Design and optimization of SPR-based binding assay for evaluation and screening of MITF-E-box binding inhibitor.

Melanin synthesis is a complex phenomenon which involves about 192 known gene products. Among them, MITF is a key transcription factor for tyrosinase, Trp1 and Trp2 proteins, which are essential for melanin biosynthesis. Thus, intervening inhibitor for the MITF-E-box complex formation can downregulate melanin synthesis. The focus of the present study is to develop a surface plasmon resonance-based system to screen the MITF-E-box complex inhibitor. The standardization of the MITF and E-box binding assay was calibrated for kinetics and specificity, in the presence of a pre-incubated 22 mer sequence containing mutated E-box (CTTGAG) along MITF. The binding assay with C17 was optimized and the steady-state kinetics was evaluated. C17 was identified as inhibitor to MITF-E-box, by virtual screening followed by in vitro assessment and EMSA assay. The k(a) and k(d) were found to be 5.5 9 103 M⁻¹ s⁻¹ and 0.0014 s⁻¹, respectively, while the steady-state association constant (K(A)) was 3.928 9 106 M⁻¹. The resonance variations after inhibition were quantified and analyzed to develop the standard method for screening of microphthalmia transcription factor-E-box binding inhibitor.