Kinases are proteins that regulate signal transduction in diseases such as Alzheimer’s, cancer, and diabetes. Given their biological significance, it is no surprise that pharmaceutical companies have expended significant R&D resources developing kinase inhibitors.
Drug makers rely on various kinase assay platforms to screen for their inhibitors. Kinase assays measure kinase activity in the presence and absence of kinase inhibitors. These platforms including biochemical, cell-based, and biophysical methods. Cell-based assays developed for high throughput are also known as a high-content screening (HCS) approach.
Biochemical and cell-based methods
Singapore-based S*BIO’s main objective is to discover and develop new anti-cancer compounds. To screen for kinase inhibitors, they use a biochemical-based luciferase-coupled ATP detection assay and the luciferase-coupled ADP detection assay. One example of a luciferase-coupled ATP detection assay is the Kinase-Glo Luminescent Kinase Assay from Promega Corporation (Madison, Wisc.), a high-throughput assay designed for multi-well formats. The assay operates by measuring the amount of ATP that remains after a kinase reaction. A proprietary luciferase enzyme then utilizes this remaining ATP to produce luminescence, which is measured by a luminometer. The amount of luminescence produced is inversely proportional to the level of kinase activity present.
Promega also produces the ADP-Glo Kinase Assay, a luciferase-coupled ADP detection assay, which directly measures kinase activity. Kinases convert ATP to ADP. A reagent in the kit converts this ADP back to ATP and the luciferase enzyme uses it to produce luminescence. The amount of luminescence produced is directly proportional to amount of kinase activity present. Inhibition of kinase activity by a specific compound reduces the amount of luminescence.
"S*BIO has used this [biochemical] approach to discover several novel kinase inhibitors as development candidates within the last four years—a CDK/Flt3 inhibitor, two JAK2 inhibitors, an mTOR/PI3K inhibitor as well as selective mTOR inhibitors," says Jan-Anders Karlsson, PhD, chief executive officer, S*BIO.
A major cell-based kinase assay approach involves HCS. According to Francesca Santini, PhD, Cellular Discovery Biology Leader at Merck, North Wales, Pa., there are three main advantages to implementing HCS for kinase inhibitor discovery in the early stages of drug discovery. First, HCS allows for a complete picture of how a compound acts on a kinase target at the cellular level. Second, the information obtained through HCS enables a better look not just at the kinase target, but also the pathway in which it functions in the cell. Finally, this high level information enables informed decisions about a compound’s potential as a lead in drug discovery. "We are using high-content screening often to monitor endogenous proteins and apply it to cells that are physiologically relevant," says Santini. HCS tools used by Santini and his team include high-content confocal microscopy, laser-scanning cytometry, and epifluorescence microscopy, all of which can be applied in a high-throughput screening format in 1536-well plates.
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This custom-designed poly-target profiling robot has three “work cells,” one dedicated to cell culture, one dedicated to compound/reagent handling, and one dedicated to assay detection. (Source: Merck) |
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One of the drug development success stories at Merck is in the discovery of Akt kinases. Merck uses Fluorescence Resonance Energy Transfer (FRET)-based HCS assays to determine the presence of Akt kinase inhibitors. Santini adds, "Using HCS, we can specifically monitor subcellular and even subpopulations of cells … we are investigating kinases involved in Alzheimer’s disease. We can screen for kinase inhibitors involved in the disease process in primary neurons, where a heterogeneous population is usually present, by using specific markers to narrow down our readout to a specific subpopulations of cells."
Biophysical methods
Caliper Life Sciences (Hopkinton, Mass.) develops kinase assays that are based on microfluidic separation of substrate and product. The assay platform works on the principle of electrophoretic separation, with a phosphorylated kinase substrate containing two additional negative charges than a non-phosphorylated substrate. "All you really need is the kinase of interest, a dye-labeled substrate and ATP," says Raj Singh, PhD, director of biology R&D at Caliper Life Sciences. "So in the interest of the expedited drug discovery process we came up with ProfilerPro Plates." ProfilerPro plates are reaction ready, 384-well plates. There are 24 columns and each column contains a kinase. The other plate contains a matched substrate and ATP. The platform consists of the two plates and all the ancillary reagents such as termination buffers and separation buffers all in a single kit. "Drug discovery scientists looking for kinase inhibitors typically profile the compounds on the enzyme by adding the substrate and ATP, letting the enzyme reaction go for some time, terminating the reaction, and then reading the reaction result on a [Caliper] platform called EZReader," says Singh. "The assay format is very easy because all we really need to do is to pipette out the right substrates and ATP to the right kinases." The plates are barcoded with all the assay and reading parameters for the EZReader.
"We have done the assay development for enzyme linearity, Km, and all the parameters that need to be determined for ATP competitive inhibitors, which are what most pharma companies are looking for," says Singh. The profiler can be used for many applications including hit-to-lead discovery, secondary confirmation, IC50 determination, potency values, and mechanism of action studies. "With the microfluidic LabChip technology you can do lots of different time course studies without stopping a reaction and having to read it manually," says Singh. "You can actually keep sipping a sample from the reaction mixture and follow the inhibition or activation without having to stop the remainder of the reaction mixture and that substantially reduces the number of wells you need for doing complicated enzyme kinetics."
These newly developed kinase assays are often compared to the gold standard, the radioactivity assay, which involves the measurement of a radiolabeled kinase substrate. The incorporation of radioactive phosphate into a kinase substrate indicates the presence of the kinase of interest. Alternatively, when screening for kinase inhibitors, the lack of incorporation indicates a hit. "However, the rationale over the last 10 to 20 years has been to move away from radioactivity and that necessitated the development of other techniques," says Singh. There are a large number of kinase screening methods currently on the market, he comments. For all these technologies you need a lot of ancillary technologies such as antibodies and beads and enzymes like luciferases or proteases for your downstream assay development," says Singh. "Our assays are closest to the gold standard because all you need is the substrate and the enzyme, which is exactly what people need for the radioactivity assay. The other benefit is that when you run it on the LabChip electrophoresis platform, we separate the products from the substrate, so it is a ratiometric readout. The benefit of ratiometric readout is exquisite data quality. And unlike other assays, there is minimal quenching because interfering compounds encountered in plate-based readers seldom electrophorese with the substrate and product during readout."
Although there are many different approaches to kinase-inhibitor screening; the high-throughput, physiological relevance, and real-time measurement capabilities of high-content screening have convinced many pharma companies to adopt the approach for drug discovery.
About the Author
James Netterwald is president and CEO of BioPharmaComm LLC, a provider of writing, editing, and consulting services to the life science, pharma-biotech, and public relations industries.
This article was published in Drug Discovery & Development
magazine: Vol. 13, No. 6, July/August, 2010, p. 14-16.
