Exploration of oxadiazole clubbed benzhydrylpiperazine pharmacophoric features as structural feature for antidepressant activity: In vitro, in vivo and in silico analysis.

Arylpiperazine clubbed various heterocyclic molecules present potential pharmacophoric structural features for the development of psychoactive drugs. There are various CNS active molecules possessing arylpiperazine moiety in their pharmacophore approved by USFDA. In the current study, we have explored the benzhydrylpiperazine moiety clubbed with various substituted oxadiazole moieties (AP1-12) for their monoamine oxidase (MAO) inhibition and antidepressant potential. Compounds AP3 and AP12 exhibited highly potent and selective MAO-A inhibition with IC50 values of 1.34 ± 0.93 µM and 1.13 ± 0.54 µM, respectively, and a selectivity index of 10- and 13-folds, respectively. Both the compounds displayed reversible binding character at the active site of MAO-A. In further in vivo evaluation, both the compounds AP3 and AP12 displayed potential antidepressant-like character in FST and TST studies via significantly reduced immobility time in comparison to non-treated animals. These compounds displayed no cytotoxicity in SH-SY5Y cell lines, which indicates that these compounds are safe for further evaluation. In silico studies reveal that synthesized compounds possess drug-likeness with minimal to no toxicity. In silico studies were conducted to understand the binding interactions and stability of compounds at the binding pocket of enzyme and observed that both the best compounds fit well at the active site of MAO-A lined by amino acid residues Tyr69, Asn181, Phe208, Ile335, Leu337, Phe352, and Tyr444 similar to standard MAO-A inhibitor clorgiline. The molecular dynamic studies demonstrated that AP3 and AP12 formed quite a stable complex at the active site of MAO-A and did not break under small abruption forces. The favourable binding interactions and appropriate ADMET properties present the benzhydrylpiperazine clubbed oxadiazole pharmacophoric features as a potential structural skeleton for further clinical evaluation and development of a new antidepressant drug molecule.

In situ Dephosphorylation Assay with Recombinant Nil Phosphatase.

The activity of numerous autophagy-related proteins depends on their phosphorylation status, which places importance on understanding the responsible kinases and phosphatases. Great progress has been made in identifying kinases regulating autophagy, but much less is known about the phosphatases counteracting their function. Genetic screens and modern proteomic approaches provide powerful tools to identify candidate phosphatases, but further experiments are required to assign direct roles for candidates. We have devised a novel protocol to test the role of purified phosphatases in dephosphorylating specific targets in situ . This approach has the potential to visualize context-specific differences in target dephosphorylation that are not easily detected by lysate-based approaches such as Western blots. Graphical abstract.

The growing concern of chlorpyrifos exposures on human and environmental health.

Chlorpyrifos (CP) and its highly electrophilic intermediates are principal toxic metabolites. The active form of CP i.e. chlorpyrifos oxon (CP-oxon) is responsible for both the insecticidal activity and is also of greater risk when present in the atmosphere. Thus, the combined effects of both CP, CP-oxan, and other metabolites enhance our understanding of the safety and risk of the insecticide CP. They cause major toxicities such as AChE inhibition, oxidative stress, and endocrine disruption. Further, it can have adverse hematological, musculoskeletal, renal, ocular, and dermal effects. Excessive use of this compound results in poisoning and potentially kills a non-target species upon exposure including humans. Several examples of reactive metabolites toxicities on plants, aquatic life, and soil are presented herein. The review covers the general overview on reactive metabolites of CP, chemistry and their mechanism through toxic effects on humans as well as on the environment. Considerable progress has been made in the replacement or alternative to CP. The different strategies including antidote mechanisms for the prevention and treatment of CP poisoning are discussed in this review. The approach analyses also the active metabolites for the pesticide activity and thus it becomes more important to know the pesticide and toxicity dose of CP as much as possible.

STING controls energy stress-induced autophagy and energy metabolism via STX17.

