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Pseudouridine (Ψ) is the most common noncanonical ribonucleoside present on mammalian noncoding RNAs (ncRNAs), including rRNAs, tRNAs, and snRNAs, where it contributes ∼7% of the total uridine level. However, Ψ constitutes only ∼0.1% of the uridines present on mRNAs and its effect on mRNA function remains unclear. Ψ residues have been shown to inhibit the detection of exogenous RNA transcripts by host innate immune factors, thus raising the possibility that viruses might have subverted the addition of Ψ residues to mRNAs by host pseudouridine synthase (PUS) enzymes as a way to inhibit antiviral responses in infected cells. Here, we describe and validate a novel antibody-based Ψ mapping technique called photo-crosslinking-assisted Ψ sequencing (PA-Ψ-seq) and use it to map Ψ residues on not only multiple cellular RNAs but also on the mRNAs and genomic RNA encoded by HIV-1. We describe 293T-derived cell lines in which human PUS enzymes previously reported to add Ψ residues to human mRNAs, specifically PUS1, PUS7, and TRUB1/PUS4, were inactivated by gene editing. Surprisingly, while this allowed us to assign several sites of Ψ addition on cellular mRNAs to each of these three PUS enzymes, Ψ sites present on HIV-1 transcripts remained unaffected. Moreover, loss of PUS1, PUS7, or TRUB1 function did not significantly reduce the level of Ψ residues detected on total human mRNA below the ∼0.1% level seen in wild-type cells, thus implying that the PUS enzyme(s) that adds the bulk of Ψ residues to human mRNAs remains to be defined., (© 2021 Martinez Campos et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Phenotypic screening identified a benzothiophene compound with activity against Leishmania donovani, the causative agent of visceral leishmaniasis. Using multiple orthogonal approaches, oxidosqualene cyclase (OSC), a key enzyme of sterol biosynthesis, was identified as the target of this racemic compound and its enantiomers. Whole genome sequencing and screening of a genome-wide overexpression library confirmed that OSC gene amplification is associated with resistance to compound 1. Introduction of an ectopic copy of the OSC gene into wild-type cells reduced susceptibility to these compounds confirming the role of this enzyme in resistance. Biochemical analyses demonstrated the accumulation of the substrate of OSC and depletion of its product in compound (S)-1-treated-promastigotes and cell-free membrane preparations, respectively. Thermal proteome profiling confirmed that compound (S)-1 binds directly to OSC. Finally, modeling and docking studies identified key interactions between compound (S)-1 and the LdOSC active site. Strategies to improve the potency for this promising anti-leishmanial are proposed., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

An in situ screening assay for UDP-galactopyranose mutase (UGM, an essential enzyme of M. tuberculosis cell wall biosynthesis) has been developed to discover novel UGM inhibitors. The approach is based on the amide-forming reaction of an amino acid core with various cinnamic acids, followed by a direct fluorescence polarization assay to identify the best UGM binders without isolation and purification of the screened ligands. This assay allows us to perform one-pot high-throughput synthesis and screening of enzyme inhibitors in a 384-well plate format. UGM ligands were successfully identified by this technology and their inhibition levels were established from pure synthetic compounds in vitro and in a whole cell antibacterial assay. This study provides a blueprint for designing enamide structures as new UGM inhibitors and anti-mycobacterial agents.

Cynaroside, a flavonoid, has been shown to have antibacterial, antifungal and anticancer activities. Here, we evaluated its antileishmanial properties and its mechanism of action through different in silico and in vitro assays. Cynaroside exhibited antileishmanial activity in time- and dose-dependent manner with 50% of inhibitory concentration (IC50) value of 49.49 ± 3.515 µM in vitro. It inhibited the growth of parasite significantly at only 20 µM concentration when used in combination with miltefosine, a standard drug which has very high toxicity. It also inhibited the intra-macrophagic parasite significantly at low doses when used in combination with miltefosine. It showed less toxicity than the existing antileishmanial drug, miltefosine at similar doses. Propidium iodide staining showed that cynaroside inhibited the parasites in G0/G1 phase of cell cycle. 2,7-dichloro dihydro fluorescein diacetate (H2DCFDA) staining showed cynaroside induced antileishmanial activity through reactive oxygen species (ROS) generation in parasites. Molecular-docking studies with key drug targets of Leishmania donovani showed significant inhibition. Out of these targets, cynaroside showed strongest affinity with uridine diphosphate (UDP)-galactopyranose mutase with -10.4 kcal/mol which was further validated by molecular dynamics (MD) simulation. The bioactivity, ADMET (absorption, distribution, metabolism, excretion and toxicity) properties, Organisation for Economic Co-operation and Development (OECD) chemical classification and toxicity risk prediction showed cynaroside as an enzyme inhibitor having sufficient solubility and non-toxic properties. In conclusion, cynaroside may be used alone or in combination with existing drug, miltefosine to control leishmaniasis with less cytotoxicity., (© 2021 The Author(s).)

