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 (Ab) pathologies. Here, we describe EHT 5372 (methyl 9-(2,4-dichlorophenylamino) thiazolo[5,4-f]quinazoline-2-carbimidate), a novel, highly potent (IC 50 = 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 Ab 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 Ab-induced Tau phosphorylation and DYRK1A-stimulated Ab production. DYRK1A is thus as a key element of Abmediated 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.

Cholinergic basal forebrain (CBF) nucleus basalis (NB) neurons display neurofibrillary tangles (NFTs) during Alzheimer's disease (AD) progression, yet the mechanisms underlying this selective vulnerability are currently unclear. Rac1, a member of the Rho family of GTPases, may interact with the proapoptotic pan-neurotrophin receptor p75(NTR) to induce neuronal cytoskeletal abnormalities in AD NB neurons. Herein, we examined the expression of Rac1b, a constitutively active splice variant of Rac1, in NB cholinergic neurons during AD progression. CBF tissues harvested from people who died with a clinical diagnosis of no cognitive impairment (NCI), mild cognitive impairment, or AD were immunolabeled for both p75(NTR) and Rac1b. Rac1b appeared as cytoplasmic diffuse granules, loosely aggregated filaments, or compact spheres in p75(NTR)-positive NB neurons. Although Rac1b colocalized with tau cytoskeletal markers, the percentage of p75(NTR)-immunoreactive neurons expressing Rac1b was significantly increased only in AD compared with both mild cognitive impairment and NCI. Furthermore, single-cell gene expression profiling with custom-designed microarrays showed down-regulation of caveolin 2, GNB4, and lipase A in AD Rac1b-positive/p75(NTR)-labeled NB neurons compared with Rac1b-negative/p75(NTR)-positive perikarya in NCI. These proteins are involved in Rac1 pathway/cell cycle progression and lipid metabolism. These data suggest that Rac1b expression acts as a modulator or transducer of various signaling pathways that lead to NFT formation and membrane dysfunction in a subgroup of CBF NB neurons in AD.

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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. Citation Format: Anne-Sophie Casagrande, Florence Bachelot, Séverine Coutadeur, Bertrand Leblond, Thierry Besson, Matthew P. Pando, Laurent Désiré. Structure activity relationship of a novel chemical class of Dyrk inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2165. doi:10.1158/1538-7445.AM2013-2165

Tumor blood vessels are an important emerging target for anticancer therapy. Here, we characterize the in vitro antiproliferative and antiangiogenic properties of the synthetic small molecule, 7-ethoxy-4-(3,4,5-trimethoxybenzyl)isoquinolin-8-amine dihydrochloride, EHT 6706, a novel microtubule-disrupting agent that targets the colchicine-binding site to inhibit tubulin polymerization. At low nM concentrations, EHT 6706 exhibits highly potent antiproliferative activity on more than 60 human tumor cell lines, even those described as being drug resistant. EHT 6706 also shows strong efficacy as a vascular-disrupting agent, since it prevents endothelial cell tube formation and disrupts pre-established vessels, changes the permeability of endothelial cell monolayers and inhibits endothelial cell migration. Genome-wide transcriptomic analysis of EHT 6706 effects on human endothelial cells shows that the antiangiogenic activity elicits gene deregulations of antiangiogenic pathways. These findings indicate that EHT 6706 is a promising tubulin-binding compound with potentially broad clinical antitumor efficacy.

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Among cytotoxics commonly used for cancer treatment, microtubule-targeting agents harbor a broad clinical anti-tumor efficacy. Microtubule-targeting agents act by their direct cytotoxic actions on tumor cells but also by their anti-angiogenic and vascular-disrupting actions. In contrast to classical antiangiogenic agents, vascular disrupting agents can, at the same time, block cell survival, prevent the formation of new vessels and damage the existing tumor vasculature. Here we describe EHT 6706, the current lead compound of a novel chemical class of tubulin targeting agents. EHT 6706 inhibits in a dose-dependent manner microtubule formation in an in vitro tubulin polymerization assay. EHT 6706 binds at or near the colchicine binding site of the tubulin-colchicine complex, hence displacing the [3]H-colchicine tracer. This binding competition is comparable to that of combretastain A-4, an established colchicine site binder with a higher affinity for tubulin than colchicine. At low concentrations, EHT 6706 decreases the amount of microtubular materials and induces alteration in tumor cell morphology. Perturbing microtubule dynamics with EHT 6706 halts the rapid cancer cells division by blocking cells in the metaphase/anaphase transition of the cell cycle and leads to apoptosis. EHT 6706 exhibits broad cytotoxicity in vitro with potency at sub-nanomolar/low nanomolar concentrations (0.3-10nM) in multiple cancer types (more than 60 cell lines, including the NCI60 panel) such as breast, melanoma, colorectal, renal, ovarian, non-small cell lung, leukaemia, as well as pancreatic cancer cells. EHT 6706 exerts this growth inhibition of human tumor cells independently of the type of main multidrug resistance mechanisms (ABC transporter MDR-1 (Pgp-1), MRP-1…). Furthermore, EHT 6706 retains cytotoxic activity against all of the resistant cell lines tested (combretastatin, vinblastin, imatinib), thus indicating the potential of this agent for the treatment of resistant tumors. EHT 6706 carries a potential for enhancing antitumor effects of radiation. In H460 and MiaPaca2 cell lines, indeed, EHT 6706 treatment inhibits clonogenic survival synergistically with ionizing radiation and exerts additive inhibition of proliferation when given in combination with conventional chemotherapeutic agents. Finally, in vitro experiments demonstrate that EHT 6706 has anti-angiogenic and vascular disrupting effects. Endothelial cell tube formation inhibition, pre-established vessels disruption, and vascular permeability increase were observed in a concentration- and time-dependent manner. These studies demonstrate that EHT 6706 has potent in vitro efficacy as a novel tubulin-targeted anti-proliferative and anti-vascular agent. EHT 6706 is a candidate for in vivo pharmacokinetic and efficacy studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2794. doi:1538-7445.AM2012-2794