What will the Next-Gen Protease activity detection be?

Fluorescent technologies enable the measurement of protease activities and the screening of compounds influencing protease activities. Fluorescence Resonance Energy Transfer (FRET) but also TR-FRET, Q-FRET together with Time Resolved Fluorescence (TRF) and Fluorescence Lifetime (FLT) are popular assays, in which protease substrates occupy a central place. These assays are based on the specific recognition and cleavage of a peptide sequence (substrate) by the protease of interest. If the substrate is not optimally designed (or too short), experimental output can be unrelevant: low selectivity and specificity, high background, false negatives or positives…). In addition, the data can also be affected by the reaction buffer (pH, compound solvent…).This lack of relevant biological information is at the origin the emergence of “Next-Gen” fluorescent protease substrates.

[Read more…]

20S Proteasome inhibitors and Leptin against obesity

The World Health Organization (WHO) estimated in 2008 that 1.4 billion adults worldwide were overweight and of these 500 million were obese with risks for developing type 2 diabetes, hypertension and cardio-vascular diseases. The discovery of the adipocyte hormone, Leptin, brought to light the possibility that its anorectic effect could be harnessed for treating the epidemic of obesity. However up until now Leptin resistance has been an unsurmountable problem and the use of this adipokine for suppressing food intake has failed. In a recent issue of Cell, Junli Liu and coworkers at Harvard Medical School report that Celastrol, a natural product isolated from the Thunder God Vine (Tripterygium Wilfordi), is a powerful antiobesity agent.

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New in vitro cellular model for human Lipodystrophy

Lipodystrophies are disorders characterized by complete or selective loss of adipose tissue from various regions of the body. They might lead to severe metabolic disorders. The development of reliable cellular experimental models mimicking such diseases in vitro is extremely challenging (1). One of the main hurdle in the design of such in vitro cellular models is the access to reliable sources of well-qualified primary cells and the identification of optimal cell culture conditions.

[Read more…]

Thiostrepton: a novel lead compound against Dengue?

Dengue Virus (DenV) is transmitted by mosquito vectors. It infects 50-100 million people each year and is at the origin of Dengue Fever and the more lethal Dengue Hemorrhagic Fever (DHF) and Shock Syndrome (DSS) leading to an estimated 500,000 cases of DHF and 22 000 deaths. The World Health Organization (WHO) estimates that 40% of the world’s population is at risk of infection.

In the June issue of Journal of Biomolecular Screening, investigators at San Diego State University (Dept of Biology) and Institute Pasteur Korea (Seoul, South Korea) developed a multiplexed cell-based assay for the identification of modulators of pre-membrane processing as a target for the discovery of DenV inhibitors. (1)

The DenV pre-membrane protein (prM) is an essential chaperone for the viral envelope protein which prevents premature fusion with vesicles during viral export. Inhibition of pre-membrane protein cleavage restricts fusion and represents, thus, a novel druggable target.

The new in vitro assay developed in this study, is the first described cell-based assay that monitors DenVprM processing within the classical secretory pathway. In a pilot screen of 1,280 small molecules on that assay, Thiostrepton, a known cyclopeptide Antibiotic and FOXM1 inhibitor,  was identified as a novel positive hit in this assay (IC50=4.94 µM).

The utility of this novel assay has been proven by the identification of Thiostrepton (available at Focus Biomolecules cat. nr 10-2108) which may be a novel lead compound for the discovery of new drugs effective against Dengue Virus.

Thiostrepton Focus Biomolecules tebu-bio Catalog # 10-2108

Thiostrepton – Antibiotic and FOXM1 inhibitor. C72H85N19O18S5 – CAS No: 1393-48-2, 98% by TLC and HPLC at Focus Biomolecules.

 

1- Stolp Z.D. et al. “A Multiplexed Cell-Based Assay for the Identification of Modulators of Pre-Membrane Processing as a Target against Dengue Virus” (2015) J. Biomol. Screen. 20:616-626. DOI: 10.1177/1087057115571247.

 

High resolution structure of the TRPA1 channel protein

One of the hottest news last month was the high resolution structure of the TRPA1 ion channel protein.

Immunohistochemical staining of TRPA1 on mouse intestine with TRPA1 polyclonal antibody (Cat. nr 157PAB11992)

IHC staining on mouse intestine with TRPA1 polyclonal antibody (Cat. nr 157PAB11992). Source: Abnova | tebu-bio.

TRPA1 is a sensor for environmental noxious agents or signaling molecules produced endogenously. The structure was solved in the presence of agonist and antagonist ligands by using single-particle electron cryo-microscopy. (1)

This smart experimental method, which has revealed several unexpected TRPA1 structural features, shows that structural  information thus discovered might greatly assist Life Researchers in the design of better antagonists which will represent the next generation analgesic and anti-inflammatory agents.

Interestingly, several TRPA1-related agents are now well characterized for further in vitro characterization.

Several useful agents for studying TRPA1 channels are available:

  • TRPA1 antagonist HC-030031 inhibits the ion channel (cat. nr 21910-1065)
  • TRPA1 antagonist A-967079 is a cell permeable TRPA1 blocker  (cat. nr 21910-1055)

    Immunogen part (green) from the 4th cystoplasmic loop of the human TRPA1 used to produce human TRPA1 specific antibody (Cat. nr OSR00130W)

    Human TRPA1 modelization. The synthetic from the 4th cytoplasmic loop of human TRPA1 (highlighted in green) was used as the antigen to produce the highly specific human TRPA1 antibody (Cat. nr 214OSR00130W). Source: Osenses | tebu-bio.

