4-(3-Phenyl-4-(3,4,5-trimethoxybenzoyl)-1H-pyrrol-1-yl)benzenesulfonamide, a Novel Carbonic Anhydrase and Wnt/ß-Catenin Signaling Pathway Dual-Targeting Inhibitor with Potent Activity against Multidrug Resistant Cancer Cells.

We synthesized new pyrrole and indole derivatives as human carbonic anhydrase (hCA) inhibitors with the potential to inhibit the Wnt/ß-catenin signaling pathway. The presence of both N1-(4-sulfonamidophenyl) and 3-(3,4,5-trimethoxyphenyl) substituents was essential for strong hCA inhibitors. The most potent hCA XII inhibitor 15 (Ki = 6.8 nM) suppressed the Wnt/ß-catenin signaling pathway and its target genes MYC, Fgf20, and Sall4 and exhibited the typical markers of apoptosis, cleaved poly(ADP-ribose)polymerase, and cleaved caspase-3. Compound 15 showed strong inhibition of viability in a panel of cancer cells, including colorectal cancer and triple-negative breast cancer cells, was effective against the NCI/ADR-RES DOX-resistant cell line, and restored the sensitivity to doxorubicin (DOX) in HT29/DX and MDCK/P-gp cells. Compound 15 is a novel dual-targeting compound with activity against hCA and Wnt/ß-catenin. It thus has a broad targeting spectrum and is an anticancer agent with specific potential in P-glycoprotein overexpressing cell lines.

Novel N-(Heterocyclylphenyl)benzensulfonamide Sharing an Unreported Binding Site with T-Cell Factor 4 at the ß-Catenin Armadillo Repeats Domain as an Anticancer Agent.

Despite intensive efforts, no inhibitors of the Wnt/ß-catenin signaling pathway have been approved so far for the clinical treatment of cancer. We synthesized novel N-(heterocyclylphenyl)benzenesulfonamides as ß-catenin inhibitors. Compounds 5-10 showed strong inhibition of the luciferase activity. Compounds 5 and 6 inhibited the MDA-MB-231, HCC1806, and HCC1937 TNBC cells. Compound 9 induced in vitro cell death in SW480 and HCT116 cells and in vivo tumorigenicity of a human colorectal cancer line HCT116. In a co-immunoprecipitation study in HCT116 cells transfected with Myc-tagged T-cell factor 4 (Tcf-4), compound 9 abrogated the association between ß-catenin and Tcf-4. The crystallographic analysis of the ß-catenin Armadillo repeats domain revealed that compound 9 and Tcf-4 share a common binding site within the hotspot binding region close to Lys508. To our knowledge, compound 9 is the first small molecule ligand of this region to be reported. These results highlight the potential of this novel class of ß-catenin inhibitors as anticancer agents.

Anticancer Activity of (S)-5-Chloro-3-((3,5-dimethylphenyl)sulfonyl)-N-(1-oxo-1-((pyridin-4-ylmethyl)amino)propan-2-yl)-1H-indole-2-carboxamide (RS4690), a New Dishevelled 1 Inhibitor.

Wingless/integrase-11 (WNT)/ß-catenin pathway is a crucial upstream regulator of a huge array of cellular functions. Its dysregulation is correlated to neoplastic cellular transition and cancer proliferation. Members of the Dishevelled (DVL) family of proteins play an important role in the transduction of WNT signaling by contacting its cognate receptor, Frizzled, via a shared PDZ domain. Thus, negative modulators of DVL1 are able to impair the binding to Frizzled receptors, turning off the aberrant activation of the WNT pathway and leading to anti-cancer activity. Through structure-based virtual screening studies, we identified racemic compound RS4690 (1), which showed a promising selective DVL1 binding inhibition with an EC50 of 0.74 ± 0.08 µM. Molecular dynamic simulations suggested a different binding mode for the enantiomers. In the in vitro assays, enantiomer (S)-1 showed better inhibition of DVL1 with an EC50 of 0.49 ± 0.11 µM compared to the (R)-enantiomer. Compound (S)-1 inhibited the growth of HCT116 cells expressing wild-type APC with an EC50 of 7.1 ± 0.6 µM and caused a high level of ROS production. These results highlight (S)-1 as a lead compound for the development of new therapeutic agents against WNT-dependent colon cancer.

