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.

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.

A Collection of Bioactive Nitrogen-Containing Molecules from the Marine Sponge Acanthostrongylophora ingens.

Thirteen nitrogen-containing molecules (1a/1b and 2-12) were isolated from the Indonesian sponge Acanthostrongylophora ingens, highlighting the richness of this organism as a source of alkaloids. Their structures were elucidated using one- and two-dimensional NMR spectroscopy and HR-ESI-MS, while the stereochemistry of the diketopiperazines was established using Marfey's method. All compounds were screened in our standard bioactivity assays, including antibacterial, antikinases, and amyloid ß-42 assays. The most interesting bioactivity result was obtained with the known acanthocyclamine A (3), which revealed for the first time a specific Escherichia coli antimicrobial activity and an inhibitory effect on amyloid ß-42 production induced by aftin-5 and no cytotoxicity at the dose of 26 µM. These results highlight the potentiality of a bipiperidine scaffold as a promising skeleton for preventing or reducing the production of amyloid ß-42, a key player in the initiation of Alzheimer's disease.

[b]-Annulated Halogen-Substituted Indoles as Potential DYRK1A Inhibitors.

Since hyperactivity of the protein kinase DYRK1A is linked to several neurodegenerative disorders, DYRK1A inhibitors have been suggested as potential therapeutics for Down syndrome and Alzheimer's disease. Most published inhibitors to date suffer from low selectivity against related kinases or from unfavorable physicochemical properties. In order to identify DYRK1A inhibitors with improved properties, a series of new chemicals based on [b]-annulated halogenated indoles were designed, synthesized, and evaluated for biological activity. Analysis of crystal structures revealed a typical type-I binding mode of the new inhibitor 4-chlorocyclohepta[b]indol-10(5H)-one in DYRK1A, exploiting mainly shape complementarity for tight binding. Conversion of the DYRK1A inhibitor 8-chloro-1,2,3,9-tetrahydro-4H-carbazol-4-one into a corresponding Mannich base hydrochloride improved the aqueous solubility but abrogated kinase inhibitory activity.

Casein kinase 1ε and 1α as novel players in polycystic kidney disease and mechanistic targets for (R)-roscovitine and (S)-CR8.

Following the discovery of (R)-roscovitine's beneficial effects in three polycystic kidney disease (PKD) mouse models, cyclin-dependent kinases (CDKs) inhibitors have been investigated as potential treatments. We have used various affinity chromatography approaches to identify the molecular targets of roscovitine and its more potent analog (S)-CR8 in human and murine polycystic kidneys. These methods revealed casein kinases 1 (CK1) as additional targets of the two drugs. CK1ε expression at the mRNA and protein levels is enhanced in polycystic kidneys of 11 different PKD mouse models as well as in human polycystic kidneys. A shift in the pattern of CK1α isoforms is observed in all PKD mouse models. Furthermore, the catalytic activities of both CK1ε and CK1α are increased in mouse polycystic kidneys. Inhibition of CK1ε and CK1α may thus contribute to the long-lasting attenuating effects of roscovitine and (S)-CR8 on cyst development. CDKs and CK1s may constitute a dual therapeutic target to develop kinase inhibitory PKD drug candidates.

Reduction of ciliary length through pharmacologic or genetic inhibition of CDK5 attenuates polycystic kidney disease in a model of nephronophthisis.

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.

TRPC6 channel translocation into phagosomal membrane augments phagosomal function.

Defects in the innate immune system in the lung with attendant bacterial infections contribute to lung tissue damage, respiratory insufficiency, and ultimately death in the pathogenesis of cystic fibrosis (CF). Professional phagocytes, including alveolar macrophages (AMs), have specialized pathways that ensure efficient killing of pathogens in phagosomes. Phagosomal acidification facilitates the optimal functioning of degradative enzymes, ultimately contributing to bacterial killing. Generation of low organellar pH is primarily driven by the V-ATPases, proton pumps that use cytoplasmic ATP to load H(+) into the organelle. Critical to phagosomal acidification are various channels derived from the plasma membrane, including the anion channel cystic fibrosis transmembrane conductance regulator, which shunt the transmembrane potential generated by movement of protons. Here we show that the transient receptor potential canonical-6 (TRPC6) calcium-permeable channel in the AM also functions to shunt the transmembrane potential generated by proton pumping and is capable of restoring microbicidal function to compromised AMs in CF and enhancement of function in non-CF cells. TRPC6 channel activity is enhanced via translocation to the cell surface (and then ultimately to the phagosome during phagocytosis) in response to G-protein signaling activated by the small molecule (R)-roscovitine and its derivatives. These data show that enhancing vesicular insertion of the TRPC6 channel to the plasma membrane may represent a general mechanism for restoring phagosome activity in conditions, where it is lost or impaired.

