Browsing by Author "Chaudhuri, Minu"
Now showing 1 - 7 of 7
- Results Per Page
- Sort Options
Item Crystal structures of Trypanosoma brucei sterol 14α-demethylase and implications for selective treatment of human infections(2010) Lepesheva, Galina I.; Park, Hee Won; Hargrove, Tatiana Y.; Vanhollebeke, Benoit; Wawrzak, Zdzislaw; Harp, Joel M.; Sundaramoorthy, Munirathinam; Nes, W. David (TTU); Pays, Etienne; Chaudhuri, Minu; Villalta, Fernando; Waterman, Michael R.Sterol 14α-demethylase (14DM, the CYP51 family of cytochrome P450) is an essential enzyme in sterol biosynthesis in eukaryotes. It serves as a major drug target for fungal diseases and can potentially become a target for treatment of human infections with protozoa. Here we present 1.9 Å resolution crystal structures of 14DM from the protozoan pathogen Trypanosoma brucei, ligand-free and complexed with a strong chemically selected inhibitor N-1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3, 4-oxadi-azol-2-yl)benzamide that we previously found to produce potent antiparasitic effects in Trypanosomatidae. This is the first structure of a eukaryotic microsomal 14DM that acts on sterol biosynthesis, and it differs profoundly from that of the water-soluble CYP51 family member from Mycobacterium tuberculosis, both in organization of the active site cavity and in the substrate access channel location. Inhibitor binding does not cause large scale conformational rearrangements, yet induces unanticipated local alterations in the active site, including formation of a hydrogen bond network that connects, via the inhibitor amide group fragment, two remote functionally essential protein segments and alters the heme environment. The inhibitor binding mode provides a possible explanation for both its functionally irreversible effect on the enzyme activity and its selectivity toward the14DM from human pathogens versus the human 14DM ortholog. The structures shed new light on 14DM functional conservation and open an excellent opportunity for directed design of novel antiparasitic drugs. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.Item Discovery of an ergosterol-signaling factor that regulates trypanosoma brucei growth(2015) Haubrich, Brad A. (TTU); Singha, Ujjal K.; Miller, Matthew B. (TTU); Nes, Craigen R. (TTU); Anyatonwu, Hosanna (TTU); Lecordier, Laurence; Patkar, Presheet (TTU); Leaver, David J. (TTU); Villalta, Fernando; Vanhollebeke, Benoit; Chaudhuri, Minu; Nes, W. David (TTU)Ergosterol biosynthesis and homeostasis in the parasitic protozoan Trypanosoma brucei was analyzed by RNAi silencing and inhibition of sterol C24β -methyltransferase ( TbSMT) and sterol 14α-demethylase [ TbSDM ( TbCYP51)] to explore the functions of sterols in T. brucei growth. Inhibition of the amount or activity of these enzymes depletes ergosterol from cells at <6 fg/cell for procyclic form (PCF) cells or <0.01 fg/cell for bloodstream form (BSF) cells and reduces infectivity in a mouse model of infection. Silencing of Tb SMT expression by RNAi in PCF or BSF in combination with 25-azalanosterol (AZA) inhibited parasite growth and this inhibition was restored completely by adding synergistic cholesterol (7.8 μM from lipid-depleted media) with small amounts of ergosterol (1.2 μM) to the medium. These observations are consistent with the proposed requirement for ergosterol as a signaling factor to spark cell proliferation while imported cholesterol or the endogenously formed cholesta-5,7,24-trienol act as bulk membrane components. To test the potential chemotherapeutic importance of disrupting ergosterol biosynthesis using pairs of mechanismbased inhibitors that block two enzymes in the post-squalene segment, parasites were treated with AZA and itraconazole at 1 μM each (ED50 values) resulting in parasite death . Taken together, our results demonstrate that the ergosterol pathway is a prime drug target for intervention in T. brucei infection. -Haubrich, B. A., U. K. Singha, M. B. Miller, C. R. Nes, H. Anyatonwu, L. Lecordier, P. Patkar, D. J. Leaver, F. Villalta, B. Vanhollebeke, M. Chaudhuri, and W. D. Nes. Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth. J. Lipid Res. 2015.Item Druggable Sterol Metabolizing Enzymes in Infectious Diseases: Cell Targets to Therapeutic Leads(2024) Nes, W. David (TTU); Chaudhuri, Minu; Leaver, David J.Sterol biosynthesis via the mevalonate-isoprenoid pathway produces ergosterol (24β-methyl cholesta-5,7-dienol) necessary for growth in a wide-range of eukaryotic pathogenic organisms in eukaryotes, including the fungi, trypanosomes and amoebae, while their animal hosts synthesize a structurally less complicated product—cholesterol (cholest-5-enol). Because phyla-specific differences in sterol metabolizing enzyme architecture governs the binding and reaction properties of substrates and inhibitors while the order of sterol metabolizing enzymes involved in steroidogenesis determine the positioning of crucial chokepoint enzymes in the biosynthetic pathway, the selectivity and effectiveness of rationally designed ergosterol biosynthesis inhibitors toward ergosterol-dependent infectious diseases varies greatly. Recent research has revealed an evolving toolbox of mechanistically distinct tight-binding inhibitors against two crucial methylation-demethylation biocatalysts—the C24 sterol methyl transferase (absent from humans) and the C14-sterol demethylase (present generally in humans and their eukaryotic pathogens). Importantly for rational drug design and development, the activities of these enzymes can be selectively blocked in ergosterol biosynthesis causing loss of ergosterol and cell killing without harm to the host organism. Here, we examine recent advances in our understanding of sterol biosynthesis and the reaction differences in catalysis for sterol methylation-demethylation enzymes across kingdoms. In addition, the novelties and nuances of structure-guided or mechanism-based approaches based on crystallographic mappings and substrate specificities of the relevant enzyme are contrasted to conventional phenotypic screening of small molecules as an approach to develop new and more effective pharmacological leads.Item Enzymatic chokepoints and synergistic drug targets in the sterol biosynthesis pathway of Naegleria fowleri(2018) Zhou, Wenxu (TTU); Debnath, Anjan; Jennings, Gareth; Hahn, Hye Jee; Vanderloop, Boden H. (TTU); Chaudhuri, Minu; Nes, W. David (TTU); Podust, Larissa M.Naegleria fowleri is a free-living amoeba that can also act as an opportunistic pathogen causing severe brain infection, primary amebic meningoencephalitis (PAM), in humans. The high mortality rate of PAM (exceeding 97%) is attributed to (i) delayed diagnosis, (ii) lack of safe and effective anti-N. fowleri drugs, and (iii) difficulty of delivering drugs to the brain. Our work addresses identification of new molecular targets that may link anti-Naegleria drug discovery to the existing pharmacopeia of brain-penetrant drugs. Using inhibitors with known mechanism of action as molecular probes, we mapped the sterol biosynthesis pathway of N. fowleri by GC-MS analysis of metabolites. Based on this analysis, we chemically validated two enzymes downstream to CYP51, sterol C24-methyltransferase (SMT, ERG6) and sterol Δ8−Δ7 -isomerase (ERG2), as potential therapeutic drug targets in N. fowleri. The sterol biosynthetic cascade in N. fowleri displayed a mixture of canonical features peculiar to different domains of life: lower eukaryotes, plants and vertebrates. In addition to the cycloartenol→ergosterol biosynthetic route, a route leading to de novo cholesterol biosynthesis emerged. Isotopic labeling of the de novo-synthesized sterols by feeding N. gruberi trophozoites on the U13C-glucose-containing growth medium identified an exogenous origin of cholesterol, while 7-dehydrocholesterol (7DHC) had enriched 13C-content, suggesting a dual origin of this metabolite both from de novo biosynthesis and metabolism of scavenged cholesterol. Sterol homeostasis in Naegleria may be orchestrated over the course of its life-cycle by a “switch” between ergosterol and cholesterol biosynthesis. By demonstrating the growth inhibition and synergistic effects of the sterol biosynthesis inhibitors, we validated new, potentially druggable, molecular targets in N. fowleri. The similarity of the Naegleria sterol Δ8−Δ7 -isomerase