A Proteomics Approach Identifies RREB1 as a Crucial Molecular Target of Imidazo-Pyrazole Treatment in SKMEL-28 Melanoma Cells.

Cutaneous melanoma is the most dangerous and deadly form of human skin malignancy. Despite its rarity, it accounts for a staggering 80% of deaths attributed to cutaneous cancers overall. Moreover, its final stages often exhibit resistance to drug treatments, resulting in unfavorable outcomes. Hence, ensuring access to novel and improved chemotherapeutic agents is imperative for patients grappling with this severe ailment. Pyrazole and its fused systems derived thereof are heteroaromatic moieties widely employed in medicinal chemistry to develop effective drugs for various therapeutic areas, including inflammation, pain, oxidation, pathogens, depression, and fever. In a previous study, we described the biochemical properties of a newly synthesized group of imidazo-pyrazole compounds. In this paper, to improve our knowledge of the pharmacological properties of these molecules, we conduct a differential proteomic analysis on a human melanoma cell line treated with one of these imidazo-pyrazole derivatives. Our results detail the changes to the SKMEL-28 cell line proteome induced by 24, 48, and 72 h of 3e imidazo-pyrazole treatment. Notably, we highlight the down-regulation of the Ras-responsive element binding protein 1 (RREB1), a member of the zinc finger transcription factors family involved in the tumorigenesis of melanoma. RREB1 is a downstream element of the MAPK pathway, and its activation is mediated by ERK1/2 through phosphorylation.

Regioselective Synthesis, Structural Characterization, and Antiproliferative Activity of Novel Tetra-Substituted Phenylaminopyrazole Derivatives.

A small library of highly functionalized phenylaminopyrazoles, bearing different substituents at position 1, 3, and 4 of the pyrazole ring, was prepared by the one-pot condensation of active methylene reagents, phenylisothiocyanate, and substituted hydrazine (namely, methyl- and benzyl-hydrazine). The identified reaction conditions proved to be versatile and efficient. Furthermore, the evaluation of alternative stepwise protocols affected the chemo- and regio-selectivity outcome of the one-pot procedure. The chemical identities of two N-methyl pyrazole isomers, selected as prototypes of the whole series, were unambiguously identified by means of NMR and mass spectrometry studies. Additionally, semiempirical calculations provided a structural rationale for the different chromatographic behavior of the two isomers. The prepared tetra-substituted phenylaminopyrazoles were tested in cell-based assays on a panel of cancer and normal cell lines. The tested compounds did not show any cytotoxic effect on the selected cell lines, thus supporting their pharmaceutical potentials.

Bicyclic Basic Merbarone Analogues as Antiproliferative Agents.

Pyrimido-pyrimidine derivatives have been developed as rigid merbarone analogues. In a previous study, these compounds showed potent antiproliferative activity and efficiently inhibited topoisomerase IIα. To further extend the structure-activity relationships on pyrimido-pyrimidines, a novel series of analogues was synthesized by a two-step procedure. Analogues 3-6 bear small alky groups at positions 1 and 3 of the pyrimido-pyrimidine scaffold whereas at position 6a (4-chloro)phenyl substituent was inserted. The basic side chains introduced at position 7 were selected on the basis of the previously developed structure-activity relationships. The antiproliferative activity of the novel compounds proved to be affected by both the nature of the basic side chain and the substituents on the pyrimido-pyrimidine moiety. Derivatives 5d and 5e were identified as the most promising molecules still showing reduced antiproliferative activity in comparison with the previously prepared pyrimido-pyrimidine analogues. In topoisomerase IIα-5d docking complex, the ligand would poorly interact with the enzyme and assume a different orientation in comparison with 1d bioactive conformation.

Fibrin Gels Entrapment of a Poly-Cyclodextrin Nanocarrier as a Doxorubicin Delivery System in an Orthotopic Model of Neuroblastoma: Evaluation of In Vitro Activity and In Vivo Toxicity.

