Inhibitor Report for: DL0410

Deficiency of the cholinergic system is thought to play a vital role in cognitive impairment of dementia. DL0410 was discovered as a dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinestease (BuChE), with potent efficiency in in-vitro experiments, but its in vivo effect on the cholinergic model has not been evaluated, and its action mechanism has also not been illustrated. In the present study, the capability of DL0410 in ameliorating the amnesia induced by scopolamine was investigated, and its effect on the cholinergic system in the hippocampus and its binding mode in the active site of AChE was also explored. Mice were administrated DL0410 (3 mg/kg, 10 mg/kg, and 30 mg/kg), and mice treated with donepezil were used as a positive control. The Morris water maze, escape learning task, and passive avoidance task were used as behavioral tests. The test results indicated that DL0410 could significantly improve the learning and memory impairments induced by scopolamine, with 10 mg/kg performing best. Further, DL0410 inhibited the AChE activity and increased acetylcholine (ACh) levels in a dose-dependent manner, and interacted with the active site of AChE in a similar manner as donepezil. However, no difference in the activity of BuChE was found in this study. All of the evidence indicated that its AChE inhibition is an important mechanism in the anti-amnesia effect. In conclusion, DL0410 could be an effective therapeutic drug for the treatment of dementia, especially Alzheimer's disease.

Cholinesterase inhibitors are first-line therapy for Alzheimer's disease (AD). DL0410 is an AChE/BuChE dual inhibitor with a novel new structural scaffold. It has been demonstrated that DL0410 could improve memory deficits in both Abeta1-42-induced and scopolamine-induced amnesia in mice. In the present study, the therapeutic effect of DL0410 and its action mechanism were investigated in APP/PS1 transgenic mice. Six-month old APP/PS1 transgenic mice were orally administered with DL0410 (3, 10, 30mg/kg/day). After 60days, several behavioural tests, including the Morris water maze and step-down tests, were used to investigate the effects of DL0410 on mice behaviours. All the behavioural experimental results showed that DL0410 significantly ameliorated memory deficits. Meanwhile, DL0410 attenuated neural cell damage and reduced senile plaques significantly in the hippocampus of APP/PS1 transgenic mice. In addition, DL0410 significantly decreased Abeta plaques, while increasing the number of synapses and the thickness of PSD in the hippocampus. We also found DL0410 decreased the expression of APP, NMDAR1B and the phosphorylation level of NMDAR2B, and increased the phosphorylation level of CAMKII and the expression of PSD-95. In this study, the results of behavioural tests demonstrated for the first time that DL0410 could improve learning and memory dysfunction in APP/PS1 transgenic mice. The mechanism of its beneficial effects might be related to cholinesterase inhibition, Abeta plaques inhibition, improvement of synapse loss by regulating of expression of proteins related to synapses. As a result, DL0410 could be considered as a candidate drug for the therapy of AD.

Butyrylcholinesterase (BCHE, EC 3.1.1.8) is an important pharmacological target for Alzheimer's disease (AD) treatment. However, the currently available BCHE inhibitor screening assays are expensive, labor-intensive, and compound-dependent. It is necessary to develop robust in silico methods to predict the activities of BCHE inhibitors for the lead identification. In this investigation, support vector machine (SVM) models and naive Bayesian models were built to discriminate BCHE inhibitors (BCHEIs) from the noninhibitors. Each molecule was initially represented in 1870 structural descriptors (1235 from ADRIANA.Code, 334 from MOE, and 301 from Discovery studio). Correlation analysis and stepwise variable selection method were applied to figure out activity-related descriptors for prediction models. Additionally, structural fingerprint descriptors were added to improve the predictive ability of models, which were measured by cross-validation, a test set validation with 1001 compounds and an external test set validation with 317 diverse chemicals. The best two models gave Matthews correlation coefficient of 0.9551 and 0.9550 for the test set and 0.9132 and 0.9221 for the external test set. To demonstrate the practical applicability of the models in virtual screening, we screened an in-house data set with 3601 compounds, and 30 compounds were selected for further bioactivity assay. The assay results showed that 10 out of 30 compounds exerted significant BCHE inhibitory activities with IC50 values ranging from 0.32 to 22.22 muM, at which three new scaffolds as BCHE inhibitors were identified for the first time. To our best knowledge, this is the first report on BCHE inhibitors using machine learning approaches. The models generated from SVM and naive Bayesian approaches successfully predicted BCHE inhibitors. The study proved the feasibility of a new method for predicting bioactivities of ligands and discovering novel lead compounds.

