Sterol 14α-Demethylase as a Potential Target for Antitrypanosomal Therapy: Enzyme Inhibition and Parasite Cell Growth

Abstract

Sterol 14alpha-demethylases (CYP51) serve as primary targets for antifungal drugs, and specific inhibition of CYP51s in protozoan parasites Trypanosoma brucei (TB) and Trypanosoma cruzi (TC) might provide an effective treatment strategy for human trypanosomiases. Primary inhibitor selection is based initially on the cytochrome P450 spectral response to ligand binding. Ligands that demonstrate strongest binding parameters were examined as inhibitors of reconstituted TB and TC CYP51 activity in vitro. Direct correlation between potency of the compounds as CYP51 inhibitors and their antiparasitic effect in TB and TC cells implies essential requirements for endogenous sterol production in both trypanosomes and suggests a lead structure with a defined region most promising for further modifications. The approach developed here can be used for further large-scale search for new CYP51 inhibitors.

Structural Insights into Inhibition of Sterol 14α-Demethylase in the Human Pathogen Trypanosoma cruzi

“…Binding and Inhibition of TC14DM with Posaconazole, Fluconazole, and VNF-Although the apparent K d s determined by spectral titration are useful for primary identification of new binding ligands, they do not always correlate with inhibition of 14DM activity (12). Thus, we found that several imidazole derivatives that reveal nanomolar K d s were easily replaced in the enzyme reaction by substrate, whereas VNF derivatives were not (12).…”

“…Thus, we found that several imidazole derivatives that reveal nanomolar K d s were easily replaced in the enzyme reaction by substrate, whereas VNF derivatives were not (12). On the other hand, some pyridine derivatives, which induce moderate spectral responses (the apparent K d s ϳ 0.2-0.5 M), turned out to be rather strong inhibitors of TC14DM (38).…”

“…Immunoblot Analysis of 14DM Expression in T. cruzi-To evaluate expression of 14DM in the three developmental stages of T. cruzi, 5 g of whole extracts of trypomastigote (12), epimastigote (12), and amastigote (34) forms of the highly invasive 20A clone of the Tulahuen strain (35) were solubilized for 10 min at 96°C in a nonreducing SDS mixture (2% SDS, 10% glycerol, 0.005% bromphenol blue, and 80 mM Tris-HCL, pH 6.8), subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with a 1:2000 dilution of mouse anti-Tbb14DM antiserum (28) or with the same dilution of preimmune serum. Bound antibodies were detected by chemiluminescence (catalog no.…”

“…Ergosterol-like molecules are required for parasite membranes, growth, development, and division (11,12). Sterol 14␣-demethylase (14DM, 3 CYP51 gene family), the membrane-bound cytochrome P450 catalyzing oxidative removal of the 14␣-methyl group from the sterol core, is a highly drug-targetable enzyme in sterol biosynthetic pathways (13).…”

“…1b) is an experimental inhibitor (12). Highly potent for trypanosomal 14DMs, this compound displays low general cytotoxicity and does not inhibit the human 14DM ortholog, which makes this carboxamide-containing inhibitory scaffold potentially advantageous in the cases of long term treatment.…”