Singh SK et al. (2012) Int J Nanomedicine "Novel microfilaricidal activity of nanosilver."

Sharma RD, Dash D, Goswami K, Singh SK, Reddy MV

[Int J Nanomedicine, 2012]

PURPOSE: The currently available drug repertoire against lymphatic filariasis, a major health hazard in the developing world, is inadequate and is fraught with serious limitations. Thus, the development of an effective antifilarial strategy has become a global research thrust mandated by the World Health Organization. Nanoparticles of silver endowed with antibacterial potency are known to induce apoptosis in eukaryotic cells. The present study was designed to investigate the possible microfilaricidal efficacy of silver nanoparticles and to establish the validity of apoptotic rationale in antifilarial drug designing. METHODS: This report analyzed the effect of nanoparticles of silver as well as gold (size range: 10-15 nm) on the microfilariae of Brugia malayi obtained from the lavage of peritoneal cavities of infected jirds (Meriones unguiculatus). The study included a microfilarial motility assay, a trypan blue exclusion test, a poly(adenosine diphosphate-ribose) polymerase activity study, ethidium bromide/acridine orange differential staining, and transmission, as well as scanning electron microscopic evaluation of ultrastructural changes in microfilariae. RESULTS: The study demonstrates that nanoparticles of silver, but not of gold, elicited significant loss in microfilarial motility. Differential staining of parasites with ethidium bromide and acridine orange, poly(adenosine diphosphate-ribose) polymerase activity in microfilarial lysate, and electron microscopic findings underscored apoptotic death of parasites attributable to nanosilver. In a trypan blue exclusion test, the 50% lethal dose of nanosilver was measured to be 101.2 M, which was higher than the recorded complete inhibitory concentration value (50.6 M), thus supporting nanosilver as a potential drug candidate against lymphatic filariasis. CONCLUSION: The present report provides the first ever conclusive proof in support of apoptosis as a novel stratagem in antifilarial drug designing and nanoscale silver as a valid lead in research on antifilarial therapeutics. The main embargo about the current drug diethylcarbamazine citrate is its empirical use without rationale. Effective microfilaricidal activity of nanosilver at relatively low concentrations as reported in this study, with evidence of the induction of apoptosis in microfilariae, projects nanosilver as a potential drug adjuvant against lymphatic filariasis. The much higher 50% lethal dose value of nanosilver compared to the complete inhibitory concentration value reported in this study argues in favor of a safe therapeutic window of this agent in its antifilarial efficacy.

Dimitriadi M et al. (2013) J Neurosci "The neuroprotective drug riluzole acts via small conductance Ca2+-activated K+ channels to ameliorate defects in spinal muscular atrophy models."

Sahin M, Hart AC, Dimitriadi M, Kalloo G, Kye MJ, Yersak JM

[J Neurosci, 2013]

Spinal muscular atrophy (SMA), a recessive neuromuscular disorder, is caused by diminished function of the Survival Motor Neuron (SMN) protein. To define the cellular processes pertinent to SMA, parallel genetic screens were undertaken in Drosophila and Caenorhabditis elegans SMA models to identify modifiers of the SMN loss of function phenotypes. One class of such genetic modifiers was the small conductance, Ca(2+)-activated K(+) (SK) channels. SK channels allow efflux of potassium ions when intracellular calcium increases and can be activated by the neuroprotective drug riluzole. The latter is the only drug with proven, albeit modest, efficacy in the treatment of amyotrophic lateral sclerosis. It is unclear if riluzole can extend life span or ameliorate symptoms in SMA patients as previous studies were limited and of insufficient power to draw any conclusions. The critical biochemical target of riluzole in motor neuron disease is not known, but the pharmacological targets of riluzole include SK channels. We examine here the impact of riluzole in two different SMA models. In vertebrate neurons, riluzole treatment restored axon outgrowth caused by diminished SMN. Additionally, riluzole ameliorated the neuromuscular defects in a C. elegans SMA model and SK channel function was required for this beneficial effect. We propose that riluzole improves motor neuron function by acting on SK channels and suggest that SK channels may be important therapeutic targets for SMA patients.