DL0410, a dualaction cholinesterase inhibitor and histamine3 receptor antagonist with a novel structural scaffold, may be a potential candidate for the treatment of Alzheimer's disease (AD). To the best of the authors' knowledge, this is the first study to demonstrate a reliable method for the measurement of DL0410 in rat plasma, brain, bile, urine and feces samples, and identification of its primary metabolites. The pharmacokinetic properties of DL0410 were analyzed by liquid chromatographymass spectrometry at oral doses of 25, 50 and 100 mg/kg and intravenous dose of 5 mg/kg. The investigation of the excretion and metabolism of DL0410 was determined following liquidliquid extraction for biliary, urinary and fecal samples. Finally, the cytochrome (CY)P450 isoforms involved in the production of DL0410 metabolites with recombinant human cytochrome P450 enzymes were characterized. The results suggested that DL0410 was not well absorbed; however, was distributed to the entorhinal cortex and hippocampus of the brain. A total of two common metabolites of the reduction of DL0140 in the bile, urine and feces were identified and CYP2D6 was involved in this reaction. The pharmacokinetic results of DL0410 provided information for the illustration of its pharmacodynamic properties, mechanism of action and promoted its continued evaluation as a therapeutic agent for AD treatment.

Deficiency of the cholinergic system is thought to play a vital role in cognitive impairment of dementia. DL0410 was discovered as a dual inhibitor of acetylcholinesterase (AChE) and butyrylcholinestease (BuChE), with potent efficiency in in-vitro experiments, but its in vivo effect on the cholinergic model has not been evaluated, and its action mechanism has also not been illustrated. In the present study, the capability of DL0410 in ameliorating the amnesia induced by scopolamine was investigated, and its effect on the cholinergic system in the hippocampus and its binding mode in the active site of AChE was also explored. Mice were administrated DL0410 (3 mg/kg, 10 mg/kg, and 30 mg/kg), and mice treated with donepezil were used as a positive control. The Morris water maze, escape learning task, and passive avoidance task were used as behavioral tests. The test results indicated that DL0410 could significantly improve the learning and memory impairments induced by scopolamine, with 10 mg/kg performing best. Further, DL0410 inhibited the AChE activity and increased acetylcholine (ACh) levels in a dose-dependent manner, and interacted with the active site of AChE in a similar manner as donepezil. However, no difference in the activity of BuChE was found in this study. All of the evidence indicated that its AChE inhibition is an important mechanism in the anti-amnesia effect. In conclusion, DL0410 could be an effective therapeutic drug for the treatment of dementia, especially Alzheimer's disease.

D-galactose has been reported to accelerate senescence in rodents, accompanied by a decline in learning and memory. We used a model of D-galactose-induced amnesia for the efficacy evaluation and pharmacologic studies of active compounds against Alzheimer's disease (AD). DL0410 is a potent inhibitor against acetylcholinesterase (AChE) and, in the present study, the effect of DL0410 was evaluated in this model. We found that DL0410 could significantly improve the learning and memory of D-galactose induced aging mice in a series of behavioral tests: novel-object recognition test, nest-building test, Morris water maze test and step-through test. Pharmacologic studies were conducted from several aspects: the cholinergic system, mitochondrial respiration, oxidative stress, neuroinflammation, apoptosis and synaptic loss. The acetylcholine level and AChE activity were not altered by D-galactose but were slightly affected by DL0410 in the brain. DL0410 could significantly improve decreased mitochondrial respiration in the NADH chain and FADH2 chain, and protect mitochondrial ultrastructure. DL0410 reduced the accumulation of advanced glycation end products (AGEs) and malondialdehyde (MDA) and increase the total antioxidant capability of the brain via an increase in activity of catalase, glutathione peroxidase (GPx) and superoxide dismutase (SOD). RAGE expression was inhibited by DL0410, followed by the decreased activation of astrocytes and microglia. Subsequent phosphorylation of NF-kappaB was also reversed by DL0410, with lower expression of cyclooxygenase-2 (COX2) and iNOS. With respect to apoptosis, the activation of caspase 3 and cleavage of PARP were downregulated significantly by DL0410, after the inhibition of phosphorylation of JNK induced by inflammation and oxidative stress. Synaptic protection by DL0410 was also demonstrated. These data suggest that mitochondrial protection has a primary role in the ameliorating effect of DL0410 on the impaired learning and memory, oxidative stress, inflammation, apoptosis and synaptic loss induced by D-galactose. DL0410 is a promising candidate for the treatment of aging-related AD, and this study lays an important foundation for its further research and development.

