Your New Best Research Partner: The Structural Genomics Consortium

Research surrounding drug discovery has historically been highly competitive and expensive. Unfortunately, many late-stage drug failures have occurred over recent years, often due to lack of efficacy. These failures have left the industry searching for new means by which to improve early drug discovery efforts aimed at understanding the drug target and its role in disease. One idea that is gaining traction is partnerships to openly share information at the early, precompetitive stages of drug discovery.

I used to think of open access only in terms of publishing data and information—online sites where you could freely access data without a subscription or membership, and without payment.

Structural Genomics Consortium logo.

Meet the Structural Genomics Consortium (SGC), the international partnership that’s taking open access to a new level in order to advance scientific research for scientists working in a variety of disciplines—structural genomics and beyond. The SGC might just become your new, best laboratory research partner. Continue reading

Don’t Let These Three Common Issues Hurt Your Luminescent Assay Results

4621CAThere is a lot riding on your luminescent assay results. Each plate represents precious time, effort and resources. Did you know that there are three things about your detection instrument that can impact how much useful information you get from each plate?  Instruments with poor sensitivity may cause you to miss low-level samples that could be the “hit” you are looking for.  Instruments with a narrow detection range limit the accuracy or reproducibility you needed to repeat your work.  Finally, instruments that let the signal from bright wells spill into adjacent wells allow crosstalk to occur and skew experimental results, costing you time and leading to failed or repeated experiments. Continue reading

The Role of the NanoLuc® Reporter in Investigating Ligand-Receptor Interactions

Luminescent reporter assays are powerful research tools for a variety of applications. Last March we presented a webinar on this topic, Understanding Luminescent Reporter Assay Design, which proved to enlighten many who registered. The webinar addressed the importance of careful experimental design when using a luminescent reporter such as Promega’s Firefly or NanoLuc® Luciferase.

Reporters provide a highly sensitive, quantifiable metric for cellular events such as gene expression, protein function and signal transduction. Luminescent reporters have become even more valuable for live, real-time measurement of various processes in living cells. This is backed by the fact that a growing number of scientific publications reference the use of the NanoLuc® Luciferase reporter and demonstrate its effectiveness as a reporter assay. Continue reading

Bioassay for Cannabinoid Receptor Agonists Designed with NanoBiT™ Techology

Cannabinoids. What are they? Sometimes, Wikipedia can give a nice definition:

Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons

Tetrahydrocannabinol (THC), a partial agonist of the CB1 and CB2 cannabinoid receptors. Wikipedia Commons

A cannabinoid is one of a class of diverse chemical compounds that acts on cannabinoid receptors in cells that alter neurotransmitter release in the brain. Ligands for these receptor proteins include the endocannabinoids (produced naturally in the body by animals), the phytocannabinoids (found in Cannabis and some other plants), and synthetic cannabinoids (manufactured artificially).

Synthetic cannabinoids (SCs) were originally created for the scientific investigation of two cannabinoid receptors, CB1 and CB2, but have made their way to the streets as “safe” and “legal” alternatives to marijuana.

The problem is that these SCs engage the cannabinoid receptors more completely and with higher affinity than anything derived from marijuana. As a result, SCs can produce serious side effects that often require medical attention. In fact, you are 30 times more likely to seek emergency medical attention following the use of an SC than with natural cannabinoid sources like marijuana. Continue reading

microRNA: The Small Molecule with a Big Story

Introduction

miR-34 precursor secondary structure. The colors indicate evolutionary conservation. Ppgardne [GFDL (http://www.gnu.org/copyleft/fdl.html) or CC BY-SA 3.0 (http://creativecommons.org/licenses/by-sa/3.0)], via Wikimedia Commons

RNA molecules have become a hot topic of research. While I was taught about messenger RNA (mRNA), ribosomal RNA (rRNA) and transfer RNA (tRNA), many more varieties have come into the nomenclature after I graduated with my science degrees. Even more interesting, these RNAs do not code for a protein, but instead have a role in regulating gene expression. From long non-coding RNA (lncRNA) to short interfering RNA (siRNA), microRNA (miRNA) and small nucleolar RNA (snoRNA), these classes of RNAs affect protein translation, whether by hindering ribosomal binding, targeting mRNA for degradation or even modifying DNA (e.g., methylation). This post will cover the topic of microRNAs, explaining what they are, how researchers understand their function and role in metabolism, cancer and cardiovascular disease, and some of the challenges in miRNA research.