Krabbendam IE et al. (2020) Cell Death Dis "SK channel-mediated metabolic escape to glycolysis inhibits ferroptosis and supports stress resistance in C. elegans."

Honrath B, Dolga AM, Branicky RS, Dilberger B, Evers B, Iannetti EF, Hekimi S, Meyer T, Bano D, Bakker BM, Krabbendam IE, Koopman WJH, Beyrath J, Dekker FJ, Eckert GP, Culmsee C, Schmidt M

[Cell Death Dis, 2020]

Metabolic flexibility is an essential characteristic of eukaryotic cells in order to adapt to physiological and environmental changes. Especially in mammalian cells, the metabolic switch from mitochondrial respiration to aerobic glycolysis provides flexibility to sustain cellular energy in pathophysiological conditions. For example, attenuation of mitochondrial respiration and/or metabolic shifts to glycolysis result in a metabolic rewiring that provide beneficial effects in neurodegenerative processes. Ferroptosis, a non-apoptotic form of cell death triggered by an impaired redox balance is gaining attention in the field of neurodegeneration. We showed recently that activation of small-conductance calcium-activated K⁺ (SK) channels modulated mitochondrial respiration and protected neuronal cells from oxidative death. Here, we investigated whether SK channel activation with CyPPA induces a glycolytic shift thereby increasing resilience of neuronal cells against ferroptosis, induced by erastin in vitro and in the nematode C. elegans exposed to mitochondrial poisons in vivo. High-resolution respirometry and extracellular flux analysis revealed that CyPPA, a positive modulator of SK channels, slightly reduced mitochondrial complex I activity, while increasing glycolysis and lactate production. Concomitantly, CyPPA rescued the neuronal cells from ferroptosis, while scavenging mitochondrial ROS and inhibiting glycolysis reduced its protection. Furthermore, SK channel activation increased survival of C. elegans challenged with mitochondrial toxins. Our findings shed light on metabolic mechanisms promoted through SK channel activation through mitohormesis, which enhances neuronal resilience against ferroptosis in vitro and promotes longevity in vivo.

Nam YW et al. (2018) Sci Rep "A V-to-F substitution in SK2 channels causes Ca2+ hypersensitivity and improves locomotion in a C. elegans ALS model."

Zhang M, Nam YW, Gazgalis D, Orfali R, Baskoylu SN, Cui M, Hart AC

[Sci Rep, 2018]

Small-conductance Ca²⁺-activated K⁺ (SK) channels mediate medium afterhyperpolarization in the neurons and play a key role in the regulation of neuronal excitability. SK channels are potential drug targets for ataxia and Amyotrophic Lateral Sclerosis (ALS). SK channels are activated exclusively by the Ca²⁺-bound calmodulin. Previously, we identified an intrinsically disordered fragment that is essential for the mechanical coupling between Ca²⁺/calmodulin binding and channel opening. Here, we report that substitution of a valine to phenylalanine (V407F) in the intrinsically disordered fragment caused a ~6 fold increase in the Ca²⁺ sensitivity of SK2-a channels. This substitution resulted in a novel interaction between the ectopic phenylalanine and M411, which stabilized PIP₂-interacting residue K405, and subsequently enhanced Ca²⁺ sensitivity. Also, equivalent valine to phenylalanine substitutions in SK1 or SK3 channels conferred Ca²⁺ hypersensitivity. An equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons, confirming that this phenylalanine substitution impacts channel function in vivo. This work for the first time provides a critical reagent for future studies: an SK channel that is hypersensitive to Ca²⁺ with increased activity in vivo.

Singh N et al. (2015) Biochem Biophys Res Commun "Identification of a novel stress regulated FERM domain containing cytosolic protein having PTP activity in Setaria cervi, a bovine filarial parasite."

