Discovery of potent and selective inhibitors of Trypanosoma brucei ornithine decarboxylase

Abstract

Human African trypanosomiasis, caused by the eukaryotic parasite Trypanosoma brucei, is a serious health problem in much of central Africa. The only validated molecular target for treatment of human African trypanosomiasis is ornithine decarboxylase (ODC), which catalyzes the first step in polyamine metabolism. Here, we describe the use of an enzymatic high throughput screen of 316,114 unique molecules to identify potent and selective inhibitors of ODC. This screen identified four novel families of ODC inhibitors, including the first inhibitors selective for the parasitic enzyme. These compounds display unique binding modes, suggesting the presence of allosteric regulatory sites on the enzyme. Docking of a subset of these inhibitors, coupled with mutagenesis, also supports the existence of these allosteric sites.

FIGURE 1.

Schematic of the polyamine biosynthetic pathway in T. brucei. The following abbreviations are used: S-adenosylmethionine decarboxylase (SAMDC), S-adenosylmethionine (S-AdoMet), 5′-methylthioadenosine (MTA), decarboxylated S-adenosylmethionine (DC-S-AdoMet), putrescine (Put), spermidine (Spd), spermidine synthase (SpdS), DFMO, glutathionyl spermidine synthase (GSS), trypanothione synthase (TryS), trypanothione reductase (TryR), and reactive oxygen species (ROS).

FIGURE 2.

Bisbiguanide inhibitor data. a and b, Lineweaver-Burk plots for ornithine versus compound 1 and PLP versus 1. ♦ = 10 μm, ▾ = 6 μm, ▴ = 4 μm, and ● = uninhibited. c, species selectivity analysis for compound 1. Data for TbODC (▾) and hODC (■) enzyme-linked assays were collected at 22 °C in 384-well plates. Data were fitted to a four-parameter sigmoidal dose response for determination of IC₅₀ values. All data were collected under isokinetic conditions at 1.5× K_m for ornithine. d, reversibility of compound 1. TbODC was incubated with inhibitor for 1 h followed by overnight dialysis into assay buffer. Data were collected as described under “Experimental Procedures.”

FIGURE 3.

Benzthiazole inhibitor data. Selectivity curves and reversibility data for these compounds can be found in the supplemental material. Lineweaver-Burk plot for ornithine versus compound 2 and PLP versus 2. ♦ = 28 μm, ▾ = 9.5 μm, ▴ = 3.2 μm, and ● = uninhibited.

FIGURE 4.

Indole inhibitor data. Selectivity curves and reversibility data for these compounds can be found in the supplemental material. Lineweaver-Burk plot for ornithine versus compound 6 and PLP versus 6. ♦ = 40 μm, ▾ = 13 μm, ▴ = 4.4 μm, and ● = uninhibited.

FIGURE 5.

Dithioamidine inhibitor data. a and b, Lineweaver-Burk plots for ornithine versus compound 8 and PLP versus 8. ♦ = 40 μm, ▾ = 13 μm, ▴ = 4.4 μm, and ● = uninhibited. c, species selectivity analysis for compound 8. Data for TbODC (▾) and hODC (■) enzyme-linked assays were collected at 22 °C in 384-well plates. Data were fitted to a four-parameter sigmoidal dose response for determination of IC₅₀ values. All data were collected under isokinetic conditions at 1.5× K_m for ornithine. d, reversibility of compound 8. TbODC was incubated with inhibitor for 1 h followed by overnight dialysis into assay buffer. Data were collected as described under “Experimental Procedures.”

FIGURE 6.

Proposed dithioamidine-binding site. a, TbODC bound to DFMO. DFMO is in purple; PLP is salmon; active site residues are white, and residues that form the proposed binding site are indicated as follows: Asp-364 is colored yellow and forms the bottom of the binding site, and magenta residues form the two possible entrances to the proposed dithioamidine-binding site. b, compound 9 docked into the proposed binding site on TbODC and superimposed with the apo human ODC structure. TbODC residues are orange, and hODC residues are light blue. Asp-364 is yellow, and active site residues are white, DFMO is purple, and PLP is salmon. Note that Ser-402 from TbODC is not conserved in hODC, and that the opening to the proposed site is completely occluded in the human enzyme. This could help account for the remarkable selectivity seen with the dithioamidine series of inhibitors.