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…]

PBMCs & drug development

Use of human samples including peripheral blood mononuclear cells (PBMCs) in drug discovery is critical for increasing the chances of success for a small molecule screen hit. A review based on recent published data!

PBMCs DD 1Several theories try to explain why many drug discovery and development projects have a moderate success rate. Even with the advances made in methods (HTS, HCA, library managment, etc), there are many hurdles for a small molecule on the long road to FDA or EMA approval.

One way that may improve predicted efficacy and toxicity of drug leads is to use human samples, such as blood, early on in drug discovery programs. Peripheral blood mononuclear cells  (PBMCs) can be easily used in a variety of ways during the drug discovery process to gain a better understanding of the effects of a small molecule. [Read more…]

PARP inhibitors involved in Cancer and Parkinson research

In a recent post (PARPs as cancer drug targets – first EC-approved drug) I wrote about Olaparib, a drug against ovarian cancer, which has been developed by AstraZeneca and was approved in December 2014 by the European commission – thus being the first drug directed against a member of the PARP family (Poly ADP-Ribose Polymerases). Furthermore I informed about the tools tebu-bio is able to provide to screen for PARP inhibitors, such as active PARP enzymes and ready-to-use inhibitor screening assays for PARP1 , 2, 3, 6, 7, 10, 11, 14, and 15 as well as for Tankyrase 1 and 2.

In this post, let’s take a look at PARP modulators/reference inhibitors especially linked to cancer and Parkinson’s disease research and screening activities. [Read more…]

PARPs as cancer drug targets – first EC-approved drug

In December 2014, the European commission approved the first drug directed against a member of the PARP familiy (Poly ADP-Ribose Polymerases). Olaparib, a drug against ovarian cancer, has been developed by AstraZeneca and might become an important medicine very soon. Olaparib blocks PARP1 (see the Figure below showing the inhibitory effect of Olaparib on PARP1 measured with the PARP1 Chemiluminescent Activity Assay), an enzyme involved in cell repair, and is designed for ovarian cancer patients with certain hereditary gene mutations. It also has promise in treating other cancers, including breast and gastric tumors, opening up a substantial market opportunity. Olaparib is able to stop cancer cell growth in patients carrying inherited faults in the BRCA1 or BRCA2 genes (BReast CAncer 1 and 2 genes). [Read more…]

Microtubule destabilization by suprafenacine: Template for novel anti-cancer drugs

Recently, B.-H. Choi et al. characterized a novel anti-mitotic molecule termed suprafenacine which destabilizes microtubules, resulting in cell cycle arrest in the G2/M phase and apoptotic cell death.  In silico screening identified several novel anti-cancer molecules based on their ability to inhibit in vitro cell proliferation and tubulin Tubulin cartoonpolymerization.  Structure Activity Relationship studies guided the synthesis of several analogues.  Of these analogues, suprafenacine was the most potent based on its in vitro ability to 1. specifically target cancer cells from multiple tumor types and 2. inhibit tubulin polymerization (IC50 = 0.38 mM).  [Read more…]

Predicting CYP induction in stable cell lines

Assessing gene transcription as an endpoint for determining induction of drug metabolizing enzymes and transporters has become the “method of choice” in the FDA’s latest draft Guidance for Industry drug interaction studies. Increased gene transcription due to NCE exposure can be determined by two distinct methods, nuclear receptor activation and changes in mRNA levels in primary hepatocyte cultures.

[Read more…]

Biomimetic Chemistry, a new route for metabolite synthesis

In 2008, the FDA released guidance for drug metabolite safety testing (MIST), emphasizing the importance of metabolite toxicity testing in the drug development process. Indeed, drug toxicity, which accounts for roughly 40% of clinical drug failures, is a leading cause of the high drug attrition rates that have contributed to the skyrocketing drug development costs witnessed over the past few decades.

Traditionally, drug metabolites have been both difficult and hugely expensive to synthesize. Conventional methods of metabolite synthesis, such as those that employ the use of microsomes (while they have proven valuable as a predictive tool, their productive capabilities could be limited by NCE stability) or synthetic chemistry, can be extremely costly and time consuming. Consequently, drugmakers often choose to forego metabolite synthesis (and subsequent metabolite toxicity testing ) early on in the drug development process, opting instead to wait until lead compounds are further along in development before carrying out these essential functions. This decision, perceived to be a calculated risk, ultimately comes at huge price, as drug makers lose millions each year on investments in lead drug candidates that eventually turn out to be failures due to toxicity.

Biomimetic Chemistry, on the other hand, possesses the advantages of both chemistry and biology and is thus a much more efficient tool for metabolite synthesis. In fact, with biomimetic chemistry, large scale metabolite generation is enabled in one step, by mimicking and optimizing the same biotransformation reactions that occur in the liver. [Read more…]

How to easily detect luciferase activity in cell based assays?

Luciferase is the general term given to a class of oxidative enzymes which catalyze reactions that give off light, a process known as bioluminescence (Fig. 1).

structure and mechanism

Fig. 1: Mechanisms of the assay

In biology, researchers take advantage of this reaction to use it as a readout for various biological processes. This has perhaps been exploited most in luciferase reporter cell lines where a promoter region from a gene of interest is placed immediately upstream of the coding sequence for luciferase. In this system, transcriptional activation of the gene of interest leads to a level of luciferase expression that is proportional to the level of gene activation.

The level of activation is then assessed by lysing the cells and adding the luciferase substrate, D-luciferin. Especially in screening processes it is crucial that the assay be accomplished in a single step, meaning the lysis buffer has to be formulated with D-luciferin. [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…]

Characterization & Applications of Human Cardiomyocytes

ReproCardio 2 Human iPSC-derived cardiomyocytes have been extensively characterized for their functional responsiveness to the known cardiotoxic compounds. In all our tests, including assays of cardiotoxic compounds not correctly identified by the hERG assay, ReproCardio has generated results in complete agreement with clinical findings.

In this post, we’ll highlight the characterisation of ReproCardio 2 cardiomyocytes and illustrate the applications they are used for. [Read more…]