Exchangeable Self-Assembled Lanthanide Antennas for PLIM Microscopy.

Lanthanides have unique photoluminescence (PL) emission properties, including very long PL lifetimes. This makes them ideal for biological imaging applications, especially using PL lifetime imaging microscopy (PLIM). PLIM is an inherently multidimensional technique with exceptional advantages for quantitative biological imaging. Unfortunately, due to the required prolonged acquisitions times, photobleaching of lanthanide PL emission currently constitutes one of the main drawbacks of PLIM. In this study, we report a small aqueous-soluble, lanthanide antenna, 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-phosphonic acid, PAnt, specifically designed to dynamically interact with lanthanide ions, serving as exchangeable dye aimed at mitigating photobleaching in PLIM microscopy in cellulo. Thus, self-assembled lanthanide complexes that may be photobleached during image acquisition are continuously replenished by intact lanthanide antennas from a large reservoir. Remarkably, our self-assembled lanthanide complex clearly demonstrated a significant reduction of PL photobleaching when compared to well-established lanthanide cryptates, used for bioimaging. This concept of exchangeable lanthanide antennas opens new possibilities for quantitative PLIM bioimaging.

Exploring the Phe-Gly dipeptide-derived piperazinone scaffold in the search for antagonists of the thrombin receptor PAR1.

A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cells. The synthetic strategy involves coupling of a protected basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a carbonylmethyl group at the N1-position, followed by formation of an aromatic urea at the exocyclic moiety linked at the C2 position of the piperazine ring and removal of protecting groups. None of the compounds showed activity in the biological evaluation.

Perfusion computed tomography-guided intravenous thrombolysis for acute ischemic stroke beyond 4.5 hours: a case-control study.

BACKGROUND: Extending the therapeutic window of intravenous thrombolysis for acute ischemic stroke beyond the established 4.5-hour limit is of critical importance in order to increase the proportion of thrombolysed stroke patients. In this setting, the capacity of MRI to select acute stroke patients for reperfusion therapies in delayed time windows has been and is being tested in clinical trials. However, whether the more available and cost-effective perfusion computed tomography (PCT) may be useful to select candidates for delayed intravenous thrombolysis remains largely unexplored. We aimed to evaluate the safety and efficacy of PCT-guided intravenous thrombolysis beyond 4.5 h after stroke onset. METHODS: We prospectively studied all consecutive acute ischemic stroke patients treated with intravenous tissue plasminogen activator (tPA) in our stroke unit between January 2008 and December 2010. Patients treated within 0- 4.5 h were treated according to non-contrast CT (NCCT) criteria. Beyond 4.5 h, patients received intravenous tPA according to PCT criteria, i.e. an infarct core on cerebral blood volume (CBV) maps not exceeding one third of the middle cerebral artery (MCA) territory and tissue at risk as defined by mean transit time-CBV mismatch greater than 20%. Predetermined primary endpoints were symptomatic hemorrhagic transformation and favorable long-term outcome, while early neurological improvement and MCA recanalization were considered secondary endpoints. Statistical analysis included bivariate comparisons between the two groups for each endpoint and logistic regression models when significance was found in bivariate analyses. This study was approved by our local ethics committee. RESULTS: A total of 245 patients received intravenous thrombolysis. After the groups were matched by baseline National Institutes of Health Stroke Scale score, 172 patients treated at <4.5 h and 43 patients treated at >4.5 h were finally included. Early and late groups were comparable regarding baseline variables; only cardioembolic etiology was more frequent in the >4.5 h group. Rates of symptomatic hemorrhagic transformation (2.9% in the <4.5 h group vs. 2.3% in the >4.5 h group; p = 1.0) and good long-term outcome (64.5 vs. 60.5%, respectively; p = 0.620) were similar between the groups. However, delayed intravenous thrombolysis was independently associated with a worse early clinical course [odds ratio (OR) 2.07, 95% confidence interval (CI) 1.04-4.1; p = 0.038] and lower 2-hour MCA recanalization rates (OR 0.4, 95% CI 0.17-0.92; p = 0.03). CONCLUSION: Primary safety and efficacy endpoints were comparable between the early and delayed thrombolysis groups. The results of our exploratory study may justify a randomized clinical trial to test the safety and efficacy of PCT-guided intravenous thrombolysis in acute ischemic stroke patients presenting beyond 4.5 h from symptom onset.

