Ehrlich's pioneering chemotherapeutic experiments published in 1904 (Ehrlich, P., and Shiga, K. (1904) Berlin Klin. Wochenschrift 20, 329-362) described the efficacy of a series of
dye molecules including
trypan blue and
trypan red to eliminate trypanosome
infections in mice. The molecular structures of the
dyes provided a starting point for the synthesis of
suramin, which was developed and used as a trypanocidal
drug in 1916 and is still in clinical use. Despite the
biological importance of these
dye-like molecules, the mode of action on trypanosomes has remained elusive. Here we present crystal structures of
suramin and three related
dyes in complex with
pyruvate kinases from Leishmania mexicana or from Trypanosoma cruzi. The phenyl sulfonate groups of all four molecules (
suramin,
Ponceau S,
acid blue 80, and
benzothiazole-2,5-disulfonic
acid) bind in the position of
ADP/
ATP at the active sites of the
pyruvate kinases (PYKs). The binding positions in the two different trypanosomatid PYKs are nearly identical. We show that
suramin competitively inhibits PYKs from humans (muscle,
tumor, and liver
isoenzymes, K(i) = 1.1-17 μM), T. cruzi (K(i) = 108 μM), and L. mexicana (K(i) = 116 μM), all of which have similar active sites. Synergistic effects were observed when examining
suramin inhibition in the presence of an allosteric effector molecule, whereby IC(50) values decreased up to 2-fold for both trypanosomatid and human PYKs. These kinetic and structural analyses provide insight into the promiscuous inhibition observed for
suramin and into the mode of action of the
dye-like molecules used in Ehrlich's original experiments.