PURPOSE: Fibrin gels (FBGs) are potential delivery vehicles for many drugs, and can be easily prepared from purified components. We previously demonstrated their applicability for the release of different doxorubicin (Dox) nanoparticles used clinically or in an experimental stage, such as its inclusion complex with the amino ß-cyclodextrin polymer (oCD-NH2/Dox). Here we extend these studies by in vitro and in vivo evaluations. METHODS: An in vitro cytotoxicity model consisting of an overlay of a neuroblastoma (NB) cell-containing agar layer above a drug-loaded FBG layer was used. Local toxicity in vivo (histology and blood analysis) was studied in a mouse orthotopic NB model (SHSY5YLuc+ cells implanted into the left adrenal gland). RESULTS: In vitro data show that FBGs loaded with oCD-NH2/Dox have a slightly lower cytotoxicity against NB cell lines than those loaded with Dox. Fibrinogen (FG), and Ca2+ concentrations may modify this activity. In vivo data support a lower general and local toxicity for FBGs loaded with oCD-NH2/Dox than those loaded with Dox. CONCLUSION: Our results suggest a possible increase of the therapeutic index of Dox when locally administered through FBGs loaded with oCD-NH2/Dox, opening the possibility of using these releasing systems for the treatment of neuroblastoma.

Geena 2, improved automated analysis of MALDI/TOF mass spectra.

BACKGROUND: Mass spectrometry (MS) is producing high volumes of data supporting oncological sciences, especially for translational research. Most of related elaborations can be carried out by combining existing tools at different levels, but little is currently available for the automation of the fundamental steps. For the analysis of MALDI/TOF spectra, a number of pre-processing steps are required, including joining of isotopic abundances for a given molecular species, normalization of signals against an internal standard, background noise removal, averaging multiple spectra from the same sample, and aligning spectra from different samples. In this paper, we present Geena 2, a public software tool for the automated execution of these pre-processing steps for MALDI/TOF spectra. RESULTS: Geena 2 has been developed in a Linux-Apache-MySQL-PHP web development environment, with scripts in PHP and Perl. Input and output are managed as simple formats that can be consumed by any database system and spreadsheet software. Input data may also be stored in a MySQL database. Processing methods are based on original heuristic algorithms which are introduced in the paper. Three simple and intuitive web interfaces are available: the Standard Search Interface, which allows a complete control over all parameters, the Bright Search Interface, which leaves to the user the possibility to tune parameters for alignment of spectra, and the Quick Search Interface, which limits the number of parameters to a minimum by using default values for the majority of parameters. Geena 2 has been utilized, in conjunction with a statistical analysis tool, in three published experimental works: a proteomic study on the effects of long-term cryopreservation on the low molecular weight fraction of serum proteome, and two retrospective serum proteomic studies, one on the risk of developing breat cancer in patients affected by gross cystic disease of the breast (GCDB) and the other for the identification of a predictor of breast cancer mortality following breast cancer surgery, whose results were validated by ELISA, a completely alternative method. CONCLUSIONS: Geena 2 is a public tool for the automated pre-processing of MS data originated by MALDI/TOF instruments, with a simple and intuitive web interface. It is now under active development for the inclusion of further filtering options and for the adoption of standard formats for MS spectra.

Matrix-assisted laser desorption/ionisation (MALDI) TOF analysis identifies serum angiotensin II concentrations as a strong predictor of all-cause and breast cancer (BCa)-specific mortality following breast surgery.

MALDI-TOF MS was used to recognise serum peptidome profiles predictive of mortality in women affected by early BCa. Mortality was analysed based on signal profiling, and appropriate statistics were used. The results indicate that four signals were increased in deceased patients compared with living patients. Three of the four signals were individually associated with all-cause mortality, but only one having mass/charge ratio (m/z) 1,046.49 was associated with BCa-specific mortality and was the only peak to maintain an independent prognostic role after multivariate analysis. Two groups exhibiting different mortality probabilities were identified after clustering patients based on the expression of the four peptides, but m/z 1,046.49 was exclusively expressed in the cluster exhibiting the worst mortality outcome, thus confirming the crucial value of this peptide. The specific role of this peak was confirmed by competing risk analysis. MS findings were validated by ELISA analysis after demonstrating that m/z 1,046.49 structurally corresponded to Angiotensin II (ATII). In fact, mortality results obtained after arbitrarily dividing patients according to an ATII serum value of 255 pg/ml (which corresponds to the 66(th) percentile value) were approximately comparable to those previously demonstrated when the same patients were analysed according to the expression of signal m/z 1,046.49. Similarly, ATII levels were specifically correlated with BCa-related deaths after competing risk analysis. In conclusion, ATII levels were increased in women who exhibited worse mortality outcomes, reinforcing the evidence that this peptide potentially significantly affects the natural history of early BCa. Our findings also confirm that MALDI-TOF MS is an efficient screening tool to identify novel tumour markers and that MS findings can be rapidly validated through less complex techniques, such as ELISA.

