EGCG-like non-competitive inhibitor of DYRK1A rescues cognitive defect in a down syndrome model.

Overexpression of the chromosome 21 DYRK1A gene induces morphological defects and cognitive impairments in individuals with Down syndrome (DS) and in DS mice models. Aging neurons of specific brain regions of patients with Alzheimer's disease, DS and Pick's disease have increased DYRK1A immunoreactivity suggesting a possible association of DYRK1A with neurofibrillary tangle pathology. Epigallocatechin-3-gallate (EGCG) displays appreciable inhibition of DYRK1A activity and, contrary to all other published inhibitors, EGCG is a non-competitive inhibitor of DYRK1A. Prenatal exposure to green tea polyphenols containing EGCG protects from brain defects induced by overexpression of DYRK1A. In order to produce more robust and possibly more active analogues of the natural compound EGCG, here we synthetized new EGCG-like molecules with several structural modifications to the EGCG skeleton. We replaced the ester boun of EGCG with a more resistant amide bond. We also replaced the oxygen ring by a methylene group. And finally, we positioned a nitrogen atom within this ring. The selected compound was shown to maintain the non-competitive property of EGCG and to correct biochemical and behavioral defects present in a DS mouse model. In addition it showed high stability and specificity.

Chemical, Biochemical, Cellular, and Physiological Characterization of Leucettinib-21, a Down Syndrome and Alzheimer's Disease Drug Candidate.

Leucettinibs are substituted 2-aminoimidazolin-4-ones (inspired by the marine sponge natural product Leucettamine B) developed as pharmacological inhibitors of DYRK1A (dual-specificity, tyrosine phosphorylation-regulated kinase 1A), a therapeutic target for indications such as Down syndrome and Alzheimer's disease. Leucettinib-21 was selected as a drug candidate following extensive structure/activity studies and multiparametric evaluations. We here report its physicochemical properties (X-ray powder diffraction, differential scanning calorimetry, stability, solubility, crystal structure) and drug-like profile. Leucettinib-21's selectivity (analyzed by radiometric, fluorescence, interaction, thermal shift, residence time assays) reveals DYRK1A as the first target but also some "off-targets" which may contribute to the drug's biological effects. Leucettinib-21 was cocrystallized with CLK1 and modeled in the DYRK1A structure. Leucettinib-21 inhibits DYRK1A in cells (demonstrated by direct catalytic activity and phosphorylation levels of Thr286-cyclin D1 or Thr212-Tau). Leucettinib-21 corrects memory disorders in the Down syndrome mouse model Ts65Dn and is now entering safety/tolerance phase 1 clinical trials.

Leucettinibs, a Class of DYRK/CLK Kinase Inhibitors Inspired by the Marine Sponge Natural Product Leucettamine B.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) recently attracted attention due to their central involvement in various pathologies. We here describe a family of DYRK/CLK inhibitors derived from Leucettines and the marine natural product Leucettamine B. Forty-five N2-functionalized 2-aminoimidazolin-4-ones bearing a fused [6 + 5]-heteroarylmethylene were synthesized. Benzothiazol-6-ylmethylene was selected as the most potent residue among 15 different heteroarylmethylenes. 186 N2-substituted 2-aminoimidazolin-4-ones bearing a benzothiazol-6-ylmethylene, collectively named Leucettinibs, were synthesized and extensively characterized. Subnanomolar IC50 (0.5-20 nM on DYRK1A) inhibitors were identified and one Leucettinib was modeled in DYRK1A and co-crystallized with CLK1 and the weaker inhibited off-target CSNK2A1. Kinase-inactive isomers of Leucettinibs (>3-10 µM on DYRK1A), named iso-Leucettinibs, were synthesized and characterized as suitable negative control compounds for functional experiments. Leucettinibs, but not iso-Leucettinibs, inhibit the phosphorylation of DYRK1A substrates in cells. Leucettinibs provide new research tools and potential leads for further optimization toward therapeutic drug candidates.

Comparative Efficacy and Selectivity of Pharmacological Inhibitors of DYRK and CLK Protein Kinases.

Dual-specificity, tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) play a large variety of cellular functions and are involved in several diseases (cognitive disorders, diabetes, cancers, etc.). There is, thus, growing interest in pharmacological inhibitors as chemical probes and potential drug candidates. This study presents an unbiased evaluation of the kinase inhibitory activity of a library of 56 reported DYRK/CLK inhibitors on the basis of comparative, side-by-side, catalytic activity assays on a panel of 12 recombinant human kinases, enzyme kinetics (residence time and Kd), in-cell inhibition of Thr-212-Tau phosphorylation, and cytotoxicity. The 26 most active inhibitors were modeled in the crystal structure of DYRK1A. The results show a rather large diversity of potencies and selectivities among the reported inhibitors and emphasize the difficulties to avoid "off-targets" in this area of the kinome. The use of a panel of DYRKs/CLKs inhibitors is suggested to analyze the functions of these kinases in cellular processes.

Discovery and Lead-Optimization of 4,5-Dihydropyrazoles as Mono-Kinase Selective, Orally Bioavailable and Efficacious Inhibitors of Receptor Interacting Protein 1 (RIP1) Kinase.

RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.

NIS-assisted aza-Friedel-Crafts reaction with α-carbamoysulfides as precursors of N-carbamoylimines.

A general and practical N-iodosuccinimide (NIS)-promoted aza-Friedel-Crafts reaction of various aromatic nucleophiles with N-acylimines generated in situ from α-amidosulfides to give a rapid access to highly functionalized amines is described. The newly developed methodology is very mild, fast, efficient, and complementary.

A comparison of ERG abnormalities in XLRS and XLCSNB.

Dark and light adapted ERGs were recorded in 19 patients with XLRS and in 15 patients with CSNB. Patients were assigned to clinical groups after identification of mutations in the RS1 (16 patients), NYX (11 patients) and CACNA1F (4 patients) genes causing XLRS, 'complete' CSNB and 'incomplete' CSNB, respectively. ERG responses were compared with those of 26 healthy volunteers. Rod responses were most severely affected in patients in the NYX group but a rod-generated b-wave could be identified in the majority of patients in this group. Rod responses were less severely affected in the CACNA1F and RS1 groups and ERGs did not differ significantly between these two groups. Cone reponses were largely unaffected in the NYX group but were abnormal in the RS1 and especially CACNA1F groups. The ERG results suggest that the RS1 and CACNA1F gene products have comparable functional consequences and that all three genes may affect multiple retinal sites.