Repurposing Study of 4-Acyl-1-phenylaminocarbonyl-2-substituted-piperazine Derivatives as Potential Anticancer Agents-In Vitro Evaluation against Breast Cancer Cells.

Breast cancer is the most common type of cancer in women. Although current treatments can increase patient survival, they are rarely curative when the disease is advanced (metastasis). Therefore, there is an urgent need to develop new cytotoxic drugs with a high selectivity toward cancer cells. Since repurposing approved drugs for cancer therapy has been a successful strategy in recent years, in this study, we screened a library of antiviral piperazine-derived compounds as anticancer agents. The compounds included a piperazine ring and aryl urea functions, which are privileged structures present in several anti-breast cancer drugs. The selective cytotoxic activity of a set of thirty-four 4-acyl-2-substituted piperazine urea derivatives against MCF7 breast cancer cells and MCF 10A normal breast cells was determined. Compounds 31, 32, 35, and 37 showed high selective anticancer activity against breast cancer cells and were also tested against another common type of cancer, non-small cell lung cancer (A549 lung cancer cells versus MRC-5 lung normal cells). Compounds 35 and 37 also showed selectivity against lung cancer cells. These results suggest that compounds 35 and 37 may be promising hit compounds for the development of new anticancer agents.

Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses.

Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases' active sites.

Design, synthesis and in vitro biological evaluation of a novel class of anti-adenovirus agents based on 3-amino-1,2-propanediol.

Nowadays there is not an effective drug for the treatment of infections caused by human adenovirus (HAdV) which supposes a clinical challenge, especially for paediatric and immunosuppressed patients. Here, we describe the design, synthesis and biological evaluation as anti-adenovirus agents of a new library (57 compounds) of diester, monoester and triazole derivatives based on 3-amino-1,2-propanediol skeleton. Seven compounds (17, 20, 26, 34, 44, 60 and 66) were selected based on their high anti-HAdV activity at low micromolar concentration (IC50 from 2.47 to 5.75 µM) and low cytotoxicity (CC50 from 28.70 to >200 µM). In addition, our mechanistic assays revealed that compounds 20 and 44 might be targeting specifically the HAdV DNA replication process, and compound 66 would be targeting HAdV E1A mRNA transcription. For compounds 17, 20, 34 and 60, the mechanism of action seems to be associated with later steps after HAdV DNA replication.

GPR120/FFAR4 Pharmacology: Focus on Agonists in Type 2 Diabetes Mellitus Drug Discovery.

The G-protein coupled receptors (GPCRs) activated by free fatty acids (FFAs) have emerged as new and exciting drug targets, due to their plausible translation from pharmacology to medicines. This perspective aims to report recent research about GPR120/FFAR4 and its involvement in several diseases, including cancer, inflammatory conditions, and central nervous system disorders. The focus is to highlight the importance of GPR120 in Type 2 diabetes mellitus (T2DM). GPR120 agonists, useful in T2DM drug discovery, have been widely explored from a structure-activity relationship point of view. Since the identification of the first reported synthetic agonist TUG-891, the research has paved the way for the development of TUG-based molecules as well as new and different chemical entities. These molecules might represent the starting point for the future discovery of GPR120 agonists as antidiabetic drugs.

In Vitro Anticancer Activity and Mechanism of Action of an Aziridinyl Galactopyranoside.

We recently screened a series of new aziridines ß-D-galactopyranoside derivatives for selective anticancer activity and identified 2-methyl-2,3-[N-(4-methylbenzenesulfonyl)imino]propyl 2,3-di-O-benzyl-4,6-O-(S)-benzylidene-ß-D-galactopyranoside (AzGalp) as the most promising compound. In this article, we explore the possible mechanisms involved in the cytotoxicity of this aziridine and evaluate its selective anticancer activity using cancer cells and normal cells from a variety of tissues. Our data show that AzGalp induces DNA damage (comet assay). Cells deficient in the nucleotide excision repair (NER) pathway were hypersensitive to the cytotoxicity of this compound. These results suggest that AzGalp induces bulky DNA adducts, and that cancer cells lacking a functional NER pathway may be particularly vulnerable to the anticancer effects of this aziridine. Several experiments revealed that neither the generation of oxidative stress nor the inhibition of glycolysis played a significant role in the cytotoxicity of AzGalp. Combinations of AzGalp with oxaliplatin or 5-fluorouracil slightly improved the ability of both anticancer drugs to selectively kill cancer cells. AzGalp also showed selective cytotoxicity against a panel of malignant cells versus normal cells; the highest selectivity was observed for two acute promyelocytic leukemia cell lines. Additional preclinical studies are necessary to evaluate the anticancer potential of AzGalp.

