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ABSTRACT Background: Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by the accumulation of Aβ peptides which elicit oxidative stress and neuronal apoptosis. Objective(s): To identify genes abnormally spliced in brain from AD patients, to pinpoint unprecedented pathways and essential targets which impact APP processing, Aβ and ROS production and related neurotoxicity, to characterize small molecules as potential drugs for the treatment of AD. Methods: ExonHit Therapeutics has used its proprietary technology DATAS™.

La presente invention concerne des composes de la formule indiquee ici, R', R 1 a R 7 et Ar etant tels que definis ici. Lesdits composes sont utiles en tant qu'inhibiteurs de la phosphodiesterase 10 (PDE10A) dans le traitement de maladies du systeme nerveux central, telles que la psychose, et par exemple dans le traitement de l'obesite, du diabete de type II, du syndrome metabolique, de l'intolerance au glucose, de la douleur et des maladies ophtalmiques.

Cette invention concerne des composes de formule (I) ou un sel pharmaceutiquement acceptable de ceux-ci - R', R 1 a R 7 et Ar etant tels que definis dans la description. Ces composes sont utiles a titre d'inhibiteurs de phosphodiesterase 10 (PDE10A) qui sont utiles pour traiter les maladies du systeme nerveux central telles que la psychose et egalement pour traiter, par exemple, l'obesite, le diabete de type II, le syndrome metabolique, l'intolerance au glucose, la douleur et les maladies opthalmiques.

La presente invention concerne le domaine de la biologie, de la genetique et de la medecine. Elle concerne notamment de nouvelles methodes pour la detection, la caracterisation et/ou le traitement (ou la prise en charge) de pathologies neurodegeneratives. L'invention concerne egalement des methodes pour l'identification ou le criblage de composes actifs dans ces pathologies. L'invention concerne egalement les composes, genes, cellules, plasmides ou compositions utiles pour la mise en oeuvre des methodes ci-dessus. L'invention decoule notamment de l'identification du role de la phosphodiesterase 4B et du recepteur peripherique aux benzodiazepines dans les pathologies degeneratives et decrit leur utilisation comme cible ou marqueur therapeutique, diagnostique ou experimental de ces desordres.

Fibroblast growth factor (FGF) 2 is a survival factor for various cell types, including retinal neurons. However, little is understood about the molecular bases of the neuroprotective role of FGF2 in the retina. In this report, FGF2 survival activity was studied in chick retinal neurons subjected to apoptosis by serum deprivation. Exogenous FGF2 supported neuronal survival after serum deprivation and increased neuronal bcl-x(L) and bcl-2 expression, through binding to its receptor R1 (FGF-R1), and subsequent extracellular signal-regulated kinase (ERK) activation. Endogenous FGF2 was transiently overexpressed after serum deprivation. Its down-regulation by antisense oligonucleotides and blockade of its signaling pathway (binding to FGF-R1, tyrosine phosphorylation, and ERK inhibition) decreased bcl-x(L) and bcl-2 levels and and enhanced apoptosis, suggesting that endogenous FGF2 supported neuronal survival through a pathway similar to that of exogenous FGF2. This pathway may serve to up-regulate, or maintain, bcl-x(L) and bcl-2 levels that normally decrease during the onset of apoptosis. Indeed, long-term ERK activation and high bcl-x(L) levels are necessary for the survival activity of both exogenous and endogenous FGF2. Because FGF2 is upregulated following retinal injury in vivo, we suggest that an injury-stimulated autocrine/paracrine FGF2 loop may serve to maintain high levels of survival proteins, such as Bcl-x(L), through ERK activation in retinal neurons.

The dual specificity tyrosine-regulated kinase 1A (DYRK1A) phosphorylates a growing number of proteins to influence various aspects of cell biology, including survival, apoptosis, proliferation, and neuronal development and function. Encoded on human chromosome 21, the DYRK1A locus resides in the Down syndrome critical region (DSCR) and has been implicated in the pathogenesis of neurocognitive deficits that result from trisomy 21. We recently demonstrated a prominent tumor-promoting role for DYRK1A in acute megakaryoblastic leukemia in children with DS (DS-AMKL). This discovery prompted us to ask whether DYRK1A is important for normal hematopoiesis, and if additional types of leukemia result from enhanced DYRK1A activity. Apart from our study in DS-AMKL, the involvement of DYRK1A in normal and malignant hematopoiesis has not been described. Dyrk1a germline knockout mice die mid-gestation; therefore, we generated conditional knockout (CKO) mice to allow inducible deletion of the kina...

