Mambalgin 1 – Blocking ASIC channels in pain pathways

Acid-Sensing Ion Channels (ASICs) are neuronal voltage-insensitive cationic channels which are activated by extracellular protons. They belong to the ENaC/Deg superfamily of ion channels. Up to now 6 members of the ASIC family have been identified: ASIC1 – ASIC 4, and the splice variants ASIC1a, 1b and 2a, 2b. The ASIC family members are trimeric and can be made up of different combinations of subunits. All ASICs are expressed in the peripheral nervous system. ASIC1a, 2a, 2b and 4 are expressed in the central nervous system. ASICs are promising drug targets for treating a wide variety of conditions linked to both the CNS and PNS especially implied in pain pathways.

Structure of Mambalgin-1

Schwarze Mamba

Fig. 1: Dendroaspis polylepis polylepis

Mambalgin-1 (from Smartox, now available through tebu-bio) was initially isolated by Sylvie Diochot and collaborators from the venom of the black mamba (Dendroaspis polylepis polylepis, Fig. 1).

Mambalgin-1 belongs to the family of three-finger toxins (Fig. 2) and has no sequence/structural homology with either PcTx1 or APETx2. Mambalgin-1 differs from mambalgin-2 by one amino acid. Both have demonstrated a similar activity.

Mambalgin 1 – a blocker of ASIC1 channels implied in pain pathways

It has been shown that Mambalgin-1 is a potent and selective blocker of ASICs implied in pain pathways (1, 2). Mambalgin-1 rapidly and reversibly inhibits recombinant homomeric ASIC1a (IC50=55 nM) and heteromeric ASIC1a+ASIC2a (IC50=246 nM) or ASIC1a+ASIC2b channels (IC50=61 nM) but also human channels hASIC1b (IC50=192 nM) and   hASIC1a+hASIC1b (IC50=72nM).

Mambalgin-1 has no effect on ASIC2a, ASIC3, ASIC1a+ASIC3 and ASIC1b+ASIC3 channels, as well as on TRPV1, P2X2, 5-HT3A, Nav1.8, Cav3.2 and Kv1.2 channels. Thus Mambalgin-1 can be used as a selective inhibitor for the above mentioned homomeric and heteromeric ASICs.

Interested in further information? Please contact me through the form below.

If you’d like an overview of the complete venomous toxins available, take a look to our special page: Synthetic peptide toxins ideal for studying ion channels

References:

(1) S, Dichot et al., Black mamba venom peptides target acid-sensing ion channels to abolish pain, Nature. 490 (7421), pages 552-5 (2012)

(2) Wen M., et al., Site-specific fluorescence spectrum detection and characterization of hASIC1a channels upon toxin mambalgin-1 binding in live mammalian cells. Chem Commun. 51 (38), pages 8153-6 (2015)

Keen to work with the best quality nucleosomes?

Histone Structure- tebu-bio

Fig. 1: Structure of a mono-nucleosome.

Nucleosomes are basis units of DNA packaging in eukaryotic cells. A core particle (mono-nucleosome) consists of a segment of DNA called core DNA (147 bp in length) wound around a histone octamer (Fig 1.). Histone octamers are made up of 2 copies of the the core histones H2A, H2B, H3, and H4. These mono-nucleosomes are connected by linker 80bp-long DNA. H1 histone, the so-called linker histone, binds to the linker DNA close to the entry and exit of the core DNA and is involved in chromatin compaction (Fig 1.). [Read more…]

9 pathway-specific screening assays in Immunotherapy

The immune system is a system of cells and organs whose function is to defend an organism from foreign pathogens. With the ability to mount a response against virtually any foreign material and return to a quiescent state following neutralization of the threat, this fascinating organ system displays remarkable specificity and plasticity. To achieve this, there is a multifaceted balancing act between the many activators and suppressors which maintains homeostasis of the body’s perhaps most complex organ system.

[Read more…]

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.

[Read more…]

TET1 – the first DNA demethylase

Graph DM

Fig. 1: Example of a DNA methyltransferase assay: DNMT1 enzyme activity, measured using the DNMT1 Assay Kit.

Methylation of DNA plays a crucial role in transcriptional regulation, imprinting, and chromatin structure. DNA methyltransferases add a methyl group to cytosine or adenine DNA nucleotides. DNA methylation is a dynamic and reversible process and alters the expression of genes during cell division and differentiation from embryonic stem cells to specific tissues. The process is usually permanent, preventing cells e.g. from re-differentiation. During zygote formation, methyl groups are removed and subsequently re-established during development.

