Biological Evaluation of Platinum(II) Sulfonamido Complexes: Synthesis, Characterization, Cytotoxicity, and Biological Imaging.

Platinum-based compounds are actively used in clinical trials as anticancer agents. In this study, two novel platinum complexes, (C1 = [PtCl2(N(SO2quin)dpa)], C2 = [PtCl2(N(SO2azobenz)dpa)]) containing quinoline and azobenzene appended dipicolylamine sulfonamide ligands were synthesized in good yield. The singlet attributable to methylene CH2 protons of the ligands of C1 and C2 appears as two doublets in 1H NMR spectra, which confirms the presence of magnetically nonequivalent protons upon coordination to platinum. Structural data of N(SO2quin)dpa (L1), N(SO2azobenz)dpa (L2) and PtCl2(N(SO2quin)dpa) confirmed the formation of the desired compounds. Time-dependent density functional theory calculations suggested that the excitation of L1 show quin-unit-based π⟶π ∗ excitations (i.e., ligand-centered charge transfer, LC), while C1 shows the metal-ligand-to-ligand charge-transfer (MLLCT) character. L1 displays intense fluorescence from the 1LC excited state, while C1 gives phosphorescence from the 3LC state. Mammalian cell toxicity of ligands and complexes was assessed with NCI-H292 nonsmall-cell lung cancer cells. Further, C1 and C2 showed significantly low IC50 values compared with N(SO2azobenz)dpa and PtCl2(N(SO2quin)dpa). Fluorescence imaging data of both ligands and complexes revealed the potential fluorescence activity of these compounds for biological imaging. All four compounds are promising novel candidates that can be further investigated on their usage as potential anticancer agents and cancer cell imaging agents.

First Complete Cytochrome B Sequences and Molecular Taxonomy of Bat Species from Sri Lanka.

This is the first report on the molecular identification and phylogeny of the Rousettus leschenaultii Desmarest, 1810, Rhinolophus rouxii Temminck, 1835, Hipposideros speoris Schneider, 1800, Hipposideros lankadiva Kelaart, 1850, and Miniopterus fuliginosus Kuhl, 1817, bat species in Sri Lanka, inferred from analyses by mitochondrially encoded cytochrome b gene sequences. Recent research has indicated that bats show enormous cryptic genetic diversity. Moreover, even within the same species, the acoustic properties of echolocation calls and morphological features such as fur color could vary in different populations. Therefore, we have used molecular taxonomy for the accurate identification of five bat species recorded in one of the largest cave populations in Sri Lanka. The bats were caught using a hand net, and saliva samples were collected non-invasively from each bat by using a sterile oral swab. Nucleic acids were extracted from the oral swab samples, and mitochondrial DNA was amplified by using primers targeting the mitochondrially encoded cytochrome b gene. This study reports the first molecular evidence for the identification of five bat species in Sri Lanka. Our findings will contribute to future conservation and systematic studies of bats in Sri Lanka. This study will also provide the basis for a genetic database of Sri Lankan bats which will contribute significantly to the investigation of potentially zoonotic bat viruses.

LcaR: a regulatory switch from Pseudomonas aeruginosa for bioengineering alkane degrading bacteria.

Application of genetically engineered bacterial strains for biodegradation of hydrocarbons is a sustainable solution for treating pollutants as well as in industrial applications. However, the process of bioengineering should be carefully carried out to optimize the output. Investigation of regulatory genes for bioengineering is essential for developing synthetic circuits for effective biocatalysts. Here we focus on LcaR, a putative transcriptional regulator affecting the expression of alkB2 and lcaR operon that has a high potential to become a tool in designing such pathways. Four LcaR dimers bind specifically to the upstream regulatory region where divergent promoters of alkB2 and lcaR genes are located with high affinity at a Kd of 0.94 ± 0.17 nM and a Hill coefficient is 1.7 ± 0.3 demonstrating cooperativity in the association. Ligand binding alters the conformation of LcaR, which releases the regulator from its cognate DNA. Tetradecanal and hexadecanal act as natural ligands of LcaR with an IC50 values of 3.96 ± 0.59 µg/ml and 0.68 ± 0.21 µg/ml, respectively. The structure and function of transcription factors homologous to LcaR have not been characterized to date. This study provides insight into regulatory mechanisms of alkane degradation with a direction towards potential applications in bioengineering for bioremediation and industrial applications.

Synthesis, characterization and biological evaluation of dipicolylamine sulfonamide derivatized platinum complexes as potential anticancer agents.

