A Novel Competitive Binding Screening Assay Reveals Sennoside B as a Potent Natural Product Inhibitor of TNF-α

Natural products (NPs) have played a significant role in drug discovery for diverse diseases, and numerous attempts have been made to discover promising NP inhibitors of tumor necrosis factor α (TNF-α), a major therapeutic target in autoimmune diseases. However, NP inhibitors of TNF-α, which have the potential to be developed as new drugs, have not been reported for over a decade. To facilitate the search for new promising inhibitors of TNF-α, we developed an efficient competitive binding screening assay based on analytical size exclusion chromatography coupled with liquid chromatography-tandem mass spectrometry. Application of this screening method to the NP library led to the discovery of a potent inhibitor of TNF-α, sennoside B, with an IC50 value of 0.32 µM in TNF-α induced HeLa cell toxicity assays. Surprisingly, the potency of sennoside B was 5.7-fold higher than that of the synthetic TNF-α inhibitor SPD304. Molecular docking was performed to determine the binding mode of sennoside B to TNF-α. In conclusion, we successfully developed a novel competition binding screening method to discover small molecule TNF-α inhibitors and identified the natural compound sennoside B as having exceptional potency.

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Crystal Structures of the 43 kDa ATPase Domain of Xanthomonas Oryzae pv. Oryzae Topoisomerase IV ParE Subunit and its Complex with Novobiocin

Crystals ◽

10.3390/cryst9110577 ◽

2019 ◽

Vol 9 (11) ◽

pp. 577 ◽

Cited By ~ 1

Author(s):

Ha Yun Jung ◽

Yong-Seok Heo

Keyword(s):

Chromosome Segregation ◽

Binding Mode ◽

Xanthomonas Oryzae ◽

Size Exclusion ◽

Atpase Domain ◽

Topoisomerase Iv ◽

Atp Binding Site ◽

Exclusion Chromatography ◽

Large Conformational Change ◽

Enzyme Functions

Topoisomerase IV, one of the best-established antibacterial targets, is an enzyme crucial for chromosome segregation and cell division by catalyzing changes in DNA topology through breaking and rejoining DNA. This enzyme functions as a heterotetramer consisting of two ParC and two ParE subunits. Aminocoumarin class inhibitors target the ParE subunit, while widely used quinolones target the ParC subunit. Here, we determined the crystal structure of the ParE 43 kDa ATPase domain from Xanthomonas oryzae pv. oryzae. Size exclusion chromatography showed that the ParE ATPase domain exists as a monomer in solution, while it dimerizes when ATP is added. Structural comparison with the structure of Escherichia coli ParE in complex with an ATP analogue showed large conformational change of the subdomains within the protein. We also determined the structure of the ParE ATPase domain in complex with novobiocin, a natural product aminocoumarin class inhibitor, revealing its binding mode and the structural change within the ATP-binding site induced by novobiocin binding. These results could provide a basis for the design of more potent topoisomerase IV inhibitors with improved antibacterial activity.

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Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

Bioscience Reports ◽

10.1042/bsr20130044 ◽

2013 ◽

Vol 33 (4) ◽

Cited By ~ 3

Author(s):

W. Mark Abbott ◽

Melanie Snow ◽

Sonia Eckersley ◽

Jonathan Renshaw ◽

Gareth Davies ◽

...

Keyword(s):

Maximum Size ◽

Size Exclusion ◽

Fab Fragment ◽

Exclusion Chromatography ◽

A Cell ◽

Factor Α ◽

Sepsis And Septic Shock ◽

Very High ◽

Necrosis Factor

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.

