Piperine directly interacts with DHODH protein
Next, we examine the interaction between piperine and DHODH by using circular dichroism (CD) spectroscopy. This technique is widely used to probe the binding of ligand to protein through analyzing the induced CD (ICD) spectrum(Micsonai et al., 2015). Purified DHODH protein was incubated with different concentrations of piperine in phosphate buffer, and detected by CD. As presented in Figure 2A, piperine induced a significant alteration of CD spectrum, indicating a changed secondary structure of DHODH protein. Considering the existing of tryptophan residues (Trp 362) in human DHODH protein, we perform an intrinsic fluorescence-quenching assay to further confirm the binding of piperine to DHODH (24). In this assay, DHODH (20 μM) was incubated with varied concentrations of piperine, and the fluorescence signals were measured. As shown in Figure 1C, DHODH protein have a maximal fluorescence intensity at 337 nm, and PIP dose-dependently induce fluorescence quenching of DHODH protein, suggesting the binding of piperine to DHODH.
Then, thermal shift assays (TSA) was used to investigate DHODH protein thermal stabilization upon compound binding(Lo et al., 2004). DHODH protein (5 μM) was incubated with various concentrations of piperine and the mixture was subjected to thermal scanning. The Melting temperature (Tm) was calculated from the Melt curve. Figure 1C shows that piperine dramatically improves the stability of DHODH protein as a reflection of the increased Tm values over 5.12 °C. Furthermore, the thermodynamic parameters of the interaction of piperine and DHODH was examined using isothermal titration calorimetry (ITC). The purified recombinant human DHODH protein was titrated with piperine and the reaction heat was recorded. As shown in Figure 1D, piperine binds to DHODH with a KD value of 1.97±0.20 μM according to the ITC titration curves, which is comparable with results of DHODH enzymatic kinetic analysis (Ki=0.71 ± 0.01 μM). Put together, these results show the potent binding potency of piperine to DHODH.