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.

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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.

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Immunotherapy Screening – IDO pathway

In previous blogs, I invited you to join me in exploring the relevance of the following pathways:

PD1 - PD-L1 - PD-L2

 

Today I’d like to focus on the IDO pathway. [Read more…]

Immunotherapy Screening – CD137:CD137L pathway

In previous blogs, I invited you to read about the relevance of the B7-1 : CD28, B7-1 : CTLA4, the BLTA:HVEM, CD47:SIRPα , the GITR:GITRL, the CD40:CD40L and the  PD-1/PD-L1/PD-L2 pathway for immunotherapy screenings and discussed the products available to work on these pathways. Today, I will focus on the CD137:CD137L pathway.

CD137 is another co-stimulatory protein that is expressed on activated T cells. Unlike CD40:CD40L signaling, which primarily involves helper T-cells, CD137 has a crucial role in the development of cytotoxic T-cells and anti-tumor immunity. Its ligand, CD137L, is mainly expressed on antigen-presenting cells, such as activated B cells, macrophages, and dendritic cells, as well as on human tumor cells.

Co-stimulation through CD137:CD137L enhances T-cell activation, promotes the rejection of cardiac allografts and skin transplants, and eradicates experimentally induced tumors in animal models. Several clinical studies are on-going that use agonistic anti-CD137 antibodies to induce an anti-cancer response to solid tumors. [Read more…]

Immunotherapy Screening – CD40:CD40L pathway

In my previous blogs, I invited you to read about the relevance of the B7-1 : CD28, B7-1 : CTLA4, the BLTA:HVEM, CD47:SIRPα , the GITR:GITRL and the PD-1/PD-L1/PD-L2 pathway for immunotherapy screenings and discussed the research products available to study these pathways. Today, I’d like to focus on the CD40:CD40L pathway.

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Immunotherapy Screening – GITR:GITRL pathway

In previous blogs, I invited you to read about the relevance of the B7-1 : CD28, B7-1 : CTLA4, the BLTA:HVEM, CD47:SIRPα , and the  PD-1/PD-L1/PD-L2 pathway for immunotherapy screenings and discussed the products available to work on these pathways. Today, let’s focus on the GITR:GITRL pathway (glucocorticoid-induced tumor-necrosis-factor-receptor-related protein).

The GITR:GITRL pathway is a very important target for immunotherapy drug discovery — in fact, the National Cancer Institute (USA) identified GITR modulation as one of the top 25 most promising research areas! GITR is expressed on most immune cells, including T-cells, B-cells, macrophages, and NK cells. Binding of GITR by the GITR ligand (found on antigen-presenting cells and many tumors) activates the immune response by stimulating macrophages and triggering T-cells to expand, proliferate, and differentiate. Using anti-GITR mAb to trigger GITR signaling is effective in treating viral, bacterial, and parasitic infections, as well in boosting immune response against tumors. Not surprisingly, clinical trials testing anti-GITR mAb in melanoma patients are already in progress (1).GITR - GITRL Figure

Of course, it’s never that simple. Stimulating GITR signaling in NK cells has the opposite effect compared to T-cells and macrophages—GITR signaling inhibits NK cell activity (2). Likewise, stimulation of T-suppressor cells by GITR signaling also down-regulates the immune response.   Researchers are using this to their advantage; down-regulation of the immune response can be used to treat autoimmune and inflammatory diseases, including arthritis, allergy, inflammatory bowel diseases, and graft rejection. Therefore, GITR-Fc fusion protein and mAb that block GITR signaling instead of stimulating the pathway are also being studied as potential drug candidates.

BPS Biosciences recently released a set of products linked to immunotherapeutically relevant targets, amongst them the human glucocorticoid-induced TNF-related ligand (GITRL), also known as AITRL, CD357L, or tumor necrosis factor ligand superfamily member 18 (TNFSF18).

If you are interested in this specific ligand or any other of the topics and reagents covered in previous blogs (there are links to them at the beginning of this post), don’t hesitate to get in touch (you can use the form below).

References:

(1) Nocentini, G., et al., Br J Pharmacol. 165(7): 2089-99 (2012)

(2) Barao, I., Front Immunol. 3: 402 (2012)

Where can I find the best PARPs?

Poly (ADP-ribose) polymerase (PARP) is a family of proteins which catalyze the NAD-dependent addition of Poly ADP-ribose (PAR) chains to nuclear proteins. PARPs play key signaling roles in apoptosis, DNA excision and repair, telomere stability, and chromatin structure. PARPs are also involved in transcriptional regulation of several signaling pathways, including genes involved in cancer and inflammation.

PARPs are composed of four domains of interest: a DNA-binding domain which contains two zinc finger motifs, a caspase-cleaved domain, an auto-modification domain, and a catalytic domain.  Upon DNA damage, the DNA-binding domain binds the damaged DNA and induce a conformational shift, subsequently the catalytic domain starts with the synthesis of a poly (ADP-ribose) chain (PAR) as a signal for the other DNA-repairing enzymes e.g. DNA ligase III. Finally the auto-modification domain is responsible for releasing the protein from the DNA after catalysis and plays an integral role in cleavage-induced inactivation.

PARP1 activity measured using the PARP1 Chemiluminescent Activity Assay Kit, (Cat. 14980569). Luminescence was measured using a Bio-Tek microplate reader. IC50 determination of the PARP inhibitor AZD2281.

PARP1 activity measured using the PARP1 Chemiluminescent Activity Assay Kit, (Cat. 14980569). Luminescence was measured using a Bio-Tek microplate reader. IC50 determination of the PARP inhibitor AZD2281.

Broadest portfolio of PARPs and PARP related products

If you’re studying enzyme kinetics or screening small molecular inhibitors for drug discovery and HTS applications, you’ll need to access PARP isozymes. BPS Biosciences provides 13 different PARP isozymes (out of 17 isozymes known). They also have a selection of 18 different kits for your activity and inhibitor screening assays, as well as a large number of PARP inhibitors and substrates.  All these products have shown to be reliable and trouble-free.

Are you working on PARP related screening projects? Share any comments or ask your questions below!

MMP-25 activity assay – the first one on the market!

With the recent release of the first commercially available and ready-to-use assay to detect MMP-25 activity , Enzium has opened a new era for screening protease activities through a robust non-FRET experimental approach. A good opportunity to take a look at the EnSens technology.

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Mutant or wild-type: 7 effective means to screen and profile Human EZH2 Methyltransferase

Histone-lysine N-methyltransferase EZH2  is a methyltransferase involved in transcriptional gene regulation. Together with with EED, RbAP48, SUZ12 and AEBP2, EZH2 forms a 5-member complex that adds 3 methyl groups to Lysine 27 of Histone H3 (H3K27). EZH2 acts on chromatin condensation and gene transcription activity.

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