Self-Assembled Lanthanide Antenna Glutathione Sensor for the Study of Immune Cells.

The small molecule 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-carboxylic acid (2b) behaves as a reactive non-fluorescent Michael acceptor, which after reaction with thiols becomes fluorescent, and an efficient Eu3+ antenna, after self-assembling with this cation in water. This behavior makes 2b a highly selective GSH biosensor, which has demonstrated high potential for studies in murine and human cells of the immune system (CD4+ T, CD8+ T, and B cells) using flow cytometry. GSH can be monitored by the fluorescence of the product of addition to 2b (445 nm) or by the luminescence of Eu3+ (592 nm). 2b was able to capture baseline differences in GSH intracellular levels among murine and human CD4+ T, CD8+ T, and B cells. We also successfully used 2b to monitor intracellular changes in GSH associated with the metabolic variations governing the induction of CD4+ naïve T cells into regulatory T cells (TREG).

Smart lanthanide antennas for sensing water.

Two new families of lanthanide antennas are described. 8-Methoxy-4,5-dihydrocyclopenta[de]quinolin-2(1H)-one phosphonates or carboxylates behave as selective antennas exhibiting Eu3+ luminescence in organic solvents, while quinolin-2(1H)-one analogues selectively sensitize the Tb3+ emission. These emissions are quenched by H2O addition. Based on this behaviour, the new lanthanide antennas can be used as highly sensitive water sensors.

Naphthalimide-based macrophage nucleus imaging probes.

The photophysical properties of naphthalimide-based fluorophores can be easily tuned by chemical manipulation of the substituents on that privileged scaffold. Replacement of a OMe group at position 6 in 2-(hydroxyl)ethyl-naphthalimide derivatives by diverse amines, including 2-(hydroxyl)ethylamine, trans-(4-acetamido)cyclohexylamine and azetidine increases the solvatochromic (ICT) character, while this replacement in 2-(dimethylamino)ethyl-naphthalimide analogues (PET fluorophores) decrease their solvent polarity sensitivity or even reversed them to solvatochromic fluorophores. These fluorophores resulted macrophage nucleus imaging probes, which bind DNA as intercalants and showed low cytotoxicity in human cancer cells.

Environment-Sensitive Probes for Illuminating Amyloid Aggregation In Vitro and in Zebrafish.

The aberrant aggregation of certain peptides and proteins, forming extracellular plaques of fibrillar material, is one of the hallmarks of amyloid diseases, such as Alzheimer's and Parkinson's. Herein, we have designed a new family of solvatochromic dyes based on the 9-amino-quinolimide moiety capable of reporting during the early stages of amyloid fibrillization. We have rationally improved the photophysical properties of quinolimides by placing diverse amino groups at the 9-position of the quinolimide core, leading to higher solvatochromic and fluorogenic character and higher lifetime dependence on the hydrophobicity of the environment, which represent excellent properties for the sensitive detection of prefibrillar aggregates. Among the different probes prepared, the 9-azetidinyl-quinolimide derivative showed striking performance in the following ß-amyloid peptide (Aß) aggregation in solution in real time and identifying the formation of different types of early oligomers of Aß, the most important species linked to cytotoxicity, using novel, multidimensional fluorescence microscopy, with one- or two-photon excitation. Interestingly, the new dye allowed the visualization of proteinaceous inclusion bodies in a zebrafish model with neuronal damage induced by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Our results support the potential of the novel fluorophores as powerful tools to follow amyloid aggregation using fluorescence microscopy in vivo, revealing heterogeneous populations of different types of aggregates and, more broadly, to study protein interactions.