The stimulator of interferon genes (STING) plays a critical role in innate immunity. Emerging evidence suggests that STING is important for DNA or cGAMP-induced non-canonical autophagy, which is independent of a large part of canonical autophagy machineries. Here, we report that, in the absence of STING, energy stress-induced autophagy is upregulated rather than downregulated. Depletion of STING in Drosophila fat cells enhances basal- and starvation-induced autophagic flux. During acute exercise, STING knockout mice show increased autophagy flux, exercise endurance, and altered glucose metabolism. Mechanistically, these observations could be explained by the STING-STX17 interaction. STING physically interacts with STX17, a SNARE that is essential for autophagosome biogenesis and autophagosome-lysosome fusion. Energy crisis and TBK1-mediated phosphorylation both disrupt the STING-STX17 interaction, allow different pools of STX17 to translocate to phagophores and mature autophagosomes, and promote autophagic flux. Taken together, we demonstrate a heretofore unexpected function of STING in energy stress-induced autophagy through spatial regulation of autophagic SNARE STX17.

A phosphoswitch at acinus-serine437 controls autophagic responses to cadmium exposure and neurodegenerative stress.

Neuronal health depends on quality control functions of autophagy, but mechanisms regulating neuronal autophagy are poorly understood. Previously, we showed that in Drosophila starvation-independent quality control autophagy is regulated by acinus (acn) and the Cdk5-dependent phosphorylation of its serine437 (Nandi et al., 2017). Here, we identify the phosphatase that counterbalances this activity and provides for the dynamic nature of acinus-serine437 (acn-S437) phosphorylation. A genetic screen identified six phosphatases that genetically interacted with an acn gain-of-function model. Among these, loss of function of only one, the PPM-type phosphatase Nil (CG6036), enhanced pS437-acn levels. Cdk5-dependent phosphorylation of acn-S437 in nil1 animals elevates neuronal autophagy and reduces the accumulation of polyQ proteins in a Drosophila Huntington's disease model. Consistent with previous findings that Cd2+ inhibits PPM-type phosphatases, Cd2+ exposure elevated acn-S437 phosphorylation which was necessary for increased neuronal autophagy and protection against Cd2+-induced cytotoxicity. Together, our data establish the acn-S437 phosphoswitch as critical integrator of multiple stress signals regulating neuronal autophagy.

Yorkie Growth-Promoting Activity Is Limited by Atg1-Mediated Phosphorylation.

The expression of multiple growth-promoting genes is coordinated by the transcriptional co-activator Yorkie with its major regulatory input provided by the Hippo-Warts kinase cascade. Here, we identify Atg1/ULK1-mediated phosphorylation of Yorkie as an additional inhibitory input independent of the Hippo-Warts pathway. Two serine residues in Yorkie, S74 and S97, are Atg1/ULK1 consensus target sites and are phosphorylated by ULK1 in vitro, thereby preventing its binding to Scalloped. In vivo, gain of function of Atg1, or its activator Acinus, caused elevated Yorkie phosphorylation and inhibited Yorkie's growth-promoting activity. Loss of function of Atg1 or Acinus raised expression of Yorkie target genes and increased tissue size. Unlike Atg1's role in autophagy, Atg1-mediated phosphorylation of Yorkie does not require Atg13. Atg1 is activated by starvation and other cellular stressors and therefore can impose temporary stress-induced constraints on the growth-promoting gene networks under the control of Hippo-Yorkie signaling.

Cdk5-mediated Acn/Acinus phosphorylation regulates basal autophagy independently of metabolic stress.

In neurons, autophagy counteracts consequences of aging. It is therefore of interest how basal rates of macroautophagy/autophagy can be controlled independently of metabolic stress. We recently investigated the regulation of basal, starvation-independent autophagy by Acn/Acinus, a multifunctional nuclear protein with proposed roles in apoptosis, alternative RNA splicing, and basal autophagy. We found that Acn is stabilized by phosphorylation of the conserved serine 437. The phosphomimetic AcnS437D mutation causes no overt developmental phenotypes, but significantly elevates levels of basal autophagy and extends life spans. An RNAi screen identified Cdk5 as a kinase targeting S437, a role confirmed by gain- and loss-of-function mutants of Cdk5 or its obligatory cofactor Cdk5r1/p35. Flies lacking Cdk5 function display reduced basal autophagy and a shortened life span. Both of these phenotypes are suppressed by the phosphomimetic AcnS437D mutation, indicating that phosphorylating serine 437 of Acn, and thereby maintaining basal levels of autophagy, is critical for Cdk5's function in maintaining neuronal health.

