Substitution of unsaturated lipid chains by thioether-containing lipid chains in cationic amphiphiles: physicochemical consequences and application for gene delivery.

The hydrophobic moiety of cationic amphiphiles plays an important role in the transfection process because its structure has an impact on both the type of the supramolecular assembly and the dynamic properties of these assemblies. The latter have to exhibit a compromise between stability and instability to efficiently compact then deliver DNA into target cells. In the present work, we report the synthesis of new cationic amphiphiles featuring a thioether function at different positions of two 18-atom length lipid chains and we study their physicochemical properties (anisotropy of fluorescence and compression isotherms) with analogues possessing either oleyl (C18:1) or stearyl (C18:0) chains. We show that the fluidity of cationic lipids featuring a thioether function located close to the middle of each lipid chain is intermediate between that of oleyl- and stearyl-containing analogues. These properties are also supported by the compression isotherm assays. When used as carriers to deliver a plasmid DNA, thioether-containing cationic amphiphiles demonstrate a good ability to transfect human-derived cell lines, with those incorporating such a moiety in the middle of the chain being the most efficient. This work supports the use of a thioether function as a possible alternative to unsaturation in aliphatic lipid chains of cationic amphiphiles to modulate physicochemical behaviours and in turn biological activities such as gene delivery ability.

Combined Virtual and Experimental Screening for CK1 Inhibitors Identifies a Modulator of p53 and Reveals Important Aspects of in Silico Screening Performance.

A compound collection of pronounced structural diversity was comprehensively screened for inhibitors of the DNA damage-related kinase CK1. The collection was evaluated in vitro. A potent and selective CK1 inhibitor was discovered and its capacity to modulate the endogenous levels of the CK1-regulated tumor suppressor p53 was demonstrated in cancer cell lines. Administration of 10 µM of the compound resulted in significant increase of p53 levels, reaching almost 2-fold in hepatocellular carcinoma cells. In parallel to experimental screening, two representative and orthogonal in silico screening methodologies were implemented for enabling the retrospective assessment of virtual screening performance on a case-specific basis. Results showed that both techniques performed at an acceptable and fairly comparable level, with a slight advantage of the structure-based over the ligand-based approach. However, both approaches demonstrated notable sensitivity upon parameters such as screening template choice and treatment of redundancy in the enumerated compound collection. An effort to combine insight derived by sequential implementation of the two methods afforded poor further improvement of screening performance. Overall, the presented assessment highlights the relation between improper use of enrichment metrics and misleading results, and demonstrates the inherent delicacy of in silico methods, emphasizing the challenging character of virtual screening protocol optimization.

Lipophosphoramidate-based bipolar amphiphiles: their syntheses and transfection properties.

Six new cationic bolaamphiphiles (also called bipolar amphiphiles, bolaform amphiphiles, or bolalipids) were readily prepared by a thiol-ene click reaction that engaged a mercapto-alcohol (mercapto-ethanol or mercapto-hexanol) and a cationic based lipophosphoramidate. The cationic lipophosphoramidates contain two lipid chains that end in an alkene group and a selected cationic polar head group (trimethylammonium, dimethyl hydroxyethyl ammonium, or methylimidazolium). These compounds were formulated in water (with or without DOPE as a colipid) to produce supramolecular aggregates. These aggregates, before (i.e. bolasomes) and after (i.e. bolaplexes) mixing with plasmid DNA (pDNA) at various charge ratios, were characterized with regard to their sizes and zeta potentials. In the case of bolasomes, the suspensions were unstable since precipitation occurred after only a few hours at room temperature. On the other hand, bolaplex formulations exhibited clearly a better colloidal stability. Then, the gene delivery properties of the cationic bolasomes were investigated using two human-derived epithelial cell lines (A549 and 16HBE). Compared to the commercially available lipofection reagent (Lipofectamine), most of the cationic bolaamphiphiles were able to efficiently transfect these cells when they were formulated with DOPE in a 1 : 1 molar ratio. We report herein that bolaamphiphiles possessing a trimethylammonium or a dimethyl hydroxyethyl ammonium head group were the most efficient in terms of transfection efficiency while exhibiting no significant cytotoxicity.

