Design,synthesis, in vitro anticancer,antioxidant and antibacterial activity; DNA/BSA binding,photoleavage and docking studies of Cu(II) ternary metal complexes

Abstract

Three mononuclear, mixed ligand ternary Cu(II) complexes of 3-((Z)-1-(2-hydroxyphenylimino)ethyl)-4-hydroxy-6-methyl-2H-pyran-2-one (HEHMP) viz; [Cu-(Phen) (HEHMP)] (1a), [Cu-(Bpy)(HEHMP)] (1?b) and [Cu-Bpy(NCS)(HEHMP)] (1c) were synthesized and characterized by data obtained from various spectral techniques. The binding affinities of these complexes with calf thymus DNA (CT-DNA) and bovine serum albumin (BSA) protein were explored by absorption and fluorescence quenching titrations. The results indicated strong affinity of the title compounds to bind with both CT-DNA and BSA. The antioxidant properties of the synthesized compounds evaluated by free-radical scavenging method using spectrophotometric technique indicated their affirmative potential activity. Gel electrophoresis experiments revealed the efficacy of metal complexes in resulting the cleavage of pBR322 supercoiled DNA. In vitro cytotoxicity studies of these complexes evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against HeLa and MCF-7 cancer cell lines indicated relatively high effectiveness of the complex 1c. Confocal microscopy signified the potential of the complexes to induce apoptosis in HeLa cell lines. In addition, the antibacterial activity of the compounds carried out by disc diffusion method revealed significantly enhanced antibacterial activity in Cu (II) ternary complexes compared to the activity of ligands in unbound form signifying the implicit role of metal ion in inducing lipophilic character.     

Synthesis and evaluation of novel azetidine analogs as potent inhibitors of vesicular [3H]dopamine uptake

Lobelane analogs that incorporate a central piperidine or pyrrolidine moiety have previously been reported by our group as potent inhibitors of VMAT2 function. Further central ring size reduction of the piperidine moiety in lobelane to a four-membered heterocyclic ring has been carried out in the current study to afford novel cis-and trans-azetidine analogs. These azetidine analogs (15a–15c and 22a–22c) potently inhibited [³H]dopamine (DA) uptake into isolated synaptic vesicles (K_i ? 66 nM). The cis-4-methoxy analog 22b was the most potent inhibitor (K_i = 24 nM), and was twofold more potent that either lobelane (2a, K_i = 45 nM) or norlobelane (2b, K_i = 43 nM). The trans-methylenedioxy analog, 15c (K_i = 31 nM), was equipotent with the cis-analog, 22b, in this assay. Thus, cis- and trans-azetidine analogs 22b and 15c represent potential leads in the discovery of new clinical candidates for the treatment of methamphetamine abuse.     

Synthesis, anticancer and antioxidant activities of some novel N-(benzo[d]oxazol-2-yl)-2-(7- or 5-substituted-2-oxoindolin-3-ylidene) hydrazinecarboxamide derivatives

A series of N-(benzo[d]oxazol-2-yl)-2-(7- or 5-substituted-2-oxoindolin-3-ylidene) hydrazinecarboxamide derivatives were synthesized by treating N-(benzoxazol-2-yl)hydrazinecarboxamide with different isatin derivatives. The newly synthesized compounds were characterized on the basis of spectral analyses. All the synthesized derivatives (Va-l) were screened for anticancer and antioxidant activities. The results showed the anticancer activity of test compounds against HeLa, IMR-32 and MCF-7 cancer cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All the synthetic compounds produced a dose-dependant inhibition of growth of the cells. The IC(50) values of some compounds were comparable with standard anticancer agent, cisplatin. All the title compounds effectively scavenged the free radical, α,α-diphenyl-β-picryl hydrazyl. The test compounds having substitution with different halides (electron withdrawing groups) at C5 position showed more potent anticancer and antioxidant activities than those at C7 position. These results indicate that C5-substituted derivatives may be useful for developing antioxidant agents that play a protective role in many pathological conditions such as cancer, diabetes and so on.     

