Efficacy of DYRK1A inhibitors in novel models of Down syndrome acute lymphoblastic leukemia.

Not available.

Increased dosage of DYRK1A leads to congenital heart defects in a mouse model of Down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21). DS is a gene dosage disorder that results in multiple phenotypes including congenital heart defects. This clinically important cardiac pathology is the result of a third copy of one or more of the approximately 230 genes on Hsa21, but the identity of the causative dosage-sensitive genes and hence mechanisms underlying this cardiac pathology remain unclear. Here, we show that hearts from human fetuses with DS and embryonic hearts from the Dp1Tyb mouse model of DS show reduced expression of mitochondrial respiration genes and cell proliferation genes. Using systematic genetic mapping, we determined that three copies of the dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1a) gene, encoding a serine/threonine protein kinase, are associated with congenital heart disease pathology. In embryos from Dp1Tyb mice, reducing Dyrk1a gene copy number from three to two reversed defects in cellular proliferation and mitochondrial respiration in cardiomyocytes and rescued heart septation defects. Increased dosage of DYRK1A protein resulted in impairment of mitochondrial function and congenital heart disease pathology in mice with DS, suggesting that DYRK1A may be a useful therapeutic target for treating this common human condition.

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.

The protein kinase CK1: Inhibition, activation, and possible allosteric modulation.

Protein kinases play a vital role in biology and deregulation of kinases is implicated in numerous diseases ranging from cancer to neurodegenerative diseases, making them a major target class for the pharmaceutical industry. However, the high degree of conservation that exists between ATP-binding sites among kinases makes it difficult for current inhibitors to be highly specific. In the context of neurodegeneration, several groups including ours, have linked different kinases such as CK1 and Alzheimer's disease for example. Strictly CK1-isoform specific regulators do not exist and known CK1 inhibitors are inhibiting the enzymatic activity, targeting the ATP-binding site. Here we review compounds known to target CK1, as well as other inhibitory types that could benefit CK1. We introduce the DNA-encoded library (DEL) technology that might represent an interesting approach to uncover allosteric modulators instead of ATP competitors. Such a strategy, taking into account known allosteric inhibitors and mechanisms, might help designing modulators that are more specific towards a specific kinase, and in the case of CK1, toward specific isoforms.

Genetic impairment of succinate metabolism disrupts bioenergetic sensing in adrenal neuroendocrine cancer.

Metabolic dysfunction mutations can impair energy sensing and cause cancer. Loss of function of the mitochondrial tricarboxylic acid (TCA) cycle enzyme subunit succinate dehydrogenase B (SDHB) results in various forms of cancer typified by pheochromocytoma (PC). Here we delineate a signaling cascade where the loss of SDHB induces the Warburg effect, triggers dysregulation of [Ca2+]i, and aberrantly activates calpain and protein kinase Cdk5, through conversion of its cofactor from p35 to p25. Consequently, aberrant Cdk5 initiates a phospho-signaling cascade where GSK3 inhibition inactivates energy sensing by AMP kinase through dephosphorylation of the AMP kinase γ subunit, PRKAG2. Overexpression of p25-GFP in mouse adrenal chromaffin cells also elicits this phosphorylation signaling and causes PC. A potent Cdk5 inhibitor, MRT3-007, reverses this phospho-cascade, invoking a senescence-like phenotype. This therapeutic approach halted tumor progression in vivo. Thus, we reveal an important mechanistic feature of metabolic sensing and demonstrate that its dysregulation underlies tumor progression in PC and likely other cancers.

Structure-Activity Relationship in the Leucettine Family of Kinase Inhibitors.