Stress-induced Cdk5 activity enhances cytoprotective basal autophagy in Drosophila melanogaster by phosphorylating acinus at serine437.

Cdk5 is a post-mitotic kinase with complex roles in maintaining neuronal health. The various mechanisms by which Cdk5 inhibits and promotes neurodegeneration are still poorly understood. Here, we show that in Drosophila melanogaster Cdk5 regulates basal autophagy, a key mechanism suppressing neurodegeneration. In a targeted screen, Cdk5 genetically interacted with Acinus (Acn), a primarily nuclear protein, which promotes starvation-independent, basal autophagy. Loss of Cdk5, or its required cofactor p35, reduces S437-Acn phosphorylation, whereas Cdk5 gain-of-function increases pS437-Acn levels. The phospho-mimetic S437D mutation stabilizes Acn and promotes basal autophagy. In p35 mutants, basal autophagy and lifespan are reduced, but restored to near wild-type levels in the presence of stabilized AcnS437D. Expression of aggregation-prone polyQ-containing proteins or the Amyloid-ß42 peptide, but not alpha-Synuclein, enhances Cdk5-dependent phosphorylation of S437-Acn. Our data indicate that Cdk5 is required to maintain the protective role of basal autophagy in the initial responses to a subset of neurodegenerative challenges.

Activated Acinus boosts basal autophagy.

Acinus (Acn) is a nuclear protein that participates in the regulation of autophagy. Loss of Acn function prevents autophagy in starving cells. Conversely, Acn activation induces basal autophagy. This enhances the quality control functions of autophagy such as the removal of misfolded proteins, thereby reducing neurodegeneration and prolonging lifespan. Acn activity is enhanced by Akt1-mediated phosphorylation, which counteracts the cleavage of Acn by a caspase-3 homolog.

Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy.

How cellular stresses up-regulate autophagy is not fully understood. One potential regulator is the Drosophila melanogaster protein Acinus (Acn), which is necessary for autophagy induction and triggers excess autophagy when overexpressed. We show that cell type-specific regulation of Acn depends on proteolysis by the caspase Dcp-1. Basal Dcp-1 activity in developing photoreceptors is sufficient for this cleavage without a need for apoptosis to elevate caspase activity. On the other hand, Acn was stabilized by loss of Dcp-1 function or by the presence of a mutation in Acn that eliminates its conserved caspase cleavage site. Acn stability also was regulated by AKT1-mediated phosphorylation. Flies that expressed stabilized forms of Acn, either the phosphomimetic Acn(S641,731D) or the caspase-resistant Acn(D527A), exhibited enhanced basal autophagy. Physiologically, these flies showed improvements in processes known to be autophagy dependent, including increased starvation resistance, reduced Huntingtin-induced neurodegeneration, and prolonged life span. These data indicate that AKT1 and caspase-dependent regulation of Acn stability adjusts basal autophagy levels.

Mechanism of amphotericin B resistance in clinical isolates of Leishmania donovani.

The clinical value of amphotericin B, the mainstay therapy for visceral leishmaniasis in sodium antimony gluconate-nonresponsive zones of Bihar, India, is now threatened by the emergence of acquired drug resistance, and a comprehensive understanding of the underlying mechanisms is the need of the hour. We have selected an amphotericin B-resistant clinical isolate which demonstrated 8-fold-higher 50% lethal doses (LD(50)) than an amphotericin B-sensitive strain to explore the mechanism of amphotericin B resistance. Fluorimetric analysis demonstrated lower anisotropy in the motion of the diphenylhexatriene fluorescent probe in the resistant strain, which indicated a higher fluidity of the membrane for the resistant strain than for the sensitive strain. The expression patterns of the two transcripts of S-adenosyl-l-methionine:C-24-Δ-sterol methyltransferase and the absence of ergosterol, replaced by cholesta-5,7,24-trien-3ß-ol in the membrane of the resistant parasite, indicate a decreased amphotericin B affinity, which is evidenced by decreased amphotericin B uptake. The expression level of MDR1 is found to be higher in the resistant strain, suggesting a higher rate of efflux of amphotericin B. The resistant parasite also possesses an upregulated tryparedoxin cascade and a more-reduced intracellular thiol level, which helps in better scavenging of reactive oxygen species produced by amphotericin B. The resistance to amphotericin B was partially reverted by the thiol metabolic pathway and ABC transporter inhibitors. Thus, it can be concluded that altered membrane composition, ATP-binding cassette transporters, and an upregulated thiol metabolic pathway have a role in conferring amphotericin B resistance in clinical isolates of Leishmania donovani.