What are microRNAs? MicroRNAs (miRNAs) are short noncoding RNAs 19–25 nucleotides long that play a role in protein expression by regulating translation initiation and degrading mRNA. miRNAs are coded as genes in DNA and transcribed by RNA polymerase as a primary transcript (pri-miRNA) that is hundreds or thousands of nucleotides long. After processing with a double-stranded RNA-specific nuclease, a 70–100 nucleotide hairpin RNA precursor (pre-miRNA) is generated and transported from the nucleus into the cytoplasm. Once in the cytoplasm, the pre-miRNA is cleaved into an 18- to 24-nucleotide duplex by ribonuclease III (Dicer). This cleaved duplex associates with the RNA-induced silencing complex (RISC), and one strand of the miRNA duplex remains with RISC to become the mature miRNA. Continue reading

Finding a Connection Between Glucose Metabolism and Macrophage Activation

Introduction to Glucose Metabolism

Macrophages. By NIAID (https://www.flickr.com/photos/niaid/17380707492/) [CC BY 2.0 (http://creativecommons.org/licenses/by/2.0)], via Wikimedia Commons

Many think of glucose as something diabetics have to test each day using a blood monitor, or a quick energy boost for someone exercising intensely. However, the simple sugar glucose, a monosaccharide, fuels most of the cells in our bodies. Disaccharides that contain glucose (e.g., sucrose is comprised of glucose and fructose) and glucose polymers (e.g., starch and glycogen) are carbohydrates that are consumed by organisms from bacteria to humans to produce energy. These carbohydrates are broken down into component monosaccharides like glucose and lactose. The process of glycolysis generates the energy currency of cells, ATP, as well as precursor molecules for nucleotides, lipids and amino acids. Because glucose is the cell fuel source, the uptake of glucose and its subsequent metabolism is increased by cells that divide rapidly like cancer cells. The more energy and precursor molecules the cancer cell can create for itself, the more rapidly the tumor can grow.

Because glucose metabolism is central to cellular functioning, changes that decrease glucose uptake or increase glycolysis have a widespread effect on on both the cells and organism. How does a simple sugar molecule create such broad effects on health? For example, diabetes results from the inability to store glucose because of a lack of insulin, a hormone that draws glucose from the blood and stores it as glycogen in the liver, muscles and adipose tissue. High levels of sugar in the blood negatively affect the body over the long term, damaging blood vessels and eyesight, making the kidneys work harder to excrete the excess sugar and increasing the risk of stroke and coronary artery disease. Because cancer cells have such a high metabolic demand for glucose, many of the mutations in cancers affect pathways that regulate glucose uptake and glucose breakdown, allowing the cancer cells to survive and grow, crowding out nearby normal cells.

Glucose metabolism is altered by processes other than mutations or an reduced production of a hormone. Throughout its life cycle, a cell will vary its requirements for glucose. For example, the cells that comprise our innate immune response are typically in a quiescent or steady state. However, when these immune cells encounter an foreign invader, they become activated and increase their demand for glucose. To respond to a potential pathogen, the activated cells need glucose to fuel cell proliferation and the production of cytokines, chemicals that activate other immune cells and initiate an inflammatory response. The typical signs of inflammation are red inflamed area that may be painful to the touch, such as a cut that becomes infected. Most inflammation resolves when the infection is eliminated, leaving behind whole skin in the instance of a cut, and the activated immune cells become quiescent again.

An Interesting Observation about Glucose Metabolism in M2 Macrophages

Glucose uptake, immunity and metabolism are cellular pathways that are intertwined such that understanding how glucose is utilized in macrophages illuminates gene induction and regulation in activated macrophages. In a recently published eLife article, Covarrubias et al. studied how activation of murine bone marrow-derived macrophages (BMDMs) by interleukin-4 (IL-4), a signaling cytokine, altered glucose metabolism in the cells and regulated a subset of genes involved in macrophage activation. Continue reading

Increasing Drug Research and Development Efficiency Using a 4-point Screening Method to Determine Molecular Mechanism of Action

Fig 4. Four point MMOA screen for tideglusib and GW8510. Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature. Experiments run with 10μM GSM peptide, 10μM ATP, and buffer. Minute preincubation (30 min) was preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. No preincubation contained inhibitor, GSM peptide, ATP, and buffer. The reaction was initiated with TbGSK3β. Reactions were run at room temperature for 5 min and stopped at 80°C. ADP formed was measured by ADP-Glo kit. Values are mean +/- standard error. N = 3 for each experiment and experiments were run in duplicates. Control reactions contained DMSO and background was determined using a zero time incubation and subtracted from all reactions. Black = 30 min preincubation Grey = No preincubation.

