Large Diversity and Original Structures of Acyl-Homoserine Lactones in Strain MOLA 401, a Marine Rhodobacteraceae Bacterium.

Quorum sensing (QS) is a density-dependent mechanism allowing bacteria to synchronize their physiological activities, mediated by a wide range of signaling molecules including N-acyl-homoserine lactones (AHLs). Production of AHL has been identified in various marine strains of Proteobacteria. However, the chemical diversity of these molecules still needs to be further explored. In this study, we examined the diversity of AHLs produced by strain MOLA 401, a marine Alphaproteobacterium that belongs to the ubiquitous Rhodobacteraceae family. We combined an original biosensors-based guided screening of extract microfractions with liquid chromatography coupled to mass spectrometry (MS), High Resolution MS/MS and Nuclear Magnetic Resonance. This approach revealed the unsuspected capacity of a single Rhodobacteraceae strain to synthesize 20 different compounds, which are most likely AHLs. Also, some of these AHLs possessed original features that have never been previously observed, including long (up to 19 carbons) and poly-hydroxylated acyl side chains, revealing new molecular adaptations of QS to planktonic life and a larger molecular diversity than expected of molecules involved in cell-cell signaling within a single strain.

Genome Sequence of the Sponge-Associated Ruegeria halocynthiae Strain MOLA R1/13b, a Marine Roseobacter with Two Quorum-Sensing-Based Communication Systems.

Ruegeria halocynthiae MOLA R1/13b is an alphaproteobacterium isolated from the Mediterranean sea sponge Crambe crambe. We report here the genome sequence and its annotation, revealing the presence of quorum-sensing genes. This is the first report of the full genome of a Ruegeria halocynthiae strain.

Genome Sequence of Maribius sp. Strain MOLA 401, a Marine Roseobacter with a Quorum-Sensing Cell-Dependent Physiology.

Maribius sp. strain MOLA401 is an alphaproteobacterium isolated from a coral reef lagoon located in New Caledonia, France. We report the genome sequence and its annotation which, interestingly, reveals the presence of genes involved in quorum sensing. This is the first report of a full genome within the genus Maribius.

CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome.

Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome.

Novel Inverse Binding Mode of Indirubin Derivatives Yields Improved Selectivity for DYRK Kinases.

DYRK kinases are involved in alternative pre-mRNA splicing as well as in neuropathological states such as Alzheimer's disease and Down syndrome. In this study, we present the design, synthesis, and biological evaluation of indirubins as DYRK inhibitors with enhanced selectivity. Modifications of the bis-indole included polar or acidic functionalities at positions 5' and 6' and a bromine or a trifluoromethyl group at position 7, affording analogues that possess high activity and pronounced specificity. Compound 6i carrying a 5'-carboxylate moiety demonstrated the best inhibitory profile. A novel inverse binding mode, which forms the basis for the improved selectivity, was suggested by molecular modeling and confirmed by determining the crystal structure of DYRK2 in complex with 6i. Structure-activity relationships were further established, including a thermodynamic analysis of binding site water molecules, offering a structural explanation for the selective DYRK inhibition.

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).

Synthesis, protein kinase inhibitory potencies, and in vitro antiproliferative activities of meridianin derivatives.

The synthesis of new meridianin derivatives is described. The indolic ring system was substituted at the C-4 to C-7 positions either by a bromine atom or by nitro or amino groups. Additionally, an iodine atom or various aryl groups were introduced at the C-5 position of the 2-aminopyrimidine ring. These compounds as well as some of their synthetic intermediates were tested for their kinase inhibitory potencies and for their in vitro antiproliferative activities. We found that this series of compounds is particularly interesting in the development of new inhibitors of DYRK1A and CLK1 kinases. The most effective compounds toward these two kinase families are the 6- and 7-bromo derivatives 30, 33, and 34 that showed more than 45-fold selectivity toward DYRK1A/CLK1 kinases over the other kinases tested. Meridianin derivatives could thus be developed toward potent and selective inhibitors of key RNA splicing regulators and potential therapeutic agents.

First BRET-based screening assay performed in budding yeast leads to the discovery of CDK5/p25 interaction inhibitors.