Anoikis potential of Entameba histolytica secretory cysteine proteases: evidence of contact independent host cell death.

Mammalian epithelial, endothelial and various other cell types, upon their detachment from the extracellular matrix (ECM) undergo a specialized kind of apoptosis, known as anoikis. Entameba histolytica cysteine proteases have been implicated in degradation of the host ECM, which may induce anoikis in host cells. To explore this hypothesis, supernatant obtained from 2 h in-vitro cultivation of E. histolytica (SRP), was used as a source of cysteine proteases. MDA-MB-231 (human mammary epithelial adenocarcinoma) cells were treated with SRP and their detachment and apoptosis was evaluated. 25 µg/ml (with respect to protein concentration), SRP was found to be the optimal concentration to dislodge over 98% MDA-MB-231 cells from monolayer in 20 min. The detachment was followed by apoptosis of at least 41.2% cells, characterized by caspase-3 dependent inter-nucleosomal DNA fragmentation. The SRP-induced apoptosis was associated exclusively with the detached fraction. Moreover, detachment preceded apoptosis. E-64 (a cysteine protease inhibitor) abolished the SRP-induced detachment as well as inter-nucleosomal DNA fragmentation. Interestingly, SRP induced a 3.21 fold increase in the JNK activity, whilst SP600125 (a JNK inhibitor) blocked the SRP-induced inter-nucleosomal DNA fragmentation. Thus, it was concluded that spontaneously released cysteine proteases of E. histolytica can induce JNK dependent anoikis of MDA-MB-231 cells, which may be implicated in contact independent host cell death during amebiasis.

Involvement of thermoplasmaquinone-7 in transplasma membrane electron transport of Entamoeba histolytica trophozoites: a key molecule for future rational chemotherapeutic drug designing.

The quinone composition of the transplasma membrane electron transport chain of parasitic protozoa Entamoeba histolytica was investigated. Purification of quinone from the plasma membrane of E. histolytica and its subsequent structural elucidation revealed the structure of the quinone as a methylmenaquinone-7 (thermoplasmaquinone-7), a napthoquinone. Membrane bound thermoplasmaquinone-7 can be destroyed by UV irradiation with a concomitant loss of plasma membrane electron transport activity. The abilities of different quinones to restore transplasma membrane electron transport activity in UV irradiated trophozoites were compared. The lost activity was recovered completely by the addition of thermoplasmaquinone-7, but ubiquinones are unable to restore the same. These findings clearly indicate that thermoplasmaquinone-7 acts as a lipid shuttle in the plasma membrane of the parasite to mediate electron transfer between cytosolic reductant and non permeable electron acceptors. This thermoplasmaquinone-7 differs from that of the mammalian host and can provide a novel target for future rational chemotherapeutic drug designing.

Hydrogen peroxide induces apoptosis-like death in Entamoeba histolytica trophozoites.