Pharmacokinetics, pharmacodynamics and efficacy on pediatric tumors of the glioma radiosensitizer KU60019.

We have recently reported that glioblastoma (GB)-initiating cells (GIC) with low expression and/or mutation of TP53 and high expression of PI3K ("responder" genetic profile) can be effectively and safely radiosensitized by the ATM inhibitor KU60019. We report here on drug's diffusion and elimination from the animal body and brain, its effects on orthotopic GB and efficacy toward pediatric GIC. Healthy mice were infused by convection enhanced delivery (CED) with KU60019 and the drug kinetics followed by high performance liquid chromatography-mass spectrometry. Already at the end of CED, KU60019 had diffused from the injection site to the ipsilateral and, to a lower extent, controlateral hemisphere. After 24 hr, no drug could be detected all over the brain or in other organs, indicating rapid draining and excretion. After intraperitoneal injection, traces only of KU60019 could be detected in the brain, indicating inability to cross the brain-blood barrier. Consistent with the induction of cell cycle progression previously observed in vitro, KU60019 stimulated proliferation of orthotopic GB cells with the highest effect observed 96 hr after drug delivery. Adult GIC with high expression of TP53 and low expression of PI3K could be radiosensitized by KU60019, although less promptly than GIC bearing the "responder" profile. Consistent with the kinetics of proliferation induction, the highest radiosensitizing effect was observed 96 hr after delivery of KU60019 to GIC. Pediatric GIC could be similarly radiosensitized after exposure to KU60019. The results indicate that ATM inhibition may allow to radiosensitize a wide range of adult and pediatric high-grade gliomas.

Fibrin gels loaded with cisplatin and cisplatin-hyaluronate complexes tested in a subcutaneous human melanoma model.

Fibrin gels are attractive biomaterials for local delivery of a variety of agents, from drugs to proteins. Similarly, polymer-anticancer-drug conjugates and nanoparticles are emerging as potential candidates for cancer treatment. Combining these different approaches, we have studied the efficacy of fibrin gels loaded with cisplatin (DDP) and a complex of DDP with hyaluronate (DDP-HA) for tumor growth inhibition in a melanoma model. Loaded gels prepared at relatively high fibrinogen concentration (22 mg/ml) showed good in vitro antiproliferative activities, prolonged release of the anticancer drug, and a long persistence (10-15 days) in vivo when implanted subcutaneously (sc) in immunodeficient mice. Gels loaded with DDP or DDP-HA containing 1/3 or even 1/6 of their systemic dose (6 mg/kg) and positioned under the tumor mass in mice bearing a sc human SK-Mel-28 tumor showed an antitumor activity better than that of the original parent compound given intraperitoneally (ip). Moreover, in an additional experiment in vivo, fibrin gels loaded with N-trimethyl chitosan-based nanoparticles containing a DDP-HA complex were assayed, resulting in a further 8 % improvement of anticancer activity, with lesser adverse systemic toxic effects. Taken together, these results suggest that the combination of fibrin gels and drugs complexed with suitable macromolecules holds great promise for loco-regional anticancer therapy of melanoma and other surgically removable cancer types.

Host-guest chemistry of a bis-calix[4]pyrrole derivative containing a trans/cis-switchable azobenzene unit with several aliphatic bis-carboxylates.

The azobenzene unit used as a photochemically and thermally switchable linker in the assembly of a bis-calix[4]pyrrole receptor provides a means to modulate the binding of bis-carboxylates of significant biological importance in cancer research. Conversely, the complexation of different bis-anionic guests has significant kinetic effects on both the photochemical and thermal trans/cis isomerization of the azobenzene unit.

A comprehensive mechanism of fibrin network formation involving early branching and delayed single- to double-strand transition from coupled time-resolved X-ray/light-scattering detection.