Indole-3-Carbonitriles as DYRK1A Inhibitors by Fragment-Based Drug Design.

Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is a potential drug target because of its role in the development of Down syndrome and Alzheimer's disease. The selective DYRK1A inhibitor 10-iodo-11H-indolo[3,2-c]quinoline-6-carboxylic acid (KuFal194), a large, flat and lipophilic molecule, suffers from poor water solubility, limiting its use as chemical probe in cellular assays and animal models. Based on the structure of KuFal194, 7-chloro-1H-indole-3-carbonitrile was selected as fragment template for the development of smaller and less lipophilic DYRK1A inhibitors. By modification of this fragment, a series of indole-3-carbonitriles was designed and evaluated as potential DYRK1A ligands by molecular docking studies. Synthesis and in vitro assays on DYRK1A and related protein kinases identified novel double-digit nanomolar inhibitors with submicromolar activity in cell culture assays.

Development of Kinase Inhibitors via Metal-Catalyzed C⁻H Arylation of 8-Alkyl-thiazolo[5,4-f]-quinazolin-9-ones Designed by Fragment-Growing Studies.

Efficient metal catalyzed C⁻H arylation of 8-alkyl-thiazolo[5,4-f]-quinazolin-9-ones was explored for SAR studies. Application of this powerful chemical tool at the last stage of the synthesis of kinase inhibitors allowed the synthesis of arrays of molecules inspired by fragment-growing studies generated by molecular modeling calculations. Among the potentially active compounds designed through this strategy, FC162 (4c) exhibits nanomolar IC50 values against some kinases, and is the best candidate for the development as a DYRK kinase inhibitor.

N-(1H-Pyrazol-3-yl)quinazolin-4-amines as a novel class of casein kinase 1δ/ε inhibitors: Synthesis, biological evaluation and molecular modeling studies.

Described herein is the design, synthesis and biological evaluation of a series of N-(1H-pyrazol-3-yl)quinazolin-4-amines against a panel of eight disease relevant protein kinases. The kinase inhibition results indicated that two compounds inhibited casein kinase 1δ/ε (CK1δ/ε) with some selectivity over related kinases, namely CDK5/p25, GSK-3α/ß, and DYRK1A. Docking studies with 3c and 3d revealed the key interactions with desired amino acids in the ATP binding site of CK1δ. Furthermore, compound 3c also elicited selective cytotoxic activity against the pancreas ductal adenocarcinoma (PANC-1) cell line. Taken together, the results of this study establish N-(1H-pyrazol-3-yl)quinazolin-4-amines especially 3c and 3d as valuable lead molecules with great potential for CK1δ/ε inhibitor development targeting neurodegenerative disorders and cancer.

Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines Inhibit Mammalian and Protozoan DYRK & CLK Kinases.

A large diversity of 2-aminoimidazolone alkaloids is produced by various marine invertebrates, especially by the marine Calcareous sponges Leucetta and Clathrina. The phylogeny of these sponges and the wide scope of 2-aminoimidazolone alkaloids they produce are reviewed in this article. The origin (invertebrate cells, associated microorganisms, or filtered plankton), physiological functions, and natural molecular targets of these alkaloids are largely unknown. Following the identification of leucettamine B as an inhibitor of selected protein kinases, we synthesized a family of analogues, collectively named leucettines, as potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. We assembled a small library of marine sponge- and ascidian-derived 2-aminoimidazolone alkaloids, along with several synthetic analogues, and tested them on a panel of mammalian and protozoan kinases. Polyandrocarpamines A and B were found to be potent and selective inhibitors of DYRKs and CLKs. They inhibited cyclin D1 phosphorylation on a DYRK1A phosphosite in cultured cells. 2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential therapeutic drug candidates acting as specific inhibitors of disease-relevant kinases, and possibly other disease-relevant targets.

Combined Virtual and Experimental Screening for CK1 Inhibitors Identifies a Modulator of p53 and Reveals Important Aspects of in Silico Screening Performance.

