Ras belongs to the family of small G-proteins and plays an important role in several signal transduction pathways involved in normal cell growth and differentiation. Over 30 years ago three isoforms of Ras – H-Ras, N-Ras, and K-Ras, have been identified for their oncogenic activation in human cancer cells. In fact aberrant Ras signaling could be shown in more than 30% of all human cancers including lung, colon, and pancreatic cancer. K-Ras has been identified as the most important Ras protein in cancer research.

As other small G proteins, Ras cycles between inactive (GDP-bound) and active state (GTP-bound) forms (see figure).

Although extensive research activities have been dedicated to Ras, no effective Ras inhibitor has been identified so far. But, there may be new hope in developing a therapeutic inhibitor to Ras proteins by targeting the upstream guanine nucleotide exchange factor (GEF) protein SOS (Son of sevenless).

In the summer of 2013, my colleague Frederic Dubor, who is in charge of our services lab, mentioned to me that he had just been asked by 3 clients within a very short time-frame, whether our laboratory would  be able to run histone binding studies for them. In fact, it wasn’t about binding to single full length histones, but all 3 clients had asked for multiplex arrays, meaning they were interested in measuring binding of either antibodies (epitope mapping) or epigenetic readers (bromodomains and methyl readers) to a set of histone derived peptides, without and with a number of modifications such as acetylation and methylation,

At that time, neither Frederic nor I could help – but these requests did make us search for a supplier offering arrays which would meet these needs. Half a year later,  we were pleased to announce a new cooperation with Epicypher, one of the market leaders in epigenetic proteomics.

But I’m running ahead a little quickly… I’d first like to summarize the potential applications of the arrays we were not able to offer one year ago.

So, what precisely were researchers looking for when asking to “run histone binding studies”?

1. The determination of the exact modification on a specific histone recognized by an antibody raised against histones. In fact, this could be meant as a strigent quality control experiment for an antibody to be used to recognize specific histone modifications. Furthermore, such a specific antibody could be used to block out the binding of any other protein to the histone target sequence. As the 4 histones which make the nucleosome’s histone octamer (H2A, H2B, H3, and H4) can be epigenetically modified by a number of chemical reactions, such as acetylation, methylation, phosphorylation, ubiquitination, sumoylation, and ADP Ribosylation, theoretically thousands of modification patterns can be found on histones.

2. The determination of specific binding sites of epigenetic “readers”, such as bromodomains (recognizing acetylated sites) and methyl readers. These “readers” can be domains of “writers” (enzymes modifying histones, e.g. methyl transferases and acetylases) or “erasers” (e.g. demethylases and deacetylases). Thus, these modifying enzymes can be targeted to their sites of action on histones.

3. Furthermore, a third application field for peptide arrays containing diverse modified and non modified histone sequences could be the analysis of  the substrate specificity of entire histone modifying enzymes.

All these applications could help in answering questions in research fields and diseases which are linked to epigenetic histone modifications such as cancer, aging, asthma, diabetes, cardiovascular diseases, neurodegeneration, autoimmune disorders, and developmental disorders.

We found arrays to answer all these questions: Precise histone binding studies are now possible with EpiCypher’s EpiTitan Histone Peptide Arrays

The EpiTitan™ Histone Peptide Arrays represent the next generation in histone peptide arrays, and are the gold standard against which all other histone peptide arrays are judged.

The arrays feature hundreds of highly purified and well-characterized histone peptides spotted onto the substrate. EpiTitan™ Histone Peptide Arrays are a dramatic improvement over arrays in which the peptides are synthesized on the substrate (SPOT synthesis), a technique that leads to peptides on the array that are of unknown quantity and quality. All the peptides on the EpiTitan™ Histone Peptide Arrays are purified by HPLC and validated by mass spectrometry prior to spotting.

The technique used in this array is quite easy.

