Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of Trypanosoma cruzi expressed in Saccharomyces cerevisiae

Abstract

ABSTRACT:Recently, a number of inhibitors of the enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), a key enzyme in sterol biosynthesis, were shown to inhibit in mammalian cells the multiplication of Trypanosoma cruzi, the parasite agent of Chagas' disease. The gene coding for the OSC of T. cruzi has been cloned and expressed in Saccharomyces cerevisiae. The expression in yeast cells could be a safe and easy model for studying the activity and the selectivity of the potential inhibitors of T. cruzi OSC. Sterols are eukaryotic membrane components that are necessary to support cell growth and differentiation. Sterol biosynthesis is a well-established chemotherapeutic target in pathogenic eukaryotes, such as fungi (1,2). The final product of the sterol biosynthetic pathway in fungi is the 24-alkyl sterol ergosterol. Ergosterol or similar 24-alkyl sterols are also the final products of the sterol synthetic pathway of some pathogenic protozoa including Trypanosoma species. These organisms cannot completely substitute these sterols with the cholesterol biosynthesized in host mammalian cells and need to synthesize at least some of their own distinctive sterols (3-5). This dependence on sterols suggests that sterol biosynthesis inhibitors may be useful antiprotozoal drugs. The sterol biosynthetic pathway ( Fig. 1) offers many potential targets for therapeutic purposes. Many effective antifungal drugs already in use act by inhibiting different enzymes of sterol biosynthesis; the allylamine antifungal drug terbinafine inhibits squalene epoxidase (6), and azole drugs such as ketoconazole and itraconazole are inhibitors of lanosterol C 14 -demethylase (7). The activity of these inhibitors can be explained as a consequence either of depletion of the ergosterol or of accumulation of toxic intermediates or side products. In addition to the antifungal activity, some of these compounds Acetyl-CoAwere shown to inhibit the growth of Trypanosoma and other kinetoplastid parasites (8). Kinetoplastid parasites have also been shown recently to be susceptible to inhibitors of squalene synthase (9,10) and oxidosqualene cyclase (11,12). The enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), which catalyzes the formation of the first cyclic precursor of sterols, is considered a good target for the inhibition of sterol biosynthesis in both humans and fungi. Many inhibitors, designed on the basis of the complex cyclization mechanism of OSC (13), are active on the enzyme and have been investigated as potential cholesterol-lowering or antifungal drugs (1,2,13). The recent crystallization of human OSC as a complex with the potent inhibitor RO48-8071 opens the way to new structurebased studies of the cyclization mechanism (14).We have designed and tested against mammalian and fungal OSC many acyclic substrate analogs modified either to mimic the high-energy carbocationic intermediates (compounds 1-3 of Fig. 2) or to bear reactive functions able to interact with the active site residues, as the methylidene (compounds 4-8), vinyl sulfide (compounds 9...

Squalene-Hopene Cyclases

“…However, the lipophilic entrance channel of OSCs rejects, for example, squalene without epoxide function before entering the active site, resulting in a substrate selection that is more specific than that of SHCs. SHCs accept both squalene as well as 2,3-oxidosqualene (57). Synthesis of hopene from squalene involves formation of five ring structures accompanied by the alteration of 13 covalent bonds and the generation of nine stereo centers.…”

Characterization of the Channel Constriction Allowing the Access of the Substrate to the Active Site of Yeast Oxidosqualene Cyclase

“…According to this hypothesis, squalene should act as a competitive inhibitor and the channel constriction could operate as an entropic trap able to prefer a properly oriented molecule of substrate, in order to reduce the risk of competitive inhibition. By testing the inhibitory effect of different concentrations of squalene on the control mutant C457D, we observed inhibitory effect only at concentration ≥0.5 mM, a very high concentration, compared with the Km of oxidosqualene which is in the micromolar range [14]. This suggests that the enzyme is not completely indifferent to the competitive action of squalene, though able to overcome its competition with substrate under physiological conditions.…”

An Insight on Targets and Patented Drugs for Chemotherapy of Chagas Disease

“…By using a recombinant T. cruzi OSC expressed in yeast, 19 inhibitors: aza, methyllidene, vinyl sulfide, and conjugated vinyl sulfide derivatives of oxidosqualene and squalene, were tested. Many inhibitors of control OSC showed comparable IC50 for T. cruzi OSC, but some phenylthiovinyl derivatives showed to be 10-100 times more effective on the T. cruzi enzyme than on the control enzymes [119]. and antiparasitic activity confirmed in a murine model of Chagas disease.…”