Identification of a Chlorogenic Ester as a Monoamine Oxidase (MAO-B) Inhibitor by Integrating "Traditional and Machine Learning" Virtual Screening and In Vitro as well as In Vivo Validation: A Lead against Neurodegenerative Disorders?

Parkinson's disease (PD) is the furthermost motor disorder of adult-onset dementia connected to memory and other cognitive abilities. Monoamine oxidases (MAOs) have gained significant attention in recent years owing to their possible therapeutic use against PD. Expression of MAO-B has been found to be elevated in PD patients for increased uptake of dopamine, producing hydrogen peroxide and finally causing neuronal injury. In this work, two new compounds have been identified as leads against MAO-B, and one of those compounds has been validated in vitro and in vivo. From the Protein Data Bank, MAO-B protein structures complexed with selegiline, 6-hydroxy-N-propargyl-1(R)-aminoindan, or a chromen derivative have been selected as templates for shape-based virtual screening (SB-VS) against the Traditional Chinese Medicinal (TCM) natural database. In parallel, using machine learning, a molecular-descriptor-based support vector model (SVM) was prepared and screened. For this purpose, naïve Bayesian, logistic regression, and random forest strategies were employed with the best specific molecular descriptor, which yielded a model with an overall accuracy (Q) of 0.81. Two common hit compounds lead-1 and lead-2 resulting from both shape and SVM screenings were analyzed through molecular docking and molecular dynamics (MD) simulation (200 ns). Also, from trajectory analysis such as molecular mechanics generalized Born surface area (MMGB/SA) and the residual interaction network (RIN) analyzer, both leads were found to bind at the active site with a favorable correlated motion, including domain movements. Lead-2, which is a chlorogenic ester, was synthesized and found to have no cytotoxic effect up to 50 µg/mL on Neuro-2A cells. The significant reactive oxygen species (ROS) scavenging activity by lead-2 could be correlated to its neuroprotective efficacy. Its capacity to inhibit human MAO-B through a competitive mode could be observed. An experimental zebra fish model confirms the neuroprotection by lead-2 by assessing the locomotor activities under malathion influence and treatment of lead-2. Also, histopathology analysis revealed that lead-2 could slow down degeneration in the brain. The present study emphasizes that integrating machine learning in parallel with traditional virtual screening may be useful to identify effective lead compounds for a given target.

Fucoidan serves a neuroprotective effect in an Alzheimer's disease model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that causes memory and cognitive deficits. The present study was carried out to evaluate the protective effects of fucoidan in monocrotophos induced AD in Drosophila melanogaster. In silico studies showed that fucoidan exhibited binding energy of -9.3 kcal with proteins. Consistent with this, fucoidan, in a dose and time-dependent fashion, had inhibitory activity against cholinergic and monoamine-metabolized enzymes in vitro. Fucoidan inhibited the increase in total mRNA and protein in monocrotophos fed flies and prevented changes in biochemicals, neurochemicals and latency time of locomotor, learning and memory induced by monocrotophos. Together, the findings show that fucoidan serves a neuroprotective effect in Alzheimer's disease model in D. melanogaster.

Corrigendum to "Counter effects of Asiaticosids-D through neurotransmission pathway on rotenone induced cerebral ganglionic injury in Lumbricus terrestris" [IBRO Rep. 6C (2019) 160-175].

[This corrects the article DOI: 10.1016/j.ibror.2019.04.003.].

Counter effects of Asiaticosids-D through putative neurotransmission on rotenone induced cerebral ganglionic injury in Lumbricus terrestris.

