Drug Affinity Responsive Target Stability (DARTS) Analysis Service
- Service Details
- Case Study
Drug Affinity Responsive Target Stability (DARTS) technology is an effective method for detecting novel small molecule protein targets.DARTS can be used to validate known small molecule-protein interactions and to find potential protein targets for natural products. When proteins are bound to small molecule compounds, their stability to protein hydrolases is significantly increased. Proteins that bind small molecules are not readily hydrolyzed by proteases. These proteins can then be detected after electrophoresis and subsequently using biomass spectroscopy specific target proteins can be identified.
Drug targets are biological macromolecules that have pharmacodynamic functions within cells and can be acted upon by drugs. Most drug target molecules belong to proteins and are capable of binding small molecule compound drugs with appropriate chemical properties and affinity. After identifying the target molecules relevant to the disease, the relevant drugs can be designed for targeted therapy based on the characteristics of the target to develop effective and safe therapeutic approaches.
For most drugs of small molecules, the key issue is to identify the molecular target, taking into account the therapeutic effect and/or adverse side effects of the drug.
Studies to discover, and identify, biological target proteins within cells through phenotypic screening are becoming increasingly popular. To a large extent, these techniques require certain chemical modifications of small molecule drugs in order to enable them to find target molecules to which they can bind. In addition, some natural product small molecules have limited modification sites due to structural factors, which also constrain the recognition of drug targets by natural products and hinder the research of small molecule drugs and their applications.
Creative Proteomics provides DARTS technology services to overcome these limitations, enabling the analysis of drug-target protein binding relationships and finding the optimal conditions for binding of target proteins to small molecule drugs.
Advantages of DARTS Technology
- Universally applicable, simple and fast
- Uses the natural, unmodified activity of small molecule drugs with affinity for interaction with target proteins for interaction analysis, rather than using downstream parameters to discover target proteins
DARTS Technology Process
DARTS is a more sensitive technique for protein identification and requires high experimental conditions. The selection and use of key reagent choices such as protease, cell lysate and buffer are all critical factors in determining the success of DARTS. We will specify targeted solutions based on specific samples. A brief process includes:
- Cell collection and lysis
- Preparation of cell lysate
- Incubation of drug with lysate in combination
- Proteolytic digestion
- Identification of bound proteins: SDS-PAGE, Western protein blotting and LC-MS/MS
- By comparing and identifying the significant differences between drug and control lysates, we can find the candidate targets for subsequent experimental confirmation.
Workflow of DARTS (Lomenick et al., 2009)
DARTS technology can identify target proteins and also detect other proteins that have some association with the target protein in the development of disease. If you are interested in our services or have any questions, please contact us.
Reference
- Lomenick, et al. (2009). Target identification using drug affinity responsive target stability (DARTS). Proceedings of the National Academy of Sciences, 106(51), 21984-21989.
Case: Identification of Actin Proteins as Cellular Targets of the Marine Natural Compound 5-epi-SNEP Using Proteomic Approaches
Background
Marine environments, constituting half of global biodiversity, are rich sources of biologically relevant compounds. This study focuses on the soft coral genus Sinularia, particularly the compound 5-epi-SNEP, known for its diverse biological activities, including anticancer and anti-virus properties. The investigation aims to identify the cellular targets of 5-epi-SNEP and understand its impact on actin proteins.
Samples
HeLa cell extracts served as the biological samples in this study. These cells were lysed, and protein extracts were used for interaction studies with the 5-epi-SNEP affinity matrix.
Technical Methods
Generation of a Functional 5-epi-SNEP Affinity Matrix:
The epoxy-activated Sepharose™ 6B matrix underwent swelling and thorough washing. A solution containing 1 μmol of 5-epi-SNEP in 50% CH3CN/50% 100 mM sodium bicarbonate, 1% triethylamine was added to the matrix. This reaction took place at room temperature for 24 hours with continuous shaking. The efficiency of immobilization was evaluated using reverse phase-high-pressure liquid chromatography (RP-HPLC).
Identification of 5-epi-SNEP Interactome:
Separate incubations of 5-epi-SNEP-bound and control beads with HeLa cell protein extracts were conducted for 24 hours at 4 °C. After isolation, the beads were washed with phosphate buffer (PBS), and bound proteins were eluted using a denaturant buffer. Eluted proteins were resolved by SDS-PAGE, and in situ trypsin digestion of gel lanes was performed. Tryptic peptides underwent nano-flow RP-HPLC MS/MS analysis on an Orbitrap LTQ XL mass spectrometer, with Mascot software used for protein identification.
Optimization of DARTS Protocol:
HeLa cell lysates were incubated with different concentrations of native 5-epi-SNEP (ranging from 0.2 ng/μL to 20 ng/μL) for 1 hour at room temperature with continuous shaking. Subtilisin proteolysis was carried out with a subtilisin to protein ratio of 1:1000 for 30 minutes at 25 °C. Samples were then boiled in SDS-PAGE sample buffer to halt digestion. Analysis included visualization through Coomassie staining and confirmation via Western blotting using an anti-actin antibody.
Biological Evaluation of 5-epi-SNEP Action:
HeLa cells were cultured and treated with varying concentrations of 5-epi-SNEP for 24 hours. Cell viability was assessed using the MTT assay. Cell cycle analysis was performed by propidium iodide staining and flow cytometry. For F-actin analysis, cells were fixed, permeabilized, and stained with TRITC-phalloidin. The effects on the actin cytoskeleton were visualized using confocal microscopy.
(A) In vitro chemical proteomics workflow based on affinity purification followed by nano-liquid chromatography (LC) tandem mass spectrometry (MS/MS) for targets identifications; (B) DARTS workflow followed by Western blotting analysis.
Results
The generation of the 5-epi-SNEP affinity matrix on Sepharose™ 6B beads was successful, and the immobilization was confirmed through HPLC analysis.
Proteomic approaches, including nano-LC-MSMS and Western blotting, identified actin proteins as the primary cellular targets of 5-epi-SNEP.
The DARTS protocol optimization demonstrated that increasing concentrations of 5-epi-SNEP protected actin from subtilisin proteolysis, confirming the direct interaction between the compound and actin.
Biological evaluations revealed that 5-epi-SNEP induced a partial disruption of the actin cytoskeleton without affecting cell viability at low concentrations.
The study establishes 5-epi-SNEP as a natural compound with a novel chemical structure capable of interfering with actin dynamics, providing insights into potential applications in understanding cytoskeleton alterations at a molecular level.
Enrichment of actin protection to protease upon 5-epi-SNEP interaction
Reference
- Morretta, Elva, et al. "Discovering the biological target of 5-epi-sinuleptolide using a combination of proteomic approaches." Marine Drugs 15.10 (2017): 312.