Alkyl cinnamates as regulator for the C1 domain of protein kinase C isoforms

The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Natural product curcumin is known to interact with PKC isoforms through the C1 domain and modulate PKC activity. The reported results demonstrate that the symmetric curcumin molecule might act as two separate units during its recognition of C1 domains. To understand the importance of the two halves of curcumin in PKC binding and to develop effective PKC regulators, we synthesized a series of alkyl cinnamates (1-8), characterized absorption and fluorescence properties and measured binding affinities with the C1b subdomains of PKC isoforms. The binding parameters of the monomeric compounds and liposomes containing compounds confirmed their interaction with the C1b subdomains of PKCδ and PKCθ. The molecular docking analysis with PKCδ and PKCθ C1b subdomains revealed that the alkyl cinnamates form hydrogen bond with the backbone of the protein at the same binding site as that of diacylglycerol and phorbol esters. The results show that the alkyl cinnamates bind to the activator binding site of PKCs and both methoxy and hydroxyl groups play important roles in the binding process.     

Spatial distribution and seasonal variation in fluoride enrichment in groundwater and its associated human health risk assessment in Telangana State,South India

Groundwater is a vital source of drinking water in Siddipet rural and urban regions of Central Telangana, South India and it is a major cause of fluoride toxicity in humans. The intake of elevated fluoride has a significant impact on human health, especially immediate problems that are seen in children's teeth. The primary aim of the study was to identify the seasonal variation in fluoride concentration and associated health risks in the residents of the study region. To assess the fluoride contamination in groundwater, a total of 158 samples were analyzed in two seasons. The mean concentrations of fluoride 1.26 mg/L and 2.21 mg/L were 1.46 and 2.8 times higher than the acceptable limit of 1.5 mg/L, before and after monsoon respectively. To estimate the human health risks due to the ingestion of elevated fluoride through drinking water, hazard quotient fluoride (HQ_Fluoride) was calculated using the United States Environmental Protection Agency method. HQ_Fluoride values were 0.44–2.44 and 0.89–4.67 for children, 0.36–2.00 and 0.73–3.82 for females, and 0.41–2.26 and 0.82–4.31 for males in pre- and post-monsoon seasons respectively, suggesting emphatically greater risk than the acceptable limits (HQ_Fluoride > 1), which generates health risks.     

Distal Lateral Subungual Onychomycosis Owing to <Emphasis Type="Italic">Tritirachium oryzae</Emphasis>: A Bystander or Invader?

The genus Tritirachium is a mitosporic fungus which inhabits in soil and decaying plant material and also a notable insect pathogen. Human infections with Tritirachium species though rare were previously reported to cause corneal ulcers, otomycosis, onychomycosis, and dermatomycosis of the scalp and hence may be considered as a potential pathogen. Here we report a case of distal lateral subungual onychomycosis involving right great toenail in a 22-year-old female, wherein direct potassium hydroxide preparations, fungal cultures, and molecular sequencing of the isolate established Tritirachium oryzae as the etiological agent. Antifungal susceptibility performed by the microbroth technique of CLSI revealed increased MICs to amphotericin B and low MICs to azoles and echinocandins. The case was managed with surgical nail avulsion followed by topical application of 2% ketoconazole cream resulting regrowth of normal nail. To the best of our knowledge, this is the first report of non-dermatophytic mold T. oryzae causing onychomycosis in India.     

Immobilization of endo-polygalacturonase from Aspergillus ustus on silica gel

Endo-polygalacturonase from Aspergillus ustus when immobilized on to modified silica gel retained 28% of its original activity. The immobilized enzyme could be re-used through 10 cycles of reaction with almost 90% retention of its original activity. It had increased thermostability over its soluble form: the half-life of the soluble enzyme at 40 °C was less than 10 h whereas the immobilized enzyme retained 82% of its activity after 10 h at 40 °C. Similarly, at 50 °C the half-life of the soluble enzyme was 30 min whereas that of the immobilized enzyme was 5 h.     

N-Naphthoyl-substituted indole thio-barbituric acid analogs inhibit the helicase activity of the hepatitis C virus NS3

The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC₅₀ values of 21–24?µM. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.     