The protein kinase DYRK1A is involved in Alzheimer's disease, Down syndrome, diabetes, viral infections, and leukemia. Leucettines, a family of 2-aminoimidazolin-4-ones derived from the marine sponge alkaloid Leucettamine B, have been developed as pharmacological inhibitors of DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). We report here on the synthesis and structure-activity relationship (SAR) of 68 Leucettines. Leucettines were tested on 11 purified kinases and in 5 cellular assays: (1) CLK1 pre-mRNA splicing, (2) Threonine-212-Tau phosphorylation, (3) glutamate-induced cell death, (4) autophagy and (5) antagonism of ligand-activated cannabinoid receptor CB1. The Leucettine SAR observed for DYRK1A is essentially identical for CLK1, CLK4, DYRK1B, and DYRK2. DYRK3 and CLK3 are less sensitive to Leucettines. In contrast, the cellular SAR highlights correlations between inhibition of specific kinase targets and some but not all cellular effects. Leucettines deserve further development as potential therapeutics against various diseases on the basis of their molecular targets and cellular effects.

Safety and pharmacokinetics of Roscovitine (Seliciclib) in cystic fibrosis patients chronically infected with Pseudomonas aeruginosa, a randomized, placebo-controlled study.

BACKGROUND: The orally available kinase inhibitor R-roscovitine has undergone clinical trials against various cancers and is currently under clinical evaluation against Cushing disease and rheumatoid arthritis. Roscovitine displays biological properties suggesting potential benefits in CF: it partially corrects F508del-CFTR trafficking, stimulates the bactericidal properties of CF alveolar macrophages, and displays anti-inflammatory properties and analgesic effects. METHODS: A phase 2 trial study (ROSCO-CF) was launched to evaluate the safety and effects of roscovitine in Pseudomonas aeruginosa infected adult CF patients carrying two CF causing mutations (at least one F508del-CFTR mutation) and harboring a FEV1 ≥40%. ROSCO-CF was a multicenter, double-blind, placebo-controlled, dose-ranging study (200, 400, 800 mg roscovitine, orally administered daily for 4 days/week/4 weeks). RESULTS: Among the 34 volunteers enrolled, randomization assigned 11/8/8/7 to receive the 0 (placebo)/ 200/400/800 mg roscovitine doses, respectively. In these subjects with polypharmacy, roscovitine was relatively safe and well-tolerated, with no significant adverse effects (AEs) other than five serious AEs (SAEs) possibly related to roscovitine. Pharmacokinetics of roscovitine were rather variable among subjects. No significant efficacy, at the levels of inflammation, infection, spirometry, sweat chloride, pain and quality of life, was detected in roscovitine-treated groups compared to the placebo-treated group. CONCLUSION: Roscovitine was relatively safe and well-tolerated in CF patients especially at the 200 and 400 mg doses. However, there were 5 subject withdrawals due to SAEs in the roscovitine group and none in the placebo group. The lack of evidence for efficacy of roscovitine (despite encouraging cellular and animal results) may be due to high pharmacokinetics variability, short duration of treatment, and/or inappropriate dosing protocol.

Design and Microwave Synthesis of New (5Z) 5-Arylidene-2-thioxo-1,3-thiazolinidin-4-one and (5Z) 2-Amino-5-arylidene-1,3-thiazol-4(5H)-one as New Inhibitors of Protein Kinase DYRK1A.

Here, we report on the synthesis of libraries of new 5-arylidene-2-thioxo-1,3-thiazolidin-4-ones 3 (twenty-two compounds) and new 2-amino-5-arylidene-1,3-thiazol-4(5H)-ones 5 (twenty-four compounds) with stereo controlled Z-geometry under microwave irradiation. The 46 designed final compounds were tested in order to determine their activity against four representative protein kinases (DYR1A, CK1, CDK5/p25, and GSK3α/ß). Among these 1,3-thiazolidin-4-ones, the molecules (5Z) 5-(4-hydroxybenzylidene)-2-thioxo-1,3-thiazolidin-4-one 3e (IC50 0.028 µM) and (5Z)-5-benzo[1,3]dioxol-5-ylmethylene-2-(pyridin-2-yl)amino-1,3-thiazol-4(5H)-one 5s (IC50 0.033 µM) were identified as lead compounds and as new nanomolar DYRK1A inhibitors. Some of these compounds in the two libraries have been also evaluated for their in vitro inhibition of cell proliferation (Huh7 D12, Caco2, MDA-MB 231, HCT 116, PC3, and NCI-H2 tumor cell lines). These results will enable us to use the 1,3-thiazolidin-4-one core as pharmacophores to develop potent treatment for neurological or oncological disorders in which DYRK1A is fully involved.

