Engineering of a Spider Peptide via Conserved Structure-Function Traits Optimizes Sodium Channel Inhibition In Vitro and Anti-Nociception In Vivo

Venom peptides are potent and selective modulators of voltage-gated ion channels that regulate neuronal function both in health and in disease. We previously identified the spider venom peptide Tap1a from the Venezuelan tarantula Theraphosa apophysis that targeted multiple voltage-gated sodium and calcium channels in visceral pain pathways and inhibited visceral mechano-sensing neurons contributing to irritable bowel syndrome. In this work, alanine scanning and domain activity analysis revealed Tap1a inhibited sodium channels by binding with nanomolar affinity to the voltage-sensor domain II utilising conserved structure-function features characteristic of spider peptides belonging to family NaSpTx1. In order to speed up the development of optimized NaV-targeting peptides with greater inhibitory potency and enhanced in vivo activity, we tested the hypothesis that incorporating residues identified from other optimized NaSpTx1 peptides into Tap1a could also optimize its potency for NaVs. Applying this approach, we designed the peptides Tap1a-OPT1 and Tap1a-OPT2 exhibiting significant increased potency for NaV1.1, NaV1.2, NaV1.3, NaV1.6 and NaV1.7 involved in several neurological disorders including acute and chronic pain, motor neuron disease and epilepsy. Tap1a-OPT1 showed increased potency for the off-target NaV1.4, while this off-target activity was absent in Tap1a-OPT2. This enhanced potency arose through a slowed off-rate mechanism. Optimized inhibition of NaV channels observed in vitro translated in vivo, with reversal of nocifensive behaviours in a murine model of NaV-mediated pain also enhanced by Tap1a-OPT. Molecular docking studies suggested that improved interactions within loops 3 and 4, and C-terminal of Tap1a-OPT and the NaV channel voltage-sensor domain II were the main drivers of potency optimization. Overall, the rationally designed peptide Tap1a-OPT displayed new and refined structure-function features which are likely the major contributors to its enhanced bioactive properties observed in vivo. This work contributes to the rapid engineering and optimization of potent spider peptides multi-targeting NaV channels, and the research into novel drugs to treat neurological diseases.

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Natural Glycoside Salidroside Ameliorates Orthopedic Surgery-Induced Cognitive Dysfunction Through Activating Adenosine 5‘-Monophosphate-Activated Protein Kinase Signaling in Mice

10.21203/rs.3.rs-745010/v1 ◽

2021 ◽

Author(s):

Cai-Long Pan ◽

Guo-Liang Dai ◽

Hui-Wen Zhang ◽

Chen-Yang Zhang ◽

Qing-Hai Meng ◽

...

Keyword(s):

Cognitive Dysfunction ◽

Orthopedic Surgery ◽

Neurological Diseases ◽

Docking Studies ◽

Morris Water Maze Test ◽

Ampk Pathway ◽

Inflammatory Phenotype ◽

Anti Inflammatory

Abstract Background: Perioperative neurocognitive disorders (PND) are the most common postoperative complications with few therapeutic options. Salidroside, a plant-derived compound, has gained increased attention as treatment for various neurological diseases and particularly modifier of microglia-mediated neuroinflammation. However, the effect of salidroside on orthopedic surgery-induced cognitive dysfunction and the underlying mechanisms are largely unknown.Methods: The Morris water maze test was used to investigate potential effects of salidroside in the animal model of tibia fracturing with intramedullary fixation. Therapeutic mechanism and related signaling pathways of salidroside in PND were further investigated with animal tissues and microglial cultures in vitro by molecular biology tests.Results: Here we found that salidroside greatly attenuated cognitive impairment in mice after orthopedic surgery. Neuroinflammation in mouse hippocampus were also attenuated by salidroside. Meanwhile, salidroside treatment induced a switch in microglia polarization to the anti-inflammatory phenotype. In vitro, salidroside suppressed the expression of pro-inflammatory cytokines and induced a switch in microglial phenotype to the anti-inflammatory phenotype. Mechanically, molecular docking studies revealed potential AMPK activation activity of salidroside. And salidroside did up-regulated the AMPK pathway proteins. Moreover, AMPK antagonist abolished the effects of salidroside in vivo and in vitro.Conclusions: Taken together, our results demonstrated that salidroside effectively suppressed PND by suppressing microglia-mediated neuroinflammation through activating AMPK pathway, and it might be a novel therapeutic approach for PND.