Top-Down N-Doped Carbon Quantum Dots for Multiple Purposes: Heavy Metal Detection and Intracellular Fluorescence.

In the present study, we successfully synthesized N-doped carbon quantum dots (N-CQDs) using a top-down approach, i.e., hydroxyl radical opening of fullerene with hydrogen peroxide, in basic ambient using ammonia for two different reaction times. The ensuing characterization via dynamic light scattering, SEM, and IR spectroscopy revealed a size control that was dependent on the reaction time, as well as a more pronounced -NH2 functionalization. The N-CQDs were probed for metal ion detection in aqueous solutions and during bioimaging and displayed a Cr3+ and Cu2+ selectivity shift at a higher degree of -NH2 functionalization, as well as HEK-293 cell nuclei marking.

Discovery of pyrrole derivatives for the treatment of glioblastoma and chronic myeloid leukemia.

Long-term survivors of glioblastoma multiforme (GBM) are at high risk of developing second primary neoplasms, including leukemia. For these patients, the use of classic tyrosine kinase inhibitors (TKIs), such as imatinib mesylate, is strongly discouraged, since this treatment causes a tremendous increase of tumor and stem cell migration and invasion. We aimed to develop agents useful for the treatment of patients with GBM and chronic myeloid leukemia (CML) using an alternative mechanism of action from the TKIs, specifically based on the inhibition of tubulin polymerization. Compounds 7 and 25, as planned, not only inhibited tubulin polymerization, but also inhibited the proliferation of both GMB and CML cells, including those expressing the T315I mutation, at nanomolar concentrations. In in vivo experiments in BALB/cnu/nu mice injected subcutaneously with U87MG cells, in vivo, 7 significantly inhibited GBM cancer cell proliferation, in vivo tumorigenesis, and tumor growth, tumorigenesis and angiogenesis. Compound 7 was found to block human topoisomerase II (hTopoII) selectively and completely, at a concentration of 100 µM.

Structure-activity relationship studies and in vitro and in vivo anticancer activity of novel 3-aroyl-1,4-diarylpyrroles against solid tumors and hematological malignancies.

Novel 3-aroyl-1,4-diarylpyrrole derivatives were synthesized to explore structure-activity relationships at the phenyls at positions 1 and 4 of the pyrrole. The presence of amino phenyl rings at positions 1 and 4 of the pyrrole ring were found to be a crucial requirement for potent antitumor activity. Several compounds strongly inhibited tubulin assembly through binding to the colchicine site. Compounds 42, 44, 48, 62 and 69 showed antitumor activity with low nanomolar IC50 values in several cancer cell lines. Compound 48 was generally more effective as an inhibitor of glioblastoma, colorectal and urinary bladder cancer cell lines; 69 consistently inhibited CML cell lines and demonstrated superiority in nilotinib and imatinib resistant LAMA84-R and KBM5-T315I cells. In animal models, compound 48 exhibited significant inhibition of the growth of T24 bladder carcinoma and ES-2 ovarian clear cell carcinoma tumors. Compounds 48 and 69 represent robust lead compounds for the design of new broad-spectrum anticancer agents active in different types of solid and hematological tumors.

Drug Design and Synthesis of First in Class PDZ1 Targeting NHERF1 Inhibitors as Anticancer Agents.