Background and Aims: Hypercholesterolemia and oxidative stress are two of the most important risk factors for atherosclerosis. The aim of the present work was to evaluate mandarin (Citrus reticulata) peel oil (MPO) in cholesterol metabolism and lipid synthesis, and its antioxidant capacity., Methods and Results: Incubation of hepatic HepG2 cells with MPO (15-60 μL/L) reduced cholesterogenesis and saponifiable lipid synthesis, demonstrated by [ 14 C]acetate radioactivity assays. These effects were associated with a decrease in a post-squalene reaction of the mevalonate pathway. Molecular docking analyses were carried out using three different scoring functions to examine the cholesterol-lowering property of all the components of MPO against lanosterol synthase. Docking simulations proposed that minor components of MPO monoterpenes, like alpha-farnesene and neryl acetate, as well the major component, limonene and its metabolites, could be partly responsible for the inhibitory effects observed in culture assays. MPO also decreased RAW 264.7 foam cell lipid storage and its CD36 expression, and prevented low-density lipoprotein (LDL) lipid peroxidation., Conclusion: These results may imply a potential role of MPO in preventing atherosclerosis by a mechanism involving inhibition of lipid synthesis and storage and the decrease of LDL lipid peroxidation., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis. In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an "oxazepino-indole" structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Molecular target identification of small molecules, so-called target deconvolution, is a major obstacle to phenotype-based drug discovery. Here, we developed an approach called perturbation-based proteomic correlation profiling (PPCP) utilizing the correlation between protein quantity and binding activity of compounds under cellular perturbation by gene silencing and successfully identified lanosterol synthase as a molecular target of TGF-β pathway inhibitor. This PPCP concept was extended to the use of a cell line panel and provides a new option for target deconvolution., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Candidiasis is the most common fungal infection in immunocompromised patients, and Candida albicans is the fourth leading agent of nosocomial infections. Mortality from this infection is significant; however, the therapeutic treatment is limited, which demands the search for new drugs and new targets. In this context, oxidosqualene cyclase (OSC) catalyzes the cyclization of the 2,3-oxidosqualene to form lanosterol, an intermediate of ergosterol biosynthesis. Therefore, this enzyme constitutes an attractive therapeutic target. Thus, the aim of this study is to identify potential inhibitors of C. albicans OSC (CaOSC) from a marketed drugs database in order to discover new antifungal agents. The CaOSC 3D model was constructed using the Swiss-Model server and important features for CaOSC inhibition were identified by molecular docking of known inhibitors using Autodock Vina 1.1.2. Subsequently, virtual screening helped to identify calcitriol, the active form of vitamin D, and other four drugs, as potential inhibitors of CaOSC. The selected drugs presented an interesting pattern of interactions with this enzyme, including hydrogen bond with Asp450, a key residue in the active site. Thus, the antifungal activity of calcitriol was evaluated in vitro against Candida spp strains. Calcitriol showed antifungal activity against C. albicans and C. tropicalis, which reinforces the potential of this compound as candidate of CaOSC inhibitor. In short, the present study provides important insights for the development of new oxidosqualene cyclase inhibitors as antifungals., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-β. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.

Oxidosqualene cyclase (OSC), a membrane-associated protein, is a key enzyme of sterol biosynthesis. Here we report a novel assay for OSC, involving reaction in aqueous solution, NMR quantification in organic solvent, and factor analysis of spectra. We evaluated one known and three novel inhibitors on OSC of Trypanosoma cruzi, a parasite causative of Chagas disease, and compared their effects on human OSC for selectivity. Among them, one novel inhibitor showed a significant parasiticidal activity.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The Mycobacterium tuberculosis (MTB) uridine diphosphogalactofuranose (UDP)-galactopyranose mutase (UGM) is an essential flavoenzyme for mycobacterial viability and an important component of cell wall. It catalyzes the interconversion of UDP-galactopyranose into UDP-galactofuranose, a key building block for cell wall construction, essential for linking the peptidoglycan and mycolic acid cell wall layers in MTB through a 2-keto intermediate. Further, as this enzyme is not present in humans, it is an excellent therapeutic target for MTB. Thus, inhibition of this UGM enzyme is a good approach to explore new anti-TB drug. This study aims to find novel and effective inhibitors against UGM from reported natural phytochemicals and ZINC database using virtual screening approach., Methods: In this study, 148 phytochemicals with reported antitubercular activity and 5280 ZINC compounds with 70% structural similarity with the natural substrate of UGM (UDP-galactopyranose and UDP-galactofuranose) were screened against UGM., Results: In virtual screening, 19 phytochemicals and 477 ZINC compounds showed comparatively better binding affinity than natural substrates. Among them, best 10 compounds from each group were proposed as potential inhibitors for UGM based on the binding energy and protein-ligand interaction analysis. Among phytochemicals, three compounds, namely, tiliacorine, amentoflavone, and 2'-nortiliacorinine showed highest binding affinity (binding energy of -10.5, -10.4, and -10.3 Kcal/mol, respectively), while among ZINC compounds, ZINC08219848 and ZINC08217649, showing highest binding affinity (binding energy of -10.0 and -9.7 Kcal/mol, respectively) toward UGM as compared to its substrates., Conclusion: These selected compounds may be proposed as potential inhibitors of UGM and need to be tested in TB culture studies in vitro to assess their anti-TB activity., Competing Interests: There are no conflicts of interest.

A practical synthesis of α-amyrin (1), β-amyrin (2), and lupeol (3) was accomplished in total yields of 32, 42, and 40% starting from easily available ursolic acid (4), oleanolic acid (5), and betulin (6), respectively. Remarkably, these three natural pentacyclic triterpenes exhibited potential inhibitory activity against human oxidosqualene cyclase., (© 2017 Deutsche Pharmazeutische Gesellschaft.)

Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Gal p ) to UDP-galactofuranose (UDP-Gal f ). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a K i of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Gal f residue levels were completely abolished in the Δ ugm strain and reduced in the Δ er strain, while overexpression of the ugm gene in the background of a Δ er strain restored Gal f levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Gal f in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs. IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Gal f , which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs., (Copyright © 2017 Ma et al.)

