Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening

Figures & data

Figure 1. Screening of CpIMPDH inhibitors. (A) Outline of HTS study. Numbers indicate the number of compounds after elimination by each screening step. (B) The counter assay was performed on 32 compounds to exclude reductase–luciferase inhibitors from specific CpIMPDH inhibitors. Dashed line showed the selection criteria, which eliminated inhibitors that inhibit less than 90% of control CpIMPDH activity in HTS assay, while also inhibit more than 50% of control reductase–luciferase activity, in the inhibitor concentration of 10 μM. ▪: disulfiram, ♦: bronopol, O: ebselen. (C) The structures of the hit compounds.

Figure 2. The inactivation of (A) CpIMPDH and (B) hIMPDH II by disulfiram. The inactivation of (C) CpIMPDH and (D) hIMPDH II by bronopol. Values are mean ± SD from two independent experiments.

Table 1. k_on values of disulfiram, bronopol and ebselen against IMPDHs (M⁻¹·s⁻¹).

	CpIMPDH	hIMPDH II	E. coli IMPDH
Disulfiram^a	1.4 × 10^4	1.8 × 10^4
Bronopol^a	2.2 × 10^4	0.7 × 10^4
Ebselen^a		>9.3 × 10^4
EICARMP^b		1.7 × 10^3	2.3 × 10^4
2-VIMP^c			7.4 × 10^3
2-FVIMP^d			2.7 × 10^4
6-Cl-IMP^e		44

^a This study. Activity was measured at 30 °C, 50 mM Tris-HCl buffer pH 8.0, 100 mM KCl, 3 mM EDTA, and 0.1 mg/ml BSA. CpIMPDH: 500 μM NAD⁺, 250 μM IMP. hIMPDH II: 100 μM NAD⁺, 250 μM IMP.

^b Activity was measured at 25 °C, 50 mM Tris-HCl buffer pH 8.0, 100 mM KCl, 3 mM EDTA, 1 mM DTT. hIMPDH II: 100 μM NAD⁺, 125 μM IMP. E. coli IMPDH: 2.5 mM NAD⁺, 1 mM IMP¹⁶.

^c Activity was measured at 25 °C, 50 mM Tris-HCl buffer pH 8.0, 150 mM KCl, 1 mM EDTA, 1 mM DTT. E. coli IMPDH: 1.5 mM NAD⁺, 500 μM IMP¹⁷.

^d Activity was measured at 25 °C, 100 mM Tris-HCl buffer pH 8.0, 150 mM KCl, 1 mM EDTA, 1 mM DTT. E. coli IMPDH: 2.5 mM NAD⁺, 1 mM IMP¹⁸.

^e Activity was measured at 25 °C, 100 mM Tris-HCl buffer pH 8.0, 100 mM KCl, 3 mM EDTA. hIMPDH II: 400 μM NAD⁺, 200 μM IMP¹⁹.

Figure 3. The ebselen inhibition of IMPDHs. (A) Ebselen inhibition in respect to NAD⁺, IMP concentration was kept constant at 250 μM. (B) Ebselen inhibition in respect to IMP, NAD⁺ concentration was kept constant at 400 μM. Circles show the assay with only vehicle and squares show the assay in the presence of 0.75 μM ebselen. Velocity (v) is in arbitrary units. (C) Ebselen irreversible inhibition to hIMPDH II. Values are mean ± SD from two independent experiments.

Table 2. The inhibition parameters of ebselen against CpIMPDH.

	CpIMPDH
Ebselen (nM)^a	706 (NC, NAD^+)
	64.6 ± 16 (Kis, MM, IMP)
	2808 ± 927 (Kii, MM, IMP)
MPA (nM)^b	9300 (UC, NAD^+)
GMP (μM)^b	46 (C, IMP)

C: competitive; UC: uncompetitive; NC: noncompetitive; MM: mixed-model inhibition.

^a This study. Activity was measured at 30 °C, 50 mM Tris-HCl buffer pH 8.0, 100 mM KCl, 3 mM EDTA, and 0.1 mg/ml BSA. Assay against NAD⁺, constant IMP at 250 μM, varied NAD⁺. Assay against IMP, constant NAD⁺ at 500 μM, varied IMP.

^b Activity was measured at 25 °C, 50 mM Tris-HCl buffer pH 8.0, 100 mM KCl, 3 mM EDTA, and 1 mM DTT. Assay against NAD⁺, constant IMP at 250 μM, varied NAD⁺ concentration. Assay against IMP, constant NAD⁺ at 500 μM, varied IMP concentration⁸.

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