Asiaticoside-D (AD) was shown to efficacy of ganglionic degenerated Lumbricus terrestris as a pioneering observation in our earlier research. Though, extract molecular mechanisms of AD for degenerative diseases (DDs) remains largely unknown. We investigated the neuroprotective effects of AD against ROT in cerebral ganglions (CGs) of degenerative L. terrestris. Worms were exposed to 0.4 ppm ROT for 7 days were subjected to co- treatment with 15 ppm of AD. After, CGs was removed. The levels oxidant, non-antioxidant, antioxidant status, ganglioside, ceramide and ceramide glycanase (CGase) were estimated. The m-RNA levels of dopamine transporter (DAT), octopamine transporter (OAT), innexins-9 (inx-9), ionotropic glutamate receptor 3 (iGlu3), heat shock proteins (hsp70), XPRLamide neuropeptide precursor, tyramine beta-hydroxylase (tbh-1) and ß- adrenergic receptor kinase-2 (ß-ARK2-3) by semi-qRT- PCR. The expression pattern of tyramine beta hydroxylase (TBH), glutamate receptor (iGluR), serotonin transporter (SERT), dopamine transporters (DAT), nerve growth factors (NGF), cytochrome C oxidase (COC), NADH dehydogenase subunit-1 (ND-1), neurotrophin receptor p75 (p75NTR), neuronal nitric oxiside synthase (nNOs) interleukin 1- beta (IL1-ß) and tumor necrosis factor alpha (TNF-α) by western blotting. Glutaminergic, serotogenic and dopaminergic toxicity variations were also performed. The levels of oxidant, non-antioxidant, antioxidant status, lipids, proteins and m-RNAs were significantly altered (p < 0.001) on ROT-induced (group II) and their levels were significantly changes (p < 0.05) by ROT+AD in CGs. The sensitive study plan concluded the neuroprotective effects of AD against ROT induced degeneration in worms and suggest that the AD deserves future studies for its use as an effective alternative medicine that could minimize the morbidity of ganglionic degenerative diseases patients.

Homology modeling identified for purported drug targets to the neuroprotective effects of levodopa and asiaticoside-D in degenerated cerebral ganglions of Lumbricus terrestris.

CONTEXT: Homology modeling plays role in determining the therapeutic targets dreadful for condition such as neurodegenerative diseases (NDD), which pose challenge in achieving the effective managements. The structures of the serotonin transporter (SERT), aquaporin (AQP), and tropomyosin receptor kinase (TrkA) which are implicated in NDD pathology are still unknown for Lumbricus terrestris, but the three-dimensional (3D) structure of the human counterpart for modeling. AIM: This study aims to generate and evaluate the 3D structure of TrkA, SERT, and AQP proteins and their interaction with the ligands, namely Asiaticoside-D (AD) and levodopa (L-DOPA) the anti-NDD agents. SUBJECTS AND METHODS: Homology modeling for SERT, AQP, and TrkA proteins of Lumbricus terrestris using SWISS-MODEL Server and the modeled structure was validated using Rampage Server. Wet-lab analysis of their correspondent m-RNA levels was also done to validate the in silico data. RESULTS: It was found that TrkA had moderately high homology (67%) to human while SERT and AQP could exhibit 58% and 42%, respectively. The reliability of the model was assessed by Ramachandran plot analysis. Interactions of AD with the SERT, AQP-4, and TrkA showed the binding energies as -9.93, 8.88, and -7.58 of Kcal/mol, respectively, while for L-DOPA did show -3.93, -5.13, and -6.0 Kcal/mol, respectively. The levels of SERT, TrkA, and AQP-4 were significantly reduced (P < 0.001) on ROT induced when compared to those of control worms. On ROT + AD supplementation group (III), m-RNA levels were significantly increased (P < 0.05) when compared to those of ROT induced worms (group II). CONCLUSION: Our pioneering docking data propose the possible of target which is proved useful for therapeutic investigations against the unconquered better of NDD.

The novel phytocomponent asiaticoside-D isolated from Centella asiatica exhibits monoamine oxidase-B inhibiting potential in the rotenone degenerated cerebral ganglions of Lumbricus terrestris.