A compound collection of pronounced structural diversity was comprehensively screened for inhibitors of the DNA damage-related kinase CK1. The collection was evaluated in vitro. A potent and selective CK1 inhibitor was discovered and its capacity to modulate the endogenous levels of the CK1-regulated tumor suppressor p53 was demonstrated in cancer cell lines. Administration of 10 µM of the compound resulted in significant increase of p53 levels, reaching almost 2-fold in hepatocellular carcinoma cells. In parallel to experimental screening, two representative and orthogonal in silico screening methodologies were implemented for enabling the retrospective assessment of virtual screening performance on a case-specific basis. Results showed that both techniques performed at an acceptable and fairly comparable level, with a slight advantage of the structure-based over the ligand-based approach. However, both approaches demonstrated notable sensitivity upon parameters such as screening template choice and treatment of redundancy in the enumerated compound collection. An effort to combine insight derived by sequential implementation of the two methods afforded poor further improvement of screening performance. Overall, the presented assessment highlights the relation between improper use of enrichment metrics and misleading results, and demonstrates the inherent delicacy of in silico methods, emphasizing the challenging character of virtual screening protocol optimization.

From Drug Screening to Target Deconvolution: a Target-Based Drug Discovery Pipeline Using Leishmania Casein Kinase 1 Isoform 2 To Identify Compounds with Antileishmanial Activity.

Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.

Synthesis and preliminary in vitro kinase inhibition evaluation of new diversely substituted pyrido[3,4-g]quinazoline derivatives.

The synthesis of new diversely substituted pyrido[3,4-g]quinazolines is described. The inhibitory potencies of prepared compounds toward a panel of five CMGC protein kinases (CDK5, CLK1, DYRK1A, CK1, GSK3), that are known to play a potential role in Alzheimer's disease, were evaluated. The best overall kinase inhibition profile was found for nitro compound 4 bearing an ethyl group at the 5-position.

5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms.

Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.

Chloromethylhalicyclamine B, a Marine-Derived Protein Kinase CK1δ/ε Inhibitor.

The halogenated alkaloid chloromethylhalicyclamine B (1), together with the known natural compound halicyclamine B (2), was isolated from the extract of the sponge Acanthostrongylophora ingens. The structure of compound 1 was determined by spectroscopic means, and it was shown that 1 is produced by reaction of 2 with CH2Cl2 used for extraction. Compound 1 was a selective CK1δ/ε inhibitor with an IC50 of 6 µM, while the natural compound 2 was inactive. The absolute configuration of 1 was determined by quantum mechanical calculation of its ECD spectrum, and this also determined the previously unknown absolute configuration of the parent halicyclamine B (2). Computational studies, validated by NOESY data, showed that compound 1 can efficiently interact with the ATP-binding site of CK1δ in spite of its globular structure, very different from the planar structure of known inhibitors of CK1δ. This opens the way to the design of a new structural type of CK1δ/ε inhibitors.

CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome.

Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome.

Synthesis of Thiazolo[5,4-f]quinazolin-9(8H)-ones as Multi-Target Directed Ligands of Ser/Thr Kinases.

A library of thirty novel thiazolo[5,4-f]quinazolin-9(8H)-one derivatives belonging to four series designated as 12, 13, 14 and 15 was efficiently prepared, helped by microwave-assisted technology when required. The efficient multistep synthesis of methyl 6-amino-2-cyano- benzo[d]thiazole-7-carboxylate (1) has been reinvestigated and performed on a multigram scale. The inhibitory potency of the final products against five kinases involved in Alzheimer's disease was evaluated. This study demonstrates that some molecules of the 12 and 13 series described in this paper are particularly promising for the development of new multi-target inhibitors of kinases.

Synthesis of Bioactive 2-(Arylamino)thiazolo[5,4-f]-quinazolin-9-ones via the Hügershoff Reaction or Cu- Catalyzed Intramolecular C-S Bond Formation.

A library of thirty eight novel thiazolo[5,4-f]quinazolin-9(8H)-one derivatives (series 8, 10, 14 and 17) was prepared via the Hügershoff reaction and a Cu catalyzed intramolecular C-S bond formation, helped by microwave-assisted technology when required. The efficient multistep synthesis of the key 6-amino-3-cyclopropylquinazolin-4(3H)-one (3) has been reinvestigated and performed on a multigram scale from the starting 5-nitroanthranilic acid. The inhibitory potency of the final products was evaluated against five kinases involved in Alzheimer's disease and showed that some molecules of the 17 series described in this paper are particularly promising for the development of novel multi-target inhibitors of kinases.