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Probing the 14-3-3 Isoform-Specificity Profile of Interactions Stabilized by Fusicoccin A

10.26434/chemrxiv.12052869.v1 ◽

2020 ◽

Author(s):

James Frederich ◽

Ananya Sengupta ◽

Josue Liriano ◽

Ewa A. Bienkiewicz ◽

Brian G. Miller

Keyword(s):

Protein Interactions ◽

Neurological Diseases ◽

Adapter Proteins ◽

Protein Protein Interactions ◽

Specific Expression ◽

Individual Client ◽

Isoform Specificity ◽

Fusicoccin A

Fusicoccin A (FC) is a fungal phytotoxin that stabilizes protein–protein interactions (PPIs) between 14-3-3 adapter proteins and their phosphoprotein interaction partners. In recent years, FC has emerged as an important chemical probe of human 14-3-3 PPIs implicated in cancer and neurological diseases. These previous studies have established the structural requirements for FC-induced stabilization of 14-3-3·client phosphoprotein complexes; however, the effect of different 14-3-3 isoforms on FC activity has not been systematically explored. This is a relevant question for the continued development of FC variants because there are seven distinct isoforms of 14-3-3 in humans. Despite their remarkable sequence and structural similarities, a growing body of experimental evidence supports both tissue-specific expression of 14-3-3 isoforms and isoform-specific functions in vivo. Herein, we report the isoform-specificity profile of FC in vitrousing recombinant human 14-3-3 isoforms and a focused library of fluorescein-labeled hexaphosphopeptides mimicking the C-terminal 14-3-3 recognition domains of client phosphoproteins targeted by FC in cell culture. Our results reveal modest isoform preferences for individual client phospholigands and demonstrate that FC differentially stabilizes PPIs involving 14-3-3s. Together, these data provide strong motivation for the development of non-natural FC variants with enhanced selectivity for individual 14-3-3 isoforms.

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Faculty Opinions recommendation of A voltage sensor-domain protein is a voltage-gated proton channel.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1032187.373891 ◽

2006 ◽

Author(s):

Michael Blatt

Keyword(s):

Voltage Sensor ◽

Proton Channel ◽

Voltage Gated ◽

Domain Protein ◽

Voltage Sensor Domain

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In-silico Studies of Isolated Phytoalkaloid Against Lipoxygenase: Study Based on Possible Correlation

Combinatorial Chemistry & High Throughput Screening ◽

10.2174/1386207321666180220125406 ◽

2018 ◽

Vol 21 (3) ◽

pp. 215-221

Author(s):

Haroon Khan ◽

Muhammad Zafar ◽

Helena Den-Haan ◽

Horacio Perez-Sanchez ◽

Mohammad Amjad Kamal

Keyword(s):

In Silico ◽

Docking Studies ◽

In Vivo Studies ◽

In Vitro Assays ◽

Chronic Obstructive ◽

Lipoxygenase Inhibitors ◽

Lipoxygenase Inhibition ◽

In Silico Studies

Aim and Objective: Lipoxygenase (LOX) enzymes play an important role in the pathophysiology of several inflammatory and allergic diseases including bronchial asthma, allergic rhinitis, atopic dermatitis, allergic conjunctivitis, rheumatoid arthritis and chronic obstructive pulmonary disease. Inhibitors of the LOX are believed to be an ideal approach in the treatment of diseases caused by its over-expression. In this regard, several synthetic and natural agents are under investigation worldwide. Alkaloids are the most thoroughly investigated class of natural compounds with outstanding past in clinically useful drugs. In this article, we have discussed various alkaloids of plant origin that have already shown lipoxygenase inhibition in-vitro with possible correlation in in silico studies. Materials and Methods: Molecular docking studies were performed using MOE (Molecular Operating Environment) software. Among the ten reported LOX alkaloids inhibitors, derived from plant, compounds 4, 2, 3 and 1 showed excellent docking scores and receptor sensitivity. Result and Conclusion: These compounds already exhibited in vitro lipoxygenase inhibition and the MOE results strongly correlated with the experimental results. On the basis of these in vitro assays and computer aided results, we suggest that these compounds need further detail in vivo studies and clinical trial for the discovery of new more effective and safe lipoxygenase inhibitors. In conclusion, these results might be useful in the design of new and potential lipoxygenase (LOX) inhibitors.

