Kinetic characterization of squalene synthase from Trypanosoma cruzi: selective inhibition by quinuclidine derivatives.

The biosynthesis of sterols is a major route for the development of antitrypanosomals. Squalene synthase (SQS) catalyzes the first step committed to the biosynthesis of sterols within the isoprenoid pathway, and several inhibitors of the enzyme have selective antitrypanosomal activity both in vivo and in vitro. The enzyme from Trypanosoma cruzi is a 404-amino-acid protein with a clearly identifiable membrane-spanning region. In an effort to generate soluble recombinant enzyme, we have expressed in Escherichia coli several truncated versions of T. cruzi SQS with a His tag attached to the amino terminus. Deletions of both the amino- and carboxyl-terminal regions generated active and soluble forms of the enzyme. The highest levels of soluble protein were achieved when 24 and 36 amino acids were eliminated from the amino and carboxyl regions, respectively, yielding a protein of 41.67 kDa. The Michaelis-Menten constants of the purified enzyme for farnesyl diphosphate and NAD (NADPH) were 5.25 and 23.34 microM, respectively, whereas the V(max) was 1,428.56 nmol min(-1)mg(-1). Several quinuclidine derivatives with antiprotozoal activity in vitro were found to be selective inhibitors of recombinant T. cruzi SQS in comparative assays with the human enzyme, with 50% inhibitory concentration values in the nanomolar range. These data suggest that selective inhibition of T. cruzi SQS may be an efficient strategy for the development of new antitrypanosomal agents.