Dual-Specificity, Tyrosine Phosphorylation-Regulated Kinases (DYRKs) and cdc2-Like Kinases (CLKs) in Human Disease, an Overview.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRK1A, 1B, 2-4) and cdc2-like kinases (CLK1-4) belong to the CMGC group of serine/threonine kinases. These protein kinases are involved in multiple cellular functions, including intracellular signaling, mRNA splicing, chromatin transcription, DNA damage repair, cell survival, cell cycle control, differentiation, homocysteine/methionine/folate regulation, body temperature regulation, endocytosis, neuronal development, synaptic plasticity, etc. Abnormal expression and/or activity of some of these kinases, DYRK1A in particular, is seen in many human nervous system diseases, such as cognitive deficits associated with Down syndrome, Alzheimer's disease and related diseases, tauopathies, dementia, Pick's disease, Parkinson's disease and other neurodegenerative diseases, Phelan-McDermid syndrome, autism, and CDKL5 deficiency disorder. DYRKs and CLKs are also involved in diabetes, abnormal folate/methionine metabolism, osteoarthritis, several solid cancers (glioblastoma, breast, and pancreatic cancers) and leukemias (acute lymphoblastic leukemia, acute megakaryoblastic leukemia), viral infections (influenza, HIV-1, HCMV, HCV, CMV, HPV), as well as infections caused by unicellular parasites (Leishmania, Trypanosoma, Plasmodium). This variety of pathological implications calls for (1) a better understanding of the regulations and substrates of DYRKs and CLKs and (2) the development of potent and selective inhibitors of these kinases and their evaluation as therapeutic drugs. This article briefly reviews the current knowledge about DYRK/CLK kinases and their implications in human disease.

LMW cyclin E and its novel catalytic partner CDK5 are therapeutic targets and prognostic biomarkers in salivary gland cancers.

Salivary gland cancers (SGCs) are rare yet aggressive malignancies with significant histological heterogeneity, which has made prediction of prognosis and development of targeted therapies challenging. In majority of patients, local recurrence and/or distant metastasis are common and systemic treatments have minimal impact on survival. Therefore, identification of novel targets for treatment that can also be used as predictors of recurrence for multiple histological subtypes of SGCs is an area of unmet need. In this study, we developed a novel transgenic mouse model of SGC, efficiently recapitulating the major histological subtype (adenocarcinomas of the parotid gland) of human SGC. CDK2 knock out (KO) mice crossed with MMTV-low molecular weight forms of cyclin E (LMW-E) mice generated the transgenic mouse models of SGC, which arise in the parotid region of the salivary gland, similar to the common site of origin seen in human SGCs. To identify the CDK2 independent catalytic partner(s) of LMW-E, we used LMW-E expressing cell lines in mass spectrometric analysis and subsequent biochemical validation in pull down assays. These studies revealed that in the absence of CDK2, LMW-E preferentially binds to CDK5. Molecular targeting of CDK5, using siRNA, resulted in inhibition of cell proliferation of human SGCs overexpressing LMW-E. We also provide clinical evidence of significant association of LMW-E/CDK5 co-expression and decreased recurrence free survival in human SGC. Immunohistochemical analysis of LMW-E and CDK5 in 424 patients representing each of the four major histological subtypes of human salivary cancers (Aci, AdCC, MEC, and SDC) revealed that LMW-E and CDK5 are concordantly (positive/positive or negative/negative) expressed in 70% of these patients. The co-expression of LMW-E/CDK5 (both positive) robustly predicts the likelihood of recurrence, regardless of the histological classification of these tumors. Collectively, our results suggest that CDK5 is a novel and targetable biomarker for the treatment of patients with SGC presenting with LMW-E overexpressing tumors.