Background: Lanosterol synthase (Oxidosqualene cyclase) is an enzyme, which plays a central role in cholesterol and sterols biosynthesis. Lanosterol synthase drugs are used to lower the level of cholesterol in the blood and treat wide variety of diseases like atherosclerosis, coronary heart diseases etc., Objective: There is a great interest in the identification of drugs that target this enzyme for anticholesteraemic agent using in silico tools., Methods: Ligand based pharmacophore model was developed using Discovery Studio 2.5. The best model was used as a tool to retrieve suitable molecule for Lanosterol synthase inhibitor from commercial database and Virtual screening of large commercially available databases to retrieve the best mole of Hypo1 using. Molecular docking was done using three different tools named as GOLD, GLIDE and AUTODOCK 4.0. Density functional theory approach and Density of State spectrum were carried out using Gaussian 09 and GAUSS SUM 3.0. Contribution of these methods in the selection of anticholesteraemic compounds has been discussed., Results: The best pharmacophore model was used to screen the commercial database. Totally 8 compounds were showed with the best orientation, binding mode and binging energy in the docking analyses. The orbital energies such as HOMO, LUMO and DOS spectrum for 8 hit compounds showed the energy gap that results in charge transfer and stability in the active site region. The results showed that our 8 potent leads could serve for further findings., Conclusion: In silico approaches, our 8 hit compounds could serve as the better understanding to design the novel lanosterol synthase inhibitors as anticholesteraemic activity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Aspergillus fumigatus is a human pathogen responsible for deadly infections in immune-compromised patients. A potential strategy for treating A. fumigatus infections is by targeting the biosynthesis of cell wall components, such as galactofuranase, which is absent in humans. Galactofuranose biosynthesis is initiated by the flavoenzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM requires the reduced form of the flavin for activity, which is obtained by reacting with NADPH. We aimed to identify inhibitors of UGM by screening a kinase inhibitor library using ThermoFAD, a flavin fluorescence thermal shift assay. The screening assay identified flavopiridol as a compound that increased the melting temperature of A. fumigatus UGM. Further characterization showed that flavopiridol is a non-competitive inhibitor of UGM and docking studies suggest that it binds in the active site. This compound does not inhibit the prokaryotic UGM from Mycobacteria tuberculosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

In this study, we report a dynamic combinatorial approach along with highly efficient in situ screening to identify inhibitors of UDP-galactopyranose mutase (UGM), an essential enzyme involved in mycobacterial cell wall biosynthesis. These two technologies converged to the identification of a new UGM inhibitor chemotype. Importantly, the best molecule not only displayed high affinity for the target enzyme but also exhibited in vitro growth inhibition against whole Mycobacterium tuberculosis cells. The strategy described here provides an avenue to explore a novel inhibitor class for UGMs and paves the way for further pharmacological studies on tuberculosis treatment.

Parasitic nematodes pose a serious threat to agriculture, livestock, and human health. Increasing resistance to antiparasitic agents underscores the need to replenish our anthelmintic arsenal. The nonpathogenic Caenorhabditis elegans, which serves as an effective model of parasitic helminths, has been used to search for new anthelmintic leads. We previously reported small-molecule inhibitors of the essential C. elegans protein UDP-galactopyranose mutase (UGM or Glf). This enzyme is required for the generation of galactofuranose (Galf)-containing glycans and is needed in nematodes for proper cuticle formation. Though our first-generation inhibitors were effective in vitro, they elicited no phenotypic effects. These findings are consistent with the known difficulty of targeting nematodes. C. elegans is recalcitrant to pharmacological modulation; typically, less than 0.02% of small molecules elicit a phenotypic effect, even at 40 μM. We postulated that the lack of activity of the UGM inhibitors was due to their carboxylic acid group, which can be exploited by nematodes for detoxification. We therefore tested whether replacement of the carboxylate with an N-acylsulfonamide surrogate would result in active compounds. UGM inhibitors with the carboxylate mimetic can phenocopy the deleterious consequences of UGM depletion in C. elegans. These findings support the use of UGM inhibitors as anthelmintic agents. They also outline a strategy to render small-molecule carboxylates more effective against nematodes.

This study reports a novel class of inhibitors of uridine 5'-diphosphate (UDP) galactopyranose mutase (UGM) derived from a screening of natural products. This enzyme is an essential biocatalyst involved in the cell wall biosynthesis of Mycobacterium tuberculosis. Flavonoids are potent inhibitors of UGM. The synthesis of novel methylated flavonoids allowed a structure-activity relationship analysis to be performed and which functional groups and structural elements were required for UGM inhibition could be determined. The binding mode of one of the best inhibitors was found to be noncompetitive. Docking simulations indicated that this molecule was likely to bind UGM in its open conformation, in a cavity recently identified as a "druggable" pocket. Importantly, two of the best inhibitors of the M. tuberculosis UGM displayed moderate activity against whole M. tuberculosis cells. This study reports the first natural products that act as inhibitor of UGM. Given the importance of natural products in medicinal chemistry, these results create new opportunities for the discovery of new antitubercular agents., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

UDP-galactopyranose mutase (Glf or UGM) catalyzes the formation of uridine 5'-diphosphate-α-d-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme is required for the production of Galf-containing glycans. UGM is absent in mammals, but members of the Corynebacterineae suborder require UGM for cell envelope biosynthesis. The need for UGM in some pathogens has prompted the search for inhibitors that could serve as antibiotic leads. Optimizing inhibitor potency, however, has been challenging. The UGM from Klebsiella pneumoniae (KpUGM), which is not required for viability, is more effectively impeded by small-molecule inhibitors than are essential UGMs from species such as Mycobacterium tuberculosis or Corynebacterium diphtheriae. Why KpUGM is more susceptible to inhibition than other orthologs is not clear. One potential source of difference is UGM ortholog conformation. We previously determined a structure of CdUGM bound to a triazolothiadiazine inhibitor in the open form, but it was unclear whether the small-molecule inhibitor bound this form or to the closed form. By varying the terminal tag (CdUGM-His 6 and GSG-CdUGM), we crystallized CdUGM to capture the enzyme in different conformations. These structures reveal a pocket in the active site that can be exploited to augment inhibitor affinity. Moreover, they suggest the inhibitor binds the open form of most prokaryotic UGMs but can bind the closed form of KpUGM. This model and the structures suggest strategies for optimizing inhibitor potency by exploiting UGM conformational flexibility.

