Supplementary Data

Gomisin A is a novel isoform-specific probe for the selective sensing of human cytochrome P450 3A4 in liver microsomes and living cells

Jing-Jing Wu, 1,2 Guang-Bo Ge, 1 Yu-Qi He, 1 Ping Wang, 1 Zi-Ru Dai, 1,2 Jing Ning, 1 Liang-Hai Hu 3 and Ling Yang 1,4

1 Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China.

2 Graduate University of Chinese Academy of Sciences, 19A Yuquanlu, Beijing 100049, China.

3 Research Center for Drug Metabolism, College of Life Science, Jilin University, Changchun 130012, China.

4 To whom correspondence should be addressed. (e-mail: )

Legends of supplemental Figures:

Fig. S1. Chemical structure of dibenzocyclooctadiene lignans involved in this study.

Fig. S2. Ramachandran plot of CYP3A5 that was homologously modeled based on the crystal structure of CYP3A4.

Fig. S3. Representative UFLC profiles of GA and its metabolites in human liver microsomes. GA (30 μM) was incubated with or without liver microsomes (0.2 mg/ml) at 37°C for 30 minutes with or without NADPH-generating system.

Fig. S4. The mass spectra of GA, M1 and M2 in positive-ion mode.

Fig. S5. Isozymes involved in the formation of DGA (M1).

Fig. S6. Cytotoxic effects of GA treatment in HepG2 cell lines. Data are shown as mean ± S.D. from three experiments carried out in duplicate.

Fig. S1

Fig. S2

Fig. S3

Fig. S4

GA:

M1:

M2:

Fig. S5

Fig. S6

Table S1 Primers used in RT-PCR Assay.

Gene / Primer Sequence (1)
CYP3A4 / Forward 5' CACAGATCCCCCTGAAATTACGCTTA 3'
Reverse 5' AAAATTCAGGCTCCACTTACGGTG 3'
CYP3A5 / Forward 5' ACAGATCCCCTTGAAATTAGACACG 3'
Reverse 5' CTTAGGGTTCCATCTCTTGAATCCA 3'
GAPDH / Forward 5' TCCTGCACCACCAACTGCTT 3'
Reverse 5' GAGGGGCCATCCACAGTCTT 3'

Table S2 Retention times (tR), UV λmax values, molecular weights (M.W.) and MS data for GA and its metabolites in HLM.

Compound / tR (min) / λmax (nm) / M.W. / Identification / Product ions (ESI+, m/z)
M1 / 3.06 / 254 / 404 / demethylene-gomisin A / 443.1 [M+K]+
M2 / 7.03 / 254 / 432 / 8-hydroxy-gomisin A / 471.1 [M+K]+
Substrate / 8.38 / 254 / 416 / gomisin A / 455.1 [M+K]+

Table S3 Proton NMR chemical shift assignments for gomisin A and its monohydroxylated metabolite.

Positions / Proton signals δ (ppm)
Gomisin A / M2 (8-HGA)
H-4 / 6.73 (1H, s) / 6.70 (1H, s)
H-11 / 6.58 (1H, s) / 6.59 (1H, s)
-OCH2O- / 6.00 (2H, d, J = 4.0 Hz) / 6.00 (2H, d, J = 4.0 Hz)
H-6a / 2.36 (1H, d, J = 8.0 Hz) / 2.38 (1H, d, J = 8.0 Hz)
H-6b / 2.23 (1H, d, J = 16.0 Hz) / 2.26 (1H, d, J = 12.0 Hz)
H-9a / 2.66 (1H, dd, J = 12.0, 4.0 Hz) / 2.58 (1H, d, J = 8.0 Hz)
H-9b / 2.22 (1H, d, J = 12.0 Hz) / 2.24 (1H, d, J = 12.0 Hz)
7-OH / 3.97 (1H, s) / 3.96 (1H, s)
H-8 / 1.66 (1H, m) / 3.61 (1H, s)
7-CH3 / 1.23 (3H, d, J = 48.0 Hz) / 1.22 (3H, d, J = 8.0 Hz)
8-CH3 / 0.69 (3H, d, J = 8.0 Hz) / 0.91 (3H, d, J = 8.0 Hz)
-OCH3 / 3.81 (3H, s), 3.72 (3H, s), 3.69 (3H, s), 3.35 (3H, s) / 3.80 (3H, s), 3.73 (3H, s), 3.69 (3H, s), 3.34 (3H, s)

All spectra were recorded on a Bruker ARX-600 spectrometer, in DMSO-d6; Abbreviations: d=doublet, m=multiplet, s=singlet, J=coupling constant.

Table S4 The average levels of CYP3A4 and CYP3A5 protein in a panel of HLMs prepared from thirteen individual donors.

HLM / Protein (pmol/mg protein) / Protein ratio
No. / Lot number / 3A4 / 3A5 / 3A4/3A5
1 / DDND / 46.5 / 7.96 / 5.84
2 / IURQ / 52.4 / 1.13 / 46.3
3 / YTQU / 32.5 / 1.25 / 26.1
4 / JJRB / 24.3 / 3.96 / 6.15
5 / BMRB / 132 / 2.28 / 58
6 / YOBL / 40.7 / 1.08 / 37.7
7* / VYIA / 61.4 / 8.87 / 6.92
8* / YCWV / 73.6 / 0.75 / 97.5
9 / MNIG / 71.8 / 0.82 / 88
10 / ZSKR / 20.1 / 2.08 / 9.69
11 / FBZR / 32.8 / 7.41 / 4.42
12 / SVKD / 85.7 / 10.5 / 8.14
13 / NHNY / 93.5 / 14.1 / 6.63

* Two individual-donor human liver microsomes were used in the inhibitory experiments.

Reference:

1. Lin YS, Dowling ALS, Quigley SD, Farin FM, Zhang J, Lamba J, et al. Co-regulation of CYP3A4 and CYP3A5 and contribution to hepatic and intestinal midazolam metabolism. Mol Pharmacol. 2002;62(1):162-72.

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