Targeted approaches aiming at modulating NHERF1 activity, rather than its overall expression, would be preferred to preserve the normal functions of this versatile protein. We focused our attention on the NHERF1/PDZ1 domain that governs its membrane recruitment/displacement through a transient phosphorylation switch. We herein report the design and synthesis of novel NHERF1 PDZ1 domain inhibitors. These compounds have potential therapeutic value when used in combination with antagonists of ß-catenin to augment apoptotic death of colorectal cancer cells refractory to currently available Wnt/ß-catenin-targeted agents.

Butyrylcholinesterase and Acetylcholinesterase polymorphisms in Multiple Sclerosis patients: implication in peripheral inflammation.

Multiple Sclerosis (MS) is an autoimmune disease, having not fully understood aetiology, and both genetic and environmental factors contribute to the pathogenesis of the disease. The cholinergic system has been indicated as a mediator of neuro-immune interactions, as well as an internal regulator of immune responses. The aim of the present research was to assess the associations between BChE and AChE genetic variations and serum cholinergic and inflammatory profiles in 102 Relapsing Remitting-MS patients and 117 healthy controls. An increased frequency of the BChE K-allele in MS patients as compared to controls was found. In addition, data showed that patients had higher BChE enzymatic activity, which is increased by the presence of the polymorphic allele and reduced amounts of circulating ACh. AChE polymorphism was significantly associated to reduced activity in both patients and controls. We propose that serum BChE and AChE activity may be used as a secondary markers to assess the role of non-neuronal cholinergic system in regulating peripheral inflammation via ACh regulation. This pilot study shed light on the role of the non-neuronal cholinergic system in immune cells to better understand MS pathogenesis. The cross-talk between the periphery and the CNS could have a new undescribed crucial role for MS, regarded as a systemic disease.

Dysregulated Homeostasis of Acetylcholine Levels in Immune Cells of RR-Multiple Sclerosis Patients.

Multiple sclerosis (MS) is characterized by pro-inflammatory cytokine production. Acetylcholine (ACh) contributes to the modulation of central and peripheral inflammation. We studied the homeostasis of the cholinergic system in relation to cytokine levels in immune cells and sera of relapsing remitting-MS (RR-MS) patients. We demonstrated that lower ACh levels in serum of RR-MS patients were inversely correlated with the increased activity of the hydrolyzing enzymes acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Interestingly, the expression of the ACh biosynthetic enzyme and the protein carriers involved in non-vesicular ACh release were found overexpressed in peripheral blood mononuclear cells of MS patients. The inflammatory state of the MS patients was confirmed by increased levels of TNFα, IL-12/IL-23p40, IL-18. The lower circulating ACh levels in sera of MS patients are dependent on the higher activity of cholinergic hydrolyzing enzymes. The smaller ratio of ACh to TNFα, IL-12/IL-23p40 and IL-18 in MS patients, with respect to healthy donors (HD), is indicative of an inflammatory environment probably related to the alteration of cholinergic system homeostasis.

Modulating intracellular acidification by regulating the incubation time of proton caged compounds.

A proton caged compound, the 1-(2-nitrophenyl)- ethylhexadecyl sulfonate (HDNS), was dosed into HEK-293 at different incubation times. Samples were irradiated with filtered UV light for inducing photolysis of the HDNS and then probed by infrared spectroscopy. The intracellular acidification reaction can be followed by monitoring the consequent CO2 peak intensity variation. The total CO2 produced is similar for all the samples, hence it is only a function of the initial HDNS concentration. The way it is achieved, though, is different for the different incubation times and follows kinetics, which results in a combination of a linear CO2 increase and a steep CO2 increase followed by a decay. This is interpreted in terms of confinement of the HDNS into intracellular vesicles of variable average size and sensitive to UV light when they reach critical dimensions.

Exogenous control over intracellular acidification: Enhancement via proton caged compounds coupled to gold nanoparticles and an alternative pathway with DMSO.

