Anthelmintic resistance in human and animal pathogenic helminths has been spreading in prevalence and severity to a point where multidrug resistance against the three major classes of anthelmintics--the benzimidazoles, imidazothiazoles and macrocyclic lactones--has become a global phenomenon in gastrointestinal nematodes of farm animals. Hence, there is an urgent need for an anthelmintic with a new mode of action. Here we report the discovery of the amino-acetonitrile derivatives (AADs) as a new chemical class of synthetic anthelmintics and describe the development of drug candidates that are efficacious against various species of livestock-pathogenic nematodes. These drug candidates seem to have a novel mode of action involving a unique, nematode-specific clade of acetylcholine receptor subunits. The AADs are well tolerated and of low toxicity to mammals, and overcome existing resistances to the currently available anthelmintics.

The human pathogen Candida albicans is responsible for a large proportion of infections in immunocompromised individuals, and the emergence of drug-resistant strains is of medical concern. Resistance to antifungal azole compounds is often due to an increase in drug efflux or an alteration of the pathway for synthesis of ergosterol, an important plasma membrane component in fungi. However, little is known about the transcription factors that mediate drug resistance. In Saccharomyces cerevisiae, two highly related transcriptional activators, Upc2p and Ecm22p, positively regulate the expression of genes involved in ergosterol synthesis (ERG genes). We have identified a homologue in C. albicans of the S. cerevisiae UPC2/ECM22 genes and named it UPC2. Deletion of this gene impaired growth under anaerobic conditions and rendered cells highly susceptible to the antifungal drugs ketoconazole and fluconazole. Conversely, overexpression of Upc2p increased resistance to ketoconazole, fluconazole, and fluphenazine. Azole-induced expression of the ERG genes was abolished in a ⌬upc2 strain, while basal levels of these mRNAs remained unchanged. Importantly, the purified DNA binding domain of Upc2p bound in vitro to putative sterol response elements in the ERG2 promoter, suggesting that Upc2p increases the expression of the ERG genes by directly binding to their promoters. These results provide an important link between changes in the ergosterol biosynthetic pathway and azole resistance in this opportunistic fungal species.

Cap1p, a transcription factor of the basic region leucine zipper family, regulates the oxidative stress response (OSR) in Candida albicans. Alteration of its C-terminal cysteine-rich domain (CRD) results in Cap1p nuclear retention and transcriptional activation. To better understand the function of Cap1p in C. albicans, we used genome-wide location profiling (chromatin immunoprecipitation-on-chip) to identify its transcriptional targets in vivo. A triple-hemagglutinin (HA 3 ) epitope was introduced at the C terminus of wild-type Cap1p (Cap1p-HA 3 ) or hyperactive Cap1p with an altered CRD (Cap1p-CSE-HA 3 ). Location profiling using wholegenome oligonucleotide tiling microarrays identified 89 targets bound by Cap1p-HA 3 or Cap1p-CSE-HA 3 (the binding ratio was at least twofold; P < 0.01). Strikingly, Cap1p binding was detected not only at the promoter region of its target genes but also at their 3 ends and within their open reading frames, suggesting that Cap1p may associate with the transcriptional or chromatin remodeling machinery to exert its activity. Overrepresented functional groups of the Cap1p targets (P < 0.02) included 11 genes involved in the OSR (CAP1, GLR1, TRX1, SOD1, CAT1, and others), 13 genes involved in response to drugs (PDR16, MDR1, FLU1, YCF1, FCR1, and others), 4 genes involved in phospholipid transport (PDR16, GIT1, RTA2, and orf19.932), and 3 genes involved in the regulation of nitrogen utilization (GST3, orf19.2693, and orf19.3121), suggesting that Cap1p has other cellular functions in addition to the OSR. Bioinformatic analyses of the bound sequences suggest that Cap1p recognizes the DNA motif 5-MTKASTMA. Finally, transcriptome analyses showed that increased expression generally accompanies Cap1p binding at its targets, indicating that Cap1p functions as a transcriptional activator.Candida albicans is an opportunistic human fungal pathogen that causes superficial infections in healthy patients. However, in patients with impaired immunity C. albicans can cause lifethreatening invasive infections, including systemic candidiasis or candidemia. In the United States, candidemia represents the fourth-most-common cause of nosocomial bloodstream infections (7). Several options for the treatment of invasive candidiasis are available to clinicians, including the administration of azole derivatives, amphotericin B preparations, or echinocandin antifungal agents, while for the treatment of mucocutaneous infections, azoles are preferred over other antifungals due to their low toxicity and increased efficacy and availability for both topical and oral use (21, 50).Azoles, including both imidazoles (e.g., ketoconazole) and triazoles (e.g., fluconazole [FLC]), inhibit the function of the lanosterol demethylase enzyme Erg11p, a component of the ergosterol biosynthesis pathway, leading to methylsterol accumulation, sterol depletion, and consequently to growth arrest (1). This fungistatic property of azoles coupled to their repeated use in the clinic renders the surviving C. albicans cells prone to the sele...

