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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.

Nuclear factor erythroid 2-related factor 2 (Nrf2) mediates the transcriptional response of cells to oxidative stress and is translocated into the nucleus following, or concomitant with, its activation by electrophiles or reactive oxygen species. The mechanism of its translocation into the nucleus is not entirely elucidated. Here we have identified two novel nuclear localization signal (NLS) motifs in murine Nrf2, one located near the N-terminal region (amino acid residues 42-53) and the other (residues 587-593) located near the C-terminal region. Imaging of green fluorescent protein (GFP)-tagged Nrf2 revealed that mutation(s) in any of these sequences resulted in decreased nuclear fluorescence intensity compared with the wild-type Nrf2 when Nrf2 activation was induced with the electrophile tert-butylhydroquinone. The mutations also impaired Nrf2-induced transactivation of antioxidant response element-driven reporter gene expression to the same extent as the Nrf2 construct bearing mutation in a previously identified bipartite NLS that maps at residues 494 -511. When linked to GFP or to GFP-PEPCK-C each of the novel NLS motifs was sufficient to drive nuclear translocation of the fusion proteins. Co-immunoprecipitation assays demonstrated that importins ␣5 and ␤1 associate with Nrf2, an interaction that was blocked by the nuclear import inhibitor SN50. SN50 also blocked tert-butylhydroquinone-induced nuclear fluorescence of GFP-Nrf2 in cells transfected with wild-type GFP-Nrf2. Overall these results reveal that multiple NLS motifs in Nrf2 function in its nuclear translocation in response to pro-oxidant stimuli and that the importin ␣-␤ heterodimer nuclear import receptor system plays a critical role in the import process.Nuclear factor erythroid 2-related factor 2 (Nrf2), 4 in association with the cytoskeleton-associated Kelch-like protein Keap1, functions as a sensor of oxidative and electrophilic stress in cells (1-4). In non-stressed cells, Nrf2 is transcriptionally inactive because of the repressive effect of Keap1 in the cytoplasm (4 -6). Reactive oxygen species or electrophilic agents induce modifications of this complex that allow Nrf2 to translocate into the nucleus to mediate activation of a variety of genes (2, 5-12). The promoters of such genes contain antioxidant response element(s) (AREs), at which Nrf2, in association with small Maf proteins or other basic region-leucine zipper transcription factors (1, 13-18), interacts to regulate gene transcription. As determined by microarray analyses, such genes include those that code for proteins that function in DNA repair, enzymes that catalyze phase II reactions in drug metabolism, signal transduction proteins, and many others that function in protein trafficking, chaperone system/stress response, and apoptosis (19,20).Electrophile-induced Nrf2 release from the Keap1-Nrf2 complex appears to involve not only modification of specific cysteine residues in Keap1 (7-10) but also switching of Cullin 3-dependent ubiquitination from Nrf2 to Keap1, leading to the de...

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The cytochrome P540 (CYP) superfamily currently includes about 9,000 proteins forming more than 800 families. The enzymes catalyze monooxygenation of a vast array of compounds and play essentially two roles. They provide biodefense (detoxification of xenobiotics, antibiotic production) and participate in biosynthesis of important endogenous molecules, particularly steroids. Based on these two roles, sterol 14|*alpha*|-demethylases (CYP51) belong to the second group of P450s. The CYP51 family, however, is very special as its members preserve strict functional conservation in enzyme activity in all biological kingdoms. At amino acid identity across the kingdoms as low as 25-30%, they all catalyze essentially the same three-step reaction of oxidative removal of the 14| *alpha*|-methyl group from the lanostane frame. This reaction is the required step in sterol biosynthesis of pathogenic microbes. We have shown that specific inhibition of protozoan CYP51 can potentially provide treatment for human trypanosomiases. Three sets of CYP51 inhibitors tested in vitro and in trypanosomal cells in this study include azoles [best results being 50% cell growth inhibition at <1 and at 1.3 µM for Trypanosoma cruzi (TC) and Trypanosoma brucei (TB), respectively], non-azole compounds (50% TC cell growth inhibition at 5 µM) and substrate analogs of the 14|*alpha*|-demethylase reaction. 32-Methylene cyclopropyl lanost-7-enol exhibited selectivity toward TC with 50% cell growth inhibition at 3 µM.