The protein kinase CDK5 (cyclin-dependent kinase 5) is activated through its association with a cyclin-like protein p35 or p39. In pathological conditions (such as Alzheimer's disease and various other neuropathies), truncation of p35 leads to the appearance of the p25 protein. The interaction of p25 with CDK5 up-regulates the kinase activity and modifies the substrate specificity. ATP-mimetic inhibitors of CDK5 have already been developed. However, the lack of selectivity of such inhibitors is often a matter of concern. An alternative approach can be used to identify highly specific inhibitors that disrupt protein interactions involving protein kinases. We have developed a bioluminescence resonance energy transfer (BRET)-based screening assay in yeast to discover protein-protein interaction inhibitors (P2I2). Here, we present the first use of BRET in yeast for the screening of small molecule libraries. This screening campaign led to the discovery of one molecule that prevents the interaction between CDK5 and p25, thus inhibiting the protein kinase activity. This molecule may give rise to high-specificity drug candidates.

Design, synthesis, and testing of an 6-O-linked series of benzimidazole based inhibitors of CDK5/p25.

Alzheimer's disease (AD) is a progressive neurodegenerative disease resulting in cognitive and behavioral impairment. The two classic pathological hallmarks of AD include extraneuronal deposition of amyloid ß (Aß) and intraneuronal formation of neurofibrillary tangles (NFTs). NFTs contain hyperphosphorylated tau. Tau is the major microtubule-associated protein in neurons and stabilizes microtubules (MTs). Cyclin dependent kinase 5 (CDK5), when activated by the regulatory binding protein p25, phosphorylates tau at a number of proline-directed serine/threonine residues, resulting in formation of phosphorylated tau as paired helical filaments (PHFs) then in subsequent deposition of PHFs as NFTs. Beginning with the structure of Roscovitine, a moderately selective CDK5 inhibitor, we sought to conduct structural modifications to increase inhibitory potency of CDK5 and increase selectivity over a similar enzyme, cyclin dependent kinase 2 (CDK2). The design, synthesis, and testing of a series of 1-isopropyl-4-aminobenzyl-6-ether-linked benzimidazoles is presented.

Development of 5-benzylpaullones and paullone-9-carboxylic acid alkyl esters as selective inhibitors of mitochondrial malate dehydrogenase (mMDH).

A collection of paullones was tested for inhibitory activity against mitochondrial malate dehydrogenase (mMDH) as a biological target for antiproliferative activity. Based on the results of this screening, 5-benzylpaullones and paullone-9-carboxylic acid alkyl esters were developed as selective mMDH inhibitors. The new derivatives did not show noteworthy antiproliferative activity when tested on a panel of cancer cell lines, suggesting that mMDH inhibition is of minor relevance for the growth inhibition caused by paullones.

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).

Synthesis and biological activities of aminopyrimidyl-indoles structurally related to meridianins.

The synthesis of new meridianin derivatives substituted at the C-5 position of the 2-aminopyrimidine ring by various aryl groups and substituted or not by a methyl group on the indole nitrogen is described. These compounds were tested for their kinase inhibitory potencies toward five kinases (CDK5/p25, CK1delta/epsilon, GSK-3alpha/beta, Dyrk1A and Erk2) as well as their in vitro antiproliferative activities toward a human fibroblast primary culture and two human solid cancer cell lines (MCF-7 and PA 1).

Pyrazolo[1,5-a]-1,3,5-triazine as a purine bioisostere: access to potent cyclin-dependent kinase inhibitor (R)-roscovitine analogue.

Pharmacological inhibitors of cyclin-dependent kinases (CDKs) have a wide therapeutic potential. Among the CDK inhibitors currently under clinical trials, the 2,6,9-trisubstituted purine (R)-roscovitine displays rather high selectivity, low toxicity, and promising antitumor activity. In an effort to improve this structure, we synthesized several bioisosteres of roscovitine. Surprisingly, one of them, pyrazolo[1,5-a]-1,3,5-triazine 7a (N-&-N1, GP0210), displayed significantly higher potency, compared to (R)-roscovitine and imidazo[2,1-f]-1,2,4-triazine 13 (N-&-N2, GP0212), at inhibiting various CDKs and at inducing cell death in a wide variety of human tumor cell lines. This approach may thus provide second generation analogues with enhanced biomedical potential.

N-&-N, a new class of cell death-inducing kinase inhibitors derived from the purine roscovitine.

