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

Revealing drug-protein interaction is highly important to select a drug candidate with improved drug-like properties in the early stages of drug discovery. This highlights the urgent need to develop assays that enable the analysis of drug-protein interaction with high speed. Herein, this purpose was realized by the development of an affinity chromatographic method with a two-fold higher speed than typical assays like frontal analysis and zonal elution. The method involved synthesis of a stationary phase by immobilizing poly(ADP-ribose) polymerase-1 (PARP 1 ) onto macroporous silica gel through a one-step bioorthogonal reaction, characterization of mutual displacement interaction of two canonical drugs to the immobilized PARP 1 , determination of the interaction between three (iniparib, rucaparib, and olaparib) drugs and the protein, and validation of these parameters by typical frontal analysis. The numbers of binding sites on the column were (2.85 ± 0.05) × 10 -7 , (1.89 ± 0.71) × 10 -6 , and (1.49 ± 0.06) × 10 -7 M for iniparib, rucaparib, and olaparib, respectively. On these sites, the association constants of the three drugs to the protein were (9.85 ± 0.56) × 10 4 , (2.85 ± 0.34) × 10 4 , and (1.07 ± 0.35) × 10 5 M -1 . The determined parameters presented a good agreement with the calculation by typical frontal analyses, which indicated that the current continuous competitive frontal analysis method was reliable for determining drug-protein interaction. Application of the methods was achieved by screening tubeimosides I and II as the bioactive compounds against breast cancer in Bolbostemma paniculatum . Their mechanism may be the interference of DNA repair via down-regulating PARP 1 and meiotic recombination 11 expressions, thus leading to oncogene mutations and death of cancer cells. The method was high speed since it allowed simultaneous determination of binding parameters between two drugs and a protein with a smaller number of experiments to be performed. Such a feature made the method an attractive alternative for high-speed analysis of drug-protein interaction or the other bindings in a binary system.