Four point MMOA screen for tideglusib and GW8510.
Time dependent inhibition was evaluated by preincubation of TbGSK3β with 60 nM tideglusib and 6 nM GW-8510 with 10μM and 100μM ATP. (A). Tideglusib [60 nM] in 10μM ATP. (B). GW8510 [60 nM] in 10μM ATP. (C.) Tideglusib [60 nM] at 100μM ATP. (D.) GW8510 [60 nM] at 100μM ATP. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature.  Black = 30 min preincubation Grey = No preincubation.

The first small-molecule kinase inhibitor approved as a cancer therapeutic, imatinib mesylate (Gleevec® treatment), has been amazingly successful. However, a thorough understanding of its molecular mechanism of action (MMOA) was not truly obtained until more than ten years after the molecule had been identified.

Understanding the MMOA for a small-molecule inhibitor can play a major role in optimizing a drug’s development. The way a drug actually works–the kinetics of binding to the target molecule and how it competes with endogenous substrates of that target–ultimately determines whether or not a a candidate therapeutic can be useful in the clinic. Drugs that fail late in development are extremely costly.

Drug research and discovery for neglected tropical diseases suffer from a lack of a large commercial market to absorb the costs of late-stage drug development failures. It becomes very important to know as much as possible, simply and quickly, about MMOA for candidate molecules for these diseases that are devastating to large populations.

One such neglected topical disease is Human African trypanosomiasis (HAT, also known as sleeping sickness). Continue reading

Inflammasome Research: A Tool to Aid Progress

Inflammasomes: A Few Basics

Inflammasomes are protein complexes composed of immune system receptors and sensor molecules. These complexes can respond to both infectious organisms and molecules derived from host proteins. When activated, a series of receptors and molecules signal via either pathogen-associate molecular patterns (PAMPs) induced by microbial pathogens, or danger-associated molecular patterns (DAMPs) induced as a result of endogenous stressors; the common next step in signaling is through pattern recognition receptors (PRR).

Inflammasome diagram.

Innate immune response in inflammation; a basic diagram.

Inflammasome activation is integral to the host immune response in mice and humans (1). The activation results in signaling that activates the caspase-1 scaffold, causing release of immune mediators such as interleukins IL-1β and IL-18. So, whether inflammation is host tissue- or pathogen-induced, inflammasome activation results in a cascade of receptor signaling and mediator release, of which caspase-1 is a critical component.

Continue reading

Making Drug Discovery More Efficient: Predicting Drug Side Effects in Early Screening Efforts

26911030-Laymans-KSPS-figure-WEB-R4Drug research and development is a complex and expensive process that begins with initial screening steps of candidate chemical compounds, and compounds that appear to have the desired potency against a specific cellular target or pathway are further evaluated. Candidate compounds that fail late in development or during clinical trials because of off-target effects are costly, and can be dangerous. Therefore drug developers not only need to ensure that a candidate compound is effective as a therapy, but also they need to predict any potential undesirable side effects due to off-target activities as early as possible in the drug discovery and development process. Continue reading

Customized Kinase Selectivity Profiling Just Got Easier

11296971-DC-CR-KinaseOff-target activities of target compounds can become costly if they aren’t discovered until late in the drug research and discovery process. Therefore, knowing the inhibitory profile of your test compounds across a broad collection of kinases as quickly as possible is highly desirable.

However, screening against many kinases at once requires a universal platform that is still sensitive enough to detect inhibitor activity and assess selectivity and potency on kinases of different classes. The luminescent ADP-Glo™ Kinase Assay is a universal platform that measures kinase activity by quantifying the amount of ADP produced during a kinase reaction.

We have used the ADP-Glo™ Chemistry to develop highly sensitive assays for more than 170 kinases across the human kinome and further enhanced the assays for ease-of-use by developing the Kinase Selectivity Profiling Systems. These systems provide an easy-to-use, reliable platform for kinase inhibitor profiling in house.

And even better, we now provide an online Kinase Profiling System Designer so that you can design a custom Kinase Selectivity Profiling System to fit your exact experimental needs. Simply drag and drop the combination of kinases you need to create an 8-kinase strip and submit your order. Continue reading