In our study, we attempted to investigate the influences of P-glycoprotein (P-gp) on DL0410, a novel synthetic molecule for Alzheimer's disease (AD) treatment, for intestinal absorption and blood-brain barrier permeability in vitro and related binding mechanisms in silico. Caco-2, MDCK, and MDCK-MDR1 cells were utilized for transport studies, and homology modelling of human P-gp was built for further docking study to uncover the binding mode of DL0410. The results showed that the apparent permeability (Papp) value of DL0410 was approximately 1 x 10(-6) cm/s, indicating the low permeability of DL0410. With the presence of verapamil, the directional transport of DL0410 disappeared in Caco-2 and MDCK-MDR1 cells, suggesting that DL0410 should be a substrate of P-gp, which was also confirmed by P-gp ATPase assay. In addition, DL0410 could competitively inhibit the transport of Rho123, a P-gp known substrate. According to molecular docking, we also found that DL0410 could bind to the drug binding pocket (DBP), but not the nucleotide binding domain (NBD). In conclusion, DL0410 was a substrate as well as a competitive inhibitor of P-gp, and P-gp had a remarkable impact on the intestine and brain permeability of DL0410, which is of significance for drug research and development.

AIM: 1,1'-([1,1'-Biphenyl]-4,4'-diyl)bis(3-(piperidin-1-yl)propan-1-one)dihydrochlorid e (DL0410) is a novel synthetic dual acetylcholinesterase (AChE)/butyrocholinesterase (BuChE) inhibitor, which has shown a potential therapeutic effect on Alzheimer's disease (AD). In this study we examined whether DL0410 produced neuroprotective effects in an AD cellular model and an Abeta1-42-induced amnesia mouse model.
METHODS: The in vitro inhibitory activities against AChE and BuChE were estimated using Ellman's assay. Copper-induced toxicity in APPsw-SY5Y cells was used as AD cellular model, the cell viability was assessed using MTS assay, and cell apoptosis was evaluated based on mitochondrial membrane potential detection. Abeta1-42-induced amnesia mouse model was made in male mice by injecting aggregated Abeta1-42 (2 mug in 2 muL 0.1% DMSO) into the right cerebral ventricle. Before and after Abeta1-42 injection, the mice were orally administered DL0410 (1, 3, 9 mg.kg-1.d-1) or rivastigmine (2 mg.kg-1.d-1) for 3 and 11 d, respectively. Memory impairments were examined using Morris water maze (MWM) test and passive avoidance test. The expression levels of APP, CREB, BDNF, JNK and Akt in the mouse brains were measured with either immunohistochemistry or Western blotting.
RESULTS: DL0410 exhibited in vitro inhibitory abilities against AChE and BuChE with IC50 values of 0.286+/-0.004 and 3.962+/-0.099 mumol/L, respectively, which were comparable to those of donepezil and rivastigmine. In APPsw-SY5Y cells, pretreatment with DL0410 (1, 3, and 10 mumol/L) decreased the phosphorylation of JNK and increased the phosphorylation of Akt, markedly decreased copper-stimulated Abeta1-42 production, reversed the loss of mitochondrial membrane potential, and dose-dependently increased the cell viability. In Abeta1-42-treated mice, DL0410 administration significantly ameliorated learning and memory deficits in MWM test and passive avoidance test. Furthermore, DL0410 administration markedly decreased Abeta1-40/42 deposits in mouse cerebral cortices, and significantly up-regulated neurotrophic CREB/BDNF. Meanwhile, Akt/JNK signaling pathway may play a key role in the neuroprotective effect of DL0410. CONCLUSION: DL0410 ameliorates cognitive deficit and exerts neuronal protection in AD models, implicating this compound as a candidate drug for the prevention and therapy of AD.

