Leucettine L41 Research Articles

DYRK1A inhibitors leucettines and TGF-β inhibitor additively stimulate insulin production in beta cells, organoids, and isolated mouse islets.

The decreased β-cell mass and impaired β-cell functionality are the primary causes of diabetes mellitus (DM). Nevertheless, the underlying molecular mechanisms by which β-cell growth and function are controlled are not fully understood. In this work, we show that leucettines, known to be DYRK1A kinase inhibitors, can improve glucose-stimulated insulin secretion (GSIS) in rodent β-cells and isolated islets, as well as in hiPSC-derived β-cells islets. We confirm that DYRK1A is expressed in murine insulinoma cells MIN6. In addition, we found that treatment with selected leucettines stimulates proliferation of β-cells and promotes MIN6 cell cycle progression to the G2/M phase. This effect is also confirmed by increased levels of cyclin D1, which is highly responsive to proliferative signals. Among other leucettines, leucettine L43 had a negligible impact on β-cell proliferation, but markedly impair GSIS. However, leucettine L41, in combination with LY364947, a, a potent and selective TGF-β type-I receptor, significantly promotes GSIS in various cellular diabetic models, including MIN6 and INS1E cells in 2D and 3D culture, iPSC-derived β-cell islets derived from iPSC, and isolated mouse islets, by increased insulin secretion and decreased glucagon level. Our findings confirm an important role of DYRK1A inhibitors as modulators of β-cells function and suggested a new potential target for antidiabetic therapy. Moreover, we show in detail that leucettine derivatives represent promising antidiabetic agents and are worth further evaluation, especially in vivo.

Cerebral Phospho-Tau Acts Synergistically with Soluble Aβ42 Leading to Mild Cognitive Impairment in AAV-AD Rats.

Alzheimer's disease (AD) is a continuum of events beginning with an increase in brain soluble Aβ42 followed by the appearance of hyperphosphorylated tau (P-tau, asymptomatic stage). Mild Cognitive Impairment (MCI) then appears (prodromal stage). However, the individual contribution of these two soluble proteins in the onset of the first cognitive symptoms remains unclear. We sought to understand the specific impact of p-tau on the development of MCI in the AAV-AD rat model, a model of late-onset Alzheimer's disease (LOAD) predementia. We specifically reduced the phosphorylation level of tau while leaving Aβ42 levels unchanged using a DYRK1A protein kinase inhibitor, Leucettine L41, in an adeno-associated virus-based Alzheimer's disease (AAV-AD) rat model. Leucettine L41 was administered by intraperitoneal injection at 20 mg/kg per day in AAV-AD rats from 9 (late asymptomatic phase) to 10 (prodromal phase) months of age. Decreased soluble forms of P-tau induced by chronic administration of Leucettine L41 did not change soluble Aβ42 levels but prevented MCI onset in 10-month-old AAV-AD rats. The present study argues that P-tau is required to induce the development of MCI. Consistent with our previous findings that soluble Aβ42 is also required for MCI onset, the data obtained in the AAV-AD rat model confirm that the transition from the asymptomatic to the prodromal stage may be caused by the combined presence of both soluble brain forms of Aβ42 and p-tau, suggesting that the development of MCI may be the consequence of their synergistic action.

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).

Inhibition of DYRK1A proteolysis modifies its kinase specificity and rescues Alzheimer phenotype in APP/PS1 mice

Recent evidences suggest the involvement of DYRK1A (dual specificity tyrosine phosphorylation-regulated kinase 1 A) in Alzheimer’s disease (AD). Here we showed that DYRK1A undergoes a proteolytic processing in AD patients hippocampus without consequences on its kinase activity. Resulting truncated forms accumulate in astrocytes and exhibit increased affinity towards STAT3ɑ, a regulator of inflammatory process. These findings were confirmed in APP/PS1 mice, an amyloid model of AD, suggesting that this DYRK1A cleavage is a consequence of the amyloid pathology. We identified in vitro the Leucettine L41 as a compound able to prevent DYRK1A proteolysis in both human and mouse protein extracts. We then showed that intraperitoneal injections of L41 in aged APP/PS1 mice inhibit STAT3ɑ phosphorylation and reduce pro-inflammatory cytokines levels (IL1- β, TNF-ɑ and IL-12) associated to an increased microglial recruitment around amyloid plaques and decreased amyloid-β plaque burden. Importantly, L41 treatment improved synaptic plasticity and rescued memory functions in APP/PS1 mice. Collectively, our results suggest that DYRK1A may contribute to AD pathology through its proteolytic process, reducing its kinase specificity. Further evaluation of inhibitors of DYRK1A truncation promises a new therapeutic approach for AD.

Correction of cognitive deficits in mouse models of Down syndrome by a pharmacological inhibitor of DYRK1A.

