Mapping of pseudouridine residues on cellular and viral transcripts using a novel antibody-based technique.

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.

Multiple unbiased approaches identify oxidosqualene cyclase as the molecular target of a promising anti-leishmanial.

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.

Identification of inhibitors of UDP-galactopyranose mutase via combinatorial in situ screening.

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 inhibits Leishmania donovani UDP-galactopyranose mutase and induces reactive oxygen species to exert antileishmanial response.

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.

Citrus reticulata peel oil as an antiatherogenic agent: Hypolipogenic effect in hepatic cells, lipid storage decrease in foam cells, and prevention of LDL oxidation.

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 [14C]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.

Synthesis and evaluation of heterocycle structures as potential inhibitors of Mycobacterium tuberculosis UGM.

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.

Virtual screening and drug repositioning as strategies for the discovery of new antifungal inhibitors of oxidosqualene cyclase.

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.

Perturbation-Based Proteomic Correlation Profiling as a Target Deconvolution Methodology.

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-ß pathway inhibitor. This PPCP concept was extended to the use of a cell line panel and provides a new option for target deconvolution.

Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells.

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-ß. 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.

NMR Biochemical Assay for Oxidosqualene Cyclase: Evaluation of Inhibitor Activities on Trypanosoma cruzi and Human Enzymes.

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 of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

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.

Identification of potential inhibitors for mycobacterial uridine diphosphogalactofuranose-galactopyranose mutase enzyme: A novel drug target through in silico approach.

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.

UDP-4-Keto-6-Deoxyglucose, a Transient Antifungal Metabolite, Weakens the Fungal Cell Wall Partly by Inhibition of UDP-Galactopyranose Mutase.

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-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a Ki 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. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf 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 Galf 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 Galf, 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.

Practical Synthesis of α-Amyrin, ß-Amyrin, and Lupeol: The Potential Natural Inhibitors of Human Oxidosqualene Cyclase.

A practical synthesis of α-amyrin (1), ß-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.

Identification of eukaryotic UDP-galactopyranose mutase inhibitors using the ThermoFAD assay.

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.

Identification of inhibitors targeting Mycobacterium tuberculosis cell wall biosynthesis via dynamic combinatorial chemistry.

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.

Deleterious Consequences of UDP-Galactopyranose Mutase Inhibition for Nematodes.

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.

Conformational Control of UDP-Galactopyranose Mutase Inhibition.

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-His6 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.

Insight Mechanism of the Selective Lanosterol Synthase Inhibitor: Molecular Modeling, Docking and Density Functional Theory Approaches.

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.