Methyl 9-anilinothiazolo[5,4-f]quinazoline-2-carbimidates 1 (EHT 5372) and 2 (EHT 1610) are strong inhibitors of DYRK's family kinases. The crystal structures of the complex revealed a noncanonical binding mode of compounds 1 and 2 in DYRK2, explaining the remarkable selectivity and potency of these inhibitors. The structural data and comparison presented here provide therefore a template for further improvement of this inhibitor class and for the development of novel inhibitors selectively targeting DYRK kinases.

EHT 0202 is a GABAA receptor modulator and a PDE4 inhibitor developed for the treatment of Alzheimer’s disease (AD). EHT 0202 is novel because it stimulates α-secretases, increasing the production of procognitive and neurotrophic sAPPα fragment of APP. Preclinical studies have shown that EHT 0202 is neuroprotective, demonstrates procognitive properties in various preclinical models and may reduce Aβ42 levels. EHT 0202 is therefore expected to offer both symptomatic and disease modifying properties for AD. Here, we provide an update on the preclinical and clinical development of EHT 0202 for AD therapy.

The dual-specificity tyrosine phosphorylated and regulated kinase Mirk/DYRK1B belongs to an evolutionary conserved family of kinases involved in the control of growth and development. DYRK1B has low level of expression in most normal cell types but is amplified or over-expressed in a number of human cancers. DYRK1B regulates the exit of cancer cells from quiescence through regulating cyclin D turnover and p27kip1 stabilization, thus participating as substantial actor in the control of cancer cell cycle progression. Diaxonhit has developed a novel class of DYRK inhibitors with potent in vitro efficacy. Among them, EHT 5372 reduces tumor growth in a PANC-1 xenograft model and reduces ascites spheroids to single cells and induces their apoptosis. Three-dimensional (3D) multicellular spheroids are symmetrical cellular aggregates that model an in vitro system of intermediate complexity between monolayer cultures and tumors in vivo. Here, we have conducted genome-wide analysis of transcri...

The invention relates to compds. of formula I that are inhibitors of phosphodiesterase 10 (PDE10A) with utility in treating central nervous system diseases such as psychosis and also in treating, for example, obesity, type II diabetes, metabolic syndrome, glucose intolerance, pain and ophthalmic diseases. Compds. of formula I (wherein R1 is H, (un) substituted (C1- C6) alkyl, (un) substituted (C3- C6) cycloalkyl and (un) substituted aryl; R2 is H; R3 and R4 are independently a (C1- C3) alkyl group; R5 is H; R6 and R7 are H; R' is H or (C1- C6) alkyl; Ar is (un) substituted fused nine- to ten- membered heteroaryl, (un) substituted benzo- fused aryl, etc.) and pharmaceutically acceptable salts thereof are claimed. Example compd. II•2HCl was prepd. from intermediate N- (3, 4- dimethoxybenzyl) - 2, 2- diethoxyethanamine and 2- naphthaldehyde. All the invention compds. were evaluated for their PDE10A inhibitory activity.

The title compds. I [X = CH or N; R1, R2 = H, halo, alkyl, etc.; R = H, OH, piperidino, morpholino, etc.] , useful for the treatment of Alzheimer's disease and other similar diseases, were prepd. E.g., a multi- step synthesis of I.3HCl [X = CH; R1, R2 = H; R = piperazino] , starting from 7- trifluoromethyl- 4- quinolinethiol and 1, 5- dibromopentane, was given. More specifically the inventive compds. I modulate (in particular, inhibit) the level of amyloid- β peptide (Aβ) exhibited by cells or tissues (Aβ peptide is a major component of the amyloid plaques found in the brains of Alzheimer's sufferers) . Exemplified compds. I were tested for inhibition of Aβ 40 prodn. in HEK- 293 cells overexpressing swAPP751 (data given for representative compds. I) . This invention also relates to the use of these inhibitors to prevent, treat or ameliorate the symptoms of Alzheimer's disease or any Amyloid- β- Peptide Related Disorder. Pharmaceutical compn. comprising the compd. I is al...