• Biotinylated peptides are spotted 12 times each per array on a surface covered with streptavidin to catch the peptides.
• Each array slide comes with two subarrays, thus two samples per slide can be evaluated.
• The protein to be investigated is added and binds to its specific target sequence and modification pattern.
• The detection of binding either starts with an antibody against the protein of interest or an anti tag antibody, should the protein carry the respective tag, and can be visualized using fluorescence scanning or standard ECL techniques – by the way the EpiTitan™ Histone Peptide Array is the only array available on the market which supports both detection methods.
• Protocols for qualitative as well as quantitative analysis of the data are provided.

Design of the Arrays and experimental steps

Interested in more information or using EpiTitan™ Histone Peptide Arrays yourself?

Just get in touch if you’d like to check the list of histone peptides and the user manual. I’ll be happy to get back to you to tell you more about these Histone arrays and companion reagents (Epigenetic readers and erases or bioactive small molecules…).

While reading this blog you were maybe already thinking about how you could apply this array in your research project…

Then I have good news for you. Our partner Epicypher just started a grant program – please have a look at our previous blog: 5 fully funded Epigenetic Grants offered by Epicypher.

The principle of the grant program is very easy – you supply a brief outline of a study which involves the role of histone modifications in physiological or pathological processes – Epicypher supplies arrays free of charge to five applicants chosen by an independent scientific committee.

The treatment of diseases by inducing, enhancing, or surpressing an immune response is referred to as Immunotherapy. T-cell activation and inactivation requires the coordination of various co-inhibitory and co-stimulatory signals which are modulated by most immunotherapeutic approaches.

Therapeutic manipulation of immunopathways has lead to promising clinical results for the treatment of a number of diseases (ex. cancer, autoimmune diseases and inflammatory diseases…). Research projects in this field are rapidly evolving as scientists seek to identify the next generation of therapies.

In an earlier post on this blog, I introduced the latest advances regarding the the B7-1 : CD28 and B7-1 : CTLA4 pathways for immunotherapy screenings. Today, I’d like to focus on the PD-1/PD-L1/PD-L2 pathway.

After a brief review of the role of this pathway in immune response, let’s take a look at some of the screening tools I’ve selected (by our partner BPS Biosciences) to investigate this pathway for drug discovery applications. Interestingly, some of these tools are the first ready-to-use assay kits made available for drug discoverers to screen for inhibitors of the PD-1/PD-L1/PD-L2pathway.

Why look at PD-1/PD-L1/PD-L2 pathways in immunotherapeutics?

PD-1 is a receptor present on the surface of activated T-cells and other immune cells.  PD-1 has two ligands, PD-L1 and PD-L2, which are overexpressed in most human cancers.  Binding of PD-L1 to PD-1 negatively regulates T cell signaling and inhibits cytotoxic T-cell activity.

Similarly, engagement of PD-1 by PD-L2 dramatically inhibits T-cell proliferation, especially in CD4+ (T helper) cells. Therefore, this upregulation of PD-L1 and PD-L2 may allow cancers to evade the host immune system.

Neutralizing antibodies can prevent PD-L1 from binding to PD-1 or to B7-1. Blockade of PD-L1 enables the activation of T-cells, restoring their ability to detect and attack tumor cells. Therefore a number of pharmaceutical companies are actively developing monoclonal antibodies targeting PD-1 and PD-L to boost the immune system for the treatment of non-small-cell lung cancer, melanoma, and bladder, renal, and triple-negative breast cancers.

The PD-1/PD-L pathway is also a therapeutic target for chronic viral-infections (including HIV and HCV). This therapeutic appraoch is based on the observation that PD-1 expression is upregulated by virus-specific T cells and that the inhibition of this loop is hoped to decrease viral load by increasing  T cell function. Conversely, since the PD-1/PD-L interaction regulates self-tolerance and immunologic homeostasis, agonists of these proteins are promising drug targets for many autoimmune disorders including multiple sclerosis, arthritis, lupus, and type I diabetes.

