For centuries, humans have been exploiting their ecological resources to heal ailments. In fact, archeological evidence reveals the use of medicinal plants such as opium poppies, ephedra, and cannabis by humans as early as 60,000 years ago. Further, written documentation for the use of plants as medicine first appears in the Egyptian culture in 1500 B.C describing the use of Ebers Papyrus to treat wounds, and to treat hair and skin disorders. These practices of early pharmacological discovery and application have become highly complex and sophisticated through scientific innovation, and are the basis of modern medicine today. Despite the discovery of many life-saving compounds via deep interrogation of physical, biochemical, and biological properties of potential molecular compounds, the full extent of drug discovery remains opaque and is an area of active investigation. Today, researchers are actively screening for novel potential drug targets by means of large
screening libraries containing an abundance of
natural and synthetic compounds alike. With seemingly infinite compounds to test and screen for, the path to
drug discovery may appear daunting. Fortunately, we provide a step-by-step guide plus tips to getting a seat at the drug discovery table and how to maximize your chances of success.
Step 1: Choose the type of molecule you want to target
Given the extensive and diverse roles kinases and phosphatases play throughout development and homeostasis of an organism, identifying targets that will modulate aberrant kinase and phosphatase pathways is advantageous toward developing a therapeutic for the treatment of many diseases. Kinases are enzymes that selectively modify other proteins, lipids, or carbohydrates by adding phosphate groups, a process called phosphorylation (Figure 1A). Conversely, phosphatases are enzymes that selectively remove these phosphate groups, a process called de-phosphorylation (Figure 1B). There are more than 500 kinases encoded in the human genome, which phosphorylate up to one-third of the proteome, and more than 100 phosphatases encoded in the human genome that remove phosphate groups throughout the proteome. Furthermore, hundreds of diseases are caused by the disruption of the exquisite balance between critical kinases and phosphatases in humans. Kinase/phosphatase abundance and importance in hundreds of biological pathways mean they are superior candidates to target in any pathway throughout your drug discovery research screen. In addition, their relevance in human disease makes them a potential gold mine for drug discovery research. Therefore,
targeting kinases and phosphatases in your drug screen will yield results. This TechNote pursues kinases and phosphatases as targets.
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figure 1. Kinases Catalyze Phosphorylation and Phosphatases Catalyze De-phosphorylation
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Step 2: Understand what we currently know about kinase and phosphatase targets, and identify knowledge gaps
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FDA-approved compounds on the market currently are targeting kinases and phosphatases that act as catalysts for signaling cascades, cellular communication, and cellular proliferation. Such pathways are essential to maintain homeostasis of tissues, and can contribute to pathogenesis of disease such as cancer when aberrantly regulated. The majority of these compounds are kinases, specifically, of which exist in different families. Families of kinases include Cyclin-Dependent Kinases (CDK) enriched in the pathogenesis of cancer; Mitogen-Activated Protein Kinases (MAPK) important for cell proliferation/differentiation and implicated in types of cancer; and Phophatidyl Inositol Kinases (PIK) facilitating the insulin-signaling pathway and linked to insulin resistance. The development of drugs from compounds that modulate kinases in these pathways are helpful in treating disease, yet the majority of kinases have no known inhibitor, with much to be discovered in the field of kinases inhibitors and drug discovery. Therefore, investigating compounds that modulate the activity of
kinases is ripe for discovery.
Step 3: Identify pitfalls in your methods early so you can proactively avoid them
Although the number of potential therapeutic compounds is plentiful, the pipeline to discover, test, and validate a compound for use in human therapeutics can be time-consuming, expensive, and labor-intensive (Figure 2). Briefly, extensive background research much be done to understand biological pathways in normal and disease contexts. You must sift through the data collected and current literature to identify a set of compounds that you hypothesize could be important in targeting the specific molecular pathway of interest. Then, traditionally low-throughput arduous experimental testing of these potential compounds against your pathway of interest is required. Advancements in
kinase assays using bioluminescence or fluorescence resonance energy transfer (BRET, FRET) and competitive binding of tracers allows the end user to visualize kinase-substrate interactions on a cellular level to profile and quantify various kinases in live cells. This method allows the investigation of these interactions in physiologically relevant conditions. If one is lucky to identify a compound that successfully targets the kinase they want, and shows potential therapeutic effect such as the inhibition of cancer growth, then the compound is purified, its structure elucidated and optimized via medicinal chemistry, combinatorial chemistry, and computational chemistry.
