Kinases are key regulators of most cellular pathways. Their association with diseases makes them significant therapeutic intervention points. According to reports, there are around 37 small molecule kinase drugs already in the market, amassing billions of dollars annually. Read our blog below to learn more about the importance of kinases in drug discovery and an interesting technique to measure compound engagement.

 

Kinases: An Overview

Kinases are enzymes that transfer phosphate groups (PO43−) from ATP (adenosine triphosphate) to other specified molecules. They play an extensive role in signal transduction and regulation of complex cellular processes vital to maintain life. They can be classified into broad groups depending on the substrate they act upon: proteins, lipids, carbohydrates, etc. Protein kinases make up most of the human kinome and are essential for cell division, metabolism, survival and apoptosis. They can further be classified into different groups (as shown below) on the basis of sequence similarity between their catalytic domains, presence of accessory domains and any known modes of regulation.

Classification of kinases

Human Kinome Map

Human Kinome Map

Kinases are highly regulated by themselves, small molecules or protein-protein interactions (PPI)*. Their dysregulation is commonly associated with various diseases, making them important therapeutic targets for small-molecule modulation.

*Follow the link to read our blog on PPI

Kinases as drug targets

Over the past two decades, kinases have become one of the most significant classes of drug targets because of their involvement in several diseases. Cancer, osteoporosis, inflammatory diseases, diabetes and neurodegeneration are some of the conditions commonly associated with dysregulation of kinases.

Fun fact- Pseudokinases

According to reports, over 150 out of 518 protein kinases are known to be mutated or mis regulated in various disease states. So far, the majority of clinical trials have targeted 43 kinases, representing a huge opportunity ahead for kinases in the drug discovery world. In order to further exploit kinases, high importance should be given to suitable bioassay development specific for individual kinases. Follow the link to our article on ‘Kinases – Application of New Assay Technologies to Shed Light on Kinase Drug Discovery’, recently published in Drug Discovery World.

 

NanoBRET™ Target Engagement Intracellular Kinase Assay:

There are several classic and emerging approaches to study kinase inhibitors. Although research relied mostly on non-cell-based assays in the past, a more physiological method is to measure functional implications as described in our poster. We have developed high throughput screening assays in living cells using the NanoBRET™ Target Engagement technique (Promega) to measure compound binding at specific target kinases. This quantitative analysis can be performed in cultured cells via energy transfer between a 19-kDa luciferase (NanoLuc, Nluc)-tagged target protein and a cell-permeable fluorescent energy transfer probe introduced to the culture medium. Addition of competing compounds results in a dose-dependent decrease in NanoBRET™ signal, which allows quantitation of the intracellular affinity of the target protein for the test compound. Unlike existing methods, this technique is compatible with analysis under equilibrium conditions, therefore allowing for determination of fractional target occupancy and affinity for various classes of ATP-competitive inhibitors in live cells. It is suitable for a wide range of throughput requirements and is a good predictor of cellular potency. Follow the link to our poster to learn more about this assay.

Compound engagement measured in a competitive format using a cell-permeable fluorescent NanoBRET™ tracer

Compound engagement measured in a competitive format using a cell-permeable fluorescent NanoBRET™ tracer

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