Bifunctional carbazole derivatives for simultaneous therapy and fluorescence imaging in prion disease murine cell models.

Prion diseases are characterized by the self-assembly of pathogenic misfolded scrapie isoforms (PrPSc) of the cellular prion protein (PrPC). In an effort to achieve a theranostic profile, symmetrical bifunctional carbazole derivatives were designed as fluorescent rigid analogues of GN8, a pharmacological chaperone that stabilizes the native PrPC conformation and prevents its pathogenic conversion. A focused library was synthesized via a four-step route, and a representative member was confirmed to have native fluorescence, including a band in the near-infrared region. After a cytotoxicity study, compounds were tested on the RML-infected ScGT1 neuronal cell line, by monitoring the levels of protease-resistant PrPSc. Small dialkylamino groups at the ends of the molecule were found to be optimal in terms of therapeutic index, and the bis-(dimethylaminoacetamido)carbazole derivative 2b was selected for further characterization. It showed activity in two cell lines infected with the mouse-adapted RML strain (ScGT1 and ScN2a). Unlike GN8, 2b did not affect PrPC levels, which represents a potential advantage in terms of toxicity. Amyloid Seeding Assay (ASA) experiments showed the capacity of 2b to delay the aggregation of recombinant mouse PrP. Its ability to interfere with the amplification of the scrapie RML strain by Protein Misfolding Cyclic Amplification (PMCA) was shown to be higher than that of GN8, although 2b did not inhibit the amplification of human vCJD prion. Fluorescent staining of PrPSc aggregates by 2b was confirmed in living cells. 2b emerges as an initial hit compound for further medicinal chemistry optimization towards strain-independent anti-prion compounds.

Improved Controlled Release and Brain Penetration of the Small Molecule S14 Using PLGA Nanoparticles.

Phosphodiesterase 7 (PDE7) is an enzyme responsible for the degradation of cyclic adenosine monophosphate (cAMP), an important cellular messenger. PDE7's role in neurotransmission, expression profile in the brain and the druggability of other phosphodiesterases have motivated the search for potent inhibitors to treat neurodegenerative and inflammatory diseases. Different heterocyclic compounds have been described over the years; among them, phenyl-2-thioxo-(1H)-quinazolin-4-one, called S14, has shown very promising results in different in vitro and in vivo studies. Recently, polymeric nanoparticles have been used as new formulations to target specific organs and produce controlled release of certain drugs. In this work, we describe poly(lactic-co-glycolic acid) (PLGA)-based polymeric nanoparticles loaded with S14. Their preparation, optimization, characterization and in vivo drug release profile are here presented as an effort to improve pharmacokinetic properties of this interesting PDE7 inhibitor.

Developing novel classes of protein kinase CK1δ inhibitors by fusing [1,2,4]triazole with different bicyclic heteroaromatic systems.

Protein kinase CK1δ expression and activity is involved in different pathological situations that include neuroinflammatory and neurodegenerative diseases. For this reason, protein kinase CK1δ has become a possible therapeutic target for these conditions. 5,6-fused bicyclic heteroaromatic systems that resemble adenine of ATP represent optimal scaffolds for the development of a new class of ATP competitive CK1δ inhibitors. In particular, a new series of [1,2,4]triazolo[1,5-c]pyrimidines and [1,2,4]triazolo[1,5-a][1,3,5]triazines was developed. Some crucial interactors have been identified, such as the presence of a free amino group able to interact with the residues of the hinge region at the 5- and 7- positions of the [1,2,4]triazolo[1,5-c]pyrimidine and [1,2,4]triazolo[1,5-a][1,3,5]triazine scaffolds, respectively; or the presence of a 3-hydroxyphenyl or 3,5-dihydroxyphenyl moiety at the 2- position of both nuclei. Molecular modeling studies identified the key interactions involved in the inhibitor-protein recognition process that appropriately fit with the outlined structure-activity relationship. Considering the fact that the CK1 protein kinase is involved in various pathologies in particular of the central nervous system, the interest in the development of new inhibitors permeable to the blood-brain barrier represents today an important goal in the pharmaceutical field. The best potent compound of the series is the 5-(7-amino-5-(benzylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)benzen-1,3-diol (compound 51, IC50 = 0.18 µM) that was predicted to have an intermediate ability to cross the membrane in our in vitro assay and represents an optimal starting point to both studies the therapeutic value of protein kinase CK1δ inhibition and to develop new more potent derivatives.