BACKGROUND: Centella asiatica (CA) is one of the most valuable herbal medicines widely being used for the treatment of various neurological ailments that are challenging for health-care providers and also is deemed to be safe and effective. PURPOSE: Monoamines (MAs) are neurotransmitters and neuromodulators that play a significant role in the neural communication, regulation of motor and cognitive functions in the brain. Neurodegeneration is associated with elevated levels of MAO-B that can lead to damaging reactive oxygen species (ROS) in the brain. The current study, evaluated the effects of asiaticoside-D (AD) from neuroprotective CA, on the levels and activities of monoamine oxidase A and B (MAO-A and B), in addition to the behavioral analysis. METHODS: Qualitative and quantitative analyses of various solvent extracts of CA were performed. The extracts were screened for antioxidant potential using 1,1-diphenyl-2-picryl hydroxyl (DPPH), 2,2'-azinobis-3-ethylbenzothiozoline-6-sulfonic acid (ABTS), hydrogen peroxide (H2O2) radical, nitric oxide (NO) radical inhibition, lipid peroxidation (LPO) and ferric reducing antioxidant power (FRAP) assays. The purification of AD was done by column, thin layer and high-performance liquid chromatographies followed by structural elucidation using IR, HR-MS, 1H and 13C NMR spectra. Docking studies were performed to assess the impact of AD on MAO-A and B.In vivo, Lumbricus terrestris were exposed to 0.4 ppm rotenone (ROT) of medium for 7 days and were subjected to co-treatment along with 15 ppm of AD from CA. At the end of experiment period, the neuronal behavior of worms was assessed. Cerebral ganglions (CGs) were removed and the m-RNA levels of MAO-A and B were analyzed by Semi Q-PCR and their activities were also analyzed. RESULTS: The ethanolic extracts exhibited higher antiradical activity against DPPH, ABTS, H2O2, LPO, FRAP, NO and vitamin C with EC50 value of 20.2, 20.9, 20.4, 22.0, 24.9, 28.1, 25.5 and 22.0 µg/ml respectively. Structural analysis by IR, HR-MS, 1H and 13C NMR spectrum have shown the structure of the isolated compound as (2α, 3ß)-2,3-dihydroxyurs-12-en-28-oicacid-O-α-L-rhamnopyranosyl-(1→4)-O-ß-d-glucopyranosyl (1→6)-ß-copyranosyl ester and was represented as AD. In silico interaction of AD with MAO-A and B residues Lys312 at distances of 1.84 Šand 2.44 Šrespectively was found to exhibit high binding energy of -9.4 and 7.4 kcal. The neuronal behavior using L. terrestris showed significant improvement against (p < 0.001) ROT impaired behavior (group II) on AD supplementation (p < 0.05). Further, the m-RNA levels and activities of MAO-A and B which were significantly altered (p < 0.001) by ROT could be effectively maintained on AD supplementation. CONCLUSION: AD was found to exert its negative impact on the levels and activities of MAO-A and B in CGs of rotenone- induced changes in L. terrestris, the property which is considered to be crucial against ROT induced neurodegenerative pathology like -Parkinsonism.

Aberrant neurotransmissional mRNAs in cerebral ganglions of rotenone-exposed Lumbricus terrestris exhibiting motor dysfunction and altered cognitive behavior.

Rotenone (ROT) was shown to affect cerebral ganglions (CGs) of Lumbricus terrestris as a pioneering observation in our earlier investigation. Though ROT is a well-known neurotoxin causing neurodegeneration (ND), the precipitation of movement dysfunction remains largely unknown. We have designed the current study to analyze motor abnormalities in worms by exposing them to different concentrations (0.0-0.4 ppm) of ROT for 7 days. GABA, cholinergic receptor, serotonin transporter (SERT), acetylcholine esterase (AchE), and dopamine-ß-hydroxylase that are well known for their involvement in neuromuscular junctions were investigated by qRT-PCR. Further, neuronal mitochondrial genes (cytochrome C oxidase-2, NADH deydrogenase-1, cytochrome-b) and actin-1 that are essential for regeneration and calreticulin (phagocytosis) were investigated. The levels of neurotransmitters, lipids, ATPase, neuronal behavior analyses, and fluorescence analysis (lipid droplets) were performed in CGs which showed significant variations at 0.3 ppm. Ultrastructural changes in lipid droplet and neuromelatonin were prominent in 0.3 ppm. Dose-dependent effect of ROT on behavior alteration and expression of m-RNAs studied suggested that at 0.3 ppm, it could deteriorate motor and cognitive functions. We predict that perhaps, by virtue of its effect on cerebral ganglionic genes and their neurotransmitting potential, ROT may cause morbidities that resemble features characteristic of hemiparkinsonic degeneration.

Neurotransmissional, structural, and conduction velocity changes in cerebral ganglions of Lumbricus terrestris on exposure to acrylamide.

Acrylamide (ACR), an environmental toxin though being investigated for decades, remains an enigma with respect to its mechanism/site of actions. We aim to explicate the changes in cerebral ganglions and giant fibers along with the behavior of worms on ACR intoxication (3.5-17.5 mg/mL of medium/7 days). Neurotransmitter analysis revealed increased levels of excitatory glutamate and inhibitory gamma amino butyrate with reduced levels of dopamine, serotonin, melatonin, and epinephrine (p < 0.001). Scanning electron microscopy showed architectural changes in cerebral ganglions at 3.5 mg/mL/ACR. The learning behavior as evidenced by Pavlovian and maze tests was also altered well at 3.5 mg/mL of ACR. Electrophysiological assessment showed a reduction in conduction velocity of the medial and lateral giant nerve fibers. We speculate that the observed dose/time-dependent changes in neurotransmission, neurosecretion, and conduction velocity on ACR intoxication at 17.5 mg/ml, possibly, could be due to its effect on nerve fibers governing motor functions. The bioaccumulation factor in the range of 0.38-0.99 mg/g of ACR causes a detrimental impact on giant fibers affecting behavior of worm. The observations made using the simple invertebrate model implicate that the cerebral ganglionic variations in the worms may be useful to appreciate the pathology of the neurological diseases which involve motor neuron dysfunction, esp where the availability of brain samples from the victims are scarce.

