A series of well-conserved nutrient-sensory protein kinases perform key roles in this signalling network: i.e. Snf1, PKA, Tor1 and Tor2, Sch9 and Pho85-Pho80. In this review, we provide a comprehensive overview on the current understanding of the signalling processes mediated via these kinases with a particular focus on how these individual pathways converge to signalling networks that ultimately ensure the dynamic translation of extracellular nutrient signals into appropriate physiological responses.

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP-dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V-ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V-ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V-ATPase is also regulated by glucose in the Min6 beta-cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose-sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N‐terminal and large natively unfolded C‐terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two‐hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N‐terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C‐terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.

Comment on: Binda M, et al. Mol Cell 2009; 35:563-73.

The target of rapamycin complex 1 (TORC1) is a central regulator of eukaryotic cell growth that is activated by a variety of hormones (e.g., insulin) and nutrients (e.g., amino acids) and is deregulated in various cancers. Here, we report that the yeast Rag GTPase homolog Gtr1, a component of the vacuolar-membrane-associated EGO complex (EGOC), interacts with and activates TORC1 in an amino-acid-sensitive manner. Expression of a constitutively active (GTP-bound) Gtr1(GTP), which interacted strongly with TORC1, rendered TORC1 partially resistant to leucine deprivation, whereas expression of a growth inhibitory, GDP-bound Gtr1(GDP), caused constitutively low TORC1 activity. We also show that the nucleotide-binding status of Gtr1 is regulated by the conserved guanine nucleotide exchange factor (GEF) Vam6. Thus, in addition to its regulatory role in homotypic vacuolar fusion and vacuole protein sorting within the HOPS complex, Vam6 also controls TORC1 function by activating the Gtr1 subunit of the EGO complex.

The four stereoisomers of 2-oxazolidinone 5-substituted with 1-methyl-2-pyrrolidinyl (1), of 1,4-benzodioxane 2-substituted with the same residue (2) and of the nor-methyl analogue of this latter (2a) were synthesized as candidate nicotinoids. Of the 12 compounds, two N-methylated pyrrolidinyl-benzodioxane stereoisomers, namely those with the same relative configuration at the pyrrolidine stereocentre as (S)-nicotine, bind at alpha4beta2 nicotinic acetylcholine receptor with submicromolar affinity. Consistently with the biological data, docking analysis enlightens significant differences in binding site interactions not only between 1 and 2, but also between 2 and 2a and between the stereoisomers of 2 accounting for the critical role played, in the case of the pyrrolidinyl-benzodioxanes, by the chirality of both the stereolabile and stereostable stereogenic atoms, namely the protonated tertiary nitrogen and the two asymmetric carbons.

The ready availability of (R)-carnitinamide, an immediate synthetic precursor of (R)-carnitine, is an ambitious goal and resolutions, due to the very low cost of racemic carnitinamide, can be the most convenient stereotechnology to reach it. We have efficiently resolved carnitinamide chloride by preferential crystallization from methanol according to simple entrainment procedure. The previous transformation of the chloride into other salts or the use of specific solvent systems was not required for successful resolution, as in the case of the reported entrainment of carnitinamide precursor, carnitine nitrile.

The methyl ester of 1,4-benzodioxane-2-carboxylic acid 1 and the mesylate of 2-hydroxymethyl-1,4-benzodioxane 2 are synthetic intermediates whose enantiomers can be advantageously used to prepare a number of enantiopure 2-substituted 1,4-benzodioxanes from readily accessible (±)-1,4-benzodioxane-2-carboxilic acid. We have previously demonstrated the conglomerate nature of the enantiomeric systems of 1 and 2. Herein, we report the resolution of their racemates by preferential crystallization according to an entrainment procedure. In particular, the entrainment resolution of 1 showed good efficiency, which makes the present method a competitive alternative to the classical resolutions of 1,4-benzodioxane-2-carboxylic acid with dehydroabietylamine and para-substituted 1-phenylethylamines that we have recently reported.