Three new Pt complexes, [PtCl2(N(SO2(2-nap))dpa)], [PtCl2(N(SO2(1-nap))dpa)] and [PtCl2(N(SO2pip)dpa)], containing a rare 8-membered ring were synthesized in good yield and high purity by utilizing the ligands N(SO2(2-nap))dpa, N(SO2(1-nap))dpa and N(SO2pip)dpa, which contain a dipicolylamine moiety. Structural studies of all three complexes confirmed that the ligands are bound in a bidentate mode via Pt-N(pyridyl) bonds forming a rare 8-membered ring. The intense fluorescence displayed by the ligands is quenched upon coordination to Pt. According to time dependent density functional theory (TDDFT) calculations, the key excitations of N(SO2(2-nap))dpa and [PtCl2(N(SO2(1-nap))dpa)] involve the 2-nap-ligand-centered π → π* excitations. While all six compounds have shown antiproliferative activity against human breast cancer cells (MCF-7), the N(SO2pip)dpa and N(SO2(2-nap))dpa ligands and [PtCl2((NSO2pip)dpa)] complex have shown significantly high cytotoxicity, directing them to be further investigated as potential anti-cancer drug leads.

Graphene Oxide-Based Nanocomposite for Sustained Release of Cephalexin.

A sustained-release carrier system for the drug cephalexin (CEF) using functionalized graphene oxide is reported. PEGylation of GO (GO-PEG) and successful loading of CEF into PEGylated graphene oxide (GO-PEG-CEF) nanoconjugate are confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, and thermogravimetric analysis. Encapsulation efficiency of 69% and a loading capacity of 19% are obtained with the optimized formulation of GO-PEG-CEF. In vitro CEF release profiles show an initial burst release followed by a more sustained release over a 96 h period with cumulative release of 80%. The half maximal inhibitory concentration (IC50) values have both dose- and time-dependent antibacterial activity for GO-PEG-CEF against both gram-positive and gram-negative bacteria while pure CEF showed only dose-dependent antibacterial activity. The minimum inhibitory concentration values of GO-PEG-CEF are 7.8 and 3.9 µg/mL against S. aureus and B. cereus, respectively, while it is 10 µg/mL with pure CEF against both gram-positive bacteria. This confirms the enhanced antibacterial activity of GO-PEG-CEF over pure CEF against gram-positive bacteria. These findings therefore show GO-PEG-CEF is promising as a sustained-release nanoantibiotic system for effective treatment against S. aureus and B. cereus infections.

Effect of natural curcuminoids-intercalated layered double hydroxide nanohybrid against Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis: A bactericidal, antibiofilm, and mechanistic study.

The study aimed to determine the antibacterial/antibiofilm effect and mechanism of interaction of curcuminoids-intercalated Mg/Al layered double hydroxide (curcuminoids-LDH) against three different bacteria. Antimicrobial effect of curcuminoids-LDH nanohybrid was investigated against P. aeruginosa, S. aureus, and E. faecalis (for both standard strains and clinical isolates), using agar well diffusion method. Minimum inhibitory concentrations (MIC) of planktonic bacteria were determined using the broth microdilution method. MIC of biofilms (MBIC50 ) and killing time for 48 hr matured biofilms were determined by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Scanning electron microscopy (SEM) was used to determine pre- and postexposure architecture of biofilms. The mechanism of the antibiofilm activity of curcuminoids-LDH was determined using UV-visible spectroscopy. All tested bacteria had given a zone of inhibition in the presence of curcuminoids-LDH. The MIC values were 0.200 g/ml for P. aeruginosa, 0.025 g/ml for S. aureus, and 0.100 g/ml for E. faecalis. The 48 hr matured biofilms were reduced by curcuminoids-LDH with an MBIC50 of 0.100 g/ml. The minimum time to achieve MBIC50 was 3 hr, and the reduction was constant until 48 hr. SEM images showed a significant reduction of biofilm cell density and exopolymer matrics for all biofilms in the presence of curcuminoids-LDH. UV-visible studies revealed the antibiofilm activity of curcuminoids-LDH as due to the auto-oxidation of curcuminoids. The oxidation products are more limited in both product concentration per unit time and the variety of products, compared to pure curcuminoids, resulting in sharper UV-visible peaks than in the case of the latter. Curcuminoids-LDH has a potential antibacterial activity against P. aeruginosa, S. aureus, and E. faecalis. An antibiofilm activity has been achieved within 3 hr of the treatment. Curcuminoids released from the LDH showed the antibacterial activity due to oxidation products interfering with bacterial cell functions, and also encapsulation in the LDH causes curcuminoids to exhibit the activity in a persistent manner compared to pure curcuminoids.