(R)-Roscovitine and CFTR modulators enhance killing of multi-drug resistant Burkholderia cenocepacia by cystic fibrosis macrophages.

Cystic fibrosis (CF) is characterized by chronic bacterial infections and heightened inflammation. Widespread ineffective antibiotic use has led to increased isolation of drug resistant bacterial strains from respiratory samples. (R)-roscovitine (Seliciclib) is a unique drug that has many benefits in CF studies. We sought to determine roscovitine's impact on macrophage function and killing of multi-drug resistant bacteria. Human blood monocytes were isolated from CF (F508del/F508del) and non-CF persons and derived into macrophages (MDMs). MDMs were infected with CF clinical isolates of B. cenocepacia and P. aeruginosa. MDMs were treated with (R)-roscovitine or its main hepatic metabolite (M3). Macrophage responses to infection and subsequent treatment were determined. (R)-roscovitine and M3 significantly increased killing of B. cenocepacia and P. aeruginosa in CF MDMs in a dose-dependent manner. (R)-roscovitine-mediated effects were partially dependent on CFTR and the TRPC6 channel. (R)-roscovitine-mediated killing of B. cenocepacia was enhanced by combination with the CFTR modulator tezacaftor/ivacaftor and/or the alternative CFTR modulator cysteamine. (R)-roscovitine also increased MDM CFTR function compared to tezacaftor/ivacaftor treatment alone. (R)-roscovitine increases CF macrophage-mediated killing of antibiotic-resistant bacteria. (R)-roscovitine also enhances other macrophage functions including CFTR-mediated ion efflux. Effects of (R)-roscovitine are greatest when combined with CFTR modulators or cysteamine, justifying further clinical testing of (R)-roscovitine or optimized derivatives.

Non-Linear Pharmacokinetics of Oral Roscovitine (Seliciclib) in Cystic Fibrosis Patients Chronically Infected with Pseudomonas aeruginosa: A Study on Population Pharmacokinetics with Monte Carlo Simulations.

Roscovitine (Seliciclib), a new protein kinase inhibitor, was administered orally to adult patients with cystic fibrosis for the first time in the ROSCO-CF trial, a dose-escalation, phase IIa, randomized, controlled trial. Extensive pharmacokinetic sampling was performed up to 12 h after the first oral dose. Roscovitine and its main metabolite M3 were quantified by liquid chromatography coupled with tandem mass spectrometry. The pharmacokinetics analyses were performed by non-linear mixed effects modelling. Monte Carlo simulations were performed to assess the impact of dose on the pharmacokinetics of oral roscovitine. Twenty-three patients received oral doses ranging from 200 to 800 mg of roscovitine and 138 data points were available for both roscovitine and M3 concentrations. The pharmacokinetics was best described by a two-compartment parent-metabolite model, with a complex saturable absorption process modelled as the sum of Gaussian inverse density functions. The Monte Carlo simulations showed a dose-dependent and saturable first-pass effect leading to pre-systemic formation of M3. The treatment with proton-pump inhibitors reduced the rate of absorption of oral roscovitine. The pharmacokinetics of oral roscovitine in adult patients with cystic fibrosis was non-linear and showed significant inter-individual variability. A repeat-dose study will be required to assess the inter-occasional variability of its pharmacokinetics.

An Overview of In Vivo and In Vitro Models for Autosomal Dominant Polycystic Kidney Disease: A Journey from 3D-Cysts to Mini-Pigs.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inheritable cause of end stage renal disease and, as of today, only a single moderately effective treatment is available for patients. Even though ADPKD research has made huge progress over the last decades, the precise disease mechanisms remain elusive. However, a wide variety of cellular and animal models have been developed to decipher the pathophysiological mechanisms and related pathways underlying the disease. As none of these models perfectly recapitulates the complexity of the human disease, the aim of this review is to give an overview of the main tools currently available to ADPKD researchers, as well as their main advantages and limitations.