Rotenone causing dysfunctional mitochondria and lysosomes in cerebral ganglions of Lumbricus terrestris degenerate giant fibers and neuromuscular junctions.

Rotenone is well-documented to cause neurodegenerative condition such as Parkinson's, in the exposed systems. However, its detrimental effect on particular sites of neuronal pathway is still under investigation. We aimed at elucidating the impact of rotenone on cerebral ganglions (CG) of Lumbricus terrestris which control movement and behaviour of the worms. Worms were exposed to 0-0.4 ppm/mL of rotenone. Mitochondrial and lysosomal integrities were found to be affected beyond 0.2 ppm/mL of rotenone. Activities of cholinergic enzymes and the expression of tyrosine hydroxylase showed an impaired neuronal transmission in CGs at the dose of 0.2 ppm/mL of rotenone. Histopathological and immunoflourescent analyses showed neuronal apoptosis, reduced nucleic acid content and inhibited of neurosecretion at 0.3 ppm/mL. Electron microscopy showed that the neurons and neuromuscular junctions were affected at 0.2 ppm/mL. Dose-dependent changes were also observed in the motor function such as burrowing behaviours and locomotion. Conduction velocity (CV) and locomotion assessment showed that the CV of lateral giant fiber (LGF) was reduced while that of MGF remains unaffected at 0.2 ppm, the dose at which the burrowing behaviour was also not affected. LGF, cholinergic enzymes and tyrosine hydroxylase are primarily targeted by rotenone affecting locomotion at 0.2 ppm/mL while MGF, neuropile and the burrowing behaviour were affected at 0.3 ppm/mL. We demonstrate, in addition to dose-dependent effects, that the bioaccumulation factors range 0.28-0.32 ppm/µg of rotenone cause degenerative impact on giant fibers affecting neuronal behaviors/locomotion of worms. We also propose worms for studying mechanisms of neuronal pathology caused by chemicals prevailing in earth's atmosphere.

Cerebral ganglionic variations and movement behaviors of Lumbricus terrestris on exposure to neurotoxin.

BACKGROUND: Invertebrate worms serve as models for understanding the features of neurological functions. Acrylamide (ACR), the well-known neurotoxin, is a water-soluble chemical widely used in various industrial and laboratory processes. ACR is also found in food items which are cooked under high temperature. PURPOSE: The study attempts to assess the neuropathological changes in cerebral ganglions along with the locomotion and neuronal behavior of Lumbricus terrestris on ACR intoxication. METHODS: The dosage of acrylamide induced neurotoxicity ranged from 0-17.5 mg/kg body weight for 7 days. The time/dose dependent changes in the oxidant and antioxidant status, activities of Na(+)/K(+)ATPase, Ca(2+)/Mg(2+) ATPase and 5' Nucleotidase were assessed along with the locomotor behavioral analysis. RESULT: The activities of super oxidase dismutase and catalases were not altered appreciably. However, the glutathione family, lipid peroxide, protein carbonyl content and vitamin C did show significant variations (p<0.001) in a dose-dependent manner, depicting more of oxidative stress, when compared to control worms. The activities of Na(+)/K(+) ATPase was significantly affected (p<0.001) at 3.5 mg/kg bw itself while those of both Ca(2+) and 5' Nucleotidase were found to be affected at 7.0 mg/kg bw of ACR. Mg(2+) ATPase showed significant reduction (p<0.001) in its activity only at 10.5 mg/kg bw of ACR. These dose dependent biochemical variations observed were found to be linked with the behavior of the worms as evident from the latency of movement in a dose-dependent manner which is less pronounced at 7.0 mg and more pronounced at 17.5 mg/kg bw of ACR. CONCLUSION: The study suggests that ACR disrupts GSSS/GSH balance and perturbs ionic homeostasis in worms and thus affect the motor function highlighting their (GSH-ions) interrelationship in influencing neuromuscular activity. These simple analyses implicate that the cerebral ganglionic variations in the worms may be useful to appreciate the pathology of the neurological diseases (provided sophisticated analyses are employed) especially which involve movement dysfunction, where the brain tissue samples from the affected human patients are scarce.