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Detection of Salivary Small Extracellular Vesicles Associated Inflammatory Cytokines Gene Methylation in Gingivitis

International Journal of Molecular Sciences ◽

10.3390/ijms21155273 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5273

Author(s):

Pingping Han ◽

Andrew Lai ◽

Carlos Salomon ◽

Sašo Ivanovski

Keyword(s):

Dna Methylation ◽

Extracellular Vesicles ◽

Size Exclusion ◽

Gene Promoters ◽

Cytokine Gene ◽

Oral Diseases ◽

Gene Methylation ◽

Tnf Α ◽

Exclusion Chromatography ◽

Necrosis Factor

Salivary small extracellular vesicles (sEV) are emerging as a potential liquid biopsy for oral diseases. However, technical difficulties for salivary sEV isolation remain a challenge. Twelve participants (five periodontally healthy, seven gingivitis patients) were recruited and salivary sEV were isolated by ultracentrifuge (UC-sEV) and size exclusion chromatography (SEC-sEV). The effect of UC and SEC on sEV yield, DNA methylation of five cytokine gene promoters (interleukin (IL)−6, tumor necrosis factor (TNF)-α, IL−1β, IL−8, and IL−10), and functional uptake by human primary gingival fibroblasts (hGFs) was investigated. The results demonstrated that SEC-sEV had a higher yield of particles and particle/protein ratios compared to UC-sEV, with a minimal effect on the detection of DNA methylation of five cytokine genes and functional uptake in hGFs (n = 3). Comparing salivary sEV characteristics between gingivitis and healthy patients, gingivitis-UC-sEV were increased compared to the healthy group; while no differences were found in sEV size, oral bacterial gDNA, and DNA methylation for five cytokine gene promoters, for both UC-sEV and SEC-sEV. Overall, the data indicate that SEC results in a higher yield of salivary sEV, with no significant differences in sEV DNA epigenetics, compared to UC.

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Characterization of the novelTrypanosoma bruceiinosine 5′-monophosphate dehydrogenase

Parasitology ◽

10.1017/s0031182012002090 ◽

2013 ◽

Vol 140 (6) ◽

pp. 735-745 ◽

Cited By ~ 7

Author(s):

TOMOAKI BESSHO ◽

SHOKO MORII ◽

TOSHIHIDE KUSUMOTO ◽

TAKAHIRO SHINOHARA ◽

MASANORI NODA ◽

...

Keyword(s):

Analytical Ultracentrifugation ◽

De Novo ◽

Binding Mode ◽

Nucleotide Metabolism ◽

Sub Saharan Africa ◽

Size Exclusion ◽

Purine Nucleotides ◽

Exclusion Chromatography ◽

Sub Saharan ◽

Monophosphate Dehydrogenase

SUMMARYThere is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy.Trypanosomalacks the enzymatic machinery for thede novosynthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5′-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinantTrypanosoma bruceiIMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculatedKmvalues of 30 and 1300 μmfor IMP and NAD, respectively. The obtainedKmvalue of TbIMPDH for NAD is approximately 20–200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies showKivalues of 3·2 μm, 21 nM and 3·3 nM for ribavirin 5′-monophosphate, mycophenolic acid and mizoribine 5′-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

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NACK and INTEGRATOR act coordinately to activate Notch-mediated transcription in tumorigenesis

Cell Communication and Signaling ◽

10.1186/s12964-021-00776-1 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Elena Shersher ◽

Mohini Lahiry ◽

Annamil Alvarez-Trotta ◽

Giulia Diluvio ◽

David J. Robbins ◽

...

Keyword(s):

Liquid Chromatography ◽

Rna Polymerase Ii ◽

Transcriptional Activation ◽

Target Genes ◽

Quantitative Polymerase Chain Reaction ◽

Size Exclusion ◽

M Cell ◽

Exclusion Chromatography ◽

Liquid Chromatography Tandem Mass ◽

Eac Cells

Abstract Background Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. Methods Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. Results We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. Conclusions This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation.

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Unbiased high-content screening reveals Aβ- and tau-independent synaptotoxic activities in human brain homogenates from Alzheimer’s patients and high-pathology controls

PLoS ONE ◽

10.1371/journal.pone.0259335 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0259335

Author(s):

Hao Jiang ◽

Thomas J. Esparza ◽

Terrance T. Kummer ◽

David L. Brody

Keyword(s):