The N-terminal tripeptide of insulin-like growth factor-I protects against beta-amyloid-induced somatostatin depletion by calcium and glycogen synthase kinase 3 beta modulation.

The protective effects of insulin-like growth factor I on the somatostatin (SRIF) system in the temporal cortex after beta-amyloid (Abeta) injury may be mediated through its N-terminal tripeptide glycine-proline-glutamate (GPE). GPE is cleaved to cyclo[Pro-Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Abeta25-35-treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Abeta25-35 accumulation, cytosolic calcium levels ([Ca(2+)](c)) and the intracellular signaling mechanisms involved. GPE and cPG did not change Abeta25-35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Abeta25-35 insult, which was coincident with Akt activation and glycogen synthase kinase 3beta inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca(2+) influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin-like growth factor I effects on the SRIF system through a mechanism independent of Abeta clearance that involves modulation of calcium and glycogen synthase kinase 3beta signaling.

Targeting the neuronal calcium sensor DREAM with small-molecules for Huntington's disease treatment.

DREAM, a neuronal calcium sensor protein, has multiple cellular roles including the regulation of Ca2+ and protein homeostasis. We recently showed that reduced DREAM expression or blockade of DREAM activity by repaglinide is neuroprotective in Huntington's disease (HD). Here we used structure-based drug design to guide the identification of IQM-PC330, which was more potent and had longer lasting effects than repaglinide to inhibit DREAM in cellular and in vivo HD models. We disclosed and validated an unexplored ligand binding site, showing Tyr118 and Tyr130 as critical residues for binding and modulation of DREAM activity. IQM-PC330 binding de-repressed c-fos gene expression, silenced the DREAM effect on KV4.3 channel gating and blocked the ATF6/DREAM interaction. Our results validate DREAM as a valuable target and propose more effective molecules for HD treatment.

Synthesis and antitumoral evaluation of indole alkaloid analogues containing an hexahydropyrrolo[1',2',3':1,9a,9]imidazo[1,2-a]indole skeleton.

The scope of acid-mediated cyclative additions of electrophiles to tryptophan-derived alpha-amino nitriles for the synthesis of 10b-substituted-1,2,4,5,10b,10c-hexahydropyrrolo[1',2',3':1,9a,9]imidazo[1,2-a]indoles analogues of indole alkaloids has been studied. The results demonstrate the high potential of the methodology for the synthesis of 10b-bromo-derivatives, by bromination with NBS, 10b-allyl-derivatives, by bromo-allyl exchange, and 10b-prenyl-derivatives, by reaction with prenyl bromide in the presence of Mg(NO(3))(2).6H(2)0. Some of the new pyrroloimidazoindole derivatives displayed moderate microM cytotoxicities in human cancer cell lines and at 10 microg/mL inhibited more than 50% EGFR or HIF-1alpha.

Highly solvatochromic and tunable fluorophores based on a 4,5-quinolimide scaffold: novel CDK5 probes.

Novel 4,5-quinolimide-based fluorophores are more solvatochromic and red-shifted than known naphthalimide analogues. Conjugation of one of these fluorophores to a peptide derived from CDK5 kinase demonstrated its sensitivity for monitoring the interaction with its regulatory partner p25. Introduction of the quinolimide-labelled peptide into living glioblastoma cells probed the interaction with endogenous p25.

Combination of molecular modeling, site-directed mutagenesis, and SAR studies to delineate the binding site of pyridopyrimidine antagonists on the human CCK1 receptor.

