Making drug discovery more efficient: predicting drug side effects in early screening efforts

Drug research and development is a complex and expensive process that begins with initial screening steps of candidate chemical compounds. 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 deleterious 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.

Fig.1. Effect of selective and non-selective drugs on cell health. Compounds that target kinases can be potent in destroying cancer cells. However, if such compounds are promiscuous towards non-target kinases, they will not only affect the cancer cells but also healthy cells, making them sick.

Cells carry out specific activities such as cell division, growth, and energy usage in response to signals from the environment, internal cues like DNA damage and even from other cells. These signals are perpetuated and amplified within the cell through biochemical events that modify proteins in the cell. One of the most common events of cell signaling is the addition of phosphate groups to proteins (phosphorylation), and phosphorylation is catalyzed by a specific group of proteins called kinases.

Because kinases are one of the largest enzyme classes in the cell and they are involved in many critical cellular processes such as growth, energy use, and cell division, any disruption in their structure (mutations) or activity leads to diseases such as cancer, inflammation or diabetes. Therefore, kinases are often targeted in drug development efforts, and many small-molecule inhibitors of kinases have already been developed for treatment of different cancers. However, many candidate compounds that show great potential also often have off-target effects that make them unsafe as therapeutics (Fig. 1). In part this is because molecules that inhibit kinase activity tend to affect the highly conserved regions of these abundant proteins.

Fig. 2. Selectivity profile of kinase inhibitors generated by the bioluminescent kinase strip method. Compounds are profiled against 8 kinases at a time and percent kinase activity remaining is reported. An example of a specific kinase inhibitor such as tofacitinib is shown. Tofacitinib is a potent and selective JAK3 kinase inhibitor FDA approved for the treatment of rheumatoid arthritis.

Developing methods for profiling the effect of a target compound on the kinase activity (selectivity assessment) is challenging, and many of the current methods are cumbersome and still rely on radioisotopes. First, to profile the selectivity of a compound, many kinases must be assayed at the same time, and one kinase assay method that is suitable for one kinase may not be appropriate for other kinases. Second, developing and optimizing assays for each kinase in the selectivity profiling panel and maintaining the stability and supply of the enzymes for the assay is difficult.

Here we describe a pre-configured and standardized kinase profiling system that can be implemented as a cost-effective in-house screening assay which can be performed by any scientist in early evaluation of candidate compounds (Fig. 2). This system uses a universal kinase assay chemistry that can reliably probe kinase activity regardless of the nature of the substrate because it monitors the production of the universal kinase reaction product, ADP.

Such an assay allows drug developers to screen candidate compounds against a wide range of kinases early in the drug development process, potentially identifying problematic off-target effects. In this paper we describe previously unreported off-target effects for two reported kinase inhibitors. Additionally the same assay can be used to gain more information about the potency of a compound toward a particular targeted kinase or family of kinases later in the screening.

Drug discovery is complex and expensive, and being able to learn as much as possible about candidate compounds early in the screening process is critical to streamlining the process and ensuring that only the most viable compounds progress to later stages of drug development. The flexible bioluminescent kinase strips that we describe here allow researchers to fully understand the activity of their candidate drugs at an earlier stage than ever before.

Michele Arduengo, and Hicham Zegzouti
Promega Corporation, Madison, WI, USA



Bioluminescent kinase strips: A novel approach to targeted and flexible kinase inhibitor profiling.
Hennek J, Alves J, Yao E, Goueli SA, Zegzouti H
Anal Biochem. 2016 Feb 15


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