D-DARTS: an alternative method for NaV1.5 affinity molecules identification based on dual-drug affinity responsive...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• Drug affinity responsive target stability (DARTS) is a label-free method for detecting target engagement.
• DARTS application to multi-transmembrane channel proteins is limited due to their instability and protease resistance.

Purpose of the Study:

• To develop a novel strategy, Dual-DARTS (D-DARTS), for evaluating target protein stability against chemical denaturation and enzymatic degradation.
• To identify affinity ligands for the NaV1.5 channel and assess the general utility of D-DARTS for multi-transmembrane proteins.

Main Methods:

• Developed Dual-DARTS (D-DARTS) using controlled proteolysis in an SDS-containing denaturing buffer.
• Applied D-DARTS to identify NaV1.5 channel binders and the mitochondrial protein VDAC1.
• Validated findings using electrophysiological assays and molecular docking.

Main Results:

• Successfully identified inhibitors and agonists for the NaV1.5 channel using D-DARTS.
• Discovered poneratoxin as a high-affinity NaV1.5 binder, with binding site predictions.
• Demonstrated D-DARTS applicability to VDAC1, confirming its broad utility.

Conclusions:

• D-DARTS enables target engagement assessment for challenging multi-transmembrane proteins.
• This method offers a simple, cost-effective alternative to electrophysiology for screening channel protein binders.
• D-DARTS is a promising strategy for target identification and active molecule screening across various transmembrane proteins.