Synthesis, Characterization, and Antimicrobial Activity of Novel Sulfonated Copper-Triazine Complexes.

Metallotriazine complexes possess interesting biological and medicinal properties, and the present study focuses on the synthesis, characterization, and antimicrobial activity of four novel copper-triazine derivatives in search of potent antibacterial and antifungal drug leads. In this study, 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4,4'-disulfonic acid monosodium salt (L1, ferrozine) and 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5,5'-disulfonic acid disodium salt (L2, ferene) have been used as ligands to study the complexation towards copper(II). The synthesized complexes, [CuCl2(ferrozine)]·7H2O·MeOH (1), [CuCl2(ferrozine)2]·5H2O·MeOH (2), [CuCl2(ferene)]·H2O·MeOH (3), and [CuCl2(ferene)2]·H2O·MeOH (4), have been characterized spectroscopically, and preliminary bioassays have been carried out. FTIR spectroscopic data have shown that N=N and C=N stretching frequencies of complexes have been shifted towards lower frequencies in comparison with that of the ligands, confirming new bond formation between Cu and N, which in turn lowers the strength of N=N and C=N bonds. In addition, a bathochromic shift has been observed for UV-visible spectra of complexes (1), (2), (3), and (4). Furthermore, elemental analysis data have been useful to obtain empirical formulas of these complexes and to establish the purity of each complex. Complexes (1) and (2) have shown antibacterial activity for both S. aureus (ATCC® 25923) and E. coli (ATCC® 25922) at 1 mg/disc concentration, and ferrozine has shown a larger inhibition zone against the clinical sample of C. albicans at 1 mg/disc concentration in comparison with the positive control, fluconazole.

Synthesis, Characterization, and BSA-Binding Studies of Novel Sulfonated Zinc-Triazine Complexes.

Four Zn(II) complexes containing a pyridyl triazine core (L1 = 3-(2-pyridyl)-5,6-di(2-furyl)-1,2,4-triazine-5',5″-disulfonic acid disodium salt and L2 = 3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4″-disulfonic acid sodium salt) were synthesized, and their chemical formulas were finalized as [Zn(L1)Cl2]·5H2O·ZnCl2 (1), [Zn(L1)2Cl2]·4H2O·2CH3OH (2), [Zn(L2)Cl2]·3H2O·CH3OH (3), and [Zn(L2)2Cl2] (4). The synthesized complexes are water soluble, making them good candidates for biological applications. All four complexes have been characterized by elemental analysis and 1H NMR, IR, and UV-Vis spectroscopy. The IR stretching frequency of N=N and C=N bonds of complexes 1-4 have shifted to lower frequencies in comparison with free ligands, and a bathochromic shift was observed in UV-Vis spectra of all four complexes. The binding studies of ligands and complexes 1-4 with bovine serum albumin (BSA) resulted binding constants (Kb) of 3.09 × 104 M-1, 12.30 × 104 M-1, and 16.84 × 104 M-1 for ferene, complex 1, and complex 2, respectively, indicating potent serum distribution via albumins.

Synthesis and characterization of novel rhenium(I) complexes towards potential biological imaging applications.

BACKGROUND: Re(I) tricarbonyl complexes exhibit immense potential as fluorescence imaging agents. However, only a handful of rhenium complexes have been utilized in biological imaging. The present study describes the synthesis of four novel rhenium complexes, their characterization and preliminary biological studies to assess their potential as biological imaging agents. RESULTS: Four facial rhenium tricarbonyl complexes containing a pyridyl triazine core, (L1 = 5,5'(3-(2-pyridyl)-1,2,4-triazine-5,6-diyl)-bis-2-furansulfonic acid disodium salt and L2 = (3-(2- pyridyl)-5,6-diphenyl-1,2,4-triazine-4',4''-disulfonic acid sodium salt) have been synthesized by utililzing two different Re metal precursors, Re(CO)5Br and [Re(CO)3(H2O)3]OTf in an organic solvent mixture and water, respectively. The rhenium complexes [Re(CO)3(H2O)L1]+ (1), Re(CO)3L1Br (2), [Re(CO)3(H2O)L2]+ (3), and Re(CO)3L2Br (4), were obtained in 70-85% yield and characterized by 1H NMR, IR, UV, and luminescence spectroscopy. In both H2O and acetonitrile, complexes display a weak absorption band in the visible region which can be assigned to a metal to ligand charge transfer excitation and fluorescent emission lying in the 650-710 nm range. Cytotoxicity assays of complexes 1, 3, and 4 were carried out for rat peritoneal cells. Both plant cells (Allium cepa bulb cells) and rat peritoneal cells were stained using the maximum non-toxic concentration levels of the compounds, 20.00 mg ml-1 for 1 and 3 and 5.00 mg ml-1 for 4 to observe under the epifluorescence microscope. In both cell lines, compound concentrated specifically in the nuclei region. Hence, nuclei showed red fluorescence upon excitation at 550 nm. CONCLUSIONS: Four novel rhenium complexes have been synthesized and characterized. Remarkable enhancement of fluorescence upon binding with cells and visible range excitability demonstrates the possibility of using the new complexes in biological applications.Graphical abstractMicrograph of rat peritoneal cells incubated with novel rhenium complex under epifluorescence microscope.