  • TRPA1 antagonist AP-18 is a selective TRPA1 channel blocker (cat. nr 21910-1127)
  • TRPA1 agonist Polygodial displays analgesic activity via desensitization of sensory neurons (cat. nr 21910-1145)

 

Thanks to our friends from Focus Biomolecules for this post !

Mechanism of action for angiogenesis inhibitor Borrelidin discovered

Originally identified as an active molecule against the Borrelia species, Borrelidin has since been described as a selective inhibitor of threonyl tRNA synthetase (ThrRS). More recent research has found that Borrelidin (also called Treponemycin, Antibiotic U 78548 or C2989)  induces the collapse of newly formed capillary tubules, exhibits anti-bacterial, -malarial, -insecticidal activities, and displays a potent anti-VEGF induced angiogenic activity (IC50=0.8 nM).

[Read more…]

Neuroregenerative effects of microtubule stabilizing Epothilone B

Epothilone B is a bioactive microtubule-stabilizing small molecule. This blood-brain barrier permeable coumpound belongs to the most popular potent cytoskeletal modeling molecules for in vitro cell based assays (see the previous post dedicated to “5 Cytoskeletal Modeling Molecules“).Epothilone B structure cat. nr 10-2133 Focus Biomolecules tebu-bio
Recently, Ruschel J. et al. have demonstrated that its delayed systemic administration in rodents promoted axonal regeneration with Central Nervous System (CNS) injuries. These findings open new therapeutic areas regarding the use of microtubule-stabilizing drug compounds, like Epothilones, in CNS recovery and neuroregeneration. [Read more…]

Which approach for measuring circulating cell-free miRNAs?

More than 2,500 human miRNAs are potentially significant biomarkers. Moreover, the use of blood circulating miRNAs as disease-specific biomarkers is one of the most valuable outputs for translational and clinical research. Nevertheless, such an analysis still requires the selection of robust technologies, huge R&D work and reproducibility studies.

During the latest AACR Meeting (April 2015, Philadelphia – USA), Nadia Normand, R&D manager at tebu-bio’s laboratories (Le Perray en Yvelines, France), presented a poster comparing various platforms for measuring circulating miRNAs. It was the opportunity to further demonstrate the robustness of  the miRNA 3D-Gene® microarray-based platform (Toray Industries).

To know more about the comparison, follow this link to access to the poster:

Mennesson E. et al. “Comparison of different highly sensitive miRNA array platforms for the investigation of circulating cell-free microRNAs in blood” (2015) – Poster AACR.

 

Live cell imaging in action: Killer T-cells hunt their target

In a recent video posted on “Cambridge University’s YouTube channel”, Professor Gillian Griffiths and co-workers prove how the Life Science Community can benefit from modern and innovative cell imaging technologies for understanding cellular activity.

In their recent study (1) and film (2), the research team captured real-time behaviour of Cytotoxic T lymphocytes (CTLs) hunting down, moving and killing cancer cells.

This study clearly illustrates the major breakthrough related to new high spatial and temporal resolution, multi-colour, 3D time-lapse imaging microscopy techniques, which image the entire cell volume and display cellular events at micrometre level.

Watch this fascinating video:

Killer T-Cell Credit: Gillian Griffiths/Jonny Settle . 

Sources:

Professor Gillian Griffiths, Dr Yukako Asano and Akex Ritter – Cambridge Institute for Medical Research – Department of Medicine of the Clinical School (NIH OxCam programme with funding from the Wellcome Trust)

1) Ritter A.T. et al. “Actin depletion initiates events leading to granule secretion at the immunological synapse” (2015) Immunity 42, 864–876. DOI: 10.1016/j.immuni.2015.04.013

2) Body’s ‘serial killers’ captured on film destroying cancer cells – See more at: http://www.cam.ac.uk/research/news

Actin and Tubulin dynamics research studies in 2015

The world of cytoskeletal dynamics studies is in mutation. By example, the first synthetic chemical targeting actin and specifically triggering its growth has been recently released for research applications (BPA). A few weeks before, SiR stains were successfully launched for Actin and Tubulin Live Cell Imaging (SiR-actin Live cell Imaging was at the JBC frontcover in Feb. 2015).

Cellular and molecular biologists have now access to a unique range of discovery tools opening new perspectives for deciphering cytoskeletal events living cells.

Branched PolyAmines (BPA)

Until now, only molecules that stabilize or destroy the cytoskeleton of actin were available. Derived from supramolecular chemistry, Branched PolyAmines (BPA) rapidly enhances the growth of lamellar networks of actin filaments.

BPA-induced actin filaments

Actin filamentous network growth 10 minutes after addition of BPA to cell cultures.Source: Pr. Daniel Riveline, Laboratory of Cell Physics, ISIS/IGBMC, Strasbourg (France).

BPA is a specific modulator for in vitro and in vivo actin dynamics studies by regulating actin nucleation and turnover in cells. This compound complements currently available molecules to study actin dynamics such as Latrunculin A , Cytochalasin D, Jasplakinolide, WiskostatinActin Binding Proteins.

Transfection-free SiR Actin and Tubulin stains

SpiroChrome’s live cell SiR actin stains are non-toxic fluorescent stains for monitoring Actin and Tubulin changes in living cells. These cell permeable SiR-Actin and SiR-Tubulin compounds stain microtubules and F-actin respectively in living cells.

SiR Tubulin tebu-bio's fluorescent dye

3D-SIM microscopy image of labeled microtubules in primary rat cortex neuron body stained with SiR Tubulin.

Novel research tools aiming at better understanding cytoskeletal reorganization have radically changed discovery frontiers. Interestingly, and despite their degree of innovation, these new reagents remain very affordable and user friendly for research applications.

tebu-bio’s experts will follow on tracking emerging cystoskeleton-based technologies.