Synthesis of new pyridazino[4,5-b]indol-4-ones and pyridazin-3(2H)-one analogs as DYRK1A inhibitors.

New pyridazino[4,5-b]indol-4-ones and pyridazin-3(2H)-one analogs were synthesized and their inhibitory activities against DYRK1A, CDK5/p25, GSK3α/ß and p110-α isoform of PI3K evaluated using harmine as reference. Both furan-2-yl 10 and pyridin-4-yl 19 from the two different series, exhibited submicromolar IC50 against DYRK1A with no activities against the three other kinases. In addition, compound 10 exhibited antiproliferative activities in the Huh-7, Caco2 and MDA-MB-231 cell lines.

Synthesis, biological evaluation and molecular modelling studies of 4-anilinoquinazoline derivatives as protein kinase inhibitors.

A series of novel 4-anilinoquinazoline derivatives (3a-3j) has been synthesized and evaluated as potential inhibitors for protein kinases implicated in Alzheimer's disease. Among all the synthesized compounds, compound 3e (N-(3,4-dimethoxyphenyl)-6,7-dimethoxyquinazolin-4-amine) exhibited the most potent inhibitory activity against CLK1 and GSK-3α/ß kinase with IC50 values of 1.5 µM and 3 µM, respectively. Docking studies were performed to elucidate the binding mode of the compounds to the active site of CLK1 and GSK-3ß. The results of our study suggest that compound 3e may serve as a valuable template for the design and development of dual inhibitors of CLK1 and GSK-3α/ß enzymes with potential therapeutic application in Alzheimer's disease.

Acridone alkaloids from Glycosmis chlorosperma as DYRK1A inhibitors.

Two new acridone alkaloids, chlorospermines A and B (1 and 2), were isolated from the stem bark of Glycosmis chlorosperma, together with the known atalaphyllidine (3) and acrifoline (4), by means of bioguided isolation using an in vitro enzyme assay against DYRK1A. Acrifoline (4) and to a lesser extent chlorospermine B (2) and atalaphyllidine (3) showed significant inhibiting activity on DYRK1A with IC50's of 0.075, 5.7, and 2.2 µM, respectively. Their selectivity profile was evaluated against a panel of various kinases, and molecular docking calculations provided structural details for the interaction between these compounds and DYRK1A.

Several human cyclin-dependent kinase inhibitors, structurally related to roscovitine, are new anti-malarial agents.

In Africa, malaria kills one child each minute. It is also responsible for about one million deaths worldwide each year. Plasmodium falciparum, is the protozoan responsible for the most lethal form of the disease, with resistance developing against the available anti-malarial drugs. Among newly proposed anti-malaria targets, are the P. falciparum cyclin-dependent kinases (PfCDKs). There are involved in different stages of the protozoan growth and development but share high sequence homology with human cyclin-dependent kinases (CDKs). We previously reported the synthesis of CDKs inhibitors that are structurally-related to (R)-roscovitine, a 2,6,9-trisubstituted purine, and they showed activity against neuronal diseases and cancers. In this report, we describe the synthesis and the characterization of new CDK inhibitors, active in reducing the in vitro growth of P. falciparum (3D7 and 7G8 strains). Six compounds are more potent inhibitors than roscovitine, and three exhibited IC50 values close to 1 µM for both 3D7 and 7G8 strains. Although, such molecules do inhibit P. falciparum growth, they require further studies to improve their selectivity for PfCDKs.

Potent inhibitors of CDK5 derived from roscovitine: synthesis, biological evaluation and molecular modelling.