Increasing Brain Permeability of PHA-767491, a Cell Division Cycle 7 Kinase Inhibitor, with Biodegradable Polymeric Nanoparticles.

A potent cell division cycle 7 (CDC7) kinase inhibitor, known as PHA-767491, has been described to reduce the transactive response DNA binding protein of 43 KDa (TDP-43) phosphorylation in vitro and in vivo, which is one of the main proteins found to aggregate and accumulate in the cytoplasm of motoneurons in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. However, the main drawback of this compound is its low permeability to the central nervous system (CNS), limiting its use for the treatment of neurological conditions. In this context, the use of drug delivery systems like nanocarriers has become an interesting approach to improve drug release to the CNS. In this study, we prepared and characterized biodegradable nanoparticles in order to encapsulate PHA-767491 and improve its permeability to the CNS. Our results demonstrate that poly (lactic-co-glycolic acid) (PLGA) nanoparticles with an average radius between 145 and 155 nm could be used to entrap PHA-767491 and enhance the permeability of this compound through the blood-brain barrier (BBB), becoming a promising candidate for the treatment of TDP-43 proteinopathies such as ALS.

Targeting nuclear protein TDP-43 by cell division cycle kinase 7 inhibitors: A new therapeutic approach for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no known cure. Aggregates of the nuclear protein TDP-43 have been recognized as a hallmark of proteinopathy in both familial and sporadic cases of ALS. Post-translational modifications of this protein, include hyperphosphorylation, cause disruption of TDP-43 homeostasis and as a consequence, promotion of its neurotoxicity. Among the kinases involved in these changes, cell division cycle kinase 7 (CDC7) plays an important role by directly phosphorylating TDP-43. In the present manuscript the discovery, synthesis, and optimization of a new family of selective and ATP-competitive CDC7 inhibitors based on 6-mercaptopurine scaffold are described. Moreover, we demonstrate the ability of these inhibitors to reduce TDP-43 phosphorylation in both cell cultures and transgenic animal models such as C. elegans and Prp-hTDP43 (A315T) mice. Altogether, the compounds described here may be useful as versatile tools to explore the role of CDC7 in TDP-43 phosphorylation and also as new drug candidates for the future development of ALS therapies.

In Vitro and In Silico Acetylcholinesterase Inhibitory Activity of Thalictricavine and Canadine and Their Predicted Penetration across the Blood-Brain Barrier.

In recent studies, several alkaloids acting as cholinesterase inhibitors were isolated from Corydalis cava (Papaveraceae). Inhibitory activities of (+)-thalictricavine (1) and (+)-canadine (2) on human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) were evaluated with the Ellman's spectrophotometric method. Molecular modeling was used to inspect the binding mode of compounds into the active site pocket of hAChE. The possible permeability of 1 and 2 through the blood⁻brain barrier (BBB) was predicted by the parallel artificial permeation assay (PAMPA) and logBB calculation. In vitro, 1 and 2 were found to be selective hAChE inhibitors with IC50 values of 0.38 ± 0.05 µM and 0.70 ± 0.07 µM, respectively, but against hBChE were considered inactive (IC50 values > 100 µM). Furthermore, both alkaloids demonstrated a competitive-type pattern of hAChE inhibition and bind, most probably, in the same AChE sub-site as its substrate. In silico docking experiments allowed us to confirm their binding poses into the active center of hAChE. Based on the PAMPA and logBB calculation, 2 is potentially centrally active, but for 1 BBB crossing is limited. In conclusion, 1 and 2 appear as potential lead compounds for the treatment of Alzheimer's disease.

