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.