The formation of a fibrin network following fibrinogen enzymatic activation is the central event in blood coagulation and has important biomedical and biotechnological implications. A non-covalent polymerization reaction between macromolecular monomers, it consists basically of two complementary processes: elongation/branching generates an interconnected 3D scaffold of relatively thin fibrils, and cooperative lateral aggregation thickens them more than 10-fold. We have studied the early stages up to the gel point by fast fibrinogen:enzyme mixing experiments using simultaneous small-angle X-ray scattering and wide-angle, multi-angle light scattering detection. The coupled evolutions of the average molecular weight, size, and cross section of the solutes during the fibrils growth phase were thus recovered. They reveal that extended structures, thinner than those predicted by the classic half-staggered, double-stranded mechanism, must quickly form. Following extensive modeling, an initial phase is proposed in which single-bonded "Y-ladder" polymers rapidly elongate before undergoing a delayed transition to the double-stranded fibrils. Consistent with the data, this alternative mechanism can intrinsically generate frequent, random branching points in each growing fibril. The model predicts that, as a consequence, some branches in these expanding "lumps" eventually interconnect, forming the pervasive 3D network. While still growing, other branches will then undergo a Ca(2+)/length-dependent cooperative collapse on the resulting network scaffolding filaments, explaining their sudden thickening, low final density, and basic mechanical properties.

Host-guest chemistry of aromatic-amide-linked bis- and tris-calix[4]pyrroles with bis-carboxylates and citrate anion.

A small library of polytopic receptors has been synthesized from meso-p- and meso-m-aminophenylcalix[4]pyrroles and p- or m-phthaloyl or trimesic chloride. Selected bis-carboxylates and the citrate anion, which either exhibit altered distribution profiles in cancerous tissues in comparison with healthy tissues or are metabolites of carcinogenic substances (for example, trans,trans-muconic acid from benzene exposure in humans) were tested as ligands. Varied affinities and binding modes were observed as a function of the number of calix[4]pyrroles and the topology of amide units present in each of the polytopic receptors. The structures of the 1:1 complexes derived by molecular modeling are in excellent agreement with the results of (1)H NMR complexation studies.

Trastuzumab quantification in serum: a new, rapid, robust ELISA assay based on a mimetic peptide that specifically recognizes trastuzumab.

Trastuzumab, a humanized monoclonal antibody directed against the epidermal growth factor receptor 2 (HER2), is a milestone in the treatment of HER2-overexpressing breast cancer patients. An enzyme-linked immunosorbent assay (ELISA) for trastuzumab has been developed for routine use in the laboratory to support clinical and pharmacokinetic studies to optimize therapy. The method relies on an antigen peptide linked to a 96-well plate via the streptavidin/biotin system. The peptide sequence mimics the extracellular portion of the HER2 receptor that is recognized by trastuzumab. The calibration range of the assay is 10 to 360 ng/mL per well, corresponding to a trastuzumab serum concentration from 5 to 180 µg/mL with a lower limit of quantification of 10 µg/mL. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum using this assay. The procedure was also tested in sera obtained from breast cancer patients to evaluate trastuzumab serum levels, confirming the applicability of method that could be a valid assay to use in daily laboratory practice.

Lipid Metabolism Reprogramming and Trastuzumab Resistance in Breast Cancer Cell Lines Overexpressing the ERBB2 Membrane Receptor.

Trastuzumab (Tz), an antibody targeting ERBB2, has significantly improved the prognosis for breast cancer (BCa) patients with overexpression of the ERBB2 receptor. However, Tz resistance poses a challenge to patient outcomes. Numerous mechanisms have been suggested to contribute to Tz resistance, and this study aimed to uncover shared mechanisms in in vitro models of acquired BCa Tz resistance. Three widely used ERBB2+ BCa cell lines, adapted to grow in Tz, were examined. Despite investigating potential changes in phenotype, proliferation, and ERBB2 membrane expression in these Tz-resistant (Tz-R) cell lines compared to wild-type (wt) cells, no common alterations were discovered. Instead, high-resolution mass spectrometry analysis revealed a shared set of differentially expressed proteins (DEPs) in Tz-R versus wt cells. Bioinformatic analysis demonstrated that all three Tz-R cell models exhibited modulation of proteins associated with lipid metabolism, organophosphate biosynthesis, and macromolecule methylation. Ultrastructural examination corroborated the presence of altered lipid droplets in resistant cells. These findings strongly support the notion that intricate metabolic adaptations, including lipid metabolism, protein phosphorylation, and potentially chromatin remodeling, may contribute to Tz resistance. The detection of 10 common DEPs across all three Tz-resistant cell lines offers promising avenues for future therapeutic interventions, providing potential targets to overcome Tz resistance and potentially improve patient outcomes in ERBB2+ breast cancer.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

A Circulating Risk Score, Based on Combined Expression of Exo-miR-130a-3p and Fibrinopeptide A, as Predictive Biomarker of Relapse in Resectable Non-Small Cell Lung Cancer Patients.