ABSTRACTGrowing evidence supports the implication of DYRK1A in the development of cognitive deficits seen in Down syndrome (DS) and Alzheimer's disease (AD). We here demonstrate that pharmacological inhibition of brain DYRK1A is able to correct recognition memory deficits in three DS mouse models with increasing genetic complexity [Tg(Dyrk1a), Ts65Dn, Dp1Yey], all expressing an extra copy of Dyrk1a. Overexpressed DYRK1A accumulates in the cytoplasm and at the synapse. Treatment of the three DS models with the pharmacological DYRK1A inhibitor leucettine L41 leads to normalization of DYRK1A activity and corrects the novel object cognitive impairment observed in these models. Brain functional magnetic resonance imaging reveals that this cognitive improvement is paralleled by functional connectivity remodelling of core brain areas involved in learning/memory processes. The impact of Dyrk1a trisomy and L41 treatment on brain phosphoproteins was investigated by a quantitative phosphoproteomics method, revealing the implication of synaptic (synapsin 1) and cytoskeletal components involved in synaptic response and axonal organization. These results encourage the development of DYRK1A inhibitors as drug candidates to treat cognitive deficits associated with DS and AD.

Leucettine L41, a DYRK1A-preferential DYRKs/CLKs inhibitor, prevents memory impairments and neurotoxicity induced by oligomeric Aβ25–35 peptide administration in mice

Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) and cdc2-like kinases (CLKs) are implicated in the onset and progression of Down syndrome (DS) and Alzheimer’s disease (AD). DYRK1A has emerged as a possible link between amyloid-β (Aβ) and Tau, the major pathological proteins in AD. We here assessed the neuroprotective potential of a novel inhibitor of DYRKs/CLKs. The Leucettine L41, acting preferentially on DYRK1A, was tested in Aβ25–35-treated mice, a nontransgenic model of AD-like toxicity. We co-injected intracerebroventricularly oligomeric Aβ25–35 peptide and L41 in Swiss male mice. After 7 days, they were submitted to behavioral tests addressing spatial and non-spatial, short- and long-term memories. The oxidative stress, apoptotic markers, kinases involved in Tau phosphorylation, and synaptic integrity were analyzed by Western blot and ELISA in the hippocampus. L41, tested at 0.4, 1.2, 4µg, prevented the Aβ25–35-induced memory deficits in the Y-maze, passive avoidance and water-maze tests, with the most active dose being 4µg. The inhibitor prevented the Aβ25–35-induced oxidative stress, as revealed by measures of lipid peroxidation levels and reactive oxygen species accumulation, and abolished Aβ25–35-induced expression of pro-apoptotic markers. L41 prevented the Aβ25–35-induced decrease of AKT activation and increase of glycogen synthase kinase-3β (GSK-3β) activation, resulting in a decrease of Tau phosphorylation. Finally, L41 restored Aβ25–35-reduced levels of synaptic markers. The novel DYRK1A-preferential inhibitor L41 therefore prevented Aβ25–35-induced memory impairments and neurotoxicity in the mouse hippocampus. These in vivo data highlighted particularly DYRK1A as a major kinase involved in Aβ pathology and suggested therapeutic developments for DYRK1A inhibitors in AD.

Design and synthesis of thiazolo[5,4-f]quinazolines as DYRK1A inhibitors, part II.

The convenient synthesis of a focused library (forty molecules) of novel 6,6,5-tricyclic thiazolo[5,4-f]quinazolines was realized mainly under microwave irradiation. A novel 6-aminobenzo[d]thiazole-2,7-dicarbonitrile (1) was used as a versatile molecular platform for the synthesis of various derivatives. Kinase inhibition, of the obtained final compounds, was evaluated on a panel of two kinases (DYRK1A/1B) together with some known reference DYRK1A and DYRK1B inhibitors (harmine, TG003, NCGC-00189310 and leucettine L41). Compound IC50 values were obtained and compared. Five of the novel thiazolo[5,4-f]quinazoline derivatives prepared, EHT 5372 (8c), EHT 6840 (8h), EHT 1610 (8i), EHT 9851 (8k) and EHT 3356 (9b) displayed single-digit nanomolar or subnanomolar IC50 values and are among the most potent DYRK1A/1B inhibitors disclosed to date. DYRK1A/1B kinases are known to be involved in the regulation of various molecular pathways associated with oncology, neurodegenerative diseases (such as Alzeimer disease, AD, or other tauopathies), genetic diseases (such as Down Syndrome, DS), as well as diseases involved in abnormal pre-mRNA splicing. The compounds described in this communication constitute a highly potent set of novel molecular probes to evaluate the biology/pharmacology of DYR1A/1B in such diseases.

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.

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.