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figure 2. The Path to Drug Discovery
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At this step, it is also important to uncover the mechanism of action of the kinase inhibitor. A recent method to evaluate this is to
measure small amounts of ADP with varying concentrations of ATP in your cell-based assay in order to identify if the inhibitor is an allosteric inhibitor or in fact modulating phosphorylation. Finally, it is subject to formulation, pharmacokinetics, and eventually undergo more specific drug metabolism studies, such as using Proteolysis-Targeting Chimeras
PROTACs to understand how the inhibitor is affecting additional domains of the kinase.
- Pitfall: Typical methods used throughout this process are low-throughput, expensive, and are not optimized for success. For example, tens of thousands of compounds need to be tested against a target, and many labs are using enzyme assays and cell proliferation assays that can only test a few compounds at a time. This creates a longer time to discovery, ultimately wasting time and money.
- How to avoid: Utilizing methods to screen more compounds in less time is of paramount importance. Combining methods of high-throughput screening with biochemical assays (such as described above) is necessary to determine accurate specificity and efficiency of potential compounds and can push your research forward.
- Pitfall: Kinases are biological actors that undergo evolution; therefore, genetic mutations in kinases can render current kinase inhibitors useless, so it is important to keep looking for new targets. It is also important to identify what genetics changed in the kinase. Going back to the drawing board is slow and arduous, and sequencing is expensive and slow.
- How to avoid: The researcher must possess a high-throughput library of compounds to test as genetic variation of kinase exists. Also harnessing next generation sequencing (NGS) technology to identify genetic variation in kinases to pursue more targeted and successful compounds in the future.
Step 4: Utilize Enzo Life Science’s solutions for drug discovery
In the field of
drug discovery, it is important to use technology that is accurate, reproducible, and scientifically sound. At Enzo Life Sciences, we offer a variety of trusted solutions for your research needs. Decades of innovation has enabled Enzo to be a leader in scientific research, specifically drug discovery. Enzo’s solutions to fast track your drug discovery program will help you save time, save money, and produce scientifically accurate and biologically relevant results that are reproducible. Highlighted below are some of Enzo’s products that will help you make the most out of your drug discovery research.
Enzo has a long and successful record of accomplishment in identifying, synthesizing and commercializing known bioactives and compounds to accelerate research. Furthermore, Enzo scientists have assembled relevant sets of focused
compounds for screening. Enzo’s
SCREEN-WELL® Kinase Inhibitor Library is a library of 80 kinase inhibitors, including inhibitors known to target CDK, MAPK, and PIK (mentioned above). The SCREEN-WELL® Kinase Inhibitor Library allows end users to run high-throughput experiments and analysis on highly relevant compounds, saving both time and money. The results obtained with the SCREEN-WELL® library are reproducible and accurate, creating confidence when you identify a hit. Compounds included in the
SCREEN-WELL® libraries are available for re-order as stand-alone products and in bulk quantities. Additional stand-alone kinase inhibitors to supplement the SCREEN-WELL assay can also be found on our website (Product Listing - Enzo Life Sciences).
Enzo’s
AKT,
PKA,
PKC, and
p70 S6K kinase activity kits supplement the SCREEN-WELL® Kinase inhibitor library. To help with your data analysis, Enzo’s kinase activity kits can be read and analyzed using our
Absorbance 96 personal compact plate reader, allowing consistent and reproducible measurements. Using these products in your pipeline can help you create a cost-effective, efficient, and consistent methodology to produce reliable and important results.
Related products to assist you in your drug discovery research and compliment the above-mentioned products are the
SCREEN-WELL® Phosphatase Inhibitor Library and
Calcineurin phosphatase assay kit. Since kinases and phosphatases work in an exquisite balance, understanding inhibitors of both enzymes is important. Similar to the stand-alone kinase solutions, Enzo has a myriad of phosphatase inhibitors and phosphatase assays available for purchase. To investigate genetic variation in your kinase or phosphatase of interest, I invite you to visit our portfolio of
NGS products that provide cost efficient, flexible, and accurate results (see our
Genomics and Molecular Biology Catalog). Do you need help designing, analyzing, or troubleshooting your drug discovery research project? The scientists at Enzo Life Sciences are eager to help you succeed. Contact our
Technical Support Team to get started!