Upc2p, a transcription factor of the zinc cluster family, is an important regulator of sterol biosynthesis and azole drug resistance in Candida albicans. To better understand Upc2p function in C. albicans, we used genomewide location profiling to identify the transcriptional targets of Upc2p in vivo. A triple hemagglutinin epitope, introduced at the C terminus of Upc2p, conferred a gain-of-function effect on the fusion protein.Location profiling identified 202 bound promoters (P < 0.05). Overrepresented functional groups of genes whose promoters were bound by Upc2p included 12 genes involved in ergosterol biosynthesis (NCP1, ERG11, ERG2, and others), 18 genes encoding ribosomal subunits (RPS30, RPL32, RPL12, and others), 3 genes encoding drug transporters (CDR1, MDR1, and YOR1), 4 genes encoding transcription factors (INO2, ACE2, SUT1, and UPC2), and 6 genes involved in sulfur amino acid metabolism (MET6, SAM2, SAH1, and others). Bioinformatic analyses suggested that Upc2p binds to the DNA motif 5-VNCGBDTR that includes the previously characterized Upc2p binding site 5-TCGTATA. Northern blot analysis showed that increased binding correlates with increased expression for the analyzed Upc2p targets (ERG11, MDR1, CDR1, YOR1, SUT1, SMF12, and CBP1). The analysis of ERG11, MDR1, and CDR1 transcripts in wild-type and upc2⌬/upc2⌬ strains grown under Upc2p-activating conditions (lovastatin treatment and hypoxia) showed that Upc2p regulates its targets in a complex manner, acting as an activator or as a repressor depending upon the target and the activating condition. Taken together, our results indicate that Upc2p is a key regulator of ergosterol metabolism. They also suggest that Upc2p may contribute to azole resistance by regulating the expression of drug efflux pump-encoding genes in addition to ergosterol biosynthesis genes.Candida albicans is an important human fungal pathogen, in terms of both its clinical significance and its use as an experimental model for scientific investigation. This opportunistic pathogen is a natural component of the human flora, colonizing skin and the gastrointestinal and genitourinary tracts (4). Although many infections involve the colonization of surface mucosal membranes (oral thrush and vaginal candidiasis, for example), immunosuppressed patients can be subject to potentially lethal systemic infections (45).Many antifungal drugs used to treat C. albicans infections function by targeting ergosterol, the analogue of cholesterol in mammalian cells and the major sterol of fungal cell membranes. Polyenes, such as amphotericin B, bind directly to ergosterol and perturb general membrane functions, resulting in low selectivity and high toxicity (2, 35). Azoles, including fluconazole and voriconazole, are more widely used and target the enzyme lanosterol demethylase (Erg11p) in the ergosterol biosynthesis pathway, with the consequence that ergosterol is depleted and replaced by unusual sterols, altering the fluidity of the membrane and the activities of membrane-bound proteins (e.g., enzymes...