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

ABSTRACT: 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.

SUBMITTER: Paradela LS

PROVIDER: S-EPMC8153249 | biostudies-literature |

REPOSITORIES: biostudies-literature

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

Project description:Samples were reduced by the addition of TCEP (25 mM final concentration) and incubated at 37 ºC for 10 min. Alkylation was carried out by the addition of iodoacetamide (25 mM final concentration) and incubated at RT for 1 h in the dark. Samples were then digested by the addition of 1:50 LysC (Wako, Japan) in 100 mM triethylammonium bicarbonate (TEAB) followed by incubation at 37 ºC for 6h and then addition of 1:50 trypsin (Thermo) followed by incubation at 37 ºC overnight. The efficiency of sample digestion efficiency was assessed by MS (LTQ XL, Thermo). The samples were then vacuum-dried and resuspended in 100 mM TEAB (100 µL). Samples were then incubated in their respective Tandem Mass Tag™ (TMT) 10-plex reagents (Thermo) for 1 h at RT with agitation. Reactions were quenched by the addition of 5% hydroxylamine for 15 min and each set of samples (treated and vehicle) were pooled in the same tube and dried overnight. The TMTTM-labelled samples were dried and kept at -85 ºC until further analysis.

2021-07-18 | PXD023780 | Pride

The cholesterol biosynthesis enzyme oxidosqualene cyclase is a new target to impair tumour angiogenesis and metastasis dissemination.

Project description: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.

| S-EPMC4357009 | biostudies-literature

Synthesis of arborane triterpenols by a bacterial oxidosqualene cyclase.

Project description:Cyclic triterpenoids are a broad class of polycyclic lipids produced by bacteria and eukaryotes. They are biologically relevant for their roles in cellular physiology, including membrane structure and function, and biochemically relevant for their exquisite enzymatic cyclization mechanism. Cyclic triterpenoids are also geobiologically significant as they are readily preserved in sediments and are used as biomarkers for ancient life throughout Earth's history. Isoarborinol is one such triterpenoid whose only known biological sources are certain angiosperms and whose diagenetic derivatives (arboranes) are often used as indicators of terrestrial input into aquatic environments. However, the occurrence of arborane biomarkers in Permian and Triassic sediments, which predates the accepted origin of angiosperms, suggests that microbial sources of these lipids may also exist. In this study, we identify two isoarborinol-like lipids, eudoraenol and adriaticol, produced by the aerobic marine heterotrophic bacterium Eudoraea adriatica Phylogenetic analysis demonstrates that the E. adriatica eudoraenol synthase is an oxidosqualene cyclase homologous to bacterial lanosterol synthases and distinct from plant triterpenoid synthases. Using an Escherichia coli heterologous sterol expression system, we demonstrate that substitution of four amino acid residues in a bacterial lanosterol synthase enabled synthesis of pentacyclic arborinols in addition to tetracyclic sterols. This variant provides valuable mechanistic insight into triterpenoid synthesis and reveals diagnostic amino acid residues to differentiate between sterol and arborinol synthases in genomic and metagenomic datasets. Our data suggest that there may be additional bacterial arborinol producers in marine and freshwater environments that could expand our understanding of these geologically informative lipids.

| S-EPMC5240688 | biostudies-literature

Oxidosqualene cyclase targeting compound in Leishmania

Project description:Overexpression library screen - Oxidosqualene cyclase targeting compound in Leishmania donovani

| PRJEB37256 | ENA

Oxidosqualene cyclase targeting compound in Leishmania

Project description:Whole genome sequencing of resistant lines - Oxidosqualene cyclase targeting compound in Leishmania donovani

| PRJEB37435 | ENA

?-Nitrostyrenes as Potential Anti-leishmanial Agents.