The purine nucleoside phosphorylase from Trichomonas vaginalis is a homologue of the bacterial enzyme

Trichomonas vaginalis is a parasitic protozoan and the causative agent of trichomoniasis. Its primary purine salvage system, consisting of a purine nucleoside phosphorylase (PNP) and a purine nucleoside kinase, presents potential targets for designing selective inhibitors as antitrichomonial drugs because of lack of de novo synthesis of purine nucleotides in this organism. cDNA encoding T. vaginalis PNP was isolated by complementation of an Escherichia coli strain deficient in PNP and expressed, and the recombinant enzyme was purified to apparent homogeneity. It bears only 28% sequence identity with that of human PNP but 57% identity with the E. coli enzyme. Gel filtration showed the enzyme in a hexameric form, similar to the bacterial PNPs. Steady-state kinetic analysis of T. vaginalis PNP-catalyzed reactions gave K(m)'s of 31.5, 59.7, and 6.1 microM for inosine, guanosine, and adenosine in the nucleosidase reaction and 45.6, 35.9, and 12.3 microM for hypoxanthine, guanine, and adenine in the direction of nucleoside synthesis. This substrate specificity appears to be similar to that of bacterial PNPs. The catalytic efficiency of this enzyme with adenine as substrate is 58-fold higher than that with either hypoxanthine or guanine, representing a distinct disparity with the mammalian PNPs, which have negligible activity with either adenine or adenosine. The kinetic mechanism of T. vaginalis PNP-catalyzed reactions, determined by product inhibition and equilibrium isotope exchange, was by random binding of substrates (purine base and ribose 1-phosphate) with ordered release of the purine nucleoside first, followed by inorganic phosphate. Formycin A, an analogue of adenosine known as an inhibitor of E. coli PNP without any effect on mammalian PNPs, was shown to inhibit T. vaginalis PNP with a K(is) of 2.3 microM by competing with adenosine. T. vaginalis PNP thus belongs to the family of bacterial PNPs and constitutes a target for antitrichomonial chemotherapy.     

Pan-genome dynamics of Pseudomonas gene complements enriched across hexachlorocyclohexane dumpsite

Background

Phylogenetic heterogeneity across Pseudomonas genus is complemented by its diverse genome architecture enriched by accessory genetic elements (plasmids, transposons, and integrons) conferring resistance across this genus. Here, we sequenced a stress tolerant genotype i.e. Pseudomonas sp. strain RL isolated from a hexachlorocyclohexane (HCH) contaminated pond (45 mg of total HCH g⁻¹ sediment) and further compared its gene repertoire with 17 reference ecotypes belonging to P. stutzeri, P. mendocina, P. aeruginosa, P. psychrotolerans and P. denitrificans, representing metabolically diverse ecosystems (i.e. marine, clinical, and soil/sludge). Metagenomic data from HCH contaminated pond sediment and similar HCH contaminated sites were further used to analyze the pan-genome dynamics of Pseudomonas genotypes enriched across increasing HCH gradient.

Results

Although strain RL demonstrated clear species demarcation (ANI ≤ 80.03%) from the rest of its phylogenetic relatives, it was found to be closest to P. stutzeri clade which was further complemented functionally. Comparative functional analysis elucidated strain specific enrichment of metabolic pathways like α-linoleic acid degradation and carbazole degradation in Pseudomonas sp. strain RL and P. stutzeri XLDN-R, respectively. Composition based methods (%codon bias and %G + C difference) further highlighted the significance of horizontal gene transfer (HGT) in evolution of nitrogen metabolism, two-component system (TCS) and methionine metabolism across the Pseudomonas genomes used in this study. An intact mobile class-I integron (3,552 bp) with a captured gene cassette encoding for dihydrofolate reductase (dhfra1) was detected in strain RL, distinctly demarcated from other integron harboring species (i.e. P. aeruginosa, P. stutzeri, and P. putida). Mobility of this integron was confirmed by its association with Tnp21-like transposon (95% identity) suggesting stress specific mobilization across HCH contaminated sites. Metagenomics data from pond sediment and recently surveyed HCH adulterated soils revealed the in situ enrichment of integron associated transposase gene (TnpA6100) across increasing HCH contamination (0.7 to 450 mg HCH g⁻¹ of soil).

Conclusions

Unlocking the potential of comparative genomics supplemented with metagenomics, we have attempted to resolve the environment and strain specific demarcations across 18 Pseudomonas gene complements. Pan-genome analyses of these strains indicate at astoundingly diverse metabolic strategies and provide genetic basis for the cosmopolitan existence of this taxon.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1488-2) contains supplementary material, which is available to authorized users.