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␤-Amyloid peptides (A␤) that form the senile plaques of Alzheimer disease consist mainly of 40-and 42-amino acid (A␤ 40 and A␤ 42) peptides generated from the cleavage of the amyloid precursor protein (APP). Generation of A␤ involves ␤-secretase and ␥-secretase activities and is regulated by membrane trafficking of the proteins involved in A␤ production. Here we describe a new small molecule, EHT 1864, which blocks the Rac1 signaling pathways. In vitro, EHT 1864 blocks A␤ 40 and A␤ 42 production but does not impact sAPP␣ levels and does not inhibit ␤-secretase. Rather, EHT 1864 modulates APP processing at the level of ␥-secretase to prevent A␤ 40 and A␤ 42 generation. This effect does not result from a direct inhibition of the ␥-secretase activity and is specific for APP cleavage, since EHT 1864 does not affect Notch cleavage. In vivo, EHT 1864 significantly reduces A␤ 40 and A␤ 42 levels in guinea pig brains at a threshold that is compatible with delaying plaque accumulation and/or clearing the existing plaque in brain. EHT 1864 is the first derivative of a new chemical series that consists of candidates for inhibiting A␤ formation in the brain of AD patients. Our findings represent the first pharmacological validation of Rac1 signaling as a target for developing novel therapies for Alzheimer disease. Alzheimer disease (AD)2 is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterized by the presence of amyloid plaques (1), intracellular neurofibrillary tangles, and pronounced cell death. The ␤-amyloid peptide (A␤) (2) is the main constituent of senile plaques found in AD brains. Furthermore, extracellular A␤ 42 appears toxic to neurons in vitro and in vivo (reviewed in Ref.3). A␤ is generated by proteolysis of an integral membrane protein, the amyloid precursor protein (APP), via at least two post-translational pathways. The amyloidogenic cleavage of APP is a sequential processing of APP initiated by ␤-secretase (BACE), which cleaves APP within the luminal domain or at the cell surface, generating the N terminus of A␤ (4). This cleavage generates several membrane-bound proteolytic C-terminal fragments (CTFs), such as the 99-residue ␤-CTF (also called C99), as well as the secreted APP ectodomain sAPP␤. The C terminus of A␤ is subsequently generated by intramembranous cleavage of CTFs by ␥-secretase, producing either A␤ 40 or A␤ 42. The cleavages at residues 40 -42 are referred to as ␥-cleavage, and the cleavages at residues 49 -52 are referred to as ⑀-cleavage (5). The nonamyloidogenic cleavage of APP, which precludes A␤ generation, is mediated by ␣-secretase, a disintegrin and metalloproteinase 10, and a disintegrin and metalloproteinase 17, in a reaction believed to occur primarily on the plasma membrane. This proteolytic cleavage by ␣-secretase occurs within the A␤ region and produces soluble APP (sAPP␣), the dominant processing product, and the residual membrane-bound 10-kDa CTF (CTF␣, also called C83). Like C99, C83 is a subs...

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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