Targeting the sterol biosynthesis pathway has been explored for the development of new bioactive compounds. Among the enzymes of this pathway, oxidosqualene cyclase (OSC) which catalyzes lanosterol cyclization from 2,3-oxidosqualene has emerged as an attractive target. In this work, we reviewed the most promising OSC inhibitors from different organisms and their potential for the development of new antiparasitic, antifungal, hypocholesterolemic and anticancer drugs. Different strategies have been adopted for the discovery of new OSC inhibitors, such as structural modifications of the natural substrate or the reaction intermediates, the use of the enzyme's structural information to discover compounds with novel chemotypes, modifications of known inhibitors and the use of molecular modeling techniques such as docking and virtual screening to search for new inhibitors. This review brings new perspectives on structural insights of OSC from different organisms and reveals the broad structural diversity of OSC inhibitors which may help evidence lead compounds for further investigations with various therapeutic applications., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

UDP-galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non-substrate-like inhibitor, MS-208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS-208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS-208 binds at an allosteric binding site (A-site) instead of the enzyme active site (S-site). A candidate A-site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A-site. Further molecular dynamics studies led us to propose that MS-208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A-site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Uridine diphosphate galactopyranose mutase (UGM also known as Glf) is a biosynthetic enzyme required for construction of the galactan, an essential mycobacterial cell envelope polysaccharide. Our group previously identified two distinct classes of UGM inhibitors; each possesses a carboxylate moiety that is crucial for potency yet likely detrimental for cell permeability. To enhance the antimycobacterial potency, we sought to replace the carboxylate with a functional group mimic-an N-acylsulfonamide group. We therefore synthesized a series of N-acylsulfonamide analogs and tested their ability to inhibit UGM. For each inhibitor scaffold tested, the N-acylsulfonamide group functions as an effective carboxylate surrogate. Although the carboxylates and their surrogates show similar activity against UGM in a test tube, several N-acylsulfonamide derivatives more effectively block the growth of Mycobacterium smegmatis. These data suggest that the replacement of a carboxylate with an N-acylsulfonamide group could serve as a general strategy to augment antimycobacterial activity.

Lymphatic filariasis, a vector-borne neglected tropical disease affects millions of population in tropical and subtropical countries. Vaccine unavailability and emerging drug resistance against standard antifilarial drugs necessitate search of novel drug targets for developing alternate drugs. Recently, UDP-galactopyranose mutases (UGM) have emerged as a promising drug target playing an important role in parasite virulence and survival. This study deals with the cloning and characterization of Brugia malayi UGM and further exploring its antifilarial drug target potential. The recombinant protein was actively involved in conversion of UDP-galactopyranose (substrate) to UDP-galactofuranose (product) revealing Km and Vmax to be ∼51.15 μM and ∼1.27 μM/min, respectively. The purified protein appeared to be decameric in native state and its 3D homology modeling using Aspergillus fumigatus UGM enzyme as template revealed conservation of active site residues. Two specific prokaryotic inhibitors (compounds A and B) of the enzyme inhibited B. malayi UGM enzymatic activity competitively depicting Ki values ∼22.68 and ∼23.0 μM, respectively. These compounds were also active in vitro and in vivo against B. malayi The findings suggest that B. malayi UGM could be a potential antifilarial therapeutic drug target., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The de novo purine biosynthesis pathway is an attractive target for antibacterial drug design, and PurE from this pathway has been identified to be crucial for Bacillus anthracis survival in serum. In this study we adopted a fragment-based hit discovery approach, using three screening methods-saturation transfer difference nucleus magnetic resonance (STD-NMR), water-ligand observed via gradient spectroscopy (WaterLOGSY) NMR, and surface plasmon resonance (SPR), against B. anthracis PurE (BaPurE) to identify active site binding fragments by initially testing 352 compounds in a Zenobia fragment library. Competition STD NMR with the BaPurE product effectively eliminated non-active site binding hits from the primary hits, selecting active site binders only. Binding affinities (dissociation constant, KD) of these compounds varied between 234 and 301μM. Based on test results from the Zenobia compounds, we subsequently developed and applied a streamlined fragment screening strategy to screen a much larger library consisting of 3000 computationally pre-selected fragments. Thirteen final fragment hits were confirmed to exhibit binding affinities varying from 14μM to 700μM, which were categorized into five different basic scaffolds. All thirteen fragment hits have ligand efficiencies higher than 0.30. We demonstrated that at least two fragments from two different scaffolds exhibit inhibitory activity against the BaPurE enzyme., (Published by Elsevier Ltd.)

The cyclization of oxidosqualene to lanosterol, catalyzed by the enzyme oxidosqualene cyclase (OSC), goes through a number of carbocationic high energy intermediates (HEI), and mimicking these intermediates is a promising approach for the development of OSC inhibitors. 3-Arylpiperidines (or tetrahydropyridines) were designed as steroidomimetic rings A + C equivalents containing two protonable amino groups for mimicking both the pro-C4 HEI and the pro-C20 HEI of the OSC-mediated cyclization cascade. Inhibitory activity is strongly dependent on the nature of the lipophilic substituent representing an equivalent of the sterol side chain. Here aromatic residues (substituted benzyl, cinnamyl, naphthylmethyl) were found to be most suitable. Docking experiments on a first optimized 3-arylpiperidine compound led to an isomeric 4-arylpiperidine with submicromolar activity on human OSC. This inhibitor reduced total cholesterol biosynthesis in a cellular assay with an IC50 value of 0.26 μM., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of cycloartenol synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished.