Serinol-Based Benzoic Acid Esters as New Scaffolds for the Development of Adenovirus Infection Inhibitors: Design, Synthesis, and In Vitro Biological Evaluation.

Over the years, human adenovirus (HAdV) has progressively been recognized as a significant viral pathogen. Traditionally associated with self-limited respiratory, gastrointestinal, and conjunctival infections, mainly in immunocompromised patients, HAdV is currently considered to be a pathogen presenting significant morbidity and mortality in both immunosuppressed and otherwise healthy individuals. Currently available therapeutic options are limited because of their lack of effectivity and related side effects. In this context, there is an urgent need to develop effective anti-HAdV drugs with suitable therapeutic indexes. In this work, we identified new serinol-derived benzoic acid esters as novel scaffolds for the inhibition of HAdV infections. A set of 38 compounds were designed and synthesized, and their antiviral activity and cytotoxicity were evaluated. Four compounds (13, 14, 27, and 32) inhibited HAdV infection at low micromolar concentrations (2.82-5.35 µM). Their half maximal inhibitory concentration (IC50) values were lower compared to that of cidofovir, the current drug of choice. All compounds significantly reduced the HAdV DNA replication process, while they did not block any step of the viral entry. Our results showed that compounds 13, 14, and 32 seem to be targeting the expression of the E1A early gene. Moreover, all four derivatives demonstrated a significant inhibition of human cytomegalovirus (HCMV) DNA replication. This new scaffold may represent a potential tool useful for the development of effective anti-HAdV drugs.

Exploration of piperazine-derived thioureas as antibacterial and anti-inflammatory agents. In vitro evaluation against clinical isolates of colistin-resistant Acinetobacter baumannii.

A. baumannii is one of the most important multidrug-resistant microorganisms in hospital units. It is resistant to many classes of antibiotics and the development of new therapeutic strategies is necessary. The aim of this study was to evaluate the antibacterial activity of a set of piperazine-derived thioureas against 13 clinical strains of colistin-resistant A. baumannii. Six derivatives were identified to inhibit bacterial growth of 46% of the A. baumannii strains at low micromolar concentrations (Minimum Inhibitory Concentration from 1.56 to 6.25 µM). A common structural feature in most active compounds was the presence of a 3,5-bis-trifluoromethyl phenyl ring at the thiourea function. In addition, the ability of the compounds to inhibit production of nitric oxide (NO) was examined in RAW 264.7 murine macrophages, highlighting the potential of piperazine-derived thioureas as promising scaffolds for the design of new combined anti-bacterial/anti-inflammatory agents.

Optimization of piperazine-derived ureas privileged structures for effective antiadenovirus agents.

In recent years, human adenovirus (HAdV) infections have shown a high clinical impact in both immunosuppressed and immunocompetent patients. The research into specific antiviral drugs for the treatment of HAdV infections in immunocompromised patients constitutes a principal objective for medicinal chemistry due to the lack of any specific secure drug to treat these infections. In this study, we report a small-molecule library (67 compounds) designed from an optimization process of piperazine-derived urea privileged structures and their biological evaluation: antiviral activity and cytotoxicity. The active compounds selected were further evaluated to gain mechanistic understanding for their inhibition. Twelve derivatives were identified that inhibited HAdV infections at nanomolar and low micromolar concentrations (IC50 from 0.6 to 5.1 µM) with low cytotoxicity. In addition, our mechanistic assays suggested differences in the way the derivatives exert their anti-HAdV activity targeting transcription, DNA replication and later steps in the HAdV replication cycle. Furthermore, eight of the 12 studied derivatives blocked human cytomegalovirus (HCMV) DNA replication at low micromolar concentrations. The data provided herein indicates that the 12 thiourea/urea piperazine derivatives studied may represent potential lead compounds for clinical evaluation and development of new anti-HAdV drugs.

Selective cytotoxic activity and DNA damage by an epoxyalkyl galactopyranoside.