Programmed cell death (PCD) is an essential process in the growth and development of multicellular organisms. However, accumulating evidence indicates that unicellular eukaryotes can also undergo PCD with apoptosis-like features. This study demonstrates that after exposure to 0.8 mM H(2)O(2) for 9 h Entamoeba histolytica presents morphological and biochemical evidence of apoptosis-like death. Morphological characteristics of apoptosis-like death including DNA fragmentation, increased vacuolization, nuclear condensation and cell rounding were observed for H(2)O(2)-exposed trophozoites with preservation of membrane integrity. Biochemical alteration in ion fluxes is also a key feature in PCD, and H(2)O(2)-exposed trophozoites showed overproduction of reactive oxygen species, increased cytosolic Ca(2+) and decreased intracellular pH. Phosphatidylserine was also found to be expressed in the outer leaflet of the plasma membrane of the H(2)O(2)-treated trophozoites. Pretreatment with the cysteine protease inhibitor E-64d, the extracellular and intracellular Ca(2+) chelators EGTA and BAPTA/AM, and the Ca(2+) influx inhibitor verapamil prior to H(2)O(2) exposure abolished DNA fragmentation. The oxidatively stressed trophozoites also showed an increased calpain activity, indicating involvement of Ca(2+)-dependent calpain-like cysteine proteases in PCD of E. histolytica. A homogeneous caspase assay showed no significant caspase activity, and administration of caspase 1 inhibitor also did not prevent the death phenotype for the oxidatively stressed trophozoites, indicating a caspase-independent apoptosis-like death. Our observations clearly demonstrate that there is a distinct calpain-dependent but caspase-independent pathway for apoptosis-like death in oxidatively stressed E. histolytica trophozoites.

The 29-kilodalton thiol-dependent peroxidase of Entamoeba histolytica is a factor involved in pathogenesis and survival of the parasite during oxidative stress.

The 29-kDa surface antigen (thiol-dependent peroxidase; Eh29) of Entamoeba histolytica exhibits peroxidative and protective antioxidant activities. During tissue invasion, the trophozoites are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS). In this investigation, attempts have been made to understand the role of the 29-kDa peroxidase gene in parasite survival and pathogenesis. Inhibition of eh29 gene expression by antisense RNA technology has shown approximately 55% inhibition in eh29 expression, maximum ROS accumulation, and significantly lower viability in 29-kDa downregulated trophozoites during oxidative stress. The cytopathic and cytotoxic activities were also found to decrease effectively in the 29-kDa downregulated trophozoites. Size of liver abscesses was substantially lower in hamsters inoculated with 29-kDa downregulated trophozoites compared to the normal HM1:IMSS. These findings clearly suggest that the 29-kDa protein of E. histolytica has a role in both survival of trophozoites in the presence of ROS and pathogenesis of amoebiasis.

Preliminary evidence on existence of transplasma membrane electron transport in Entamoeba histolytica trophozoites: a key mechanism for maintaining optimal redox balance.

Entamoeba histolytica, an amitochondriate parasitic protist, was demonstrated to be capable of reducing the oxidized form of alpha-lipoic acid, a non permeable electron acceptor outside the plasma membrane. This transmembrane reduction of non permeable electron acceptors with redox potentials ranging from -290 mV to +360 mV takes place at neutral pH. The transmembrane reduction of non permeable electron acceptors was not inhibited by mitochondrial electron transport inhibitors such as antimycin A, rotenone, cyanide and azide. However, a clear inhibition with complex III inhibitor, 2-(n-heptyl)-4-hydroxyquinoline-N-oxide; modifiers of sulphydryl groups and inhibitors of glycolysis was revealed. The iron-sulphur centre inhibitor thenoyltrifluoroacetone failed to inhibit the reduction of non permeable electron acceptors whereas capsaicin, an inhibitor of energy coupling NADH oxidase, showed substantial inhibition. p-trifluromethoxychlorophenylhydrazone, a protonophore uncoupler, resulted in the stimulation of alpha-lipoic acid reduction but inhibition in oxygen uptake. Mitochondrial electron transport inhibitors substantially inhibited the oxygen uptake in E. histolytica. Transmembrane reduction of alpha-lipoic acid was strongly stimulated by anaerobiosis and anaerobic stimulation was inhibited by 2-(n-heptyl)-4-hydroxyquinoline-N-oxide. Transmembrane redox system of E. histolytica was also found to be sensitive to UV irradiation. All these findings clearly demonstrate the existence of transplasma membrane electron transport system in E. histolytica and possible involvment of a naphthoquinone coenzyme in transmembrane redox of E. histolytica which is different from that of mammalian host and therefore can provide a novel target for future rational chemotherapeutic drug designing.