Cyclin-dependent kinases (CDKs) and their regulators show frequent abnormalities in tumors. Ten low molecular weight pharmacologic inhibitors of CDKs are currently in clinical trials against various cancers, including the 2,6,9-trisubstituted purine (R)-roscovitine (CYC202/Seliciclib). We here report the characterization of N-&-N1, a bioisoster of roscovitine displaying improved antitumoral properties. N-&-N1 shows exquisite selectivity for CDKs, with 2- to 3-fold enhanced potency compared with (R)-roscovitine. Inhibition of retinoblastoma protein phosphorylation and RNA polymerase II Ser2 phosphorylation in neuroblastoma SH-SY5Y cells exposed to N-&-N1 indicates that N-&-N1 is able to inhibit CDKs in a cellular context. N-&-N1 also down-regulates the expression of RNA polymerase. Cocrystal structures of N-&-N1 and (R)-roscovitine in complex with CDK2/cyclin A reveal that both inhibitors adopt similar binding modes. A competitive assay shows that, compared with (R)-roscovitine, N-&-N1 has reduced affinity for Erk2 and pyridoxal kinase. N-&-N1 triggers cell death in a panel of diverse cell lines. Cell death is accompanied by events characteristic of apoptosis: cytochrome c release, activation of effector caspases, and poly(ADP-ribose) polymerase cleavage. Induction of p53 and p21CIP1 and down-regulation of the Mcl-1 antiapoptotic factor were also observed. Studies in mice show that N-&-N1 has pharmacokinetics properties similar to those of (R)-roscovitine. Altogether, these results show that analogues of (R)-roscovitine can be designed with improved antitumor potential.

Soluble 3',6-substituted indirubins with enhanced selectivity toward glycogen synthase kinase -3 alter circadian period.

Glycogen synthase kinase -3 (GSK-3) is a key enzyme involved in numerous physiological events and in major diseases, such as Alzheimer's disease, diabetes, and cardiac hypertrophy. Indirubins are bis-indoles that can be generated from various natural sources or chemically synthesized. While rather potent and selective as GSK-3 inhibitors, most indirubins exhibit low water solubility. To address the issue of solubility, we have designed novel analogues of 6-bromo-indirubin-3'-oxime with increased hydrophilicity based on the GSK-3/indirubins cocrystal structures. The new derivatives with an extended amino side chain attached at position 3' showed potent GSK-3 inhibitory activity, enhanced selectivity, and dramatically increased water solubility. Furthermore, some of them displayed little or no cytotoxicity. The new indirubins inhibit GSK-3 in a cellular reporter model. They alter the circadian period measured in rhythmically expressing cell cultures, suggesting that they might constitute tools to investigate circadian rhythm regulation.

Roscovitine-derived, dual-specificity inhibitors of cyclin-dependent kinases and casein kinases 1.

Cyclin-dependent kinases (CDKs) and casein kinases 1 (CK1) are involved in the two key molecular features of Alzheimer's disease, production of amyloid-beta peptides (extracellular plaques) and hyper-phosphorylation of Tau (intracellular neurofibrillary tangles). A series of 2,6,9-trisubstituted purines, structurally related to the CDK inhibitor roscovitine, have been synthesized. They mainly differ by the substituent on the C-6 position. These compounds were screened for kinase inhibitory activities and antiproliferative effects. Several biaryl derivatives displayed potent inhibition of both CDKs and CK1. In particular, derivative 13a was a potent inhibitor of CDK1/cyclin B (IC 50: 220 nM), CDK5/p25 (IC 50: 80 nM), and CK1 (IC 50: 14 nM). Modeling of these molecules into the ATP-binding pocket of CK1delta provided a rationale for the increased selectivity toward this kinase. 13a was able to prevent the CK1-dependent production of amyloid-beta in a cell model. CDK/CK1 dual-specificity inhibitors may have important applications in Alzheimer's disease and cancers.

Photoreactivity of indirubin derivatives.