Preclinical Research & Development Several clinically useful anticancer drugs selectively kill cancer cells by inducing DNA damage; the genomic instability and DNA repair defects of cancer cells make them more vulnerable than normal cells to the cytotoxicity of DNA-damaging agents. Because epoxide-containing compounds can induce DNA damage, we have used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate the selective cytotoxicity of three epoxyalkyl galactopyranosides against A549 lung cancer cells and MRC-5 lung normal cells. Compound (2S,3S)-2,3-epoxydecyl 4,6-O-(S)-benzylidene-ß-d-galactopyranoside (EDBGP) showed the highest selective anticancer activity and was selected for mechanistic studies. After observing that EDBGP induced cellular DNA damage (comet assay), we found that cells deficient in nucleotide excision repair were hypersensitive to the cytotoxicity of this compound; this suggests that EDBGP may induce bulky DNA adducts. EDBGP did not inhibit glycolysis (glucose consumption and lactate production). Pretreatment of lung cancer cells with several antioxidants did not reduce the cytotoxicity of EDBGP, thereby indicating that reactive oxygen species do not participate in the anticancer activity of this compound. Finally, EDBGP was screened against a panel of cancer cells and normal cells from several tissues, including three genetically modified skin fibroblasts with increasing degree of malignancy. Our results suggest that epoxyalkyl galactopyranosides are promising lead compounds for the development of new anticancer agents.

New 4-Acyl-1-phenylaminocarbonyl-2-phenylpiperazine Derivatives as Potential Inhibitors of Adenovirus Infection. Synthesis, Biological Evaluation, and Structure-activity Relationships.

The search for human adenovirus (HAdV)-specific antiviral drugs for the treatment of HAdV infections in immunocompromised patients continues to be a challenging goal for medicinal chemistry. Here, we report the synthesis, biological evaluation, and structure-activity relationships of a small molecules library. We have identified six phenylpiperazine derivatives that significantly inhibited HAdV infection. These six compounds showed the capacity to block HAdV and, in addition, human cytomegalovirus (HCMV) replications at low micromolar concentration, with little or no cytotoxicity. On the basis of our biological studies, these molecules block HAdV and HCMV infections in different phases of their life cycle, providing potential candidates for the development of a new family of antiviral drugs for the treatment of infections by DNA viruses.

THDP17 decreases ammonia production through glutaminase inhibition. A new drug for hepatic encephalopathy therapy.

Ammonia production is implicated in the pathogenesis of hepatic encephalopathy (HE), being intestinal glutaminase activity the main source for ammonia. Management of ammonia formation can be effective in HE treatment by lowering intestinal ammonia production. The use of glutaminase inhibitors represents one way to achieve this goal. In this work, we have performed a search for specific inhibitors that could decrease glutaminase activity by screening two different groups of compounds: i) a group integrated by a diverse, highly pure small molecule compounds derived from thiourea ranging from 200 to 800 Daltons; and ii) a group integrated by commonly use compounds in the treatment of HE. Results shown that THDP-17 (10 µM), a thiourea derivate product, could inhibit the intestinal glutaminase activity (57.4±6.7%). Inhibitory effect was tissue dependent, ranging from 40±5.5% to 80±7.8% in an uncompetitive manner, showing Vmax and Km values of 384.62 µmol min(-1), 13.62 mM with THDP-17 10 µM, respectively. This compound also decreased the glutaminase activity in Caco-2 cell cultures, showing a reduction of ammonia and glutamate production, compared to control cultures. Therefore, the THDP-17 compound could be a good candidate for HE management, by lowering ammonia production.

Aziridines from alkenyl-ß-D-galactopyranoside derivatives: Stereoselective synthesis and in vitro selective anticancer activity.

A series of new aziridines ß-D-galactopyranoside derivatives were synthesized from alkenyl ß-D-galactopyranosides employing Sharpless conditions. The structures of the compounds were established by (1)H NMR, (13)C NMR, MS, HRMS and elemental analysis. The stereoselectivity of the reaction and the structural requirements of the alkenyl precursor for improving diastereoisomeric excesses of the direct aziridination reaction were also studied. The new compounds were subjected to a preliminary screening for cytotoxic activity against human lung cancer cells vs. human non-malignant lung cells. Terminal aziridine derivatives showed activity and, most notably, selectivity. One of the most active and selective compounds was also evaluated against breast cancer cells, melanoma cells, and non-malignant cells from the same origin. Its cytotoxic activity was similar to that of the positive controls, displaying a highly selective cytotoxic activity against both types of cancer cells.

Temperature- and salinity-decoupled overproduction of hydroxyectoine by Chromohalobacter salexigens.

Hydroxyectoine overproduction by the natural producer Chromohalobacter salexigens is presented in this study. Genetically engineered strains were constructed that at low salinity coexpressed, in a vector derived from a native plasmid, the ectoine (ectABC) and hydroxyectoine (ectD) genes under the control of the ectA promoter, in a temperature-independent manner. Hydroxyectoine production was further improved by increasing the copies of ectD and using a C. salexigens genetic background unable to synthesize ectoines.