The most advanced PD-1/PD-L1/PD-L2 screening reagents

These new therapeutical advances highlight the importance of reliable R&D tools in Drug discovery approaches and translation medicine programs. Access to robust research reagents covering the needs of Drug discoverers from target validation to screening (HTS or secondary) or even DMPK studies is crucial. Below, I’ve made a selection of some of the latest releases for scientists interested in the PD-1/PD-L1/PD-L2 pathway.

         #1 – PD-1  – a receptor for PD-L1

PD-1 is the receptor for PD-L1. Formation of the receptor-ligand complex leads to an inhibitory signal which reduces proliferation of CD8x T cells.

PD-1 is available as a Fc Fusion (Id. nr 71106), as a FLAG-tagged variant (Id. nr 71115), and as a Biotin labeled variant (Id. nr 71109)

        #2 – PD-L1 – a ligand for PD-1 receptor

PL-L1 is one of the ligands for PD-1, which plays a role in surpressing the immune response especially during pregnancy, tissue allografts, autoimmune diseases and diseases such as Hepatitis.

PL-L1 is available as a non labeled variant (Id. nr 71104) and a Biotin-labeled variant (Id. nr 71105).

        #3 – PD-L2 –  aligand for PD-2 receptor

PL-L2 is another ligand for PD-1 which, upon complex formation T cell proliferation, especially of CD4+ (T helper) cells, is dramatically decreased.

PL-L2 is available as a non labeled variant (Id. nr 71107) and a Biotin-labeled variant (Id. nr 71108).

        #4 – PD1-neutralizing antibody

This antibody (Id. nr 71120) can be used as a control inhibitor using the PD-1:PD-L1[Biotinylated] Inhibitor Screening Assay Kit (see below).

Ready-to-use PD-1/PD-L1/PD-L2 screening assay kits

Recently, BPS Biosciences have released 3 unique ready-to-use assay kits to screen and profile inhibitors for the PD-1/PD-L1/PD-L2 signaling pathways

PD-1:PD-L1[Biotinylated] Inhibitor Screening Assay Kit (Id. nr 72003)

PD-1:PD-L2[Biotinylated] Inhibitor Screening Assay Kit Id. nr 72004)

Both are 96-well format assay kits and come with biotinylated PD-L1 or PD-L2, purified PD-1,  streptavidin labeled HRP for 100 binding reactions.

Typical data obtained with the BPS’ Ready-to-use Inhibitor Screening assays.

PD-L2 Inhibitor Screening Assay Kit (Id. nr 72006).

This 96-well format assay kits comes with biotinylated PD-1, purified PD-L2,  streptavidin labeled HRP  for 100 binding reactions.

Looking for PD-1/PD-L1/PD-L2 testings?

Choosing the right reagents or screening kits to raise the value of your compounds of interest by looking to PD-1/PD-L1/PD-L2 pathway can be challenging.

Research tools reviewed in this second post (belonging to a series of 3 posts dedicated to  Immunotherapy screening) will definitely help you there. Otherwise, you can simply contact me with the form below.

Recently released Immunotherapy Screening posts on the blog “being bio-reactive”:

• Immunotherapy Screening – Part 1:  B7-1:CD28 and B7-1:CTLA4 Pathways

Upcoming Immunotherapy Screening posts on the blog “being bio-reactive”:

• Immunotherapy Screening – Part 3:  BLTA:HVEM and CD47:SIRPalpha Pathways

The treatment of diseases by inducing, enhancing, or surpressing an immune response is referred to as Immunotherapy. T-cell activation and inactivation requires the coordination of various co-inhibitory and co-stimulatory signals and most immunotherapies modulate these signals.

Therapeutic manipulation of immunopathways has lead to promising clinical results for the treatment of a number of diseases such as cancer, autoimmune diseases and inflammatory diseases. Research in this field is rapidly evolving as scientists seek to identify the next generation of therapies.

In the first post of a series of three,  I will describe the B7-1 : CD28 and B7-1 : CTLA4 pathways and summarize the portfolio of tools currently available to researchers to investigate these pathways. In fact, these are the first kits on the market to screen for inhibitors of these pathways.