Cyclin dependent kinase 5 (CDK5) is a serine/threonine kinase belonging to the cyclin dependent kinase (CDK) family. CDK5 is involved in numerous neuronal diseases (including Alzheimer's or Parkinson's diseases, stroke, traumatic brain injury), pain signaling and cell migration. In the present Letter, we describe syntheses and biological evaluations of new 2,6,9-trisubstituted purines, structurally related to roscovitine, a promising CDK inhibitor currently in clinical trials (CDK1/Cyclin B, IC(50)=350 nM; CDK5/p25, IC(50)=200 nM). These new molecules were synthesized using an original Buchwald-Hartwig catalytic procedure; several compounds (3j, 3k, 3l, 3e, 4k, 6b, 6c) displayed potent kinase inhibitory potencies against CDK5 (IC(50) values ranging from 17 to 50 nM) and showed significant cell death inducing activities (IC(50) values ranging from 2 to 9 µM on SH-SY5Y). The docking of the inhibitors into the ATP binding domain of the CDK5 catalytic site highlighted the discriminatory effect of a hydrogen bond involving the CDK5 Lys-89. In addition, the calculated final energy balances for complexation measured for several inhibitors is consistent with the ranking of the IC(50) values. Lastly, we observed that several compounds exhibit submicromolar activities against DYRK1A (dual specificity, tyrosine phosphorylation regulated kinase 1A), a kinase involved in Down syndrome and Alzheimer's disease (3g, 3h, 4m; IC(50) values ranging from 300 to 400 nM).

Natural aristolactams and aporphine alkaloids as inhibitors of CDK1/cyclin B and DYRK1A.

In an effort to find potent inhibitors of the protein kinases DYRK1A and CDK1/Cyclin B, a systematic in vitro evaluation of 2,500 plant extracts from New Caledonia and French Guyana was performed. Some extracts were found to strongly inhibit the activity of these kinases. Four aristolactams and one lignan were purified from the ethyl acetate extracts of Oxandra asbeckii and Goniothalamus dumontetii, and eleven aporphine alkaloids were isolated from the alkaloid extracts of Siparuna pachyantha, S. decipiens, S. guianensis and S. poeppigii. Among these compounds, velutinam, aristolactam AIIIA and medioresinol showed submicromolar IC50 values on DYRK1A.

Catalyst-free synthesis of quinazolin-4-ones from (hetero)aryl-guanidines: application to the synthesis of pyrazolo[4,3-f]quinazolin-9-ones, a new family of DYRK1A inhibitors.

A small library of heterocycle-fused quinazolin-4-ones was prepared and evaluated as kinase inhibitors. The key step of the two-step process involves the environmental friendly thermolysis of N-ethoxycarbonyl-N'-(hetero) arylguanidines at 130 °C in water. The cyclization is fully regioselective. The most active molecules, 7-(2-hydroxyethylamino)- and 7-(3-hydroxypropylamino)-pyrazolo[4,3-f]quinazolin-9-ones, inhibit DYRK1A and CLK1 at submicromolar concentrations, indicating the potential interest of this new heterocycle in drug design.

A practical approach to new (5Z) 2-alkylthio-5-arylmethylene-1-methyl-1,5-dihydro-4H-imidazol-4-one derivatives.

A practical protocol for the preparation of (5Z)-2-alkylthio-5-arylmethylene-1-methyl-1,5-dihydro-4H-imidazol-4-one derivatives is reported. The new compounds were obtained in good yield and stereoselectivity in two steps, namely a solvent-free Knoevenagel condensation under microwave irradiation, followed by an S-alkylation reaction with various halogenoalkanes.

Design and Microwave Synthesis of New (5Z) 5-Arylidene-2-thioxo-1,3-thiazolinidin-4-one and (5Z) 2-Amino-5-arylidene-1,3-thiazol-4(5H)-one as New Inhibitors of Protein Kinase DYRK1A.