Isoprenyl-thiourea and urea derivatives as new farnesyl diphosphate analogues: synthesis and in vitro antimicrobial and cytotoxic activities.

A series of new isoprenyl-thiourea and urea derivatives were synthesized by the reaction of alkyl or aryl isothiocyanate or isocyanate and primary amines. The structures of the compounds were established by (1)H NMR, (13)C NMR, MS, HRMS and elemental analysis. The new compounds were screened for in vitro antimicrobial activity against seven strains representing different types of gram-positive and gram-negative bacteria. More than a third of the synthesized compounds showed variable inhibition activities against the tested strains. Best antimicrobial activities were found for those thiourea analogues with 3-methyl-2-butenyl, isobutyl or isopentyl groups and aromatic rings possessing electron withdrawing substituents. The new compounds were also subjected to a preliminary screening for antitumoral activity. The presence of a highly lipophilic group and an electron withdrawing group in the aromatic rings enhanced anticancer activity of the synthesized compounds, showing in most cases more activity than that of the controls.

Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli.

BACKGROUND: The compatible solute trehalose is involved in the osmostress response of Rhizobium etli, the microsymbiont of Phaseolus vulgaris. In this work, we reconstructed trehalose metabolism in R. etli, and investigated its role in cellular adaptation and survival to heat and desiccation stress under free living conditions. RESULTS: Besides trehalose as major compatible solute, R. etli CE3 also accumulated glutamate and, if present in the medium, mannitol. Putative genes for trehalose synthesis (otsAB/treS/treZY), uptake (aglEFGK/thuEFGK) and degradation (thuAB/treC) were scattered among the chromosome and plasmids p42a, p42c, p42e, and p42f, and in some instances found redundant. Two copies of the otsA gene, encoding trehalose-6-P-synthase, were located in the chromosome (otsAch) and plasmid p42a (otsAa), and the latter seemed to be acquired by horizontal transfer. High temperature alone did not influence growth of R. etli, but a combination of high temperature and osmotic stress was more deleterious for growth than osmotic stress alone. Although high temperature induced some trehalose synthesis by R. etli, trehalose biosynthesis was mainly triggered by osmotic stress. However, an otsAch mutant, unable to synthesize trehalose in minimal medium, showed impaired growth at high temperature, suggesting that trehalose plays a role in thermoprotection of R. etli. Desiccation tolerance by R. etli wild type cells was dependent of high trehalose production by osmotic pre-conditioned cells. Cells of the mutant strain otsAch showed ca. 3-fold lower survival levels than the wild type strain after drying, and a null viability after 4 days storage. CONCLUSIONS: Our findings suggest a beneficial effect of osmotic stress in R. etli tolerance to desiccation, and an important role of trehalose on the response of R. etli to high temperature and desiccation stress.

Role of trehalose in salinity and temperature tolerance in the model halophilic bacterium Chromohalobacter salexigens.

The disaccharide trehalose is considered as a universal stress molecule, protecting cells and biomolecules from injuries imposed by high osmolarity, heat, oxidation, desiccation and freezing. Chromohalobacter salexigens is a halophilic and extremely halotolerant γ-proteobacterium of the family Halomonadaceae. In this work, we have investigated the role of trehalose as a protectant against salinity, temperature and desiccation in C. salexigens. A mutant deficient in the trehalose-6-phosphate synthase gene (otsA::Ω) was not affected in its salt or heat tolerance, but double mutants ectoine- and trehalose-deficient, or hydroxyectoine-reduced and trehalose-deficient, displayed an osmo- and thermosensitive phenotype, respectively. This suggests a role of trehalose as a secondary solute involved in osmo- (at least at low salinity) and thermoprotection of C. salexigens. Interestingly, trehalose synthesis was osmoregulated at the transcriptional level, and thermoregulated at the post-transcriptional level, suggesting that C. salexigens cells need to be pre-conditioned by osmotic stress, in order to be able to quickly synthesize trehalose in response to heat stress. C. salexigens was more sensitive to desiccation than E. coli and desiccation tolerance was slightly improved when cells were grown at high temperature. Under these conditions, single mutants affected in the synthesis of trehalose or hydroxyectoine were more sensitive to desiccation than the wild-type strain. However, given the low survival rates of the wild type, the involvement of trehalose and hydroxyectoine in C. salexigens response to desiccation could not be firmly established.