Project description:Development of new therapeutic approach to treat leishmaniasis has become a priority. In the present study, the antileishmanial effect of ?-nitrostyrenes was investigated against in vitro promastigotes and amastigotes. A series of ?-nitrostyrenes have been synthesized by using Henry reaction and were evaluated for their antimicrobial activities by broth microdilution assay and in vitro antileishmanial activities against Leishmania donovani promastigotes by following standard guidelines. The most active compounds were futher evaluated for their in vitro antileishmanial activities against intracellular amastigotes. Among the tested ?-nitrostyrenes, compounds 7, 8, 9, 12, and 17 exhibited potential activities (MICs range, 0.25-8 ?g/mL) against clinically significant human pathogenic fungi. However, the microbactericidal concentrations (MBCs) and the microfungicidal concentrations (MFCs) were found to be either similar or only two-fold greater than the MICs. Anti-leishmanial results demonstrated that compounds 9, 12, 14, and 18 were found to be most active among the tested samples and exhibited 50% inhibitory concentration (IC50) by 23.40 ± 0.71, 37.83 ± 3.74, 40.50 ± 1.47, 55.66 ± 2.84 nM against L. donovani promastigotes and 30.5 ± 3.42, 21.46 ± 0.96, 26.43 ± 2.71, and 61.63 ± 8.02 nM respectively against intracellular L. donovani promastigotes amastigotes respectively which are comparable with standard AmB (19.60 ± 1.71 nM against promastigotes and 27.83 ± 3.26 nM against amastigotes). Compounds 9, 12, 14, and 18 were found to have potent in vitro leishmanicidal activity against L. donovani and found to be non-toxic against mammalian macrophages even at a concentration of 25 ?M. Nitric oxide (NO) estimation studies reveals that these compounds are moderately inducing NO levels.

| S-EPMC5007854 | biostudies-literature

Anti-leishmanial and Anti-inflammatory Agents from Endophytes: A Review.

Project description:Leishmaniases and chronic inflammatory diseases are the cause of millions of deaths in the world each year. The treatment of leishmaniasis is facing serious drawbacks particularly due to the limited number of effective medicines, the resistance, and the toxicity of available drugs. On the other hand, many drugs are used for the management of inflammatory disorders. However, the most commonly prescribed although efficient is highly toxic with multiples side effects. New leads compounds for the development of new anti-leishmanial and anti-inflammatory drugs are needed. Over the past decade, several studies on the potential of endophytes to produce bioactive metabolites have been reported. We are presenting in the present review the status of research from 2000 to 2019 on the anti-leishmanial and anti-inflammatory metabolites isolated from endophytes from diverse habitats. An emphasis was put on existing gaps in the literature to inspire and guide future investigations. We hope that this review will help accelerate the drug discovery against leishmaniases and inflammation-associated disorders.

| S-EPMC6814666 | biostudies-literature

Multiple chemical scaffolds inhibit a promising Leishmania drug target.

Project description:The need for an effective oral therapy for leishmaniasis is addressed through the study of the target N-myristoyltransferase from Leishmania major.

| S-EPMC4107919 | biostudies-literature

Proteomic and genetic approaches identify Syk as an AML target.

Project description:Cell-based screening can facilitate the rapid identification of compounds inducing complex cellular phenotypes. Advancing a compound toward the clinic, however, generally requires the identification of precise mechanisms of action. We previously found that epidermal growth factor receptor (EGFR) inhibitors induce acute myeloid leukemia (AML) differentiation via a non-EGFR mechanism. In this report, we integrated proteomic and RNAi-based strategies to identify their off-target, anti-AML mechanism. These orthogonal approaches identified Syk as a target in AML. Genetic and pharmacological inactivation of Syk with a drug in clinical trial for other indications promoted differentiation of AML cells and attenuated leukemia growth in vivo. These results demonstrate the power of integrating diverse chemical, proteomic, and genomic screening approaches to identify therapeutic strategies for cancer.

| S-EPMC2803063 | biostudies-literature

Arginase Flavonoid Anti-Leishmanial in Silico Inhibitors Flagged against Anti-Targets.

Project description:Arginase, a drug target for the treatment of leishmaniasis, is involved in the biosynthesis of polyamines. Flavonoids are interesting natural compounds found in many foods and some of them may inhibit this enzyme. The MetIDB database containing 5667 compounds was screened using an EIIP/AQVN filter and 3D QSAR to find the most promising candidate compounds. In addition, these top hits were screened in silico versus human arginase and an anti-target battery consisting of cytochromes P450 2a6, 2c9, 3a4, sulfotransferase, and the pregnane-X-receptor in order to flag their possible interactions with these proteins involved in the metabolism of substances. The resulting compounds may have promise to be further developed for the treatment of leishmaniasis.

| S-EPMC6274217 | biostudies-literature

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