Isoquinoline Alkaloids from Berberis vulgaris as Potential Lead Compounds for the Treatment of Alzheimer's Disease.

Three new alkaloids, bersavine (3), muraricine (4), and berbostrejdine (8), together with seven known isoquinoline alkaloids (1-2, 5-7, 9, and 10) were isolated from an alkaloidal extract of the root bark of Berberis vulgaris. The structures of the isolated compounds were determined by spectroscopic methods, including 1D and 2D NMR techniques, HRMS, and optical rotation, and by comparison of the obtained data with those in the literature. The NMR data of berbamine (5), aromoline (6), and obamegine (7) were completely assigned employing 2D NMR experiments. Alkaloids isolated in sufficient amounts were evaluated for their in vitro acetylcholinesterase, butyrylcholinesterase (BuChE), prolyl oligopeptidase, and glycogen synthase kinase-3ß inhibitory activities. Selected compounds were studied for their ability to permeate through the blood-brain barrier. Significant human BuChE ( hBuChE) inhibitory activity was demonstrated by 6 (IC50 = 0.82 ± 0.10 µM). The in vitro data were further supported by computational analysis that showed the accommodation of 6 in the active site of hBuChE.

Computer-aided molecular design of pyrazolotriazines targeting glycogen synthase kinase 3.

Numerous studies have highlighted the implications of the glycogen synthase kinase 3 (GSK-3) in several processes associated with Alzheimer's disease (AD). Therefore, GSK-3 has become a crucial therapeutic target for the treatment of this neurodegenerative disorder. Hereby, we report the design and multistep synthesis of ethyl 4-oxo-pyrazolo[4,3-d][1-3]triazine-7-carboxylates and their biological evaluation as GSK-3 inhibitors. Molecular modelling studies allow us to develop this new scaffold optimising the chemical structure. Potential binding mode determination in the enzyme and the analysis of the key features in the catalytic site are also described. Furthermore, the ability of pyrazolotriazinones to cross the blood-brain barrier (BBB) was evaluated by passive diffusion and those who showed great GSK-3 inhibition and permeation to the central nervous system (CNS) showed neuroprotective properties against tau hyperphosphorylation in a cell-based model. These new brain permeable pyrazolotriazinones may be used for key in vivo studies and may be considered as new leads for further optimisation for the treatment of AD.

A Triazolotriazine-Based Dual GSK-3ß/CK-1δ Ligand as a Potential Neuroprotective Agent Presenting Two Different Mechanisms of Enzymatic Inhibition.

Glycogen synthase kinase 3ß (GSK-3ß) and casein kinase 1δ (CK-1δ) are emerging targets for the treatment of neuroinflammatory disorders, including Parkinson's disease. An inhibitor able to target these two kinases was developed by docking-based design. Compound 12, 3-(7-amino-5-(cyclohexylamino)-[1,2,4]triazolo[1,5-a][1,3,5]triazin-2-yl)-2-cyanoacrylamide, showed combined inhibitory activity against GSK-3ß and CK-1δ [IC50 (GSK-3ß)=0.17 µm; IC50 (CK-1δ)=0.68 µm]. In particular, classical ATP competition was observed against CK-1δ, and a co-crystal of compound 12 inside GSK-3ß confirmed a covalent interaction between the cyanoacrylamide warhead and Cys199, which could help in the development of more potent covalent inhibitors of GSK-3ß. Preliminary studies on in vitro models of Parkinson's disease revealed that compound 12 is not cytotoxic and shows neuroprotective activity. These results encourage further investigations to validate GSK-3ß/CK-1δ inhibition as a possible new strategy to treat neuroinflammatory/degenerative diseases.

1-(Benzo[d]thiazol-2-yl)-3-phenylureas as dual inhibitors of casein kinase 1 and ABAD enzymes for treatment of neurodegenerative disorders.