Author Correction: A Small Compound Targeting Prohibitin with Potential Interest for Cognitive Deficit Rescue in Aging mice and Tau Pathology Treatment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exploring Kinase Inhibition Properties of 9H-pyrimido[5,4-b]- and [4,5-b]indol-4-amine Derivatives.

We previously highlighted the interest in 6,5,6-fused tricyclic analogues of 4-aminoquinazolines as kinase inhibitors in the micromolar to the nanomolar range of IC50 values. For the generation of chemical libraries, the formamide-mediated cyclization of the cyanoamidine precursors was carried out under microwave irradiation in an eco-friendly approach. In order to explore more in-depth the pharmacological interest in such tricyclic skeletons, the central five member ring, i.e., thiophène or furan, was replaced by a pyrrole to afford 9H-pyrimido[5,4-b]- and [4,5-b]indol-4-amine derivatives inspired from harmine. The inhibitory potency of the final products was determined against four protein kinases (CDK5/p25, CK1/ε, GSK3 and DYRK1A). As a result, we have identified promising compounds targeting CK1/ε and DYRK1A and displaying micromolar and submicromolar IC50 values.

Long-Term Fipronil Treatment Induces Hyperactivity in Female Mice.

Fipronil is an insecticide widely used for veterinary and agricultural purposes. While its insecticidal properties mostly rely on its high affinity antagonistic activity on insect γ aminobutyric acid (GABA) receptors, fipronil and its main metabolite fipronil sulfone nevertheless display non-negligible affinity for mammalian GABAA receptor. As several environmental toxicants have been shown to raise the risk of developing various neurodegenerative disorders, the aim of this study was to evaluate whether long-term low dose administration of fipronil could lead to cognitive deficiencies. Our results indicate that long-term fipronil treatment leads to behavioral perturbations in mice, indicating an accumulative effect of sustained exposure to low dose of fipronil. Although no memory impairment was observed during the course of our study, we noticed a significant hyperlocomotion behavior after 43 weeks of weekly fipronil administration, which is consistent with its direct effect on the GABAergic system.

A Small Compound Targeting Prohibitin with Potential Interest for Cognitive Deficit Rescue in Aging mice and Tau Pathology Treatment.

Neurodegenerative diseases, including Alzheimer's and Parkinson's disease, are characterized by increased protein aggregation in the brain, progressive neuronal loss, increased inflammation, and neurogenesis impairment. We analyzed the effects of a new purine derivative drug, PDD005, in attenuating mechanisms involved in the pathogenesis of neurodegenerative diseases, using both in vivo and in vitro models. We show that PDD005 is distributed to the brain and can rescue cognitive deficits associated with aging in mice. Treatment with PDD005 prevents impairment of neurogenesis by increasing sex-determining region Y-box 2, nestin, and also enhances synaptic function through upregulation of synaptophysin and postsynaptic density protein 95. PDD005 treatment also reduced neuro-inflammation by decreasing interleukin-1ß expression, activation of astrocytes, and microglia. We identified prohibitin as a potential target in mediating the therapeutic effects of PDD005 for the treatment of cognitive deficit in aging mice. Additionally, in the current study, glycogen synthase kinase appears to attenuate tau pathology.

Biological Characterization of 8-Cyclopropyl-2-(pyridin-3-yl)thiazolo[5,4-f]quinazolin-9(8H)-one, a Promising Inhibitor of DYRK1A.

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) hyperactivity has been linked to the development of a number of human malignancies. DYRK1A is the most studied family member, and the discovery of novel specific inhibitors is attracting considerable interest. The 8-cyclopropyl-2(pyridin-3-yl)thiazolo[5,4-f]quinazolin-9(8H)-one (also called FC162) was found to be a promising inhibitor of DYRK1A and was characterized in biological experiments, by western transfer and flow cytometry on SH-SY5Y and pre-B cells. Here, the results obtained with FC162 are compared to well-characterized known DYRK1A inhibitors (e.g., Leucettine L41 and EHT1610).