Affinity Chromatographic Method for Determining Drug-Protein Interaction with Enhanced Speed Than Typical Frontal...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Drug Discovery

Background:

• Drug-protein interactions are critical for identifying effective drug candidates early in the discovery process.
• Existing assays for drug-protein interaction analysis often lack the required speed for high-throughput screening.
• There is a need for rapid and reliable methods to quantify drug-protein binding affinities.

Purpose of the Study:

• To develop a high-speed affinity chromatographic method for analyzing drug-protein interactions.
• To immobilize poly(ADP-ribose) polymerase-1 (PARP1) onto macroporous silica gel for use as a stationary phase.
• To determine the binding parameters of PARP1 inhibitors (iniparib, rucaparib, olaparib) and screen natural compounds.

Main Methods:

• Synthesis of a stationary phase by immobilizing PARP1 onto macroporous silica gel via bioorthogonal reaction.
• Utilizing a continuous competitive frontal analysis method for high-speed drug-protein interaction determination.
• Validation of the method by comparing results with traditional frontal analysis techniques.

Main Results:

• The developed method demonstrated a two-fold increase in speed compared to conventional assays.
• Accurate determination of binding site numbers and association constants for iniparib, rucaparib, and olaparib to PARP1.
• Successful screening of tubeimosides I and II from Bolbostemma paniculatum, revealing potential anti-cancer mechanisms involving PARP1 inhibition.

Conclusions:

• The developed continuous competitive frontal analysis method is a reliable and high-speed alternative for analyzing drug-protein interactions.
• This method facilitates efficient screening of drug candidates and natural bioactive compounds.
• The approach offers a valuable tool for accelerating the early stages of drug discovery and development.