Tau accumulates in Crohn's disease gut.

A sizeable body of evidence has recently emerged to suggest that gastrointestinal (GI) inflammation might be involved in the development of Parkinson's disease (PD). There is now strong epidemiological and genetical evidence linking PD to inflammatory bowel diseases and we recently demonstrated that the neuronal protein alpha-synuclein, which is critically involved in PD pathophysiology, is upregulated in inflamed segments of Crohn's colon. The microtubule associated protein tau is another neuronal protein critically involved in neurodegenerative disorders but, in contrast to alpha-synuclein, no data are available about its expression and phosphorylation patterns in inflammatory bowel diseases. Here, we examined the expression levels of tau isoforms, their phosphorylation profile and truncation in colon biopsy specimens from 16 Crohn's disease (CD) and 6 ulcerative colitis (UC) patients and compared them to samples from 16 controls. Additional experiments were performed in full thickness segments of colon of five CD and five control subjects, in primary cultures of rat enteric neurons and in nuclear factor erythroid 2-related factor (Nrf2) knockout mice. Our results show the upregulation of two main human tau isoforms in the enteric nervous system (ENS) in CD but not in UC. This upregulation was not transcriptionally regulated but instead likely resulted from a decrease in protein clearance via an Nrf2 pathway. Our findings, which provide the first detailed characterization of tau in CD, suggest that the key proteins involved in neurodegenerative disorders such as alpha-synuclein and tau, might also play a role in CD.

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.

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.

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.

cdc-like/dual-specificity tyrosine phosphorylation-regulated kinases inhibitor leucettine L41 induces mTOR-dependent autophagy: implication for Alzheimer's disease.

Leucettines, a family of pharmacological inhibitors of dual-specificity tyrosine phosphorylation regulated kinases and cdc-like kinases (CLKs), are currently under investigation for their potential therapeutic application to Down syndrome and Alzheimer's disease. We here report that leucettine L41 triggers bona fide autophagy in osteosarcoma U-2 OS cells and immortalized mouse hippocampal HT22 cells, characterized by microtubule-associated protein light chain 3 membrane translocation and foci formation. Leucettine L41-triggered autophagy requires the Unc-51-like kinase and is sensitive to the phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and 3-methyladenine, suggesting that it acts through the mammalian target of rapamycin (mTOR)/PI3K-dependent pathway. Leucettine L41 does not act by modifying the autophagic flux of vesicles. Leucettine L41-induced autophagy correlates best with inhibition of CLKs. Leucettine L41 modestly inhibited phosphatidylinositol-3-phosphate 5-kinase, FYVE domain-containing activity as tested both in vitro and in vivo, which may also contribute to autophagy induction. Altogether these results demonstrate that leucettines can activate the autophagic mTOR/PI3K pathway, a characteristic that may turn advantageous in the context of Alzheimer's disease treatment.

Pharmacological assessment defines Leishmania donovani casein kinase 1 as a drug target and reveals important functions in parasite viability and intracellular infection.

Protein kinase inhibitors have emerged as new drugs in various therapeutic areas, including leishmaniasis, an important parasitic disease. Members of the Leishmania casein kinase 1 (CK1) family represent promising therapeutic targets. Leishmania casein kinase 1 isoform 2 (CK1.2) has been identified as an exokinase capable of phosphorylating host proteins, thus exerting a potential immune-suppressive action on infected host cells. Moreover, its inhibition reduces promastigote growth. Despite these important properties, its requirement for intracellular infection and its chemical validation as a therapeutic target in the disease-relevant amastigote stage remain to be established. In this study, we used a multidisciplinary approach combining bioinformatics, biochemical, and pharmacological analyses with a macrophage infection assay to characterize and define Leishmania CK1.2 as a valid drug target. We show that recombinant and transgenic Leishmania CK1.2 (i) can phosphorylate CK1-specific substrates, (ii) is sensitive to temperature, and (iii) is susceptible to CK1-specific inhibitors. CK1.2 is constitutively expressed at both the promastigote insect stage and the vertebrate amastigote stage. We further demonstrated that reduction of CK1 activity by specific inhibitors, such as D4476, blocks promastigote growth, strongly compromises axenic amastigote viability, and decreases the number of intracellular Leishmania donovani and L. amazonensis amastigotes in infected macrophages. These results underline the potential role of CK1 kinases in intracellular survival. The identification of differences in structure and inhibition profiles compared to those of mammalian CK1 kinases opens new opportunities for Leishmania CK1.2 antileishmanial drug development. Our report provides the first chemical validation of Leishmania CK1 protein kinases, required for amastigote intracellular survival, as therapeutic targets.