Involvement of EupR, a response regulator of the NarL/FixJ family, in the control of the uptake of the compatible solutes ectoines by the halophilic bacterium Chromohalobacter salexigens.

BACKGROUND: Osmosensing and associated signal transduction pathways have not yet been described in obligately halophilic bacteria. Chromohalobacter salexigens is a halophilic bacterium with a broad range of salt tolerance. In response to osmotic stress, it synthesizes and accumulates large amounts of the compatible solutes ectoine and hydroxyectoine. In a previous work, we showed that ectoines can be also accumulated upon transport from the external medium, and that they can be used as carbon sources at optimal, but not at low salinity. This was related to an insufficient ectoine(s) transport under these conditions. RESULTS: A C. salexigens Tn1732-induced mutant (CHR95) showed a delayed growth with glucose at low and optimal salinities, could not grow at high salinity, and was able to use ectoines as carbon sources at low salinity. CHR95 was affected in the transport and/or metabolism of glucose, and showed a deregulated ectoine uptake at any salinity, but it was not affected in ectoine metabolism. Transposon insertion in CHR95 caused deletion of three genes, Csal0865-Csal0867: acs, encoding an acetyl-CoA synthase, mntR, encoding a transcriptional regulator of the DtxR/MntR family, and eupR, encoding a putative two-component response regulator with a LuxR_C-like DNA-binding helix-turn-helix domain. A single mntR mutant was sensitive to manganese, suggesting that mntR encodes a manganese-dependent transcriptional regulator. Deletion of eupR led to salt-sensitivity and enabled the mutant strain to use ectoines as carbon source at low salinity. Domain analysis included EupR as a member of the NarL/FixJ family of two component response regulators. Finally, the protein encoded by Csal869, located three genes downstream of eupR was suggested to be the cognate histidine kinase of EupR. This protein was predicted to be a hybrid histidine kinase with one transmembrane and one cytoplasmic sensor domain. CONCLUSIONS: This work represents the first example of the involvement of a two-component response regulator in the osmoadaptation of a true halophilic bacterium. Our results pave the way to the elucidation of the signal transduction pathway involved in the control of ectoine transport in C. salexigens.

The extremely halophilic bacterium Salicola marasensis IC10 accumulates the compatible solute betaine.

The extremely halophilic bacterium strain IC10 was isolated from a solar saltern on Isla Cristina (southern Spain). Phylogenetic, genotypic and phenotypic data supported the inclusion of this strain in the species Salicola marasensis. An analysis of intracellular organic osmotic solutes showed glycine betaine to be present, contributing to the overall osmotic balance, and this was the only compatible solute accumulated when S. marasensis IC10 was grown over a wide range of external NaCl concentrations (10-25%, w/v).

Biosynthesis of compatible solutes in rhizobial strains isolated from Phaseolus vulgaris nodules in Tunisian fields.

BACKGROUND: Associated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T. RESULTS: The most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order) by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the beta-1,2-cyclic glucan from R. tropici CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined. CONCLUSIONS: The soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain R. tropici CIAT 899.

Interplay between iron homeostasis and the osmotic stress response in the halophilic bacterium Chromohalobacter salexigens.

In this study, the connection between iron homeostasis and the osmostress response in the halophile Chromohalobacter salexigens was investigated. A decrease in the requirement for both iron and histidine and a lower level of siderophore synthesis were observed at high salinity, and these findings were correlated with a lower protein content in salt-stressed cells. A six-gene operon (cfuABC-fur-hisI-orf6 operon) located downstream of the ectABC ectoine synthesis genes was characterized. A fur strain (in which the ferric iron uptake regulator Fur was affected) had the Mn resistance phenotype typical of fur mutants, was deregulated for siderophore production, and displayed delayed growth under iron limitation conditions, indicating that fur encodes a functional iron regulator. hisI was essential for histidine synthesis, which in turn was necessary for siderophore production. Fur boxes were found in the promoters of the cfuABC-fur-hisI-orf6 and ectABC operons, suggesting that Fur directly interacts with DNA in these regions. Fur mediated the osmoregulated inhibition of cfuABC-fur-hisI-orf6 operon expression by iron and functioned as a positive regulator of the ectABC genes under high-salinity conditions, linking the salt stress response with iron homeostasis. Excess iron led to a higher cytoplasmic hydroxyectoine content, suggesting that hydroxyectoine protects against the oxidative stress caused by iron better than ectoine. This study provides the first evidence of involvement of the iron homeostasis regulator Fur as part of the complex circuit that controls the response to osmotic stress in halophilic bacteria.