Here, we report on the synthesis of libraries of new 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones 3 (twenty-two compounds) and new 2-amino-5-arylidene-1,3-thiazol-4(5H)-ones 5 (twenty-four compounds) with stereo controlled Z-geometry under microwave irradiation. The 46 designed final compounds were tested in order to determine their activity against four representative protein kinases (DYR1A, CK1, CDK5/p25, and GSK3α/ß). Among these 1,3-thiazolidin-4-ones, the molecules (5Z) 5-(4-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one 3e (IC50 0.028 µM) and (5Z)-5-benzo[1,3]dioxol-5-ylmethylene-2-(pyridin-2-yl)amino-1,3-thiazol-4(5H)-one 5s (IC50 0.033 µM) were identified as lead compounds and as new nanomolar DYRK1A inhibitors. Some of these compounds in the two libraries have been also evaluated for their in vitro inhibition of cell proliferation (Huh7 D12, Caco2, MDA-MB 231, HCT 116, PC3, and NCI-H2 tumor cell lines). These results will enable us to use the 1,3-thiazolidin-4-one core as pharmacophores to develop potent treatment for neurological or oncological disorders in which DYRK1A is fully involved.

Concise synthesis and CDK/GSK inhibitory activity of the missing 9-azapaullones.

A remarkably concise, chromatography-free route to the parent compound of the paullone family of cyclin-dependent kinase (CDK) inhibitors is reported. A similar strategy allowed the synthesis of the hitherto missing 9-azapaullone and its protonated, N-oxidised and N-alkylated derivatives. Screening studies identified an active and strongly selective inhibitor of CDK9/cyclin T.

Synthesis and biological evaluation of new penta- and heptacyclic indolo- and quinolinocarbazole ring systems obtained via Pd(0) catalysed reductive N-heteroannulation.

A short route, involving a tetramolecular condensation reaction and a Pd/C catalyst-H(2)-mediated reductive N-heteroannulation as the key-steps, has been found for the synthesis of some new penta- and heptacyclic indolo- (12), quinolino- (13) and indoloquinolinocarbazole (11) derivatives. HF-DFT (B3LYP) energy profiles and NMR calculations were carried out to help in the understanding of the experimental results. N-Alkylated indoloquinolinocarbazoles (16b, 17a, 17b and 18) were prepared and screened essentially toward some cancer-(G-quadruplex, DNA, topoisomerase I) and CNS-related (kinases) targets. Biological results evidenced 13 as a potent CDK-5 and GSK-3ß kinases inhibitor, while di- or triaminopropyl-substituted indoloquinolinocarbazoles 17b or 18 targeted rather DNA-duplex or telomeric G-quadruplex structures, respectively.

Synthesis and biological evaluation of 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives as protein kinase inhibitors.

The synthesis and biological evaluation of a number of differently substituted 3,6-diamino-1H-pyrazolo[3,4-b]pyridine derivatives are reported. From the inhibition results on a selection of disease-relevant protein kinases [IC(50) (microM) DYRK1A=11; CDK5=0.41; GSK-3=1.5] we have observed that 3,6-diamino-4-phenyl-1H-pyrazolo[3,4-b]pyridine-5-carbonitrile (4) constitutes a potential new and simple lead compound in the search of drugs for the treatment of Alzheimer's disease.

Identification of potential cellular targets of aloisine A by affinity chromatography.

Affinity chromatography was used to identify potential cellular targets of aloisine A (7-n-butyl-6-(4'-hydroxyphenyl)-5H-pyrrolo[2,3b]pyrazine), a potent inhibitor of cyclin-dependent kinases. This technique is based on the immobilization of the drug on a solid matrix, followed by identification of specifically bound proteins. To this end, both aloisine A and the protein-kinase inactive control N-methyl aloisine, bearing extended linker chains have been synthesized. We present the preparation of such analogues having the triethylene glycol chain at different positions of the molecule, as well as their immobilization on an agarose-based matrix. Affinity chromatography of various biological extracts on the aloisine matrices allowed the identification of both protein kinases and non-kinase proteins as potential cellular targets of aloisine.

Synthesis and kinase inhibitory activity of novel substituted indigoids.