New 5-ylidene rhodanine derivatives based on the dispacamide A model.

A practical approach for the preparation of (5Z) 5-ylidene rhodanine derivatives bearing the (4,5-dihalogeno-pyrrol-2-yl)carbamoyl fragment of dispacamide A is reported. The new compounds were obtained in good yields (19-88 %) by Knoevenagel condensation according to a solution-phase microwave dielectric heating protocol in the presence of organic bases (piperidine, TEA, and AcONa) from a set of N-substituted rhodanines 2(a-i). The ten synthetic products 3(a-j) have been synthesized with a Z-geometry about their exocyclic double bond and the structure of one of these compounds (3) was confirmed by a single X-ray diffraction analysis. The new (5Z) 5-ylidene rhodanine derivatives 3(a-j) were tested against eight protein kinases.

Role of ICAM-1 in the Adhesion of T Cells to Enteric Glia: Perspectives in the Formation of Plexitis in Crohn's Disease.

BACKGROUND & AIMS: The presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn's disease. To decipher the mechanisms leading to their formation, T-cell interactions with enteric neural cells were studied in vitro and in vivo. METHODS: T cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn's disease. The mechanisms involved in T-cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro but also in vivo in Winnie mice. RESULTS: The mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn's disease (2.42 ± 0.5) as compared with controls (0.36 ± 0.08, P = .0007). Co-culture experiments showed that exposure to proinflammatory cytokines enhanced T-cell adhesion to glia and increased intercellular adhesion molecule-1 (ICAM-1) expression in glia. We next demonstrated that T-cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/lymphocyte function-associated antigen-1 (LFA-1) with lifitegrast reduced colitis severity and decreased T-cell infiltration in the myenteric plexus. CONCLUSIONS: Our present work argues for a role of glia-T-cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1 and suggests that deciphering the functional consequences of glia-T-cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn's disease.

An efficient approach to dispacamide A and its derivatives.

Dispacamide A and new analogs of this marine alkaloid were prepared in seven steps with an overall yield ranging from 12 to 33%. The key step of the strategy was a stereocontrolled Knoevenagel condensation under microwave dielectric heating in the last step. In this condensation, the 2-aminoimidazolin-4-one hydrochloride partners 10a-c were synthesized in three steps with good overall yields (33-79%) via the ring closure of N-guanidino acetic acids 9a-c and the aldehydes 5a,b as the two others building-blocks, in 3 steps with 60-66% overall yields. The six synthetic products have been obtained with a Z geometry about their exocyclic bond on the basis of (13)C/(1)H long-range coupling constants using a gHSQMBC experiment.

Library-based discovery of DYRK1A/CLK1 inhibitors from natural product extracts.

The dual specificity tyrosine-phosphorylation-regulated kinase DYRK1A possesses diverse roles in neuronal development and adult brain physiology, and increased activity has been linked to neurodegenerative diseases. Very few inhibitors of this kinase have been reported up to now. Screening of a library of > 900 plant and fungal extracts afforded 25 extracts with IC50s < 10 µg/mL against DYRK1A. To identify the active constituents, the extracts were submitted to a process integrating physicochemical data with biological information, referred to as HPLC-based activity profiling. Follow-up investigation of four extracts led to the targeted isolation of harmine (1, IC50 0.022 µM) from Peganum harmala, emodin (3, IC50 4.2 µM) from Cassia nigricans, kaempferol (4, IC50 0.91 µM) from Cuscuta chinensis, and 3,8-di-O-methylherbacetin (11, IC50 8.6 µM), 3,3',4'-tri-O-methylmyricetin (12, IC50 7.1 µM) and ombuin (15, IC50 1.7 µM) from Larrea tridentata as the active constituents. Active extracts and compounds were also tested on the closely related cdc2-like kinase CLK1. Finally, the selectivity profile of compounds was evaluated by including other members of the DYRKs and CLKs families. While the flavonoids and emodin did not show significant differences in the potency of their activities, harmine (1) was most active against DYRK1A, CLK1, and CLK4, and less potent against the other kinases, with selectivity ranging from 2- to 20-fold.

Intestinal Epithelial AMPK Deficiency Causes Delayed Colonic Epithelial Repair in DSS-Induced Colitis.