The effect of an inhibitor of cycloartenol synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC-MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.

The synthesis of 1-[5-O-(α-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione and 1-[(5-O-(β-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione as non-ionic substrate mimics of UDP-Galp are described. UDP-Galp is a precursor of Galf, which is a primary component of the cell-wall glycans of several microorganisms. The interconversion of UDP-Galp and UDP-Galf is catalyzed by UDP galactopyranose mutase (UGM); its inhibition comprises a mode of compromising the microorganisms. The nonionic polyhydroxylated chain was intended to mimic the ionic pyrophosphate group and the ribose moiety in UDP-Galp and increase the bioavailabilities of the candidate inhibitors. Inhibition assays with UGM of Mycobacterium tuberculosis showed only weak inhibition of the enzyme by these compounds., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Many developing tissues display regenerative capability that allows them to compensate cell loss and preserve tissue homeostasis. Because of their remarkable regenerative capability, Drosophila wing discs are extensively used for the study of regenerative phenomena. We thus used the developing wing to investigate the role played in tissue homeostasis by the evolutionarily conserved eukaryotic H/ACA small nucleolar ribonucleoprotein pseudouridine synthase. Here we show that localized depletion of this enzyme can act as an endogenous stimulus capable of triggering apoptosis-induced proliferation, and that context-dependent effects are elicited in different sub-populations of the silenced cells. In fact, some cells undergo apoptosis, whereas those surrounding the apoptotic foci, although identically depleted, overproliferate. This overproliferation correlates with ectopic induction of the Wg and JAK-STAT (Janus kinase-signal transducer and activator of transcription) mitogenic pathways. Expression of a p35 transgene, which blocks the complete execution of the death program and generates the so-called 'undead cells', amplifies the proliferative response. Pseudouridine synthase depletion also causes loss of apicobasal polarity, disruption of adherens cell junctions and ectopic induction of JNK (c-Jun N-terminal kinase) and Mmp1 (matrix metalloproteinase-1) activity, leading to a significant epithelial reorganization. Unexpectedly, cell-nonautonomous effects, such as epithelial mesenchymal transition in the contiguous unsilenced squamous epithelium, are also promoted. Collectively, these data point out that cell-cell communication and long-range signaling can take a relevant role in the response to pseudouridine synthase decline. Considering that all the affected pathways are highly conserved throughout evolution, it is plausible that the response to pseudouridine synthase depletion has been widely preserved. On this account, our results can add new light on the still unexplained tumor predisposition that characterizes X-linked dyskeratosis, the human disease caused by reduced pseudouridine synthase activity.

UDP-galactopyranose mutase (UGM) is an enzyme involved in the biosynthesis of the Mycobacterium tuberculosis cell wall, and is essential for the growth and survival of the organism. A micromolar inhibitor developed by tetrafluorination of the UGM substrate has been previously studied by saturation transfer difference (STD) NMR spectroscopy. To elucidate the bioactive conformation of the inhibitor bound to UGM, we employ molecular dynamics (MD) simulations to construct a structural model. The MD model is subsequently validated by a good fit between experimental and theoretical STD effects, the latter calculated by a complete relaxation and conformational exchange matrix (CORCEMA) analysis. This structural model is used to explain the relative binding affinities of the inhibitor and the parent substrate., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Aberrant cholesterol homeostasis and biosynthesis has been observed in different tumour types. This paper investigates the role of the post-squalenic enzyme of cholesterol biosynthesis, oxidosqualene cyclase (OSC), in regulating tumour angiogenesis and metastasis dissemination in mouse models of cancer. We showed that Ro 48-8071, a selective inhibitor of OSC, reduced vascular density and increased pericyte coverage, with a consequent inhibition of tumour growth in a spontaneous mouse model of pancreatic tumour (RIP-Tag2) and two metastatic mouse models of human colon carcinoma (HCT116) and pancreatic adenocarcinoma (HPAF-II). Remarkably, the inhibition of OSC hampered metastasis formation in HCT116 and HPAF-II models. Ro 48-8071 induced tumour vessel normalization and enhanced the anti-tumoral and anti-metastatic effects of 5-fluorouracil (5-FU) in HCT116 mice. Ro 48-8071 exerted a strong anti-angiogenic activity by impairing endothelial cell adhesion and migration, and by blocking vessel formation in angiogenesis assays. OSC inhibition specifically interfered with the PI3K pathway. According to in vitro results, Ro 48-8071 specifically inhibited Akt phosphorylation in both cancer cells and tumour vasculature in all treated models. Thus, our results unveil a crucial role of OSC in the regulation of cancer progression and tumour angiogenesis, and indicate Ro 48-8071 as a potential novel anti-angiogenic and anti-metastatic drug.

UDP-Galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the reversible conversion of UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. A soluble, active form of UGM from Mycobacterium tuberculosis (MtUGM) was obtained from a dual His6-MBP-tagged MtUGM construct. We present the first complex structures of MtUGM with bound substrate UDP-Galp (both oxidized flavin and reduced flavin). In addition, we have determined the complex structures of MtUGM with inhibitors (UDP and the dideoxy-tetrafluorinated analogues of both UDP-Galp (UDP-F4-Galp) and UDP-Galf (UDP-F4-Galf)), which represent the first complex structures of UGM with an analogue in the furanose form, as well as the first structures of dideoxy-tetrafluorinated sugar analogues bound to a protein. These structures provide detailed insight into ligand recognition by MtUGM and show an overall binding mode similar to those reported for other prokaryotic UGMs. The binding of the ligand induces conformational changes in the enzyme, allowing ligand binding and active-site closure. In addition, the complex structure of MtUGM with UDP-F4-Galf reveals the first detailed insight into how the furanose moiety binds to UGM. In particular, this study confirmed that the furanoside adopts a high-energy conformation ((4)E) within the catalytic pocket. Moreover, these investigations provide structural insights into the enhanced binding of the dideoxy-tetrafluorinated sugars compared to unmodified analogues. These results will help in the design of carbohydrate mimetics and drug development, and show the enormous possibilities for the use of polyfluorination in the design of carbohydrate mimetics.