A rational combination of site-directed mutagenesis studies, structure-activity relationships, and dynamic-based docking of pyridopyrimidine-derived CCK1R antagonists into a refined three-dimensional model of the CCK1R allowed us to identify the receptor residues and the ligand functional groups implicated in the molecular recognition process. Our results provided unambiguous evidence that the binding site of these antagonists is overlapping that of the C-terminal tetrapeptide of CCK. In particular, Asn333 and Arg336 residues of the CCK1R are essential for high-affinity binding of these ligands. Moreover, the 2-aryl group in the pyridopyrimidine derivatives shares the same binding pocket as the C-terminal Phe side chain of CCK. Our [pyridopyrimidine.CCK1R] complex model is consistent with previous suggestions concerning the molecular basis that governs functional activity and provides useful considerations about the high CCK1 versus CCK2 selectivity of our derivatives and could contribute to fine-tune the rational design of new molecules with optimized properties.

From Multiple PAR1 Receptor/Protein Interactions to their Multiple Therapeutic Implications.

PAR1, member of the family of protease-activated receptors, is a GPCR whose activation requires a proteolytic cleavage at its extracellular N-terminus to unveil a tethered activating ligand. Although thrombin is the main activator of this receptor, diverse other proteases can also activate and disarm PAR1. Besides, tethered activating ligand-based peptides (PAR-APs) can also activate the receptor. PAR1 mainly signals via G proteins but, it can also signal using ß-arrestin pathways and by transactivation of other receptors. This complex PAR1 interactome is completed with the receptor desensitization, trafficking, and degradation. PAR1 has shown species-, cellular-, and physiological or pathological state-dependent specificity. This review try to give an overview on the complex PAR1 interactome, its therapeutic impact upon the cardiovascular, immune and nervous systems, inflammation and cancer, as well as, on its modulation with agonists and antagonists.

5-(tryptophylamino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-based cholecystokinin receptor antagonists: reversal of CCK1 receptor subtype selectivity toward CCK2 receptors.

With the aim of reversing selectivity or antagonist/agonist functionality in the 5-(tryptophylamino)-1,3-dioxoperhydropyrido[1,2-c]pyrimidine-derived potent and highly selective CCK(1) antagonists, a series of 4-benzyl and 4-methyl derivatives have been synthesized. Whereas the introduction of the benzyl group led, in all cases, to complete loss of the binding affinity, the incorporation of the methyl group gave a different result depending on the stereochemistry of the 1,3-dioxoperhydropyrido[1,2-c]pyrimidine scaffold. Thus, the introduction of the methyl group into the (4aS,5R)-diastereoisomers, giving a (4S)-configuration, produced a 3-fold increase in the CCK(1) binding potency and selectivity. However, the same structural manipulation in the opposite (4aR,5S)-stereochemistry, leading to a (4R,4aR,5S)-configuration, produced reversal of the selectivity for CCK(1) to the CCK(2) receptors. The replacement of the Boc group at the tryptophan moiety by a 2-adamantyloxycarbonyl group also contributed to that reversal. The resulting compounds displayed moderate CCK(2) antagonist activity in rat and human receptors, and a very small partial agonist effect on the production of inositol phosphate in COS-7 cells transfected with the wild-type human CCK(2) receptor.

Synthesis, conformational analysis, and cytotoxicity of conformationally constrained aplidine and tamandarin A analogues incorporating a spirolactam beta-turn mimetic.

With the aim of studying the contribution of the beta II turn conformation at the side chain of didemnins to the bioactive conformation responsible for their antitumoral activity, conformationally restricted analogues of aplidine and tamandarin A, where the side chain dipeptide Pro8-N-Me-d-Leu7 is replaced with the spirolactam beta II turn mimetic (5R)-7-[(1R)-1-carbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.4]nonane, were prepared. Additionally, restricted analogues, where the aplidine (pyruvyl9) or tamandarin A [(S)-Lac9] acyl groups are replaced with the isobutyryl, Boc, and 2-methylacryloyl groups, were also prepared. These structural modifications were detrimental to cytotoxic activity, leading to a decrease of 1-2 orders of magnitude with respect to that exhibited by aplidine and tamandarin A. The conformational analysis of one of these spirolactam aplidine analogues, by NMR and molecular modeling methods, showed that the conformational restriction caused by the spirolactam does not produce significant changes in the overall conformation of aplidine, apart from preferentially stabilizing the trans rotamer at the pyruvyl9-spirolactam amide bond, whereas in aplidine both cis and trans rotamers at the pyruvyl9-Pro8 amide bond are more or less equally stabilized. These results seem to indicate a preference for the cis form at that amide bond in the bioactive conformation of aplidine. The significant influence of this cis/trans isomerism upon the cytotoxicity suggests a possible participation of a peptidylprolyl cis/trans isomerase in the mechanism of action of aplidine.

