Drug Affinity Responsive Target Stability (Darts)

Identifying the molecular targets for beneficial or detrimental effects of small molecule drugs is an important process and currently unmet challenge in drug development. As a professional supplier in the biochemical market, Creative Biolabs has utilized a novel method, drug affinity responsive target stability (Darts), to analysis binding interactions between small molecule compounds and cellular macromolecules such as proteins. This technology has been used in many drug profiling projects.

• Identify hit targets and decipher mechanisms of drug action (target deconvolution)
• Investigate the off-target effects of active compounds
• Evaluate the safety of small molecular drugs
• Support drug repositioning in new therapeutic fields

Darts can identify direct binding interactions using native, unmodified compounds, but can also be applied to validate potential protein-ligand interactions detected by other means and to estimate the affinity of interactions. This tool overcomes the limitation of current target identification methods and is particularly useful for the initial identification of protein targets of small molecules.

• How Does Darts work?

Darts is a relatively quick and easy-to-use approach to identify potential protein targets for small molecules. The technique is based on the principle that the stabilization of protein structure upon drug binding leads to target proteins with resistance to protease degradation. When a small molecule is incubated with protein lysates, it binds and protects specific proteins from proteolysis. The degradation of unspecific proteins won’t be affected. Through proteomics, desired protein targets are then identified by their enrichment in the compound-treated sample versus a control sample post-proteolysis. Darts is universally applicable since it requires no modification of the drug and is independent of the molecule's mechanism of drug action or chemical structure.

• Darts Measurement Services at Creative Biolabs

Small molecule target identification is a considerably monumental task. Darts represents a straightforward technology to identify small molecules’ protein targets and assist in the characterization of interactions among small molecules and putative targets. It is a simple and favorable approach that can be performed using crude cell lysates and other complex protein mixtures. The greatest advantage of Darts is being able to exploit the native small molecule without having to modify it by incorporation of biotin, fluorescent, radioisotope, or affinity labels.

Fig.2 Experimental scheme of Darts using mammalian cell lysates. (Lomenick, 2011)

• Workflow

At Creative Biolabs, Darts is conducted by simply treating aliquots of cell lysate with the compound of interest and vehicle control, followed by limited digestion of proteins in the cell lysate with proteases. Next, the sample is separated by SDS-PAGE and stained to exhibit protein bands that are protected from proteolysis. Mass spectrometry (MS) is subsequently used to identify the proteins in each band. The tested molecules involve small bioactive molecules and drugs under development or market, and our standard workflow is summarized as below.

• Strategic Planning

A few general key choices must be made for each Darts and we have worked out a number of strategies to support this experiment.

1. The source of protein. In theory, any cell type that is sensitive to the biological effects of a small molecule can be utilized. Here, we have successfully accomplished Darts with lysates from a wide range of mammalian cells, including HeLa, HEK293, A549, Raw264.7, MEFs, and Jurkat cells, as well as from the yeast cells (Saccharomyces cerevisiae).
2. The concentration range of small molecules. Generally, any small molecule that is believed to bind proteins should be suitable for Darts. Given that the targets of most small molecules used for Darts are unknown, the binding affinities will also unknown. Therefore, we suggest to estimate the binding affinity based on EC50 of the compound, and the initial concentration of the compound could be 10-fold higher than the EC50.

3. The type of proteases. There are many proteases available for Darts experiments. Now, we take advantage of two major types: pronase and thermolysin. Pronase is a mixture of multiple proteases that can digest both folded and unfolded proteins, and is the preferred choice of an enzyme. Thermolysin is a metalloendopeptidase with low cleavage specificity, that can only digest unfolded proteins.
4. Identification methods. In order to identify novel targets of small molecules by Darts, there’re several proteomic techniques after the proteolysis, including but not limited to immunoblotting, SDS-PAGE, 2D-PAGE, and gel-free MS-based proteomics.

Advantages

• Flexible, cost-effective, and time-saving target identification services
• Detection of molecular bindings, even weak small molecule-protein interactions
• No additional modifications or tagging of small molecule compounds
• Full sophisticated bioinformatics analysis of results

Knowledge of binding interactions between molecules is crucial for unraveling their biological properties and mechanisms of action. Remarkably, Darts is emerging as a general methodology for identifying and studying protein-ligand interactions. With rich expertise in chemical proteomics, Creative Biolabs offers Darts services to help clients characterize the most relevant interactions between small molecules and proteins, as well as guide the development of therapies for different diseases. Our deliverables contain the list of identified protein partners, the interaction domain on each protein, a confidence score, and any other information you required. For more details, please don’t hesitate to contact us.

Reference

1. Lomenick, B.; et al. Target identification using drug affinity responsive target stability (DARTS). Curr Protoc Chem Biol. 2011, 3(4): 163-180.

For Research Use Only.