To determine chemical-protein interactions (CPI) is costly, time-consuming, and labor-intensive. In silico prediction of CPI can facilitate the target identification and drug discovery. Although many in silico target prediction tools have been developed, few of them could predict active molecules against multitarget for a single disease. In this investigation, naive Bayesian (NB) and recursive partitioning (RP) algorithms were applied to construct classifiers for predicting the active molecules against 25 key targets toward Alzheimer's disease (AD) using the multitarget-quantitative structure-activity relationships (mt-QSAR) method. Each molecule was initially represented with two kinds of fingerprint descriptors (ECFP6 and MACCS). One hundred classifiers were constructed, and their performance was evaluated and verified with internally 5-fold cross-validation and external test set validation. The range of the area under the receiver operating characteristic curve (ROC) for the test sets was from 0.741 to 1.0, with an average of 0.965. In addition, the important fragments for multitarget against AD given by NB classifiers were also analyzed. Finally, the validated models were employed to systematically predict the potential targets for six approved anti-AD drugs and 19 known active compounds related to AD. The prediction results were confirmed by reported bioactivity data and our in vitro experimental validation, resulting in several multitarget-directed ligands (MTDLs) against AD, including seven acetylcholinesterase (AChE) inhibitors ranging from 0.442 to 72.26 muM and four histamine receptor 3 (H3R) antagonists ranging from 0.308 to 58.6 muM. To be exciting, the best MTDL DL0410 was identified as an dual cholinesterase inhibitor with IC50 values of 0.442 muM (AChE) and 3.57 muM (BCHE) as well as a H3R antagonist with an IC50 of 0.308 muM. This investigation is the first report using mt-QASR approach to predict chemical-protein interaction for a single disease and discovering highly potent MTDLs. This protocol may be useful for in silico multitarget prediction of other diseases.

Cholinesterase inhibitors are first-line therapy for Alzheimer's disease (AD). DL0410 is an AChE/BuChE dual inhibitor with a novel new structural scaffold. It has been demonstrated that DL0410 could improve memory deficits in both Abeta1-42-induced and scopolamine-induced amnesia in mice. In the present study, the therapeutic effect of DL0410 and its action mechanism were investigated in APP/PS1 transgenic mice. Six-month old APP/PS1 transgenic mice were orally administered with DL0410 (3, 10, 30mg/kg/day). After 60days, several behavioural tests, including the Morris water maze and step-down tests, were used to investigate the effects of DL0410 on mice behaviours. All the behavioural experimental results showed that DL0410 significantly ameliorated memory deficits. Meanwhile, DL0410 attenuated neural cell damage and reduced senile plaques significantly in the hippocampus of APP/PS1 transgenic mice. In addition, DL0410 significantly decreased Abeta plaques, while increasing the number of synapses and the thickness of PSD in the hippocampus. We also found DL0410 decreased the expression of APP, NMDAR1B and the phosphorylation level of NMDAR2B, and increased the phosphorylation level of CAMKII and the expression of PSD-95. In this study, the results of behavioural tests demonstrated for the first time that DL0410 could improve learning and memory dysfunction in APP/PS1 transgenic mice. The mechanism of its beneficial effects might be related to cholinesterase inhibition, Abeta plaques inhibition, improvement of synapse loss by regulating of expression of proteins related to synapses. As a result, DL0410 could be considered as a candidate drug for the therapy of AD.

Butyrylcholinesterase (BCHE, EC 3.1.1.8) is an important pharmacological target for Alzheimer's disease (AD) treatment. However, the currently available BCHE inhibitor screening assays are expensive, labor-intensive, and compound-dependent. It is necessary to develop robust in silico methods to predict the activities of BCHE inhibitors for the lead identification. In this investigation, support vector machine (SVM) models and naive Bayesian models were built to discriminate BCHE inhibitors (BCHEIs) from the noninhibitors. Each molecule was initially represented in 1870 structural descriptors (1235 from ADRIANA.Code, 334 from MOE, and 301 from Discovery studio). Correlation analysis and stepwise variable selection method were applied to figure out activity-related descriptors for prediction models. Additionally, structural fingerprint descriptors were added to improve the predictive ability of models, which were measured by cross-validation, a test set validation with 1001 compounds and an external test set validation with 317 diverse chemicals. The best two models gave Matthews correlation coefficient of 0.9551 and 0.9550 for the test set and 0.9132 and 0.9221 for the external test set. To demonstrate the practical applicability of the models in virtual screening, we screened an in-house data set with 3601 compounds, and 30 compounds were selected for further bioactivity assay. The assay results showed that 10 out of 30 compounds exerted significant BCHE inhibitory activities with IC50 values ranging from 0.32 to 22.22 muM, at which three new scaffolds as BCHE inhibitors were identified for the first time. To our best knowledge, this is the first report on BCHE inhibitors using machine learning approaches. The models generated from SVM and naive Bayesian approaches successfully predicted BCHE inhibitors. The study proved the feasibility of a new method for predicting bioactivities of ligands and discovering novel lead compounds.