Regulatory requirements for proper safety testing of a new drug requires the identification of the metabolites that humans are exposed during treatment. EHT 0202 is a compound currently being tested in a phase IIa clinical trial as an adjunctive therapy to an acetylcholinesterase inhibitor in mild to moderate Alzheimer's disease. In order to identify and characterize the EHT 0202 metabolites, this compound was incubated in the presence of human, monkey, dog, rabbit and rat liver microsomes.The identification, synthesis and characterization of the metabolites as well as the parent compound will be presented.

The present invention relates generally to substituted isoquinolines of general formula I (wherein X is N or N+- Z, wherein Z is C1- 6 alkyl, aryl or acyl; R1 is H, CN, C1- 5 alkyl, etc.; R3 is H, CN, OH, etc.; R7 is C1- 5 alkyl, C1- 5 alkoxy, C1- 5 alkylthio, etc.; R8 is H, halo, NO2, etc.; or R8 and R7 together form part of a 5- 6- membered heterocyclic ring; R'2 is H, halo, C1- 5 alkyl, etc.; R'3 and R'5 are independently halo, NH2, NO2, etc.; R'4 is H, halo, NH2, etc.; L is CH2, C=O, CF2, etc.) and their use as tubulin polymn. inhibitors for treating cancer, inflammation, and disorders caused by unwanted neovascularization. The present invention also provides compns. contg. I. Synthetic procedures for prepg. I are exemplified. Example compd. II, prepd. by oxidizing and hydrolyzing intermediate III, had an IC50 of 1.64 nM against K562 tumor cells assessed using an MTT assay and caused 71.29 % inhibition of tubulin polymn. at 5 μM in a spectrophotometric assay.

Dual-specificity tyrosine-phosphorylated and regulated kinases (DYRK) is a family of conserved protein kinases which mediate survival and differentiation in normal tissues like skeletal muscle for Mirk/Dyrk1B or neuronal cells for Dyrk1A. Among the five Dyrk proteins, Mirk/Dyrk1B has very low level of expression in most normal cell types but has been found to be upregulated in solid tumors and to mediate cell survival in colon cancer, pancreatic ductal adenocarcinoma, rhabdomyosarcomas, lung and ovarian cancer. Mirk/Dyrk1B expression and abundance varies during the cell cycle with the highest levels found in quiescent G0 phase where it mediates G0 tumor cells prolonged survival (through increasing expression of a cohort of antioxidant genes). Dyrk1A also promotes quiescence and senescence through DREAM complex assembly by phosphorylating the DREAM subunit LIN52. The ability of cells to exit from the cell cycle and enter into the G0 or quiescence state is important for cell different...

Pre-B and pre-T lymphocytes must orchestrate a transition from a highly proliferative state to a quiescent one during development. Cyclin D3 is essential for these cells' proliferation, but little is known about its posttranslational regulation at this stage. Here, we show that the dual specificity tyrosine-regulated kinase 1A (DYRK1A) restrains Cyclin D3 protein levels by phosphorylating T283 to induce its degradation. Loss of DYRK1A activity, via genetic inactivation or pharmacologic inhibition in mice, caused accumulation of Cyclin D3 protein, incomplete repression of E2F-mediated gene transcription, and failure to properly couple cell cycle exit with differentiation. Expression of a nonphosphorylatable Cyclin D3 T283A mutant recapitulated these defects, whereas inhibition of Cyclin D:CDK4/6 mitigated the effects of DYRK1A inhibition or loss. These data uncover a previously unknown role for DYRK1A in lymphopoiesis, and demonstrate how Cyclin D3 protein stability is negatively...

Biomarkers have gained an increased importance in the past years in helping physicians to diagnose Alzheimer's disease (AD). This study was designed to identify a blood-based, transcriptomic signature that can differentiate AD patients from control subjects. The performance of the signature was then evaluated for robustness in an independent blinded sample population. RNA was extracted from 177 blood samples (90 AD patients and 87 controls) and gene expression profiles were generated using the human Genome-Wide Splice Array™. These profiles were used to establish a signature to differentiate AD patients from controls. Subsequently, prediction results were optimized by establishing grey zone boundaries that discount prediction scores near the disease status threshold. Signature validation was then performed on a blinded independent cohort of 209 individuals (111 AD and 98 controls). The AclarusDx™ signature consists of 170 probesets which map to 136 annotated genes, a significant...

The dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) gene is located within the Down Syndrome (DS) critical region on chromosome 21 and is implicated in the generation of Tau and amyloid pathologies that are associated with the early onset Alzheimer's Disease (AD) observed in DS. DYRK1A is also found associated with neurofibrillary tangles in sporadic AD and phosphorylates key AD players (Tau, amyloid precursor, protein, etc). Thus, DYRK1A may be an important therapeutic target to modify the course of Tau and amyloid beta (Aβ) pathologies. Here, we describe EHT 5372 (methyl 9-(2,4-dichlorophenylamino) thiazolo[5,4-f]quinazoline-2-carbimidate), a novel, highly potent (IC50  = 0.22 nM) DYRK1A inhibitor with a high degree of selectivity over 339 kinases. Models in which inhibition of DYRK1A by siRNA reduced and DYRK1A over-expression induced Tau phosphorylation or Aβ production were used. EHT 5372 inhibits DYRK1A-induced Tau phosphorylation at multiple AD-relevant sites in biochemical and cellular assays. EHT 5372 also normalizes both Aβ-induced Tau phosphorylation and DYRK1A-stimulated Aβ production. DYRK1A is thus as a key element of Aβ-mediated Tau hyperphosphorylation, which links Tau and amyloid pathologies. EHT 5372 and other compounds in its class warrant in vivo investigation as a novel, high-potential therapy for AD and other Tau opathies. Inhibition of the dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) is a new high-potential therapeutic approach for Alzheimer disease. Here we describe EHT 5372, a novel potent and selective DYRK1A inhibitor. EHT 5372 inhibits DYRK1A-induced Tau phosphorylation, Aβ production and Aβ effects on phospho-Tau, including Tau aggregation.

ABSTRACT Dual-specificity tyrosine-phosphorylated and regulated kinases (DYRK) is a family of conserved protein kinases which mediate survival and differentiation in normal tissues like skeletal muscle for Mirk/Dyrk1B or neuronal cells for Dyrk1A. Among the five Dyrk proteins, Mirk/Dyrk1B has very low level of expression in most normal cell types but has been found to be upregulated in solid tumors and to mediate cell survival in colon cancer, pancreatic ductal adenocarcinoma, rhabdomyosarcomas, lung and ovarian cancer. Mirk/Dyrk1B expression and abundance varies during the cell cycle with the highest levels found in quiescent G0 phase where it mediates G0 tumor cells prolonged survival (through increasing expression of a cohort of antioxidant genes). Dyrk1A also promotes quiescence and senescence through DREAM complex assembly by phosphorylating the DREAM subunit LIN52. The ability of cells to exit from the cell cycle and enter into the G0 or quiescence state is important for cell differentiation, tissue development, and prevention of tumorigenesis. Tumors may contain a fraction of quiescent cells responsible for resistance to chemotherapy and radiation, serving as a reservoir for tumor repopulation post-therapy. Thus, pharmacological Dyrk inhibition would reduce the ability of cells to enter into quiescence and sensitize cancer cells to conventional chemotherapeutic agents. Exonhit has developed a novel class of DYRK inhibitors. Lead optimization led to the synthesis of new chemical entities with subnanomolar kinase inhibitory activities associated with a high degree of selectivity over 400 kinases. Most active compounds were evaluated in various cellular models, demonstrating good correlation between cellular activity and amplification/expression of Mirk/Dyrk1B. Similarly, in accordance with the expression levels of Mirk/Dyrk1B during the cell cycle, cellular potency of pharmacological inhibitors was improved 3 to 11 fold in SW620 quiescent cultures when compared to cycling cultures. We analyzed the effects of Dyrk inhibitors alone and in combination with gemcitabine in the Panc1 pancreatic cancer cell line multicellular tumor spheroid model described to be largely quiescent and enriched in Mirk/Dyrk1B kinase and confirmed that Dyrk inhibitors sensitize cells to gemcitabine with a synergistic effect of the combination. Among the 120 NCEs from this series, we identified lead compounds having potent in vitro efficacy as Dyrk inhibitors. These compounds are being further characterized in various cellular studies and show promising in vivo activities in patient-derived ovarian cancer ascites spheroids and Panc1 xenografts model without detectable toxicity in mice, establishing the usefulness of these inhibitors for targeting cancer cells with high Dyrk kinase activity.