Synthesis, Characterization, and Biological Studies of a Piperidinyl Appended Dipicolylamine Ligand and Its Rhenium Tricarbonyl Complex as Potential Therapeutic Agents for Human Breast Cancer.

A novel ligand bearing a central piperidinyl sulfonamide group, N(SO2pip)dpa, and its corresponding Re tricarbonyl complex, [Re(CO)3(N(SO2pip)dpa)]+, have been synthesized in good yield. The methylene CH2 signal seen as a singlet (4.54 ppm) in a 1H NMR spectrum of the ligand in DMSO-d6 appears as two doublets (5.39, 5.01 ppm) in a spectrum of the [Re(CO)3(N(SO2pip)dpa)]+ complex and confirms the presence of magnetically nonequivalent protons upon coordination to Re. Structural results revealed that the Re-N bond lengths fall within the normal range establishing coordination of ligand to metal. The presence of intraligand π → π⁎ and n → π⁎ transitions is indicated by the absorption peaks around 200-250 nm in UV-visible spectra. Absorption peaks in UV-visible spectra around 300 nm for metal complexes were identified as MLCT transitions. The S-N stretch observed as a strong peak at 923 cm-1 for N(SO2pip)dpa appeared at a shorter frequency, at 830 cm-1 in an FTIR spectrum of the [Re(CO)3(N(SO2pip)dpa)]+. The intense fluorescence displayed by the N(SO2pip)dpa ligand has quenched upon coordination to Re. Relatively low IC50 values given by human breast cancer cells, MCF-7, (N(SO2pip)dpa = 139 µM, [Re(CO)3(N(SO2pip)dpa)]+ = 360 µM) indicate that N(SO2pip)dpa and [Re(CO)3(N(SO2pip)dpa)]+ are promising novel compounds that can be further investigated on their usage as potential anticancer agents.

Lethal and sub-lethal effects on the Asian common toad Duttaphrynus melanostictus from exposure to hexavalent chromium.

Chromium discharged in industrial effluents frequently occurs as an environmental pollutant, but the lethal and sub-lethal effects the heavy metal might cause in animals exposed to it have been insufficiently investigated. Selecting the amphibian Duttaphrynus melanostictus, we carried out laboratory tests to investigate the effects of short and long term exposure to hexavalent chromium (Cr(VI)) in both tadpoles and adult toads. The concentrations used were 0.002, 0.02, 0.2, 1.0 and 2.0mg/L, the first three corresponding to field levels. In vitro exposures were also carried out using toad erythrocytes and Cr(VI) concentrations of 0.0015, 0.003, 0.015, 0.03, 0.15mg/L. Mortality, growth retardation, developmental delays and structural aberrations were noted in the metal-treated tadpoles, with increasing incidence corresponding to increase in Cr(VI) level and duration of exposure. Many of the sub-lethal effects were evident with long term exposure to environmentally relevant levels of the toxicant. Changes in selected blood parameters and erythrocyte morphometry were also detected in Cr(VI) exposed toads, indicating anaemic and leucopenic conditions. In the genotoxicity study, DNA damage indicated by comet assay and increased micronuclei frequency, occurred at the low Cr(VI) concentrations tested. The multiple deleterious effects of exposure to chromium signal the need for monitoring and controlling the discharge of chromium to the environment. The dose-dependency and genotoxic effects observed in this widely distributed Asian toad indicates its suitability for monitoring heavy metal pollution in aquatic systems.

MarR homologs with urate-binding signature.