Dysfunctions in the intestinal barrier, associated with an altered paracellular pathway, are commonly observed in inflammatory bowel disease (IBD). The AMP-activated protein kinase (AMPK), principally known as a cellular energy sensor, has also been shown to play a key role in the stabilization and assembly of tight junctions. Here, we aimed to investigate the contribution of intestinal epithelial AMPK to the initiation, progression and resolution of acute colitis. We also tested the hypothesis that protection mediated by metformin administration on intestinal epithelium damage required AMPK activation. A dextran sodium sulfate (DSS)-induced colitis model was used to assess disease progression in WT and intestinal epithelial cell (IEC)-specific AMPK KO mice. Barrier integrity was analyzed by measuring paracellular permeability following dextran-4kDa gavage and pro-inflammatory cytokines and tight junction protein expression. The deletion of intestinal epithelial AMPK delayed intestinal injury repair after DSS exposure and was associated with a slower re-epithelization of the intestinal mucosa coupled with severe ulceration and inflammation, and altered barrier function. Following intestinal injury, IEC AMPK KO mice displayed a lower goblet cell counts with concomitant decreased Muc2 gene expression, unveiling an impaired restitution of goblet cells and contribution to wound healing process. Metformin administration during the recovery phase attenuated the severity of DSS-induced colitis through improvement in intestinal repair capacity in both WT and IEC AMPK KO mice. Taken together, these findings demonstrate a critical role for IEC-expressed AMPK in regulating mucosal repair and epithelial regenerative capacity following acute colonic injury. Our studies further underscore the therapeutic potential of metformin to support repair of the injured intestinal epithelium, but this effect is conferred independently of intestinal epithelial AMPK.

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.

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.

Identification and structure-activity relationship of 8-hydroxy-quinoline-7-carboxylic acid derivatives as inhibitors of Pim-1 kinase.

Pim-1 kinase is a cytoplasmic serine/threonine kinase that controls programmed cell death by phosphorylating substrates that regulate both apoptosis and cellular metabolism. A series of 2-styrylquinolines and quinoline-2-carboxamides has been identified as potent inhibitors of the Pim-1 kinase. The 8-hydroxy-quinoline 7-carboxylic acid moiety appeared to be a crucial pharmacophore for activity. Molecular modeling indicated that interaction of this scaffold with Asp186 and Lys67 residues within the ATP-binding pocket might be responsible for the kinase inhibitory potency.

Characterisation of tau in the human and rodent enteric nervous system under physiological conditions and in tauopathy.

Tau is normally a highly soluble phosphoprotein found predominantly in neurons. Six different isoforms of tau are expressed in the adult human CNS. Under pathological conditions, phosphorylated tau aggregates are a defining feature of neurodegenerative disorders called tauopathies. Recent findings have suggested a potential role of the gut-brain axis in CNS homeostasis, and therefore we set out to examine the isoform profile and phosphorylation state of tau in the enteric nervous system (ENS) under physiological conditions and in tauopathies. Surgical specimens of human colon from controls, Parkinson's disease (PD) and progressive supranuclear palsy (PSP) patients were analyzed by Western Blot and immunohistochemistry using a panel of anti-tau antibodies. We found that adult human ENS primarily expresses two tau isoforms, localized in the cell bodies and neuronal processes. We did not observe any difference in the enteric tau isoform profile and phosphorylation state between PSP, PD and control subjects. The htau mouse model of tauopathy also expressed two main isoforms of human tau in the ENS, and there were no apparent differences in ENS tau localization or phosphorylation between wild-type and htau mice. Tau in both human and mouse ENS was found to be phosphorylated but poorly susceptible to dephosphorylation with lambda phosphatase. To investigate ENS tau phosphorylation further, primary cultures from rat enteric neurons, which express four isoforms of tau, were pharmacologically manipulated to show that ENS tau phosphorylation state can be regulated, at least in vitro. Our study is the first to characterize tau in the rodent and human ENS. As a whole, our findings provide a basis to unravel the functions of tau in the ENS and to further investigate the possibility of pathological changes in enteric neuropathies and tauopathies.

Induction of Amyloid-ß42 Production by Fipronil and Other Pyrazole Insecticides.