The reducing effect of gene expression is mediated by methylation at the 5 position of cytosine and it is present on all vertebrates. In adult somatic cells methylation is seen in a CgG context, while non-CpG methylation is current in embryogenic stem cells. Enzymes that catalyze DNA methylation are well characterized and already used as target enzymes for drug discovery screenings. BPS Biosciences offers a range of active DNA methyltransferases and DNA methyltransferase assays for inhibitor screenings (see Fig.1). [Read more…]

Trapping PARP-DNA complexes – anti-cancer drug screening

PARPs (Poly ADP ribose polymerases) are found in the nucleus of the cell and they are involved in SSB repair (single-strand DNA breaks). PARP is known to bind damaged DNA through its N-terminal zinc finger domain. Subsequently it starts to synthesize a poly (ADP-ribose) chain which serves as a signal for other DNA-repairing enzymes.

SYNTHETIC_ LEATHALITY

Fig. 1: Principle of synthetic lethality

PARP inhibitors are considered to be promising  candidates as anti cancer drugs (recently Olaparib, the first drug directed against PARP1, has been approved by the European commission). One of the reasons is that some tumors are more dependent on PARP than regular cells. These cancer cells are mutated in BRCA1 or BRCA2 – both genes which are involved in key DNA damage repair mechanisms. In healthy cells PARPs can function as a kind of back-up system and let the cells survive even without functional BRCA gene products. When PARPs are inhibited the cells do not possess any functional SSB repair mechanism anymore and are bound to die (Fig. 1; take a look at PARPs as cancer drug targets – first EC-approved drug to learn more about the concept of “synthetic lethality”). [Read more…]

Cellular models to study the Cardiac System (part II)

In a previous post, I introduced several models to study the cardiac human system. This first post introduced human aortic, brachiocephalic, carotid artery and coronary artery cells isolated by Cell Applications Inc. Here is the second part of this inventory of cellular models to study the human cardiac system, where I’ll be highlighting human internal thoracic artery, pulmonary artery, subclavian artery cells, cardiac fibroblasts and cardiomyocytes.

Later on, I’ll conclude this series by part III, referring to animal cellular models for studying the cardiac system. But let’s now concentrate on today’s topic! [Read more…]

Caspases as pharmaceutical targets – screening for inhibitors?

Caspases (cysteine-dependent aspartate-directed proteases) belong to the family of cysteine proteases and are involved in networks controlling cell death (apoptosis and necrosis) and inflammation. 12 human caspases have been described so far (1.). Human Caspases have been classified according to their roles in apoptosis (Caspase-3, -6, -7, -8, and -9) and inflammation (Caspase-1, -4, -5, and -12). Caspase-2, -10, and -14 can be less easily classified concerning the function (for an overview see 2.).

So let’s take a further look at their role, and some of the tools available to investigate and screen compounds modifying Caspase activities.

[Read more…]

Optimal substrate design for protease screening!

EnSens assays, a new protease activity assay technology, which are produced by  Enzium, were recently introduced to the European market. A number of  assays are available from catalog, such as activity assays for MMP-2, MMP-9, MMP-14, MMP-25, ADAM10, ADAM17, Factor Xa, Furin, and Thrombin.

But what about if what you’re looking for is not available? In this post, we’ll take a look at how you can get an optimal substrate for your protease of interest, even if it is not yet obtainable from catalog.

EnSens technology

First, let’s take a look at the EnSens assay technology itself. It’s based on a non-FRET technology and makes use of a protein substrate expressed in E. coli which is a modular dumbbell comprising two domains separated by a linker that contains a highly specific protease recognition sequence. One domain is the dye-binding domain, and the other is a blocking domain. When the linker is cleaved by a protease, separating the two domains, the binding domain is free to bind dye, constraining it and causing it to fluoresce (see Figure below).

EnSens principle

EnSens assays overcome limitations of FRET based protease assays

  • the substrate can incorporate full recognition site sequences – in FRET assays the recognition sites usually have to be shorter which can result in a number of false positives and negatives because the recognition site might not be highly selective.
  • The substrate is an scFv-based protein (single-chain variable fragment, an artificially generated antibody fragment) which confers high stability in vitro and in vivo.
  • The EnSens se-Red fluorophore is resistant to photobleaching up to 48 hours
  • The long emission wavelength (665nm) reduces interference from other assay components
  • The assay works stably in wide range of pH, salt, & solvent conditions
  • Substrate and fluorophore are separate in the systems, so the fluorophore is constantly renewed in solution
  • The system is characterized by high signal-to-noise ratios: 6-20x
  • The read-out can be done in standard plate or cuvette fluorimeters
  • The EnSens assay gives repeatable and reproducible kinetic activity curves from reagent to reagent

and finally (and this is where it gets interesting for non-catalog items!):

  • The substrate is modular, allowing easy swapping of protease recognition sites – thus customized substrates can be obtained if you know the recognition site of your protease of interest

So, you can get your customized protease substrate!

In other words: If you want to screen for modulators/inhibitors of a protease with a known recognition site, you simply need to provide the sequence of the site – and the substrate and the complete EnSens assay can be designed for you in cooperation with Enzium.

Interested in a highly specific protease assay for your screening? Get in touch through the form below, I’m sure a tailored solution can be found to match your needs exactly!