To date, the 5-year overall survival rate of 60% for early-stage non-small cell lung cancer (NSCLC) is still unsatisfactory. Therefore, reliable prognostic factors are needed. Growing evidence shows that cancer progression may depend on an interconnection between cancer cells and the surrounding tumor microenvironment; hence, circulating molecules may represent promising markers of cancer recurrence. In order to identify a prognostic score, we performed in-depth high-throughput analyses of plasma circulating markers, including exosomal microRNAs (Exo-miR) and peptides, in 67 radically resected NSCLCs. The miRnome profile selected the Exo-miR-130a-3p as the most overexpressed in relapsed patients. Peptidome analysis identified four progressively more degraded forms of fibrinopeptide A (FpA), which were depleted in progressing patients. Notably, stepwise Cox regression analysis selected Exo-miR-130a-3p and the greatest FpA (2-16) to build a score predictive of recurrence, where high-risk patients had 18 months of median disease-free survival. Moreover, in vitro transfections showed that higher levels of miR-130a-3p lead to a deregulation of pathways involved in metastasis and angiogenesis, including the coagulation process and metalloprotease increase which might be linked to FpA reduction. In conclusion, by integrating circulating markers, the identified risk score may help clinicians predict early-stage NSCLC patients who are more likely to relapse after primary surgery.

Substitution of the human αC region with the analogous chicken domain generates a fibrinogen with severely impaired lateral aggregation: fibrin monomers assemble into protofibrils but protofibrils do not assemble into fibers.

Fibrin polymerization occurs in two steps: the assembly of fibrin monomers into protofibrils and the lateral aggregation of protofibrils into fibers. Here we describe a novel fibrinogen that apparently impairs only lateral aggregation. This variant is a hybrid, where the human αC region has been replaced with the homologous chicken region. Several experiments indicate this hybrid human-chicken (HC) fibrinogen has an overall structure similar to normal. Thrombin-catalyzed fibrinopeptide release from HC fibrinogen was normal. Plasmin digests of HC fibrinogen produced fragments that were similar to normal D and E; further, as with normal fibrinogen, the knob 'A' peptide, GPRP, reversed the plasmin cleavage associated with addition of EDTA. Dynamic light scattering and turbidity studies with HC fibrinogen showed polymerization was not normal. Whereas early small increases in hydrodynamic radius and absorbance paralleled the increases seen during the assembly of normal protofibrils, HC fibrinogen showed no dramatic increase in scattering as observed with normal lateral aggregation. To determine whether HC and normal fibrinogen could form a copolymer, we examined mixtures of these. Polymerization of normal fibrinogen was markedly changed by HC fibrinogen, as expected for mixed polymers. When the mixture contained 0.45 µM normal and 0.15 µM HC fibrinogen, the initiation of lateral aggregation was delayed and the final fiber size was reduced relative to normal fibrinogen at 0.45 µM. Considered altogether, our data suggest that HC fibrin monomers can assemble into protofibrils or protofibril-like structures, but these either cannot assemble into fibers or assemble into very thin fibers.

The application of atmospheric pressure matrix-assisted laser desorption/ionization to the analysis of long-term cryopreserved serum peptidome.

Although most time-of-flight (TOF) mass spectrometers come equipped with vacuum matrix-assisted laser desorption/ionization (MALDI) sources, the atmospheric pressure MALDI (API-MALDI) source is an attractive option because of its ability to be coupled to a wide range of analyzers. This article describes the use of an API-MALDI source coupled to a TOF mass spectrometer for evaluation of the effects of medium- and long-term storage on peptidomic profiles of cryopreserved serum samples from healthy women. Peptides were purified using superparamagnetic beads either from fresh sera or after serum storage at -80°C for 18 months or at -20°C for 8 years. Data were preprocessed using newly developed bioinformatic tools and then were subjected to statistical analysis and class prediction. The analyses showed a dramatic effect of storage on the abundance of several peptides such as fibrinopeptides A and B, complement fractions, bradykinin, and clusterin, indicated by other authors as disease biomarkers. Most of these results were confirmed by shadow clustering analysis, able to classify each sample in the correct group. In addition to demonstrating the suitability of the API-MALDI technique for peptidome profiling studies, our data are of relevance for retrospective studies that involve frozen sera stored for many years in biobanks.