Rathaur S, Heneberg P, Singh SK, Singh N

[Biochem Biophys Res Commun, 2015]

A 67 kDa cytosolic FERM domain containing protein having significant protein tyrosine phosphatases activity (PTPL) has been purified to homogeneity from Setaria cervi, a bovine filarial parasite. The MALDI-MS/MS analysis of the purified protein revealed 16 peptide peaks showing nearest match to Brugia malayi Moesin/ezrin/radixin homolog 1 protein and one peptide showing significant similarity with a region lying in the catalytic domain of human PTPD1. PTPL showed significant cross reactivity with the human PTP1B antibody and colocalize with actin in the coelomyrian cells of hypodermis in the parasite. PTPL was stress regulated as it showed marked decrease in the expression when exposed to Aspirin, an antifilarial drug and Phenylarsine Oxide, PTP inhibitor.

Huang H et al. (2017) Bio Protoc "Measuring Caenorhabditis elegans Sleep During the Transition to Adulthood Using a Microfluidics-based System."

Hart AC, Singh K, Huang H

[Bio Protoc, 2017]

C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski & Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During lethargus, animals cycle between periods of immobility (sleep bouts) and periods of active locomotion (motion bouts). Sleep bouts resemble sleep in other species based on behavioral criteria, including cessation of feeding and locomotion, increased arousal threshold for response to sensory stimulation, rapid reversibility, and homeostatic response to sleep loss. Several assays have been developed to study sleep in C. elegans (Belfer et al., 2013; Bringmann, 2011; Nelson et al., 2013; Raizen et al., 2008). Here, we contribute a detailed protocol for assessment of C. elegans sleep during lethargus, which has been used successfully by many research groups, incorporating simple microfluidic chambers, a low cost camera with lighting system, and computational analysis based on image subtraction. We note that this system could be easily adapted to assess sleep in any small animal.

Gupta S et al. (2016) Int J Biol Macromol "Investigating the folding pathway and substrate induced conformational changes in B. malayi Guanylate kinase."

Yadav S, Saxena JK, Gupta S, Singh SK, Suryanarayanan V

[Int J Biol Macromol, 2016]

Guanylate kinase is one of the key enzymes in nucleotide biosynthesis. The study highlights the structural and functional properties of Brugia malayi Guanylate kinase (BmGK) in the presence of chemical denaturants. An inactive, partially unfolded, dimeric intermediate was observed at 1-2M urea while GdnCl unfolding showed monomer molten globule like intermediate at 0.8-1.0M. The results also illustrate the protective role of substrates in maintaining the integrity of the enzyme. The thermo stability of protein was found to be significantly enhanced in the presence of the substrates. Furthermore, binding of the substrates, GMP and ATP to BmGK changed its GdnCl induced unfolding pattern. Docking and molecular dynamic simulation performed for native BmGK, BmGK bound to GMP and GMP+ATP showed change in the fluctuation in the region between 130-150 residues. Arg134 lost its interaction with GMP and Arg145 interaction shifted to ATP after 40ns simulation upon binding of ATP to BmGK-GMP complex. We, thus, propose the importance of specific rearrangements contributed by binding of substrates which participate in the overall stability of the protein. The work here emphasizes on detailed biophysical characterization of BmGK along with the significant role of substrates in modulating the structural and functional properties of BmGK.

Gupta S et al. (2017) Int J Biol Macromol "Delineating the role of ionic interactions in structural and functional integrity of B. malayi Guanylate Kinase."

