In vitro inhibitory effect of lysionotin on the activity of cytochrome P450 enzymes

Figures & data

Figure 1. The chemical structure of lysionotin.

Table 1. Isoforms tested, marker reactions, incubation conditions, and K_m used in the inhibition study.

CYPs	Marker reactions	Substrate concentration (μM)	Protein concentration (mg/mL)	Incubation time (min)	Estimated Km (μM)
1A2	phenacetin O-deethylation	40	0.2	30	48
3A4	testosterone 6β-hydroxylation	50	0.5	10	53
2A6	coumarin 7-hydroxylation	1.0	0.1	10	1.5
2E1	chlorzoxazone 6-hydroxylation	120	0.4	30	126
2D6	dextromethorphan O-demethylation	25	0.25	20	4.8
2C9	diclofenac 4′-hydroxylation	10	0.3	10	13
2C19	S-Mephenytoin 4-hydroxylation	100	0.2	40	105
2C8	paclitaxel 6α-hydroxylation	10	0.5	30	16

Table 2. HPLC analysis conditions for mentioned CYP isoforms.

CYP isoforms	Detection wavelength (nm)	Mobile phase gradient
1A2	254	Methanol (A): Phosphate buffer (pH = 3.0, 50 mmol/L) (B) = 34:66
2A6	340	Acetonitrile (A): Acetic acid (0.1%, v/v) (B) = 35:65
3A4	248	Methanol (A): Water (B) = 52:48
2C8	230	Methanol (A): Water (B) = 65:35
2C9	280	Acetonitrile (A): Phosphate buffer (pH = 7.4, 100 mmol/L) (B) = 32:68,
2C19	211	Methanol (A): Acetonitrile (B) = 69:33
2D6	244	Acetonitrile (A): Phosphate buffer (pH = 3.0, 50 mmol/L ) (B) = 25:75
2E1	281	Acetonitrile (A): Acetic acid (0.5%, v/v) (B) = 22:78

Figure 2. Effects of lysionotin on the activity of CYPs, including CYP1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8. *p < 0.05. Negative control: incubation without lysionotin or specific inhibitors. Lysionotin: incubation with 100 μM lysionotin. Positive control: incubation with specific inhibitors.

Figure 3. The dose-inhibition curves of lysionotin on CYP3A4 (A), 2C19 (B), and 2C8 (C).

Figure 4. Lineweaver-Burk plots (A) and the secondary plot for Ki (B) of inhibition of lysionotin on CYP3A4 catalysed reactions (testosterone 6β-hydroxylation) in pooled HLM. Data were obtained from a 30 min incubation with testosterone (20–100 μM) in the absence or presence of lysionotin (0–30 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 5. Lineweaver-Burk plots (A) and the secondary plot for Ki (B) of inhibition of lysionotin on CYP2C19 catalysed reactions (S-mephenytoin 4-hydroxylation) in pooled HLM. Data were obtained from a 30 min incubation with mephenytoin (50–200 μM) in the absence or presence of lysionotin (0–50 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 6. Lineweaver-Burk plots (A) and the secondary plot for Ki (B) of inhibition of lysionotin on CYP2C8 catalysed reactions (paclitaxel 6α-hydroxylation) in pooled HLM. Data were obtained from a 30 min incubation with paclitaxel (5–20 μM) in the absence or presence of lysionotin (0–50 μM). All data represent the mean of the incubations (performed in triplicate).

Figure 7. Effect of incubation time on the inhibition of CYP3A4, 2C19, and 2C8.

Figure 8. Time and concentration-inactivation of microsomal CYP3A4 activity by lysionotin in the presence of NADPH. The initial rate constant of inactivation of CYP3A4 by each concentration (Kobs) was determined through linear regression analysis of the natural logarithm of the percentage of remaining activity versus pre-incubation time (A). The K_I and K_inact values were determined through non-linear analysis of the Kobs versus the pachymic acid concentration (B).