Selectivity, Cocrystal Structures, and Neuroprotective Properties of Leucettines, a Family of Protein Kinase Inhibitors Derived from the Marine Sponge Alkaloid Leucettamine B

DYRKs (dual specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases) are implicated in the onset and development of Alzheimer's disease and Down syndrome. The marine sponge alkaloid leucettamine B was recently identified as an inhibitor of DYRKs/CLKs. Synthesis of analogues (leucettines) led to an optimized product, leucettine L41. Leucettines were cocrystallized with DYRK1A, DYRK2, CLK3, PIM1, and GSK-3β. The selectivity of L41 was studied by activity and interaction assays of recombinant kinases and affinity chromatography and competition affinity assays. These approaches revealed unexpected potential secondary targets such as CK2, SLK, and the lipid kinase PIKfyve/Vac14/Fig4. L41 displayed neuroprotective effects on glutamate-induced HT22 cell death. L41 also reduced amyloid precursor protein-induced cell death in cultured rat brain slices. The unusual multitarget selectivity of leucettines may account for their neuroprotective effects. This family of kinase inhibitors deserves further optimization as potential therapeutics against neurodegenerative diseases such as Alzheimer's disease.

Leucettines, a family of pharmacological inhibitors of DYRKs & CLKs kinases derived from the marine sponge Leucettamine B

We here report on leucettines, a family of kinase inhibitors derived from the marine sponge leucettamine B. Stepwise synthesis of analogues, followed by activity testing on 8 purified kinases led to highly potent inhibitors of CLKs & DYRKs, two families of kinases involved in pre-mRNA splicing and Alzheimer's disease. Leucettine L41 was co-crystallized with DYRK1A, -2, CLK3 and PIM1. Leucettine L41 inhibits phosphorylation of pre-RNA splicing regulating Ser/Arg-rich proteins. Leucettine L41 modulates alternative pre-mRNA splicing in a cellular systems. The selectivity of Leucettine L41 was extensively characterized. Leucettine L41 provides protection against glutamate-induced cell death in cultured HT22 hippocampal cells. It also provides neuroprotection against APP-induced cell death in mouse brain slices. Finally it prevents in vivo cognitive impairments due to icv injection of amyloid-β 25–35. Leucettines should be further explored as pharmacological tools to study and modulate pre-RNA splicing. Leucettines should also be investigated as potential therapeutic drugs in Alzheimer's disease and in diseases involving abnormal pre-mRNA splicing.

P3‐352: Leucettines, a family of pharmacological inhibitors of DYRKs and CLKs derived from the marine sponge: Natural product Leucettamine B

We here report on the synthesis, optimization and biological characterization of leucettines, a family of kinase inhibitors derived from the marine sponge leucettamine B. Stepwise synthesis of analogues starting from the natural structure, guided by activity testing on 8 purified kinases, led to highly potent inhibitors of CLKs and DYRKs, two families of kinases involved in alternative premRNA splicing and Alzheimer’s disease / Down syndrome. Leucettine L41 was co-crystallized with DYRK1A, DYRK2, CLK3 and PIM1. It interacts with key residues located within the ATPbinding pocket of the kinase. Leucettine L41 inhibits the phosphorylation of serine/arginine-rich proteins (SRp), a family of proteins regulating pre-RNA splicing. Indeed leucettine L41 was demonstrated to modulate alternative pre-mRNA splicing in a cell based reporting system. The selectivity of Leucettine L41 was extensively studied (kinase activity and interaction assays, affinity chromatography). Leucettine L41 provides protection against glutamate -induced cell death in cultured mouse HT22 hippocampal cells. It also provides neuroprotection against APP-induced cell death in mouse brain slices. Finally it prevents in vivo cognitive impairments due to intracerebroventricular injection of amyloid-# 25-35. Leucettines should be further explored as pharmacological tools to study and modulate preRNA splicing. Leucettines should also be investigated as potential therapeutic drugs in Alzheimer’s disease and in diseases involving abnormal pre-mRNA splicing.

Leucettines, a Class of Potent Inhibitors of cdc2-Like Kinases and Dual Specificity, Tyrosine Phosphorylation Regulated Kinases Derived from the Marine Sponge Leucettamine B: Modulation of Alternative Pre-RNA Splicing

We here report on the synthesis, optimization, and biological characterization of leucettines, a family of kinase inhibitors derived from the marine sponge leucettamine B. Stepwise synthesis of analogues starting from the natural structure, guided by activity testing on eight purified kinases, led to highly potent inhibitors of CLKs and DYRKs, two families of kinases involved in alternative pre-mRNA splicing and Alzheimer's disease/Down syndrome. Leucettine L41 was cocrystallized with CLK3. It interacts with key residues located within the ATP-binding pocket of the kinase. Leucettine L41 inhibits the phosphorylation of serine/arginine-rich proteins (SRp), a family of proteins regulating pre-RNA splicing. Indeed leucettine L41 was demonstrated to modulate alternative pre-mRNA splicing, in a cell-based reporting system. Leucettines should be further explored as pharmacological tools to study and modulate pre-RNA splicing. Leucettines may also be investigated as potential therapeutic drugs in Alzheimer's disease (AD) and in diseases involving abnormal pre-mRNA splicing.