Unprecedented stereospecific synthesis of a novel tetracyclic ring system, a hybrid of tetrahydropyrrolo[2,3-b]indole and tetrahydroimidazo[1,2-a]indole, via a domino reaction upon a tryptophan-derived amino nitrile.

Compounds containing a novel tetracyclic ring system, a hybrid of tetrahydropyrrolo[2,3-b]indole and tetrahydroimidazo[1,2-a]indole, are synthesized via an acid-mediated stereospecific domino tautomerization of a tryptophan-derived alpha-amino nitrile. Characterization of these new compounds and preliminary studies on the reactivity of the tetracyclic heterocyclic system are reported. [reaction: see text]

Solvent-free synthesis of α-amino nitrile-derived ureas.

An efficient and environmentally friendly methodology for the solvent-free synthesis of α-amino nitrile derived ureas from α-amino acid based amino nitriles has been developed. At room temperature no epimerization was observed in the resulting ureas, but under microwave heating, epimerization occurred at the chiral center bearing the cyano group.

Highly functionalized 2-oxopiperazine-based peptidomimetics: an approach to PAR1 antagonists.

A series of pseudodipeptide-based chiral 1,3,4,5-tetrasubstituted-2-oxopiperazines has been designed and synthesized as potential PAR1 antagonists. These highly functionalized piperazines were synthesized from aromatic and basic amino acid derived Ψ[CH(CN)NH]pseudodipeptides through a four step pathway that involves reduction of the cyano group to build the 2-oxopiperazine ring, followed by selective functionalization at the N4-, N1-positions, and at the exocyclic moiety at position C5. This regioselective functionalization required the fine tuning of reaction conditions. All new compounds were screened as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cell lines. Some of the compounds displayed moderate PAR1 antagonist activity, while, others were cytotoxic at µM concentration. No correlation was observed between both types of activities.

Design, synthesis and biological evaluation of new peptide-based ureas and thioureas as potential antagonists of the thrombin receptor PAR1.

By applying a diversity oriented synthesis strategy for the search of new antagonists of the thrombin receptor PAR1, a series of peptide-based ureas and thioureas, including analogues of the PAR1 reference antagonist RWJ-58259, has been designed and synthesized. The general synthetic scheme involves reduction of basic amino acid-derived amino nitriles by hydrogen transfer from hydrazine monohydrate in the presence of Raney Ni, followed by reaction with diverse isocyanates and isothiocyanates, and protecting group removal. All new compounds have been evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN. Some protected peptide-based ureas displayed significant antagonist activity.

Latent tuberculosis seems not to reactivate in multiple sclerosis patients on natalizumab.

Current safety recommendations for multiple sclerosis (MS) patients who are considered for natalizumab do not specify how to screen for latent tuberculosis (LTB). Only chest X-ray is recommended as a routine, and follow-up depends on its results. The incidence of TB in Spain is high and the risk of a LTB reactivation due to natalizumab is unknown. Our objective is to describe in our clinical practice if following the current recommendations for the MS population on natalizumab allows identifying patients with a LTB, as well as the risk for TB reactivation. Our study demonstrated that, in our environment, current recommendations are not sensitive enough to identify cases of LTB, though no cases of active TB were observed. Considering the lack of documented active TB cases worldwide among natalizumab patients, we suggest that these safety measures are probably unnecessary, even in countries with a high TB incidence.