Generation of amyloid-ß peptides (Aßs) by proteolytic cleavage of the amyloid-ß protein precursor (AßPP), especially increased production of Aß42/Aß43 over Aß40, and their aggregation as oligomers and plaques, represent a characteristic feature of Alzheimer's disease (AD). In familial AD (FAD), altered Aß production originates from specific mutations of AßPP or presenilins 1/2 (PS1/PS2), the catalytic subunits of γ-secretase. In sporadic AD, the origin of altered production of Aßs remains unknown. We hypothesize that the 'human chemical exposome' contains products able to favor the production of Aß42/Aß43 over Aß40 and shorter Aßs. To detect such products, we screened a library of 3500 + compounds in a cell-based assay for enhanced Aß42/Aß43 production. Nine pyrazole insecticides were found to induce a ß- and γ-secretase-dependent, 3-10-fold increase in the production of extracellular Aß42 in various cell lines and neurons differentiated from induced pluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showed increased production of Aßs cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and shorter. Strongly supporting a direct effect on γ-secretase activity, pyrazoles shifted the cleavage pattern of another γ-secretase substrate, alcadeinα, and shifted the cleavage of AßPP by highly purified γ-secretase toward Aß42/Aß43. Focusing on fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production of Aß42/Aß43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolized rapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widely used pyrazole insecticides enhance the production of toxic, aggregation prone Aß42/Aß43 peptides, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in sporadic AD.

Specific Triazine Herbicides Induce Amyloid-ß42 Production.

Proteolytic cleavage of the amyloid-ß protein precursor (AßPP) by secretases leads to extracellular release of amyloid-ß (Aß) peptides. Increased production of Aß42 over Aß40 and aggregation into oligomers and plaques constitute an Alzheimer's disease (AD) hallmark. Identifying products of the 'human chemical exposome' (HCE) able to induce Aß42 production may be a key to understanding some of the initiating causes of AD and to generate non-genetic, chemically-induced AD animal models. A cell model was used to screen HCE libraries for Aß42 inducers. Out of 3500+ compounds, six triazine herbicides were found that induced a ß- and γ-secretases-dependent, 2-10 fold increase in the production of extracellular Aß42 in various cell lines, primary neuronal cells, and neurons differentiated from human-induced pluripotent stem cells (iPSCs). Immunoprecipitation/mass spectrometry analyses show enhanced production of Aß peptides cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and lower, a characteristic of AD. Neurons derived from iPSCs obtained from a familial AD (FAD) patient (AßPP K724N) produced more Aß42 versus Aß40 than neurons derived from healthy controls iPSCs (AßPP WT). Triazines enhanced Aß42 production in both control and AD iPSCs-derived neurons. Triazines also shifted the cleavage pattern of alcadeinα, another γ-secretase substrate, suggesting a direct effect of triazines on γ-secretase activity. In conclusion, several widely used triazines enhance the production of toxic, aggregation prone Aß42/Aß43 amyloids, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in late-onset AD.

Dispacamide E and other bioactive bromopyrrole alkaloids from two Indonesian marine sponges of the genus Stylissa.

Chemical investigation of methanolic extracts of the two Indonesian marine sponges Stylissa massa and Stylissa flabelliformis yielded 25 bromopyrrole alkaloids including 2 new metabolites. The structures of all isolated compounds were unambiguously elucidated based on extensive 1D and 2D NMR, LR-MS and HR-MS analyses. All isolated compounds were assayed for their antiproliferative and protein kinase inhibitory activities. Several of the tested compounds revealed selective activity(ies) which suggested preliminary SARs of the isolated bromopyrrole alkaloids.

Chemical synthesis and biological validation of immobilized protein kinase inhibitory Leucettines.

Leucettines, a family of marine sponge-derived 2-aminoimidazolone alkaloids, are potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). They constitute promising pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. In order to investigate the scope of potential targets of Leucettine L41, a representative member of the chemical class, we designed an affinity chromatography strategy based on agarose-immobilized leucettines. A synthesis protocol for the attachment of a polyethylene (3 or 4 units) linker to L41 was first established. The linker attachment site on L41 was selected on the basis of the co-crystal structure of L41 with several kinases. L41 was then covalently bound to agarose beads through the primary amine located at the end of the linker. Control, kinase inactive Leucettine was also immobilized, as well as free linker devoid of ligand. Extracts of several mouse tissues revealed a complex pattern of interacting proteins, some of which probably resulting from non-specific, hydrophobic binding, while others representing bona fide Leucettine-interacting proteins. DYRK1A and GSK-3 (glycogen synthase kinase-3) were confirmed as interacting targets by Western blotting in various mouse tissues. The Leucettine affinity chromatography resin constitutes a powerful tool to purify and identify the targets of this new promising therapeutic class of molecules.