Targeting defective sphingosine kinase 1 in Niemann-Pick type C disease with an activator mitigates cholesterol accumulation.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.

Hydroxynaphthalenecarboxamides and substituted piperazinylpropandiols, two new series of BRAF inhibitors. A theoretical and experimental study.

The oncogenic mutated kinase BRAFV600E is an attractive molecular target because it is expressed in several human cancers, including melanoma. To present, only three BRAF small inhibitors are approved by the FDA for the treatment of patients with metastatic melanoma: Vemurafenib, Dabrafenib and Encorafenib. Although many protocol treatments have been probed in clinical trials, BRAF inhibition has a limited effectiveness because patients invariably develop resistance and secondary toxic effects associated with the therapy. These limitations highlight the importance of designing new and better inhibitors with different structures that could establish different interactions in the active site of the enzyme and therefore decrease resistance progress. Considering the data from our previous report, here we studied two series of derivatives of structural scaffolds as potential BRAF inhibitors: hydroxynaphthalenecarboxamides and substituted piperazinylpropandiols. Our results indicate that structural analogues of substituted piperazinylpropandiols do not show significantly better activities to that previously reported. In contrast, the hydroxynaphthalenecarboxamides derivatives significantly inhibited cell viability and ERK phosphorylation, a measure of BRAF activity, in Lu1205 BRAFV600E melanoma cells. In order to better understand these experimental results, we carried out a molecular modeling study using different combined techniques: docking, MD simulations and quantum theory of atoms in molecules (QTAIM) calculations. Thus, by using this approach we determined that the molecular interactions that stabilize the different molecular complexes are closely related to Vemurafenib, a well-documented BRAF inhibitor. Furthermore, we found that bi-substituted compounds may interact more strongly respect to the mono-substituted analogues, by establishing additional interactions with the DFG-loop at the BRAF-active site. On the bases of these results we synthesized and tested a new series of hydroxynaphthalenecarboxamides bi-substituted. Remarkably, all these compounds displayed significant inhibitory effects on the bioassays performed. Thus, the structural information reported here is important for the design of new BRAFV600E inhibitors possessing this type of structural scaffold.

Searching new structural scaffolds for BRAF inhibitors. An integrative study using theoretical and experimental techniques.

The identification of the V600E activating mutation in the protein kinase BRAF in around 50% of melanoma patients has driven the development of highly potent small inhibitors (BRAFi) of the mutated protein. To date, Dabrafenib and Vemurafenib, two specific BRAFi, have been clinically approved for the treatment of metastatic melanoma. Unfortunately, after the initial response, tumors become resistant and patients develop a progressive and lethal disease, making imperative the development of new therapeutic options. The main objective of this work was to find new BRAF inhibitors with different structural scaffolds than those of the known inhibitors. Our study was carried out in different stages; in the first step we performed a virtual screening that allowed us to identify potential new inhibitors. In the second step, we synthesized and tested the inhibitory activity of the novel compounds founded. Finally, we conducted a molecular modelling study that allowed us to understand interactions at the molecular level that stabilize the formation of the different molecular complexes. Our theoretical and experimental study allowed the identification of four new structural scaffolds, which could be used as starting structures for the design and development of new inhibitors of BRAF. Our experimental data indicate that the most active compounds reduced significantly ERK½ phosphorylation, a measure of BRAF inhibition, and cell viability. Thus, from our theoretical and experimental results, we propose new substituted hydroxynaphthalenecarboxamides, N-(hetero)aryl-piperazinylhydroxyalkylphenylcarbamates, substituted piperazinylethanols and substituted piperazinylpropandiols as initial structures for the development of new inhibitors for BRAF. Moreover, by performing QTAIM analysis, we are able to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analysis indicates which portion of the different molecules must be changed in order to obtain an increase in the binding affinity of these new ligands.

Synthesis, Analysis, Cholinesterase-Inhibiting Activity and Molecular Modelling Studies of 3-(Dialkylamino)-2-hydroxypropyl 4-[(Alkoxy-carbonyl)amino]benzoates and Their Quaternary Ammonium Salts.