Human Brain ◽

Brain Regions ◽

Size Exclusion ◽

Screening Assay ◽

Synaptic Loss ◽

Synapse Loss ◽

Brain Extracts ◽

Exclusion Chromatography ◽

Cultured Neuron

Alzheimer’s disease (AD) is tightly correlated with synapse loss in vulnerable brain regions. It is assumed that specific molecular entities such as Aβ and tau cause synapse loss in AD, yet unbiased screens for synaptotoxic activities have not been performed. Here, we performed size exclusion chromatography on soluble human brain homogenates from AD cases, high pathology non-demented controls, and low pathology age-matched controls using our novel high content primary cultured neuron-based screening assay. Both presynaptic and postsynaptic toxicities were elevated in homogenates from AD cases and high pathology non-demented controls to a similar extent, with more modest synaptotoxic activities in homogenates from low pathology normal controls. Surprisingly, synaptotoxic activities were found in size fractions peaking between the 17–44 kDa size standards that did not match well with Aβ and tau immunoreactive species in these homogenates. The fractions containing previously identified high molecular weight soluble amyloid beta aggregates/”oligomers” were non-toxic in this assay. Furthermore, immunodepletion of Aβ and tau did not reduce synaptotoxic activity. This result contrasts with previous findings involving the same methods applied to 3xTg-AD mouse brain extracts. The nature of the synaptotoxic species has not been identified. Overall, our data indicates one or more potential Aβ and tau independent synaptotoxic activities in human AD brain homogenates. This result aligns well with the key role of synaptic loss in the early cognitive decline and may provide new insight into AD pathophysiology.

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Demyelination and Neurological Sequelae in Etanercept and Infliximab Therapies: A Review of the Literature

Psoriasis Forum ◽

10.1177/247553030511a00103 ◽

2005 ◽

Vol 11a (1) ◽

pp. 6-8

Author(s):

Vandana K. Madkan ◽

John Y.M. Koo

Keyword(s):

Multiple Sclerosis ◽

Tumor Necrosis ◽

New Drugs ◽

Review Of The Literature ◽

Neurological Sequelae ◽

Tnf Α ◽

Cytokine Tumor Necrosis Factor ◽

Factor Α ◽

Necrosis Factor ◽

New Onset

Etanercept (Enbrel) and infliximab (Remicade) are two relatively new drugs, referred to as biologics, which target the cytokine tumor necrosis factor-α (TNF-α). Both are used in the treatment of psoriasis and psoriatic arthritis. Recently, there have been rare reports of demyelinating and neurological sequelae in patients while on these therapies. The authors sought to systematically review the reported cases of new-onset neurological symptoms and exacerbation of established multiple sclerosis (MS) found in the literature to determine the causality, if any, of demyelinating events by these therapies. Although these two medications may uncover latent multiple sclerosis in patients who are prone to the disease, no cause-and-effect relationship can currently be established.

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A Comparison of Blood Plasma Exosome Enrichment Strategies for Proteomic Analysis

10.21203/rs.3.rs-819042/v1 ◽

2021 ◽

Author(s):

Natalie P. Turner ◽

Pevindu Abeysinghe ◽

Keith A. Kwan Cheung ◽

Kanchan Vaswani ◽

Jayden Logan ◽

...

Keyword(s):

Blood Plasma ◽

Proteomic Analysis ◽

Protein Identification ◽

Biomarker Discovery ◽

Bos Taurus ◽

Size Exclusion ◽

Gold Standard Method ◽

Exclusion Chromatography ◽

Liquid Chromatography Tandem Mass ◽

Enrichment Methods

Abstract Proteomic analysis of exosomes (EX) poses a significant challenge. A ‘gold-standard’ method for plasma EX enrichment for downstream proteomic analysis is yet to be established. Our group has performed a comprehensive study of multi-dimensional enrichment methods to determine their efficiency for protein isolation. Methods were evaluated for their capacity to a) successfully isolate and enrich EX from blood plasma, b) minimise the presence of highly abundant plasma proteins, and c) result in the optimum representation of EX proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS). Blood plasma from four animals (Bos taurus) of similar physical attributes and genetics were used. Three methods of EX enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These enrichment methods were combined to create four groups for methodological evaluation: UC+SEC, UC+SEC+UF, SEC+UC and SEC+UF. UC+SEC yielded the highest number of protein IDs. Plasma protein identification was the least in SEC+UC, but this method yielded the lowest number of protein IDs overall. UC+SEC+UF decreased EX protein ID and did not improve purity compared to UC+SEC. Our data suggest that the method and sequence of EX enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

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