Singh SK, Suryanarayanan V, Gupta S, Yadav S, Saxena JK

[Int J Biol Macromol, 2017]

The present work deals with investigating the role of ionic interactions in the native conformation of BmGK by altering pH and salt concentration as well as by disruption of inter-subunit region. The study on structural and functional properties of BmGK as a function of pH showed that the secondary and tertiary elements of the protein were disturbed at low pH with loss of its native oligomerization and functional activity. High concentration of NaCl also changed the native conformation of BmGK with dissociation of its dimeric form. We also mutated dimeric interface of BmGK and identified intersubunit residues, Arg105 and Glu140, essential for dimer stability as double mutation at both positions hinders dimerization. The quaternary structure is found to be essential for full enzymatic activity and stability. In vitro results were supported by in silico molecular dynamics simulation studies through conformational stability analysis. Thus, the work carried out points toward new approach of targeting dimeric interface of BmGK in lieu of its similar active site region to its counterpart human enzyme. This may lead to the design of inhibitors targeted to key parasitic enzyme (BmGK) specifically.

Yu N et al. (2013) Peptides "Analogs of sulfakinin-related peptides demonstrate reduction in food intake in the red flour beetle, Tribolium castaneum, while putative antagonists increase consumption."

Zabrocki J, Benzi V, Smagghe G, Staljanssens D, Kaczmarek K, Yu N, Nachman RJ, Zotti MJ

[Peptides, 2013]

The insect sulfakinins (SKs) constitute a family of neuropeptides that display both structural and functional similarities to the mammalian hormones gastrin and cholecystokinin (CCK). As a multifunctional neuropeptide, SKs are involved in muscle contractions as well as food intake regulation in many insects. In the red flour beetle Tribolium castaneum, the action on food intake by a series of synthetic SK analogs and one putative antagonist was investigated by injection in beetle adults. The most remarkable result was that both sulfated and non-sulfated SKs [FDDY(SO3H)GHMRFamide] inhibited food intake by about 70%. Strong activity observed for SK analogs featuring a residue that mimics the acidic nature of Tyr(SO3H) but lack the phenyl ring of Tyr, indicate that aromaticity is not a critical characteristic for this position of the peptide. SK demonstrated considerable tolerance to Ser and Ala substitution in position 8 (basic Arg), as analogs featuring these uncharged substitutions retained almost all of the food intake inhibitory activity. Also, the Phe in position 1 could be replaced by Ser without complete loss of activity. Conversely, substitution of Met by Nle in position 7 led to inactive compounds. Finally, the Caenorhabditis elegans sulfated neuropeptide-like protein-12 (NLP-12), that shares some sequence similarities with the SKs but features a Gln-Phe-amide rather than an Arg-Phe-amide at the C-terminus, elicited increased food intake in T. castaneum, which may indicate an antagonist activity. Co-injection of NLP-12 with nsSK blocked the food intake inhibitory effects of nsSK.

Doharey PK et al. (2016) Int J Biol Macromol "Insights into the structure-function relationship of Brugia malayi thymidylate kinase (BmTMK)."

Verma P, Doharey PK, Saxena JK, Singh SK, Rathaur S, Verma A

[Int J Biol Macromol, 2016]

Lymphatic filariasis is a debilitating disease caused by lymph dwelling nematodal parasites like Wuchereria bancrofti, Brugia malayi and Brugia timori. Thymidylate kinase of Brugia malayi is a key enzyme in the de novo and salvage pathways for thymidine 5'-triphosphate (dTTP) synthesis. Therefore, Brugia malayi thymidylate kinase (BmTMK) is an essential enzyme for DNA biosynthesis and an important drug target to rein in filariasis. In the present study, the structural and functional changes associated with recombinant BmTMK, in the presence of protein denaturant GdnHCl, urea and pH were studied. GdnHCl and urea induced unfolding of BmTMK is non-cooperative and influence the functional property of the enzyme much lower than their Cm values. Thestudy delineate that BmTMK is more prone to ionic perturbation. The dimeric assembly of BmTMK is an absolute requirement for enzymatic acitivity and any subtle change in dimeric conformation due to denaturation leads to loss of enzymatic activity. The pH induced changes on structure and activity suggests that selective modification of active site microenvironment pertains to difference in activity profile. This study also envisages that chemical moieties which acts by modulating oligomeric assembly, could be used for better designing of inhibitors against BmTMK enzyme.