Twenty-nine analogs of indirubin, an isomer of indigo, have been synthesized to optimize its promising kinase inhibitory scaffold. These compounds being also pigmented, have been tested for their photoreactivity. Absorption maxima were between 485 nm and 560 nm. Addition of fetal calf serum induced fluorescence and time dependent absorption modifications. Appropriate illumination induced Reactive Oxygen Species (ROS) production for nineteen compounds out of twenty-nine. The relationship between fluorescence and ROS production is discussed. Six compounds showed an important toxicity on F98 cells, a murine glioma cell line. Three of these were found to be also phototoxic, as four other non-toxic compounds. All but one phototoxic compounds were detected as ROS producers by in vitro tests. Photoreactivity assessment is important to anticipate adverse reactions for compounds that might be clinically developed. The experimental assay was found to be the only way to evaluate the photoreactivity of this family of compounds since no predictive criteria on structures could be found. Combining the vascular tumor growth inhibition induced by kinase inhibitors with the massive local blood flow arrest following photodynamic treatment may be an efficient anti-cancer strategy. These data could orientate further syntheses of either non-photoreactive compounds or compounds displaying both kinase inhibitory activity and strong phototoxicity.

9-cyano-1-azapaullone (cazpaullone), a glycogen synthase kinase-3 (GSK-3) inhibitor activating pancreatic beta cell protection and replication.

Recently, the serine/threonine kinase glycogen synthase kinase-3 (GSK-3) emerged as a regulator of pancreatic beta cell growth and survival. On the basis of the previous observation that GSK-3 inhibitors like 1-azakenpaullone promote beta cell protection and replication, paullone derivatives were synthesized including 1-aza-, 2-aza-, and 12-oxapaullone scaffolds. In enzymatic assays distinct 1-azapaullones were found to exhibit selective GSK-3 inhibitory activity. Within the series of 1-azapaullones, three derivatives stimulated INS-1E beta cell replication and protected INS-1E cells against glucolipotoxicity induced cell death. Cazpaullone (9-cyano-1-azapaullone), the most active compound in the protection assays, also stimulated the replication of primary beta cells in isolated rat islets. Furthermore, cazpaullone showed a pronounced transient stimulation of the mRNA expression of the beta cell transcription factor Pax4, an important regulator of beta cell development and growth. These features distinguish cazpaullone as a unique starting point for the development of beta cell regenerative agents which might be useful in the treatment of diabetes.

Meriolins (3-(pyrimidin-4-yl)-7-azaindoles): synthesis, kinase inhibitory activity, cellular effects, and structure of a CDK2/cyclin A/meriolin complex.

We report the synthesis and biological characterization of 3-(pyrimidin-4-yl)-7-azaindoles (meriolins), a chemical hybrid between the natural products meridianins and variolins, derived from marine organisms. Meriolins display potent inhibitory activities toward cyclin-dependent kinases (CDKs) and, to a lesser extent, other kinases (GSK-3, DYRK1A). The crystal structures of 1e (meriolin 5) and variolin B (Bettayeb, K.; Tirado, O. M.; Marionneau-Lambert, S.; Ferandin, Y.; Lozach, O.; Morris, J.; Mateo-Lozano, S.; Drückes, P.; Schächtele, C.; Kubbutat, M.; Liger, F.; Marquet, B.; Joseph, B.; Echalier, A.; Endicott, J.; Notario, V.; Meijer, L. Cancer Res. 2007, 67, 8325-8334) in complex with CDK2/cyclin A reveal that the two inhibitors are orientated in very different ways inside the ATP-binding pocket of the kinase. A structure-activity relationship provides further insight into the molecular mechanism of action of this family of kinase inhibitors. Meriolins are also potent antiproliferative and proapoptotic agents in cells cultured either as monolayers or in spheroids. Proapoptotic efficacy of meriolins correlates best with their CDK2 and CDK9 inhibitory activity. Meriolins thus constitute a promising class of pharmacological agents to be further evaluated against the numerous human diseases that imply abnormal regulation of CDKs including cancers, neurodegenerative disorders, and polycystic kidney disease.

Anticancer alkaloid lamellarins inhibit protein kinases.

Lamellarins, a family of hexacyclic pyrrole alkaloids originally isolated from marine invertebrates, display promising anti-tumor activity. They induce apoptotic cell death through multi-target mechanisms, including inhibition of topoisomerase I, interaction with DNA and direct effects on mitochondria. We here report that lamellarins inhibit several protein kinases relevant to cancer such as cyclin-dependent kinases, dual-specificity tyrosine phosphorylation activated kinase 1A, casein kinase 1, glycogen synthase kinase-3 and PIM-1. A good correlation is observed between the effects of lamellarins on protein kinases and their action on cell death, suggesting that inhibition of specific kinases may contribute to the cytotoxicity of lamellarins. Structure/activity relationship suggests several paths for the optimization of lamellarins as kinase inhibitors.