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

Abstract

Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with k_on values of 0.7 × 10⁴ to 9.3 × 10⁴ M^-1·s^-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.

Keywords: Drug repurposing; IMP dehydrogenase; irreversible inhibitors; purine metabolic pathway.

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.

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.