Tertiary amines 3-(dialkylamino)-2-hydroxypropyl 4-[(alkoxycarbonyl)amino]benzoates and their quaternary ammonium salts were synthesized. The final step of synthesis of quaternary ammonium salts was carried out by microwave-assisted synthesis. Software-calculated data provided the background needed to compare fifteen new resulting compounds by their physicochemical properties. The acid dissociation constant (pKa) and lipophilicity index (log P) of tertiary amines were determined; while quaternary ammonium salts were characterized by software-calculated lipophilicity index and surface tension. Biological evaluation aimed at testing acetylcholinesterase and butyrylcholinesterase-inhibiting activity of synthesized compounds. A possible mechanism of action of these compounds was determined by molecular modelling study using combined techniques of docking; molecular dynamics simulations and quantum mechanics calculations.

Synthesis and Determination of Physicochemical Properties of New 3-(4-Arylpiperazin-1-yl)-2-hydroxypropyl 4-Alkoxyethoxybenzoates.

Nine new dihydrochloride salts of 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-alkoxyethoxybenzoates were designed and synthesized. The physicochemical properties such as lipophilicity index (log kw) and dissociation constant (pKa) were experimentally determined and compared to the software calculated data. The lipophilicity index was determined by means of reversed-phase high performance liquid chromatography (RP-HPLC). The pKa values were determined by means of capillary zone electrophoresis. The "drug-likeness" properties according to the Lipinski Rule of Five and prediction of possible blood-brain barrier penetration were computed and discussed.

Synthesis and Characterization of New 3-(4-Arylpiperazin-1-yl)-2-hydroxypropyl 4-Propoxybenzoates and Their Hydrochloride Salts.

Five new 3-(4-arylpiperazin-1-yl)-2-hydroxypropyl 4-propoxybenzoates were designed and synthesized as potential dual antihypertensive agents. The compounds were prepared as free bases and subsequently transformed to hydrochloride salts. The position of protonation of nitrogen atoms in the piperazine ring of hydrochloride salts was determined by means of (13)C-CP/MAS and (15)N-CP/MAS NMR and IR spectroscopy. Using these solid-state analytical techniques, it was found that both nitrogen atoms were protonated when excess hydrogen chloride was used for preparation of salts. On the other hand, when the equimolar amount of hydrogen chloride was used, piperazine nitrogen substituted by aryl was protonated.

(Hetero)Aryloxyaminopropanols with N-Phenylpiperazine Structural Fragment - Review of Cardiovascular Activity.

Aryloxyphenylpiperazinylpropanols are a group of compounds exhibiting a wide range of biological activities, affecting the central nervous system and many cardiovascular mechanisms among them. As cardiovascular agents, aryloxyphenylpiperazinylpropanols work as antihypertensives, antiarrhythmics, cardiotonics or antiaggregants. The mechanism of action is almost always an α1-adrenolytic or combined α1- and ß-adrenolytic effect, but sometimes other mechanisms (e.g., Ca2+ antagonism or phosphodiesterase inhibition) can positively participate. In some cases, compounds with a small modification of the connecting chain also exhibit the desired cardiovascular effects. Several studies dealt with chirality of aryloxyphenylpiperazinylpropanols and determined the differences between the particular activities of racemic and enantiomeric compounds.

Indol-2-Carboxylic Acid Esters Containing N-Phenylpiperazine Moiety - Preparation and Cholinesterase-inhibiting Activity.

BACKGROUND: The indole derivatives and the N-phenylpiperazine fragment represent interesting molecular moieties suitable for the research of new potentially biologically active compounds. This study was undertaken to identify if indol-2-carboxylic acid esters containing N-phenylpiperazine moiety possess acetylcholinesterase and butyrylcholinesterase inhibitory activity. MATERIALS AND METHODS: The study dealt with the synthesis of a novel series of analogs of 1H-indole-2- carboxylic acid and 3-methyl-1H-indole-2-carboxylic acid. The structure of the derivatives was represented by the indolylcarbonyloxyaminopropanol skeleton with the attached N-phenylpiperazine or diethylamine moiety, which formed a basic part of the molecule. The final products were synthesized as dihydrochloride salts, fumaric acid salts, and quaternary ammonium salts. The first step of the synthetic pathway led to the preparation of esters of 1H-indole-2-carboxylic acid from the commercially available 1H-indole-2-carboxylic acid. The Fischer indole synthesis was used to synthesize derivatives of 3-methyl-1H-indole-2-carboxylic acid. RESULTS AND DISCUSSION: Final 18 indolylcarbonyloxyaminopropanols in the form of dihydrochlorides, fumarates, and quaternary ammonium salts were prepared using various optimization ways. The very efficient way for the formation of 3-methyl-1H-indole-2-carboxylate (Fischer indole cyclization product) was the one-pot synthesis of phenylhydrazine with methyl 2-oxobutanoate with acetic acid and sulphuric acid as catalysts. CONCLUSION: Most of the derivatives comprised of an attached N-phenylpiperazine group, which formed a basic part of the molecule and in which the phenyl ring was substituted in position C-2 or C-4. The synthesized compounds were subjected to cholinesterase-inhibiting activity evaluation, by modified Ellman method. Quaternary ammonium salt of 1H-indole-2-carboxylic acid which contain N-phenylpiperazine fragment with nitro group in position C-4 (7c) demonstrated the most potent activity against acetylcholinesterase.

