[Development and application of screening method for steroid dihydrofolate reductase inhibitors by high performance...

A novel screening method for dihydrofolate reductase inhibitors (DHFRI) using high performance capillary electrophoresis (HPCE) was developed. The separation was performed in a fused silica capillary column using disodium tetraborate (50 mmol/L, pH 9. 18) buffer solution with 0. 002% Brij-35. The separation temperature was controlled at 25 degrees C and a voltage of 25 kV was applied. The detection wavelength was 280 nm. Five microL of various concentrations of inhibitor was added to the enzyme reaction system consisting of 25 micrpL of 50 mmol/L pH 6. 0 potassium phosphate buffer, 10 microL of 0. 025 mg/mL dihydrofolate (FH2), 10 microL of dihydrofolate reductase (DHFR) (0. 25 unit) and 5 microL of 0. 5 mg/mL NADPH in a 300-miL sample tube, then mixed for 1 min and incubated for 30 min at room temperature. The reaction mixture injections were performed by pressure at 3.45 kPa (0. 5 psi) for 10 s. With the developed CE method the substrate and product were baseline resolved within 19 min. The time course of the reaction was studied to show excellent correlation. Quantitative analysis of DHFR inhibition was performed by determining its dynamic parameters. The difference of peak areas between FH2 and FH4 was used to calculate the inhibitory rate. Three inhibitors, amethopterin, trimethoprim and folic acid, were used in the enzyme reaction system to test this method and their IC50 values determined were close to the literature values. It was demonstrated that the developed method is suitable for screening DHFRIs.