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Mouse models to study von Willebrand factor structure-function relationships in vivo

Hämostaseologie ◽

10.1055/s-0037-1616933 ◽

2009 ◽

Vol 29 (01) ◽

pp. 17-20 ◽

Cited By ~ 2

Author(s):

I. Marx ◽

I. Badirou ◽

R. Pendu ◽

O. Christophe ◽

C. V. Denis

Keyword(s):

Structure Function ◽

Transient Expression ◽

Von Willebrand Factor ◽

Large Size ◽

Mouse Hepatocytes ◽

Function Relationship ◽

Von Willebrand ◽

Willebrand Factor

SummaryVon Willebrand factor (VWF) structure-function relationship has been studied only through in vitro approaches. The VWF-deficient mouse model has been extremely useful to examine the in vivo function of VWF but does not allow a more subtle analysis of the relative importance of its different domains. However, considering the large size of VWF and its capacity to interact with various ligands in order to support platelet adhesion and aggregation, the necessity to evaluate independently these interactions appeared increasingly crucial. A recently developed technique, known as hydrodynamic injection, which allows transient expression of a transgene by mouse hepatocytes, proved very useful in this regard. Indeed, transient expression of various VWF mutants in VWF-deficient mice contributed to improve our knowledge about the role of VWF interaction with subendothelial collagens and with platelets receptors in VWF roles in haemostasis and thrombosis. These findings can provide new leads in the development of anti-thrombotic therapies.

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The effect of dipeptidyl peptidase IV on disease-associated microglia phenotypic transformation in epilepsy

Journal of Neuroinflammation ◽

10.1186/s12974-021-02133-y ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Zhicheng Zheng ◽

Peiyu Liang ◽

Baohua Hou ◽

Xin Lu ◽

Qianwen Ma ◽

...

Keyword(s):

Signaling Pathway ◽

Neurological Diseases ◽

Dipeptidyl Peptidase ◽

Dipeptidyl Peptidase Iv ◽

Sequencing Data ◽

Migration Ability ◽

Dpp4 Inhibitor ◽

Phenotypic Transformation

Abstract Background Accumulating evidence suggests that disease-associated microglia (DAM), a recently discovered subset of microglia, plays a protective role in neurological diseases. Targeting DAM phenotypic transformation may provide new therapeutic options. However, the relationship between DAM and epilepsy remains unknown. Methods Analysis of public RNA-sequencing data revealed predisposing factors (such as dipeptidyl peptidase IV; DPP4) for epilepsy related to DAM conversion. Anti-epileptic effect was assessed by electroencephalogram recordings and immunohistochemistry in a kainic acid (KA)-induced mouse model of epilepsy. The phenotype, morphology and function of microglia were assessed by qPCR, western blotting and microscopic imaging. Results Our results demonstrated that DPP4 participated in DAM conversion and epilepsy. The treatment of sitagliptin (a DPP4 inhibitor) attenuated KA-induced epilepsy and promoted the expression of DAM markers (Itgax and Axl) in both mouse epilepsy model in vivo and microglial inflammatory model in vitro. With sitagliptin treatment, microglial cells did not display an inflammatory activation state (enlarged cell bodies). Furthermore, these microglia exhibited complicated intersections, longer processes and wider coverage of parenchyma. In addition, sitagliptin reduced the activation of NF-κB signaling pathway and inhibited the expression of iNOS, IL-1β, IL-6 and the proinflammatory DAM subset gene CD44. Conclusion The present results highlight that the DPP4 inhibitor sitagliptin can attenuate epilepsy and promote DAM phenotypic transformation. These DAM exhibit unique morphological features, greater migration ability and better surveillance capability. The possible underlying mechanism is that sitagliptin can reduce the activation of NF-κB signaling pathway and suppress the inflammatory response mediated by microglia. Thus, we propose DPP4 may act as an attractive direction for DAM research and a potential therapeutic target for epilepsy.

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Synthesis and Evaluation of Novel α-Aminoamides Containing Benzoheterocyclic Moiety for the Treatment of Pain

Molecules ◽

10.3390/molecules26061716 ◽

2021 ◽

Vol 26 (6) ◽

pp. 1716

Author(s):

Kun Tong ◽

Ruotian Zhang ◽

Fengzhi Ren ◽

Tao Zhang ◽

Junlin He ◽

...