Several neurodegenerative disorders including Alzheimer's disease (AD) have been connected with deregulation of casein kinase 1 (CK1) activity. Inhibition of CK1 therefore presents a potential therapeutic strategy against such pathologies. Recently, novel class of CK1-specific inhibitors with N-(benzo[d]thiazol-2-yl)-2-phenylacetamide structural scaffold has been discovered. 1-(benzo[d]thiazol-2-yl)-3-phenylureas, on the other hand, are known inhibitors amyloid-beta binding alcohol dehydrogenase (ABAD), an enzyme also involved in pathophysiology of AD. Based on their tight structural similarity, we decided to evaluate series of previously published benzothiazolylphenylureas, originally designed as ABAD inhibitors, for their inhibitory activity towards CK1. Several compounds were found to be submicromolar CK1 inhibitors. Moreover, two compounds were found to inhibit both, ABAD and CK1. Such dual-activity could be of advantage for AD treatment, as it would simultaneously target two distinct pathological processes involved in disease's progression. Based on PAMPA testing both compounds were suggested to permeate the blood-brain barrier, which makes them, together with their unique dual activity, interesting lead compounds for further development.

Chameleon-like behavior of indolylpiperidines in complex with cholinesterases targets: Potent butyrylcholinesterase inhibitors.

Alzheimer's disease (AD) is the most common form of dementia worldwide with an increasing prevalence for the next years. The multifactorial nature of AD precludes the design of new drugs directed to a single target being probably one of the reasons for recent failures. Therefore, dual binding site acetylcholinesterase (AChE) inhibitors have been revealed as cognitive enhancers and ß-amyloid modulators offering an alternative in AD therapy field. Based on the dual ligands NP61 and donepezil, the present study reports the synthesis of a series of indolylpiperidines hybrids to optimize the NP61 structure preserving the indole nucleus, but replacing the tacrine moiety of NP61 by benzyl piperidine core found in donepezil. Surprisingly, this new family of indolylpiperidines derivatives showed very potent and selective hBuChE inhibition. Further studies of NMR and molecular dynamics have showed the capacity of these hybrid molecules to change their bioactive conformation depending on the binding site, being capable to inhibit with different shapes BuChE and residually AChE.

Leucine rich repeat kinase 2 (LRRK2) inhibitors based on indolinone scaffold: Potential pro-neurogenic agents.

Leucine-rich repeat kinase 2 (LRRK2) is one of the most pursued targets for Parkinson's disease (PD) therapy. Moreover, it has recently described its role in regulating Wnt signaling and thus, it may be involved in adult neurogenesis. This new hypothesis could give rise to double disease-modifying agents firstly by the benefits of inhibiting LRRK2 and secondly by promoting adult neurogenesis. Herein we report, the design, synthesis, biological evaluation, SAR and potential binding mode of indoline-like LRRK2 inhibitors and their preliminary neurogenic effect in neural precursor cells isolated from adult mice ventricular-subventricular zone. These results open new therapeutic horizons for the use of LRRK2 inhibitors as neuroregenerative agents. Moreover, the indolinone derivatives here prepared, inhibitors of the kinase activity of LRRK2, may be considered as pharmacological probes to study the potential neuroregeneration of the damaged brain.

3-(Benzyloxy)-1-(5-[18F]fluoropentyl)-5-nitro-1H-indazole: a PET radiotracer to measure acetylcholinesterase in brain.

AIM: Noninvasive studies of the acetylcholinesterase (AChE) level in Alzheimer's disease (AD) patients can contribute to a better understanding of the disease and its therapeutic. We propose 3-(benzyloxy)-1-(5-[18F]fluoropentyl)-5-nitro-1H-indazole, [18F]-IND1, structurally related to the AChE-inhibitor CP126,998, as a new positron emission tomography-radiotracer. EXPERIMENTAL: Radiosynthesis, with 18F, stability, lipophilicity and protein binding of [18F]-IND1 were studied. In vivo behavior, in normal mice and on AD mice models, were also analyzed. RESULTS: [18F]-IND1 was obtained in good radiochemical yield, was stable for at least 2 h in different conditions, and had adequate lipophilicity for blood-brain barrier penetration. Biodistribution studies, in normal mice, showed that [18F]-IND1 was retained in the brain after 1 h. In vivo tacrine-blocking experiments indicated this uptake could be specifically due to AChE interaction. Studies in transgenic AD mice showed differential, compared with normal mice, binding in many brain regions. CONCLUSION: [18F]-IND1 can be used to detect AChE changes in AD patients.