Chorismate-utilizing enzymes (CUE) such as chorismate mutase, anthranilate synthase, chorismate pyruvate-lyase, 4-amino-4-deoxychorismate synthase, isochorismate synthase and salicylate synthase are responsible for converting chorismate into various products necessary for the survival of bacteria. The absence of these enzymes in humans and their importance in the virulence and survival of bacteria make them suitable targets for potential antimicrobial compounds. Furthermore, the CUE have significant structural homology and similar catalytic mechanisms, enabling the strategy of affecting multiple enzymes with one single inhibitor. This review follows up the investigation of mechanisms of CUE-catalysed reactions and the concurrent development of CUE inhibitors. Many active compounds were found amongst the structures mimicking the transition state of chorismate during the reaction. Most recently, high nanomolar and low micromolar inhibitors against isochorismate-pyruvate lyase were identified, which were also effective against chorismate mutase and salicylate synthase and belong to the most active inhibitors reported up to date.

Breast cancer cells express enzymes that convert cholesterol, the synthetic precursor of steroid hormones, into estrogens and androgens, which then drive breast cancer cell proliferation. In the present study, we sought to determine whether oxidosqualene cyclase (OSC), an enzyme in the cholesterol biosynthetic pathway, may be targeted to suppress progression of breast cancer cells. In previous studies, we showed that the OSC inhibitor RO 48-8071 (RO) may be a ligand which could potentially be used to control the progression of estrogen receptor-α (ERα)-positive breast cancer cells. Herein, we showed, by real-time PCR analysis of mRNA from human breast cancer biopsies, no significant differences in OSC expression at various stages of disease, or between tumor and normal mammary cells. Since the growth of hormone-responsive tumors is ERα-dependent, we conducted experiments to determine whether RO affects ERα. Using mammalian cells engineered to express human ERα or ERβ protein, together with an ER-responsive luciferase promoter, we found that RO dose-dependently inhibited 17β-estradiol (E2)-induced ERα responsive luciferase activity (IC50 value, ~10 µM), under conditions that were non-toxic to the cells. RO was less effective against ERβ-induced luciferase activity. Androgen receptor (AR) mediated transcriptional activity was also reduced by RO. Notably, while ERα activity was reduced by atorvastatin, the HMG-CoA reductase inhibitor did not influence AR activity, showing that RO possesses broader antitumor properties. Treatment of human BT-474 breast cancer cells with RO reduced levels of estrogen-induced PR protein, confirming that RO blocks ERα activity in tumor cells. Our findings demonstrate that an important means by which RO suppresses hormone-dependent growth of breast cancer cells is through its ability to arrest the biological activity of ERα. This warrants further investigation of RO as a potential therapeutic agent for use against hormone-dependent breast cancers.

In most human breast cancers, tumor cell proliferation is estrogen dependent. Although hormone-responsive tumors initially respond to anti-estrogen therapies, most of them eventually develop resistance. Our goal was to identify alternative targets that might be regulated to control breast cancer progression. Sulforhodamine B assay was used to measure the viability of cultured human breast cancer cell lines exposed to various inhibitors. Protein expression in whole-cell extracts was determined by Western blotting. BT-474 tumor xenografts in nude mice were used for in vivo studies of tumor progression. RO 48-8071 ([4'-[6-(Allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate]; RO), a small-molecule inhibitor of oxidosqualene cyclase (OSC, a key enzyme in cholesterol biosynthesis), potently reduced breast cancer cell viability. In vitro exposure of estrogen receptor (ER)-positive human breast cancer cells to pharmacological levels of RO or a dose close to the IC50 for OSC (nM) reduced cell viability. Administration of RO to mice with BT-474 tumor xenografts prevented tumor growth, with no apparent toxicity. RO degraded ERα while concomitantly inducing the anti-proliferative protein ERβ. Two other cholesterol-lowering drugs, Fluvastatin and Simvastatin, were less effective in reducing breast cancer cell viability and were found not to induce ERβ. ERβ inhibition or knockdown prevented RO-dependent loss of cell viability. Importantly, RO had no effect on the viability of normal human mammary cells. RO is a potent inhibitor of hormone-dependent human breast cancer cell proliferation. The anti-tumor properties of RO appear to be in part due to an off-target effect that increases the ratio of ERβ/ERα in breast cancer cells.

The multistep synthesis of a novel UDP-C-cyclohexene, designed as a high energy intermediate analogue of the UDP-galactopyranose mutase (UGM) catalyzed isomerization reaction, is reported. The synthesis of the central carbasugar involved the preparation of a galactitol derivative bearing two olefins necessary for the construction of the cyclohexene ring by a ring-closing metathesis as a key step. Further successive phosphonylation, deprotection, and UMP coupling provided the target molecule. The final molecule was assayed against UGM and compared with UDP-C-Galf, the C-glycosidic UGM substrate analogue.