Keyword(s):

Ion Channels ◽

Patch Clamp ◽

Formalin Test ◽

Sodium Ion ◽

Inhibitory Potency ◽

Voltage Gated ◽

Patch Clamp Electrophysiology ◽

Sodium Ion Channels

Novel α-aminoamide derivatives containing different benzoheterocyclics moiety were synthesized and evaluated as voltage-gated sodium ion channels blocks the treatment of pain. Compounds 6a, 6e, and 6f containing the benzofuran group displayed more potent in vivo analgesic activity than ralfinamide in both the formalin test and the writhing assay. Interestingly, they also exhibited potent in vitro anti-Nav1.7 and anti-Nav1.8 activity in the patch-clamp electrophysiology assay. Therefore, compounds 6a, 6e, and 6f, which have inhibitory potency for two pain-related Nav targets, could serve as new leads for the development of analgesic medicines.

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The Potential Neuroprotective Role of Free and Encapsulated Quercetin Mediated by miRNA against Neurological Diseases

Nutrients ◽

10.3390/nu13041318 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1318

Author(s):

Tarek Benameur ◽

Raffaella Soleti ◽

Chiara Porro

Keyword(s):

Inflammatory Responses ◽

Pathological Condition ◽

Neurological Diseases ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

In Vivo Studies ◽

Innovative Drug ◽

Chronic Neuroinflammation

Chronic neuroinflammation is a pathological condition of numerous central nervous system (CNS) diseases such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, amyotrophic lateral sclerosis and many others. Neuroinflammation is characterized by the microglia activation and concomitant production of pro-inflammatory cytokines leading to an increasing neuronal cell death. The decreased neuroinflammation could be obtained by using natural compounds, including flavonoids known to modulate the inflammatory responses. Among flavonoids, quercetin possess multiple pharmacological applications including anti-inflammatory, antitumoral, antiapoptotic and anti-thrombotic activities, widely demonstrated in both in vitro and in vivo studies. In this review, we describe the recent findings about the neuroprotective action of quercetin by acting with different mechanisms on the microglial cells of CNS. The ability of quercetin to influence microRNA expression represents an interesting skill in the regulation of inflammation, differentiation, proliferation, apoptosis and immune responses. Moreover, in order to enhance quercetin bioavailability and capacity to target the brain, we discuss an innovative drug delivery system. In summary, this review highlighted an important application of quercetin in the modulation of neuroinflammation and prevention of neurological disorders.

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Hypoglycemic, hepatoprotective and molecular docking studies of 5-[(4-chlorophenoxy) methyl]-1, 3, 4-oxadiazole-2-thiol

Bangladesh Journal of Pharmacology ◽

10.3329/bjp.v13i2.35514 ◽

2018 ◽

Vol 13 (2) ◽

pp. 149 ◽

Cited By ~ 1

Author(s):

Naureen Shehzadi ◽

Khalid Hussain ◽

Nadeem Irfan Bukhari ◽

Muhammad Islam ◽

Muhammad Tanveer Khan ◽

...

Keyword(s):

Molecular Docking ◽

Video Clip ◽

Serum Levels ◽

Docking Studies ◽

Normal Serum ◽

Yeast Cells ◽

Drug Candidate ◽

Molecular Docking Studies

The present study aimed at the evaluation of anti-hyperglycemic and hepatoprotective potential of a new drug candidate, 5-[(4-chlorophenoxy) methyl]-1,3,4-oxadiazole-2-thiol (OXCPM) through in vitro and in vivo assays, respectively. The compound displayed excellent dose-dependent ɑ-amylase (28.0-92.0%), ɑ-glucosidase (40.3-93.1%) and hemoglobin glycosylation (9.0%-54.9%) inhibitory effects and promoted the uptake of glucose by the yeast cells (0.2 to 26.3%). The treatment of the isoniazid- and rifampicin- (p.o., 50 mg/kg of each) intoxicated rats with OXCPM (100 mg/kg, p.o.) resulted in restoring the normal serum levels of the non-enzymatic (total bilirubin, total protein and albumin) and bringing about a remarkable decrease in the levels of enzymatic (alanine transaminases, aspartate transaminases and alkaline phosphatase) biomarkers. The molecular docking studies indicated high binding affinity of the compound for hyperglycemia-related protein targets; fructose-1,6-bisphosphatase, beta2-adrenergic receptors and glucokinase. The results indicate that OXCPM may not only reduce hyperglycemia by enzyme inhibition but also the disease complications through protection of hemoglobin glycosylation and hepatic injury.Video Clip of Methodology:Glucose uptake by yeast cells: 4 min 51 sec <a href="https://www.youtube.com/v/8cJkuMtV0Wc">Full Screen</a> <a href="https://www.youtube.com/watch?v=8cJkuMtV0Wc">Alternate</a>

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