GeenaR: A Web Tool for Reproducible MALDI-TOF Analysis.

Mass spectrometry is a widely applied technology with a strong impact in the proteomics field. MALDI-TOF is a combined technology in mass spectrometry with many applications in characterizing biological samples from different sources, such as the identification of cancer biomarkers, the detection of food frauds, the identification of doping substances in athletes' fluids, and so on. The massive quantity of data, in the form of mass spectra, are often biased and altered by different sources of noise. Therefore, extracting the most relevant features that characterize the samples is often challenging and requires combining several computational methods. Here, we present GeenaR, a novel web tool that provides a complete workflow for pre-processing, analyzing, visualizing, and comparing MALDI-TOF mass spectra. GeenaR is user-friendly, provides many different functionalities for the analysis of the mass spectra, and supports reproducible research since it produces a human-readable report that contains function parameters, results, and the code used for processing the mass spectra. First, we illustrate the features available in GeenaR. Then, we describe its internal structure. Finally, we prove its capabilities in analyzing oncological datasets by presenting two case studies related to ovarian cancer and colorectal cancer. GeenaR is available at http://proteomics.hsanmartino.it/geenar/.

Blocking glutamate mGlu5 receptors with the negative allosteric modulator CTEP improves disease course in SOD1G93A mouse model of amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: The pathogenesis of amyotrophic lateral sclerosis (ALS) is not fully clarified, although excessive glutamate (Glu) transmission and the downstream cytotoxic cascades are major mechanisms for motor neuron death. Two metabotropic glutamate receptors (mGlu1 and mGlu5 ) are overexpressed in ALS and regulate cellular disease processes. Expression and function of mGlu5 receptors are altered at early symptomatic stages in the SOD1G93A mouse model of ALS and knockdown of mGlu5 receptors in SOD1G93A mice improved disease progression. EXPERIMENTAL APPROACH: We treated male and female SOD1G93A mice with 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP), an orally available mGlu5 receptor negative allosteric modulator (NAM), using doses of 2 mg·kg-1 per 48 h or 4 mg·kg-1 per 24 h from Day 90, an early symptomatic disease stage. Disease progression was studied by behavioural and histological approaches. KEY RESULTS: CTEP dose-dependently ameliorated clinical features in SOD1G93A mice. The lower dose increased survival and improved motor skills in female mice, with barely positive effects in male mice. Higher doses significantly ameliorated disease symptoms and survival in both males and females, females being more responsive. CTEP also reduced motor neuron death, astrocyte and microglia activation, and abnormal glutamate release in the spinal cord, with equal effects in male and female mice. No differences were also observed in CTEP access to the brain. CONCLUSION AND IMPLICATIONS: Our results suggest that mGlu5 receptors are promising targets for the treatment of ALS and highlight mGlu5 receptor NAMs as effective pharmacological tools with translational potential.

Hydrodynamic and mass spectrometry analysis of nearly-intact human fibrinogen, chicken fibrinogen, and of a substantially monodisperse human fibrinogen fragment X.

The shape and solution properties of fibrinogen are affected by the location of the C-terminal portion of the Aalpha chains, which is presently still controversial. We have measured the hydrodynamic properties of a human fibrinogen fraction with these appendages mostly intact, of chicken fibrinogen, where they lack 11 characteristic 13-amino acids repeats, and of human fragment X, a plasmin early degradation product in which they have been removed. The human fibrinogen/fragment X samples were extensively characterized by SDS-PAGE/Western blotting and mass spectrometry, allowing their composition to be precisely determined. The solution properties of all samples were then investigated by analytical ultracentrifugation and size-exclusion HPLC coupled with multi-angle light scattering and differential pressure viscometry detectors. The measured parameters suggest that the extra repeats have little influence on the overall fibrinogen conformation, while a significant change is brought about by the removal of the C-terminal portion of the Aalpha chains beyond residue Aalpha200.