The small intestine plays a fundamentally important role in regulating whole body cholesterol balance and plasma lipoprotein composition. This is articulated through the interplay of a constellation of genes that ultimately determines the net amount of chylomicron cholesterol delivered to the liver. Major advances in our insights into regulation of the cholesterol absorption pathway have been made using genetically manipulated mouse models and agents such as ezetimibe. One unresolved question is how a sustained pharmacological inhibition of intestinal cholesterol synthesis in vivo may affect cholesterol handling by the absorptive cells. Here we show that the lanosterol cyclase inhibitor, Ro 48-8071, when fed to BALB/c mice in a chow diet (20 mg/day/kg body weight), leads to a rapid and sustained inhibition (>50%) of cholesterol synthesis in the whole small intestine. Sterol synthesis was also reduced in the large intestine and stomach. In contrast, hepatic cholesterol synthesis, while markedly suppressed initially, rebounded to higher than baseline rates within 7 days. Whole body cholesterol synthesis, fractional cholesterol absorption, and fecal neutral and acidic sterol excretion were not consistently changed with Ro 48-8071 treatment. There were no discernible effects of this agent on intestinal histology as determined by H&E staining and the level of Ki67, an index of proliferation. The mRNA expression for multiple genes involved in intestinal cholesterol regulation including NPC1L1 was mostly unchanged although there was a marked rise in the mRNA level for the PXR target genes CYP3A11 and CES2A., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Tetrafluorinated analogues of both UDP-galactopyranose and UDP-galactofuranose have been synthesized and assayed against UDP-galactopyranose mutase, a key enzyme for Mycobacterium tuberculosis cell wall biosynthesis. Competition assays and STD-NMR spectroscopy techniques have evidenced not only the first unambiguous case of affinity enhancement through local sugar polyfluorination, but also showed that tetrafluorination can still have a beneficial effect on binding when monofluorination at the same position does not., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that represents one of the major health challenges of the Latin American countries. Successful efforts were made during the last few decades to control the transmission of this disease, but there is still no treatment for the 10 million adults in the chronic phase of the disease. In T. cruzi, as well as in other pathogens, the flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, a precursor of the cell surface β-galactofuranose that is involved in the virulence of the pathogen. The fact that UGM is not present in humans makes inhibition of this enzyme a good approach in the design of new Chagas therapeutics. By performing a series of computer simulations of T. cruzi UGM in the presence or absence of an active site ligand, we address the molecular details of the mechanism that controls the uptake and retention of the substrate. The simulations suggest a modular mechanism in which each moiety of the substrate controls the flexibility of a different protein loop. Furthermore, the calculations indicate that interactions with the substrate diphosphate moiety are especially important for stabilizing the closed active site. This hypothesis is supported with kinetics measurements of site-directed mutants of T. cruzi UGM. Our results extend our knowledge of UGM dynamics and offer new alternatives for the prospective design of drugs., (© 2013 The Protein Society.)

A series of aminopropylindenes, designed as mimics of a cationic high energy intermediate in the oxidosqualene cyclase(1) (OSC)-mediated cyclization of 2,3-oxidosqualen to lanosterol was prepared from Grundmann's ketone. Screening on OSCs from five different organisms revealed interesting activities and selectivities of some of the compounds. A N,N-dimethylaminopropyl derivative showed promising inhibition of Trypanosoma cruzi OSC in combination with low cytotoxicity, and showed significant reduction of cholesterol biosynthesis in a human cell line., (Copyright © 2013. Published by Elsevier Masson SAS.)

β-Amyrin, a natural triterpene, is widely distributed in the plant kingdom, and its pentacyclic skeleton is produced by oxidosqualene cyclase (OSC). OSC enzymes are classified as membrane proteins, and they catalyze the polycyclization reaction of (3S)-2,3-oxidosqualene to yield nearly 150 different cyclic triterpene skeletons. To date, no report has described the successful purification and characterization of plant β-amyrin synthase. The β-amyrin synthase from Euphorbia tirucalli (EtAS) was expressed as a polyhistidine-tagged protein in Saccharomyces cerevisiae GIL77, which lacks the lanosterol synthase gene. The expression yield, determined by western blotting analysis, was 5-7 mg. By Ni(2+) -nitrilotriacetic acid affinity column chromatography and careful selection of the proper imidazole concentration during the purification processes of washing and elution, a single band was successfully obtained on SDS/PAGE. We then tested the effects of four detergents on the enzyme activity. Supplementation with Triton X-100 at a concentration of 0.05% yielded the highest activity. The optimal pH and temperature were 7.0 and 30 °C, respectively. The kinetic parameters, K(m) and k(cat) , were determined to be 33.8 ± 0.53 μm and 46.4 ± 0.68 min(-1), respectively. To the best of our knowledge, there are no reports describing both K(m) and k(cat) for OSCs except for two examples of rat and bovine lanosterol synthases. The β-amyrin synthase purified in this study showed a significantly higher catalytic efficiency (k(cat)/K(m)) (~ 10(3)-fold) than those of the two reported lanosterol synthases. Gel-filtration HPLC indicated that the OSC exists as a monomer, and the eluted OSC retained its activity. Furthermore, the inhibition constants K(i) and IC(50) and types of inhibition by iminosqualene, Ro48-8071 and U18666A were determined, and indicated that iminosqualene and Ro48-8071 are potent inhibitors. Additionally, this is the first report of the kinetic data of the mutated enzymes targeted for the DCTAE(485-489) motif, which is a putative initiation site for the polycyclization reaction. No activity of the D485N variant and significantly decreased activity of the C564A variant were found, definitively demonstrating that the acidic carboxyl residue Asp485 serves as a proton donor to initiate the polycyclization reaction, and that Cys564 is involved in hydrogen bond formation with the carboxyl residue Asp458 to enhance the acidity. The CD spectrum is the first to be reported for OSCs, and the CD spectra of the wild-type and the mutated EtASs were almost the same, indicating that the protein architecture was not altered by these mutations., (© 2013 The Authors Journal compilation © 2013 FEBS.)