Subtly Modulating Glycogen Synthase Kinase 3 ß: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases.

Glycogen synthase kinase 3 ß (GSK-3ß) is a central target in several unmet diseases. To increase the specificity of GSK-3ß inhibitors in chronic treatments, we developed small molecules allowing subtle modulation of GSK-3ß activity. Design synthesis, structure-activity relationships, and binding mode of quinoline-3-carbohydrazide derivatives as allosteric modulators of GSK-3ß are presented here. Furthermore, we show how allosteric binders may overcome the ß-catenin side effects associated with strong GSK-3ß inhibition. The therapeutic potential of some of these modulators has been tested in human samples from patients with congenital myotonic dystrophy type 1 (CDM1) and spinal muscular atrophy (SMA) patients. We found that compound 53 improves delayed myogenesis in CDM1 myoblasts, while compounds 1 and 53 have neuroprotective properties in SMA-derived cells. These findings suggest that the allosteric modulators of GSK-3ß may be used for future development of drugs for DM1, SMA, and other chronic diseases where GSK-3ß inhibition exhibits therapeutic effects.

Pharmacological tools based on imidazole scaffold proved the utility of PDE10A inhibitors for Parkinson's disease.

AIM: Since neuroinflammation is partially mediated by cAMP levels and PDE10A enzyme is able to regulate these levels being highly expressed in striatum, its inhibitors emerged as useful drugs to mitigate this inflammatory process and hence the neuronal death associated with Parkinson's disease (PD). Methodology & results: To study the utility of PDE10A as a pharmacological target for PD, in this work we propose the search and development of new PDE10A inhibitors that could be useful as pharmacological tools in models of the disease and presumably as potential drug candidates. By using different medicinal chemistry approaches we have discovered imidazole-like PDE10A inhibitors and showed their neuroprotective actions. CONCLUSION: Here, we demonstrate the neuroprotective effect of PDE10A inhibitors in cellular models of PD. [Formula: see text].

Drugs in clinical development for the treatment of amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a fatal motor neuron progressive disorder for which no treatment exists to date. However, there are other investigational drugs and therapies currently under clinical development may offer hope in the near future. Areas covered: We have reviewed all the ALS ongoing clinical trials (until November 2016) and collected in Clinicaltrials.gov or EudraCT. We have described them in a comprehensive way and have grouped them in the following sections: biomarkers, biological therapies, cell therapy, drug repurposing and new drugs. Expert opinion: Despite multiple obstacles that explain the absence of effective drugs for the treatment of ALS, joint efforts among patient's associations, public and private sectors have fueled innovative research in this field, resulting in several compounds that are in the late stages of clinical trials. Drug repositioning is also playing an important role, having achieved the approval of some orphan drug applications, in late phases of clinical development. Endaravone has been recently approved in Japan and is pending in USA.

Biological and Pharmacological Characterization of Benzothiazole-Based CK-1δ Inhibitors in Models of Parkinson's Disease.

Parkinson's disease (PD), an age-related neurodegenerative disorder that results from a progressive loss of dopaminergic neurons has an enormous economical and human cost. Unfortunately, only symptomatic treatment such as dopamine replacement therapy is available. Therefore, drugs with new mechanisms of action able to protect against neuronal cell death are an urgent need. We here report the in vivo efficacy on dopaminergic neuronal protection in a PD mouse model and the lack of toxicity in zebrafish and Ames test of benzothiazole-based casein kinase-1δ (CK-1δ) nanomolar inhibitors. On the basis of these results, we propose protein kinase CK-1δ inhibitors as the possible disease-modifying drugs for PD, benzothiazole 4 being a promising drug candidate for further development as a new therapy of this neurodegenerative disease.