Proton caged compounds exhibit a characteristic behavior when directly dosed into cells or being coupled to gold nanoparticles prior to the dosing. When irradiated in the near ultraviolet region, they release protons that interact with intracellular HCO3 (-) to yield H2CO3. The dissociation of carbonic acid, then, releases CO2 that can be distinctively singled out in infrared spectra. In the process of searching a pathway to augment the intracellular uptake of proton caged compounds, we probed the association of 1-(2-nitrophenyl)-ethylhexadecyl sulfonate (HDNS) with DMSO, an agent to enhance the membrane permeability. We found out a different UV-induced protonation mechanism that opens up to new conduits of employing of proton caged compounds. Here, we report the infrared data we collected in this set of experiments.

Exogenous control over intracellular acidification: Enhancement via proton caged compounds coupled to gold nanoparticles.

The pH regulation has a fundamental role in several intracellular processes and its variation via exogenous compounds is a potential tool for intervening in the intracellular processes. Proton caged compounds (PPCs) release protons upon UV irradiation and may efficiently provoke intracellular on-command acidification. Here, we explore the intracellular pH variation, when purposely synthesized PCCs are coupled to gold nanoparticles (AuNPs) and dosed to HEK-293 cells. We detected the acidification process caused by the UV irradiation by monitoring the intensity of the asymmetric stretching mode of the CO(2) molecule at 2343 cm(-1). The comparison between free and AuNPs functionalized proton caged compound demonstrates a highly enhanced CO(2) yield, hence pH variation, in the latter case. Finally, PCC functionalized AuNPs were marked with a purposely synthesized fluorescent marker and dosed to HEK-293 cells. The corresponding fluorescence optical images show green grains throughout the whole cytoplasm.

Attractiveness and affordance shape tools neural coding: insight from ERPs.

The relation between attractiveness and motor affordance is a key topic in design and has not yet been investigated electrophysiologically. In this respect, action affordance and attractiveness represent two crucial dimensions in object processing (specifically for tools). In light of this evidence, Event Related Potentials (ERPs) enabled us to gain new insights into the time course of the interaction between these two dimensions during an explicit tool evaluation task. Behaviorally, tools that were judged as high affording and high attractive yielded faster response times than those judged as low affording and low attractive. The ERP results showed that early processes related to sensory gating and feature extraction (N100) were sensitive to both affordance and attractiveness; the P200 was dominated by affordance, indexing a facilitated access to motor action representation. The N300, P300 and the Late Positive Potential (LPP) showed enhanced responses for highly affording/attractive tools, reflecting the interconnection between attractiveness and affordance. Later responses were entirely affected by attractiveness, suggesting additional affective responses evoked by desirable tools. We are showing that things that are perceived as more functional and attractive have a privileged neural activation in the time course of tool evaluation, for the first time.

Direct HPLC enantioseparation of chiral aptazepine derivatives on coated and immobilized polysaccharide-based chiral stationary phases.

The direct HPLC enantioseparation of Mianserin and a series of aptazepine derivatives is accomplished on polysaccharide-based chiral stationary phases (CSPs). The resolutions are performed on the coated-type Chiralcel OD and Chiralpak AD CSPs and on the first commercially available immobilized-type Chiralpak IA CSP, in normal-phase and polar-organic modes. The complete separation of enantiomers of all racemates investigated was successfully achieved under at least one of CSP/eluent combinations employed. Pure alcohols such ethanol or 2-propanol, with a fixed percentage of DEA added, serve as valuable alternatives to the more common n-hexane-based normal-phase eluents in resolution of Mianserin on the AD CSP. In order to study the chiroptical properties of aptazepine derivatives, chromatographic resolutions are carried out at semipreparative scale using Chiralpak AD and Chiralpak IA as CSPs. Nonconventional dichloromethane-based eluents have permitted to expand the chiral resolving ability of the immobilized Chiralpak IA CSP and to perform mg-scale enantioseparations with an analytical-size column. Assignment of the absolute configuration of the separated enantiomers is empirically established by comparing their chiroptical data with those of structurally related Mianserin.