to the human non-opioid σ1 receptor, implicated in human CNS conditions such as addiction, amnesia, pain and depression, provides an incentive to assess structurally diverse small-molecule brain-penetrant drugs targeting the human receptor for anti-Naegleria activity.Item Steroidal antibiotics are antimetabolites of Acanthamoeba steroidogenesis with phylogenetic implications(2019) Zhou, Wenxu (TTU); Ramos, Emilio (TTU); Zhu, Xunlu (TTU); Fisher, Paxtyn M. (TTU); Kidane, Medhanie E. (TTU); Vanderloop, Boden H. (TTU); Thomas, Crista D. (TTU); Yan, Juqiang (TTU); Singha, Ujjal; Chaudhuri, Minu; Nagel, Michael T. (TTU); David Nes, W. (TTU)Pathogenic organisms may be sensitive to inhibitors of sterol biosynthesis, which carry antimetabolite properties, through manipulation of the key enzyme, sterol methyltransferase (SMT). Here, we isolated natural suicide substrates of the ergosterol biosynthesis pathway, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetraenol (ERGT), and demonstrated their interference in Acanthamoeba castellanii steroidogenesis: CHT and ERGT inhibit trophozoite growth (EC50 of 51 nM) without affecting cultured human cell growth. Washout experiments confirmed that the target for vulnerability was SMT. Chemical, kinetic, and protein-binding studies of inhibitors assayed with 24-AcSMT [catalyzing C28-sterol via 24(28)-olefin production] and 28-AcSMT [catalyzing C29-sterol via 25(27)-olefin production] revealed interrupted partitioning and irreversible complex formation from the conjugated double bond system in the side chain of either analog, particularly with 28-AcSMT. Replacement of active site Tyr62 with Phe or Leu residues involved in cation- interactions that model product specificity prevented protein inactivation. The alkylating properties and high selective index of 103 for CHT and ERGT against 28-AcSMT are indicative of a new class of steroidal antibiotic that, as an antimetabolite, can limit sterol expansion across phylogeny and provide a novel scaffold in the design of amoebicidal drugs. Animal studies of these suicide substrates can further explore the potential of their antibiotic properties.Item Steroidal Antimetabolites Protect Mice against Trypanosoma brucei(2022) Chaudhuri, Minu; Singha, Ujjal K.; Vanderloop, Boden H. (TTU); Tripathi, Anuj; Nes, W. David (TTU)Trypanosoma brucei, the causative agent for human African trypanosomiasis, is an emerging ergosterol-dependent parasite that produces chokepoint enzymes, sterol methyltransferases (SMT), not synthesized in their animal hosts that can regulate cell viability. Here, we report the lethal effects of two recently described natural product antimetabolites that disrupt Acanthamoeba sterol methylation and growth, cholesta-5,7,22,24-tetraenol (CHT) and ergosta-5,7,22,24(28)-tetrae-nol (ERGT) that can equally target T. brucei. We found that CHT/ERGT inhibited cell growth in vitro, yielding EC50 values in the low nanomolar range with washout experiments showing cidal activity against the bloodstream form, consistent with their predicted mode of suicide inhibition on SMT activity and ergosterol production. Antimetabolite treatment generated altered T. brucei cell morphology and death rapidly within hours. Notably, in vivo ERGT/CHT protected mice infected with T. brucei, doubling their survival time following daily treatment for 8–10 days at 50 mg/kg or 100 mg/kg. The current study demonstrates a new class of lead antibiotics, in the form of common fungal sterols, for antitrypanosomal drug development.Item Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design(2017) Kidane, Medhanie E. (TTU); Vanderloop, Boden H. (TTU); Zhou, Wenxu (TTU); Thomas, Crista D. (TTU); Ramos, Emilio (TTU); Singha, Ujjal; Chaudhuri, Minu; Nes, W. David (TTU)Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C28- and C29-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC50 values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding Ki 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.—Kidane, M. E., B. H. Vanderloop, W. Zhou, C. D. Thomas, E. Ramos, U. Singha, M. Chaudhuri, and W. D. Nes. Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design.