The bis-indole indigoids are a promising protein kinase inhibitor scaffold to be further evaluated against the numerous human diseases that imply abnormal regulation of kinases including neurodegenerative disorders. In an effort to identify new pharmacological inhibitors of disease-relevant protein kinases with increased potency and selectivity, we designed, synthesized new 5,7-disubstituted or 6-substituted bis-indole derivatives. On the basis of our previous synthetic work, 22 selected compounds were tested on CDK1/cyclin B, CDK5/p25, DYRK1A, CK1, and GSK-3alpha/beta kinases, five kinases involved in Alzheimer's disease. Some of them were also evaluated for their cytotoxic and antiproliferative activities. 6-Nitro-3'-N-oxime-indirubin and 5-amino-3'-N-oxime-indirubin derivatives exhibited inhibitory activity in a submicromolar range against CDK1/cyclin B (0.18 and 0.1 microM, respectively), CK1 (0.6 microM and 0.13 microM) and GSK3 (0.04 microM and 0.36 microM).

Meriolins, a new class of cell death inducing kinase inhibitors with enhanced selectivity for cyclin-dependent kinases.

Protein kinases represent promising anticancer drug targets. We describe here the meriolins, a new family of inhibitors of cyclin-dependent kinases (CDK). Meriolins represent a chemical structural hybrid between meridianins and variolins, two families of kinase inhibitors extracted from various marine invertebrates. Variolin B is currently in preclinical evaluation as an antitumor agent. A selectivity study done on 32 kinases showed that, compared with variolin B, meriolins display enhanced specificity toward CDKs, with marked potency on CDK2 and CDK9. The structures of pCDK2/cyclin A/variolin B and pCDK2/cyclin A/meriolin 3 complexes reveal that the two inhibitors bind within the ATP binding site of the kinase, but in different orientations. Meriolins display better antiproliferative and proapoptotic properties in human tumor cell cultures than their parent molecules, meridianins and variolins. Phosphorylation at CDK1, CDK4, and CDK9 sites on, respectively, protein phosphatase 1alpha, retinoblastoma protein, and RNA polymerase II is inhibited in neuroblastoma SH-SY5Y cells exposed to meriolins. Apoptosis triggered by meriolins is accompanied by rapid Mcl-1 down-regulation, cytochrome c release, and activation of caspases. Meriolin 3 potently inhibits tumor growth in two mouse xenograft cancer models, namely, Ewing's sarcoma and LS174T colorectal carcinoma. Meriolins thus constitute a new CDK inhibitory scaffold, with promising antitumor activity, derived from molecules initially isolated from marine organisms.

Lipid-Based Quaternary Ammonium Sophorolipid Amphiphiles with Antimicrobial and Transfection Activities.

Twelve new quaternary ammonium sophorolipids with long alkyl chains on the nitrogen atom were synthesized starting from oleic and petroselinic acid-based sophorolipids. These novel derivatives were evaluated for their antimicrobial activity against selected Gram-negative and Gram-positive bacteria and their transfection efficacies on three different eukaryotic cell lines in vitro as good activities were demonstrated for previously synthesized derivatives. Self-assembly properties were also evaluated. All compounds proved to possess antimicrobial and transfection properties, and trends could be observed based on the length of the nitrogen substituent and the total length of the sophorolipid tail. Moreover, all long-chain quaternary ammonium sophorolipids form micelles, which proved to be a prerequisite to induce antimicrobial activity and transfection capacity. These results are promising for future healthcare applications of long-chained quaternary ammonium sophorolipids.

Rebeccamycin derivatives as dual DNA-damaging agents and potent checkpoint kinase 1 inhibitors.

Rebeccamycin is an indolocarbazole class inhibitor of topoisomerase I. In the course of structure-activity relationship studies on rebeccamycin derivatives, we have synthesized analogs with the sugar moiety attached to either one or both indole nitrogens. Some analogs, especially those with substitutions at the 6' position of the carbohydrate moiety, exhibit potent inhibitory activity toward checkpoint kinase 1 (Chk1), a kinase that has a major role in the G(2)/M checkpoint in response to DNA damage. Some of these compounds retained a genotoxic activity either through intercalation into the DNA and/or by topoisomerase I-mediated DNA cleavage. We explored the structure-activity relationship between these compounds and their multiple targets. These rebeccamycin derivatives represent a novel class of potential antitumor agents that have a dual effect and might selectively induce the death of cancer cells.