ABSTRACT The pivotal role of Rac1, a member of the Rho family of smallGTPases, has been well characterized in the genesis of many cancers. The aberrant activation of Rac1 promotes uncontrolled proliferation, invasion and metastatic properties of human cancer cells. Via ExonHit Therapeutics' alternative splicing discovery plateform DATASTM, we identified a repertoire of genes deregulated in cancer. Among them, Rac1b, an alternative splice variant of Rac1, was found deregulated and overexpressed in diverse cancer types, including breast and colon. Rac1b is constitutively active and transforming, and since it was shown to sustain cancer cell survival, such as for colorectal cancer cells, there is considerable interest in the development of small molecule inhibitors of Rac1/Rac1b. We previously synthesized and fully characterized EHT 1864 as a pharmacological tool to study Rac1 functions (Shutes A et al.(2007). J Biol Chem. 282(49):35666-78). Then, using molecular docking algorithms and ExonHit's screening platform for nucleotide binding inhibitors, novel compounds were synthesized and characterized for Rac1/Rac1b inhibition and Rac1 versus Rac1b selectivity. Here, we describe new Rac1/Rac1b inhibitors with better pharmacological profile than EHT 1864 and better potency in various in vitro cancer cell models. Some of these pharmacological tools are good candidates for subsequent in vivo cancer studies to study Rac1 and Rac1b mediated cellular functions and to modulate pathological conditions in which Rac1 or Rac1b deregulation may play a role.

The potential of EHT 6706, a novel tubulin-binding agent, was investigated in combination with ionizing radiation (IR) and with conventional cytotoxic chemotherapy agents. Cell proliferation, cell cycle, apoptosis and clonogenic assays were performed in five human cancer cell lines: H460 (non small cell lung carcinoma, NSCLC), HCT116 and HCT116 p53-/- (colorectal cancer), MDA-MB-231 (breast cancer), and MiaPaca2 cells (pancreatic cancer). The drug inhibited cell proliferation in all cell lines. This effect was associated with G2/M arrest and activation of apoptosis in a dose-dependent manner. The drug was then tested in combination with chemotherapy and IR in vitro. Effects on proliferation and clonogenic survival were analyzed. EHT 6706 treatment inhibited clonogenic survival synergistically with IR in H460 and MiaPaca2 cell lines. In the remaining cell lines, the effects of EHT 6706 and IR were additive. For H460 and MiaPaca2 cell lines, the highest effect was seen when cells were exposed for 20 h to EHT 6706 before being irradiated. EHT 6706 also exerted additive inhibition of proliferation when given in combination with conventional chemotherapy agents, such as oxaliplatin, cisplatin and gemcitabine in H460 and MiaPaca2 tumor cell lines. These data show that EHT 6706 could act synergistically with IR and additively with chemotherapy in tumor cell lines in vitro. This provides a good rationale to further assess EHT 6706 in combination protocols and confirm these effects in vivo.

During retinal differentiation, fibroblast growth factor 2 (FGF2) expression increases in retinal neurons following the sequential appearance of the neuronal layers. The function of the developmental increase of endogenous FGF2 in the developing chick retina was investigated by using an antisense strategy, using both optic vesicle cultures and in ovo-intravitreal microinjections. The former model allowed us to study the consequences of FGF2 down-regulation on early ganglion cell differentiation, whereas, in the latter model, subsequent development stages and terminal maturation of the retina were studied. FGF2 inhibition resulted in reduced ganglion cell differentiation, as visualized by the expression of the ganglion cell-specific RA4 and Islet-1 markers in optic vesicle cultures. Eyes intravitreally injected with the FGF2-specific antisense oligonucleotide exhibited profound retinal differentiation defects: thinning of the ganglion and outer nuclear (photoreceptors) cell layers and increased cell death in ganglion cell and inner nuclear layers. These results indicate that the loss of endogenous FGF2 cannot be compensated for in the retina and suggest that, although many other sources of FGF exist in the eye, the main role of the increase in endogenous FGF2 observed during retinal development is to intrinsically stimulate neuron differentiation and to protect neurons against cell death.

Mutations in the gene encoding phospholipase C-γ2 (PLCγ2) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLCγ2 an ∼2-3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca(2+) following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLCγ2 mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells. Enhanced "basal" activity of PLCγ2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLCγ2(F897Q) mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLCγ2 activity. ...