UDP-Galactopyranose mutase (UGM) is a unique flavin-dependent enzyme that catalyzes the conversion of UDP-galactopyranose(UDP-Galp) to UDP-galactofuranose (UDP-Galf). The product of this reaction is the precursor to Galf, a major component of the cell wall and of cell surface glycoproteins and glycolipids in many eukaryotic and prokaryotic human pathogens. The function of UGM is important in the virulence of fungi, parasites, and bacteria. Its role in virulence and its absence in humans suggest that UGM is an ideal drug target. Significant structural and mechanistic information has been accumulated on the prokaryotic UGMs; however, in the past few years the research interest has shifted to UGMs from eukaryotic human pathogens such as fungi and protozoan parasites. It has become clear that UGMs from prokaryotic and eukaryotic organisms have different structural and mechanistic features. The amino acid sequence identity between these two classes of enzymes is low, resulting in differences in oligomeric states, substrate binding, active site flexibility, and interaction with redox partners. However, the unique role of the flavin cofactor in catalysis is conserved among this enzyme family. In this review, recent findings on eukaryotic UGMs are discussed and presented in comparison with prokaryotic UGMs.

A series of UDP-galactitols were designed as analogues of high-energy intermediates of the UDP-galactopyranose mutase (UGM) catalyzed furanose/pyranose interconversion, an essential step of Mycobacterium tuberculosis cell wall biosynthesis. The final compounds structurally share the UDP and the galactitol substructures that were connected by four distinct electrophilic connections (epoxide, lactone and Michael acceptors). All molecules were synthesized from a common perbenzylated acyclic galactose precursor that was derivatized by alkenylation, alkynylation and cyclopropanation. The inhibition study against UGM could clearly show that slight changes in the relative orientation of the UDP and the galactitol moieties resulted in dramatic variations of binding properties. Compared to known inhibitors, the epoxide derivative displayed a very tight, reversible, inhibition profile. Moreover, a time-dependent inactivation study showed that none of these electrophilic structures could react with UGM, or its FAD cofactor, the catalytic nucleophile of this still intriguing reaction., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Riboflavin, called generally vitamin B12, is the precursor of cofactor flavin adenine dinucleotide (FAD) and flavin mononucleotide, which plays crucial roles in the biosynthesis of organisms. Bacteria need to synthesize riboflavin to maintain their survival and proliferation if they cannot obtain flavin from the surroundings. 3,4-Dihydroxy-2-butanone4-phosphate synthase (DHBPs) is one of the key enzymes in biosynthesis of riboflavin. In the presence of Mg2+, DHBPs can degrade ribulose-5-phosphate (Ru5P) into formate and 3,4-dihydroxy-2-butanone-4-phosphate (DHBP). Potentially, these enzymes related to biosynthesis and metabolism of riboflavin, including DHBPs, may serve as the target for new antibacterial drug. In order to determine the three-dimensional structure and screen small molecule of inhibitor of DHBPs, we expressed and purified DHBPs from Streptococcus pneumonia (S. pn), and characterized the activity of this enzyme., Method: DHBPs gene was amplified by PCR, and over-expression plasmid pW28-DHBPs was constructed. pW28-DHBPs was transformed into Escherichia coli BL21 (DE3) to express DHBPs; the recombinant protein was purified by nickel column and ion-exchange column. The enzymatic activity was tested using spectroscopy., Results: The plasmid of pW28-DHBPs was verified by restrictive enzyme digestion and sequencing. Soluble DHBPs was expressed in E. coli BL21, and purified with 95% purity. The result of size exclusion chromatography indicates that DHBPs was dimer in solution. Additionally, the recombinant protein has activity to hydrolyze Ru5P into formate and DHBP in the conditions of pH 7.5 and 25 degrees C and in the presence of Mg2+., Conclusions: DHBPs could be highly expressed in soluble form in E. coli BL21 strain, and the recombinant protein has activity to hydrolyze Ru5P.

Artificial influence: We describe a semi-artificial adenosylpeptide B(12) that behaves as a cofactor in B(12)-dependent enzymatic reactions and demonstrate that the peptide backbone influences its chemical properties and modulates its bioactivity in vitro and in vivo. Inhibition of the growth of L. delbrueckii is demonstrated, thus providing a potentially powerful approach for the development of antibacterial and antiproliferative compounds., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3 Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45% identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10-50, primarily by decreasing k(cat). Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

Ten oxidosqualene cyclase inhibitors with high efficacy as cholesterol-lowering agents and of different chemical structure classes were evaluated as potential anticancer agents against human cancer cells from various tissue origins and nontumoral human-brain-derived endothelial cells. Inhibition of cancer cell growth was demonstrated at micromolar concentrations, comparable to the concentrations of statins necessary for antitumor effect. Human glioblastoma cells were among the most sensitive cells. These compounds were also able to decrease the proliferation of angiogenic brain-derived endothelial cells, as a model of tumor-induced neovasculation. Additive effects in human glioblastoma cells were also demonstrated for oxidosqualene cyclase inhibitors in combination with atorvastatin while maintaining selectivity against endothelial cells. Thus, not only statins targeting the 3-hydroxy-3-methylglutaryl coenzyme A reductase but also inhibitors of oxidosqualene cyclase decrease tumor growth, suggesting new therapeutic opportunities of combined anti-cholesterol agents for dual treatment of glioblastoma.

Five human 2,3-oxidosqualnene cyclase (OSC) inhibitors were discovered using the combination of a virtual screening based on a docking study and an isotope-free assay system. All of these inhibitors were revealed to have activities comparable or superior to that of LDAO, a known OSC inhibitor. The computational study of the newly identified inhibitors suggested that CH/π interactions with F444 and W581 contribute to the binding, and these interactions are candidates for additional structural filters in the next generation of virtual screening., (Copyright © 2011 Elsevier Ltd. All rights reserved.)