Bacteria that associate with living hosts require intricate mechanisms to detect and respond to host defenses. Part of the early host defense against invading bacteria is the production of reactive oxygen species, and xanthine oxidase is one of the main producers of such agents. The end-product of the enzymatic activity of xanthine oxidase, urate, was previously shown to be the natural ligand for Deinococcus radiodurans-encoded HucR and it was shown to attenuate DNA binding by Agrobacterium tumefaciens-encoded PecS and Burkholderia thailandensis-encoded MftR, all members of the multiple antibiotic resistance regulator (MarR) family. We here show that residues involved in binding urate and eliciting the DNA binding antagonism are conserved in a specific subset of MarR homologs. Although HucR controls endogenous urate levels by regulating a uricase gene, almost all other homologs are predicted to respond to exogenous urate levels and to regulate a transmembrane transport protein belonging to either the drug metabolite transporter (DMT) or the major facilitator superfamily (MFS), as further evidenced by the presence of conserved binding sites for the cognate transcription factor within the respective promoter regions. These data suggest the use of orthologous genes for different regulatory purposes. We propose the designation UrtR (urate responsive transcriptional regulator) for this distinct subfamily of MarR homologs based on their common mechanism of urate-mediated attenuation of DNA binding.

Urate is a ligand for the transcriptional regulator PecS.

PecS is a member of the MarR (multiple antibiotic resistance regulator) family, which has been shown in Erwinia to regulate the expression of virulence genes. MarR homologs typically bind a small molecule ligand, resulting in attenuated DNA binding. For PecS, the natural ligand has not been identified. We have previously shown that urate is a ligand for the Deinococcus radiodurans-encoded MarR homolog HucR (hypothetical uricase regulator) and identified residues responsible for ligand binding. We show here that all four residues involved in urate binding and propagation of conformational changes to DNA recognition helices are conserved in PecS homologs, suggesting that urate is the ligand for PecS. Consistent with this prediction, Agrobacterium tumefaciens PecS specifically binds urate, and urate attenuates DNA binding in vitro. PecS binds two operator sites in the intergenic region between the divergent pecS gene and pecM genes, one of which features two partially overlapping repeats to which PecS binds as a dimer on opposite faces of the duplex. Notably, urate dissociates PecS from cognate DNA, allowing transcription of both genes in vivo. Taken together, our data show that urate is a ligand for PecS and suggest that urate serves a novel function in signaling the colonization of a host plant.

Molecular mechanisms of ligand-mediated attenuation of DNA binding by MarR family transcriptional regulators.

Bacteria and archaea encode members of the large multiple antibiotic resistance regulator (MarR) family of transcriptional regulators. Generally, MarR homologs regulate activity of genes involved in antibiotic resistance, stress responses, virulence or catabolism of aromatic compounds. They constitute a diverse group of transcriptional regulators that includes both repressors and activators, and the conventional mode of regulation entails a genetic locus in which the MarR homolog and a gene under its regulation are encoded divergently; binding of the MarR homolog to the intergenic region typically represses transcription of both genes, while binding of a specific ligand to the transcription factor results in attenuated DNA binding and hence activated gene expression. For many homologs, the natural ligand is unknown. Crystal structures reveal a common architecture with a characteristic winged helix domain for DNA binding, and recent structural information of homologs solved both in the absence and presence of their respective ligands, as well as biochemical data, is finally converging to illuminate the mechanisms by which ligand-binding causes attenuated DNA binding. As MarR homologs regulate pathways that are critical to bacterial physiology, including virulence, a molecular understanding of mechanisms by which ligands affect a regulation of gene activity is essential. Specifying the position of ligand-binding pockets further has the potential to aid in identifying the ligands for MarR homologs for which the ligand remains unknown.

Mechanism for attenuation of DNA binding by MarR family transcriptional regulators by small molecule ligands.

Members of the multiple antibiotic resistance regulator (MarR) family control gene expression in a variety of metabolic processes in bacteria and archaea. Hypothetical uricase regulator (HucR), which belongs to the ligand-responsive branch of the MarR family, regulates uricase expression in Deinococcus radiodurans by binding a shared promoter region between uricase and HucR genes. We show here that HucR responds only to urate and, to a lesser extent, to xanthine by attenuated DNA binding, compared to other intermediates of purine degradation. Using molecular-dynamics-guided mutational analysis, we identified the ligand-binding site in HucR. Electrophoretic mobility shift assays and intrinsic Trp fluorescence have identified W20 from the N-terminal helix and R80 from helix 3, which serves as a scaffold for the DNA recognition helix, as being essential for ligand binding. Using structural data combined with in silico and in vitro analyses, we propose a mechanism for the attenuation of DNA binding in which a conformational change initiated by charge repulsion due to a bound ligand propagates to DNA recognition helices. This mechanism may apply generally to MarR homologs that bind anionic phenolic ligands.