Small GSK-3 Inhibitor Shows Efficacy in a Motor Neuron Disease Murine Model Modulating Autophagy.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron degenerative disease that has no effective treatment up to date. Drug discovery tasks have been hampered due to the lack of knowledge in its molecular etiology together with the limited animal models for research. Recently, a motor neuron disease animal model has been developed using ß-N-methylamino-L-alanine (L-BMAA), a neurotoxic amino acid related to the appearing of ALS. In the present work, the neuroprotective role of VP2.51, a small heterocyclic GSK-3 inhibitor, is analysed in this novel murine model together with the analysis of autophagy. VP2.51 daily administration for two weeks, starting the first day after L-BMAA treatment, leads to total recovery of neurological symptoms and prevents the activation of autophagic processes in rats. These results show that the L-BMAA murine model can be used to test the efficacy of new drugs. In addition, the results confirm the therapeutic potential of GSK-3 inhibitors, and specially VP2.51, for the disease-modifying future treatment of motor neuron disorders like ALS.

Development of Blood-Brain Barrier Permeable Nitrocatechol-Based Catechol O-Methyltransferase Inhibitors with Reduced Potential for Hepatotoxicity.

Recent efforts have been focused on the development of centrally active COMT inhibitors, which can be valuable assets for neurological disorders such as Parkinson's disease, due to the severe hepatotoxicity risk associated with tolcapone. New nitrocatechol COMT inhibitors based on naturally occurring caffeic acid and caffeic acid phenethyl ester were developed. All nitrocatechol derivatives displayed potent inhibition of peripheral and cerebral COMT within the nanomolar range. Druglike derivatives 13, 15, and 16 were predicted to cross the blood-brain barrier in vitro and were significantly less toxic than tolcapone and entacapone when incubated at 50 µM with rat primary hepatocytes. Moreover, their unique acidity and electrochemical properties decreased the chances of formation of reactive quinone-imines and, as such, the potential for hepatotoxicity. The binding mode of 16 confirmed that the major interactions with COMT were established via the nitrocatechol ring, allowing derivatization of the side chain for future lead optimization efforts.

Targeting TDP-43 phosphorylation by Casein Kinase-1δ inhibitors: a novel strategy for the treatment of frontotemporal dementia.

BACKGROUND: Mutations in the progranulin gene (GRN) are the most common cause of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). TDP-43 pathology is characterized by the hyperphosphorylation of the protein at Serine 409/410 residues. Casein kinase-1δ (CK-1δ) was reported to phosphorylate TDP-43 directly. Previous works from our laboratory described the presence of CDK6/pRb-dependent cell cycle alterations, and cytosolic accumulation of TDP-43 protein in lymphoblast from FTLD-TDP patients carriers of a loss-of function mutation in GRN gene (c.709-1G > A). In this work, we have investigated the effects of two brain penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27) designed and synthetized in our laboratory on cell proliferation, TDP-43 phosphorylation and subcellular localization, as well as their effects on the known nuclear TDP-43 function repressing the expression of CDK6. RESULTS: We report here that both CK-1δ inhibitors (IGS-2.7 and IGS-3.27) normalized the proliferative activity of PGRN-deficient lymphoblasts by preventing the phosphorylation of TDP-43 fragments, its nucleo-cytosol translocation and the overactivation of the CDK6/pRb cascade. Moreover, ours results show neuroprotective effects of CK-1δ inhibitors in a neuronal cell model of induced TDP-43 phosphorylation. CONCLUSIONS: Our results suggest that modulating CK-1δ activity could be considered a novel therapeutic approach for the treatment of FTLD-TDP and other TDP-43 proteinopathies.