Drug Affinity Responsive Target Stability (DARTS) Assay for Determining Drug-Target Interactions

The discovery of novel drug targets is a critical step in the development of effective drugs. Drug targets are usually proteins or nucleic acids that are involved in the pathogenesis of a disease. The identification of a target is only the first step in the drug development process. It is equally important to validate the target and determine the affinity of the drug toward the target.

Four Types of Drug Targets

In the domain of pharmaceuticals, drug targets hold immense importance, and there exist four distinct types of them: enzymes, ion channels, G-protein coupled receptors (GPCRs), and nuclear receptors.

Enzymes are remarkable proteins that catalyze biochemical reactions in the body, contributing to various cellular processes like DNA replication, metabolism, and signal transduction. Proteases, kinases, and phosphatases are a few instances of enzyme targets.

Ion channels, another type of drug targets, allow the transmission of ions across cell membranes. They have a crucial role in generating and propagating electrical signals in neurons and muscles. Sodium channels, potassium channels, and calcium channels are examples of the various ion channel targets that researchers study.

GPCRs, a family of proteins that play a crucial role in transmitting extracellular signals to the interior of cells, are implicated in a wide range of physiological processes, such as sensory perception, neurotransmission, and hormone signaling. Adrenergic receptors, serotonin receptors, and dopamine receptors are among the GPCR targets that scientists investigate.

Nuclear receptors, the final type of drug targets, are a family of proteins that regulate gene expression in response to ligand binding. Their critical role in various cellular processes, including metabolism, development, and reproduction, makes them a compelling target for researchers. Examples of nuclear receptor targets include estrogen receptors, androgen receptors, and glucocorticoid receptors.

Major protein families as drug targets (Santos et al., 2017).

Drug-Target Affinity

Drug-target affinity refers to the strength of the interaction between a drug and its target. It is a crucial factor in determining the efficacy and selectivity of a drug. A drug with high affinity for its target will bind tightly and produce a more significant effect, while a drug with low affinity will bind weakly and produce a weaker effect.

The affinity between a drug and its target is usually quantified using the dissociation constant (Kd). The Kd is the concentration of the drug required to occupy 50% of the target. The lower the Kd value, the higher the affinity of the drug for the target.

Drug Target Validation

The intricate and complex process of drug target validation encompasses a series of meticulously designed experiments and assessments, aimed at deciphering the feasibility and potential therapeutic utility of a given target. Such validation is pivotal in determining whether the target in question can indeed be harnessed for therapeutic purposes.

The validation process entails a comprehensive evaluation of the biological function and pathological relevance of the target, utilizing a gamut of sophisticated experimental techniques. This assessment is carried out through a multifaceted approach that includes in vitro experiments, such as cell-based assays and biochemical assays, aimed at unraveling the target's intricate nuances.

Following this, the target undergoes a rigorous evaluation in animal models, where its safety and efficacy are meticulously scrutinized, and its potential as a drug target is assessed. This meticulous and painstaking process of target validation serves as a vital criterion in selecting the most promising and viable targets for drug development, thereby laying the foundation for novel therapeutic interventions.

Drug Affinity Responsive Target Stability (DARTS) Assay

The Drug Affinity Responsive Target Stability (DARTS) assay is a biochemical assay used to determine the binding affinity of a drug for its target. The assay is based on the principle that binding a drug to a target can increase its stability in protease digestion. The assay is also relatively insensitive to non-specific binding.

Scheme of DARTS (Lomenick et al., 2011)

DARTS Assay Protocol

The DARTS assay protocol involves several steps:

• Protein purification: The target protein is purified from a suitable source, such as bacterial or mammalian cells.
• Drug incubation: The purified protein is incubated with increasing concentrations of the drug for a suitable period.
• Protease digestion: After drug incubation, the protein is treated with a protease, such as trypsin or chymotrypsin.
• SDS-PAGE analysis: The protein is analyzed by SDS-PAGE or Western blotting to determine the extent of proteolysis.
• Data analysis: The drug affinity is quantified by comparing the stability of the protein in the presence and absence of the drug.

Advantages of DARTS Assay

• Speed: The assay is completed in a relatively short period, making it suitable for high-throughput screening.
• Sensitivity: Relatively insensitive to non-specific binding.
• Versatility: Can be used to determine the binding affinity of drugs to a wide range of targets, including enzymes, receptors, and transcription factors.

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