Automated approach for the evaluation of glutathione-S-transferase P1-1 inhibition by organometallic anticancer compounds

Figures & data

Figure 1. Illustration of the SIA manifold used. CS – Carrier solution; HC – holding coil; PP – peristaltic pump; SV – Selection valve; DMSO/INIB – dimethylsulfoxide/inhibitor; CDNB- 1-Chloro-2,4-dinitrobenzene; GSH – Glutathione reduced; GST P1-1 – Glutathione S-transferase P1-1; PB – Phosphate buffer; RC – Reaction coil; D – Detector and W – Waste.

Table 1. Analytical cycle used to perform the GST P1-1 inhibition assays.

Step	Position valve	Reagent	Volume (µL)	Time (s)	Flow rate (mL min ^-1)	Action
1	8	CDNB	10	1.2	0.5	Aspiration of CDNB
2	6	DMSO/ inhibitor	10	1.2	0.5	Aspiration of DMSO/inhibitor
3	2	GST P1-1	20	2.4	0.5	Aspiration of GST P1-1
4	7	GSH	20	2.4	0.5	Aspiration of GSH
5	4	Mixture	333	20	1	Propulsion to the reactor coil
6	4		–	240	0	Stop period in reactor coil
7	4	Mixture	2000	60	2	Propulsion to the detector

Figure 2. GST P1-1 enzymatic reaction.

Table 2. SIA system optimisation.

Condition	Range	Selected value
Stop period (min)	0–5	4
Aspiration order	GSH – DMSO/inhibitor – GST P1-1 – CDNB GSH – GST P1-1 – DMSO/inhibitor – CDNB CDNB – DMSO/inhibitor – GST P1-1 – GSH CDNB – GST P1-1- DMSO/inhibitor – GSH	CDNB – DMSO/inhibitor – GST P1-1 – GSH
Temperature (°C)	25–37	37
GST volume (µL)	10–25	20
GSH volume (µL)	15–25	20

Figure 3. Optimisation of the GST P1-1 reaction time.

Table 3. Analytical features of the calibration curve.

Analytical features	Values
Detection limit	0.26 mM
Quantification limit	0.85 mM
R^2	0.99
Slope	0.09
Intercept	0.14
Standard error of slope (Sb)	0.006
Standard error of intercept (Sa)	0.007
Standard error of regression	0.008
Sum of squares of the regression	0.01
Sum of squares of the residuals	0.0002

Table 4. GST P1-1 inhibition of ethacrynic acid and a series of organometallic compounds.

Compound	Structure^a	IC50 (µM ± SD)
1 Ethacrynic acid		11.3 ± 0.8
2a		57 ± 4
2b		76 ± 6
2c		24.7 ± 1.4
2d		61 ± 5
3a		235 ± 2
4a		275 ± 9
4b		219 ± 8
4c		113 ± 5
4d		72 ± 5
5a		83 ± 9
5b		91 ± 9
5c		46.8 ± 2.8
5d		34.0 ± 4.3
5e		30.3 ± 8.3
6a		17.4 ± 2.8
6b		165 ± 4
7a		181 ± 7
7b		152 ± 6
7c		138 ± 12
7d		6.7 ± 0.7
7e		12.1 ± 1.8

^[a] Cationic diiron complexes as CF₃SO₃⁻ salts; cationic Ru/Ir complexes (3a, 7d, 7e) as NO₃⁻ salt.

Table 5. Assessment of SIA and batch IC₅₀ values.

Compounds	IC50 batch ± SD (µM)	IC50 SIA ± SD (µM)
1	13.58 ± 0.02 ^(1)	11.3 ± 0.8
4a	226 ± 3	275 ± 9
4c	61 ± 3	113 ± 5.3
4d	33 ± 6	72 ± 5
6a	11.5 ± 0.7	17 ± 3
7e	11.6 ± 0.5	12 ± 2

IC₅₀ value obtained using the same batch method in reference⁴¹.

Biancalana L, Batchelor LK, Pereira SAP, et al. Bis-conjugation of bioactive molecules to cisplatin-like complexes through (2,2'-Bipyridine)-4,4'-dicarboxylic acid with optimal cytotoxicity profile provided by the combination ethacrynic acid/flurbiprofen. Chem Eur J 2020;26:17525–17535.

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