The Repurposed Drug Disulfiram Inhibits Urease and Aldehyde Dehydrogenase and Prevents In Vitro Growth of the Oomycete Pythium insidiosum

Abstract

Pythium insidiosum is an oomycete microorganism that causes a life-threatening infectious disease, called pythiosis, in humans and animals. The disease has been increasingly reported worldwide. Conventional antifungal drugs are ineffective against P. insidiosum Treatment of pythiosis requires the extensive removal of infected tissue (i.e., eye and leg), but inadequate surgery and recurrent infection often occur. A more effective treatment is needed for pythiosis patients. Drug repurposing is a promising strategy for the identification of a U.S. Food and Drug Administration-approved drug for the control of P. insidiosum Disulfiram has been approved to treat alcoholism, but it exhibits antimicrobial activity against various pathogens. In this study, we explored whether disulfiram possesses an anti-P. insidiosum activity. A total of 27 P. insidiosum strains, isolated from various hosts and geographic areas, were susceptible to disulfiram in a dose-dependent manner. The MIC range of disulfiram against P. insidiosum (8 to 32 mg/liter) was in line with that of other pathogens. Proteogenomic analysis indicated that several potential targets of disulfiram (i.e., aldehyde dehydrogenase and urease) were present in P. insidiosum By homology modeling and molecular docking, disulfiram can bind the putative aldehyde dehydrogenase and urease of P. insidiosum at low energies (i.e., -6.1 and -4.0 Kcal/mol, respectively). Disulfiram diminished the biochemical activities of these enzymes. In conclusion, disulfiram can inhibit the growth of many pathogenic microorganisms, including P. insidiosum The drug can bind and inactivate multiple proteins of P. insidiosum, which may contribute to its broad antimicrobial property. Drug repurposing of disulfiram could be a new treatment option for pythiosis.

Keywords: Pythium insidiosum; disulfiram; drug repurposing; pythiosis; susceptibility.

FIG 1

Agar-based susceptibility of P. insidiosum against disulfiram. The radial growths (%) of the organism (27 strains) after exposure to various concentrations of disulfiram (range, 0 to 128 mg/liter) are plotted. A straight line indicates mean growth. An asterisk represents significant growth reduction in relation to the no-drug control.

FIG 2

Complexes of the ligand “disulfiram” and the predicted 3D structures of the urease (A to C) and aldehyde dehydrogenase (D to F) of P. insidiosum. Panels A and D show ribbon structure models, while panels B and E depict enlarged structure showing molecular docking of disulfiram. Panels C and F show the 2D interactions of disulfiram and the predicted binding sites of urease and aldehyde dehydrogenase, respectively. The interacting amino acids and types are shown in figures B, C, E, and F. The structures and complex interaction images were generated using the UCSF Chimera and Discovery Studio Visualizer (see Materials and Methods).

FIG 3

Biochemical effect of disulfiram on urease activity. The urease test tube assays were tested against DMSO (a diluent of disulfiram; this served as a control) (A), Jack bean urease alone (0.1 mg/ml) (B), and a preincubated mixture of Jack bean urease (0.1 mg/ml) and disulfiram (128 mg/liter) (C).