Activity of N-Phenylpiperazine Derivatives Against Bacterial and Fungal Pathogens.

BACKGROUND: As the bacterial resistance to antibacterial chemotherapeutics is one of the greatest problems in modern medicine, efforts are made to develop new antimicrobial drugs. Compounds with a piperazine ring have proved to be promising agents against various pathogens. OBJECTIVE: The aim of the study was to prepare a series of new N-phenylpiperazines and determine their activity against various pathogens. METHOD: Target compounds were prepared by multi-step synthesis starting from an appropriate substituted acid to an oxirane intermediate reacting with 1-(4-nitrophenyl)piperazine. Lipophilicity and pKa values were experimentally determined. Other molecular parameters were calculated. The inhibitory activity of the target compounds against Staphylococcus aureus, four mycobacteria strains, Bipolaris sorokiniana, and Fusarium avenaceum was tested. In vitro antiproliferative activity was determined on a THP-1 cell line, and toxicity against plant was determined using Nicotiana tabacum. RESULTS: In general, most compounds demonstrated only moderate effects. 1-(2-Hydroxy-3-{[4-(propan- 2-yloxy)benzoyl]oxy}propyl)-4-(4-nitrophenyl)piperazinediium dichloride and 1-{3-[(4-butoxybenzoyl)- oxy]-2-hydroxypropyl}-4-(4-nitrophenyl)piperazinediium dichloride showed the highest inhibition activity against M. kansasii (MIC = 15.4 and 15.0 µM, respectively) and the latter also against M. marinum (MIC = 15.0 µM). 1-(2-Hydroxy-3-{[4-(2-propoxyethoxy)benzoyl]oxy}propyl)-4-(4-nitrophenyl)piperazinediium dichloride had the highest activity against F. avenaceum (MIC = 14.2 µM). All the compounds showed only insignificant toxic effects on human and plant cells. CONCLUSION: Ten new 1-(4-nitrophenyl)piperazine derivatives were prepared and analyzed, and their antistaphylococcal, antimycobacterial, and antifungal activities were determined. The activity against M. kansasii was positively influenced by higher lipophilicity, the electron-donor properties of substituent R and a lower dissociation constant. The exact mechanism of action will be investigated in follow-up studies.

An integrative study to identify novel scaffolds for sphingosine kinase 1 inhibitors.

Sphingosine kinase 1 (SphK1), the enzyme that produces the bioactive sphingolipid metabolite, sphingosine-1-phosphate, is a promising new molecular target for therapeutic intervention in cancer and inflammatory diseases. In view of its importance, the main objective of this work was to find new and more potent inhibitors for this enzyme possessing different structural scaffolds than those of the known inhibitors. Our theoretical and experimental study has allowed us to identify two new structural scaffolds (three new compounds), which could be used as starting structures for the design and then the development of new inhibitors of SphK1. Our study was carried out in different steps: virtual screening, synthesis, bioassays and molecular modelling. From our results, we propose a new dihydrobenzo[b]pyrimido[5,4-f]azepine and two alkyl{3-/4-[1-hydroxy-2-(4-arylpiperazin-1-yl)ethyl]phenyl}carbamates as initial structures for the development of new inhibitors. In addition, our molecular modelling study using QTAIM calculations, allowed us to describe in detail the molecular interactions that stabilize the different Ligand-Receptor complexes. Such analyses indicate that the cationic head of the different compounds must be refined in order to obtain an increase in the binding affinity of these ligands.