The Evaluation of Metal Co-ordinating Bis-Thiosemicarbazones as Potential Anti-malarial Agents.

BACKGROUND: The emergence of resistance to the artemisinins which are the current mainstays for antimalarial chemotheraphy has created an environment where the development of new drugs acting in a mechanistally discrete manner is a priority. OBJECTIVE: The goal of this work was to synthesize ane evaluate bis-thiosemicarbazones as potential antimalarial agents. METHODS: Fifteen compounds were generated using two condensation protocols and evaluated in vitro against the NF54 (CQ sensitive) strain of Plasmodium falciparum. A preliminary assessment of the potential for human toxicity was conducted in vitro against the MRC5 human lung fibroblast line. RESULTS: The activity of the bis-thiosemicarbazones was highly dependent on the nature of the arene at the core of the structure. The inclusion of a non-coordinating benzene core resulted in inactive compounds, while the inclusion of a pyridyl core resulted in compounds of moderate or potent antimalarial activity (4 compounds showing IC50 < 250 nM). CONCLUSION: Bis-thiosemicarbazones containing a central pyridyl core display potent antimalarial activity in vitro. Sequestration and activation of ferric iron appears to play a significant role in this activity. Ongoing studies are aimed at further development of this series as potential antimalarials.

Cytotoxic activity of expanded coordination bis-thiosemicarbazones and copper complexes thereof.

A series of bis-thiosemicarbazone agents with coordinating groups capable of multiple metal coordination modes has been generated and evaluated for potential cytotoxic effects against melanoma (MelRm) and breast adenocarcinoma (MCF-7) cell lines. The bis-thiosemicarbazones in this study generally demonstrated superior cytotoxic activity against MelRm than MCF-7 in the absence of metal ion supplementation, but in most cases could not be considered superior to the reference thiosemicarbazone Dp44mT. The key structural features for the cytotoxic activity were the central metal binding atom on the aromatic core, the thiocarbonyl residue and the nature of substitution on the N4-terminus in terms of size and lipophilicity. The cytotoxicity of bis-thiosemicarbazone ligands improved significantly with Cu(II) supplementation, particularly against MCF-7 cells. The mechanism of cytotoxicity of bis-thiosemicarbazones was proposed to be dependent on the combined effect of metal mobilisation and ROS generation which is so called a "double-punch effect".

Investigation of the cytotoxic implications of metal chelators against melanoma and approaches to improve the cytotoxicity profiles of metal coordinating agents.

The cytotoxic activity of thiosemicarbazones (TSC) and thiocarbohydrazones was investigated against the MelRm melanoma cell line. In general, the melanoma line was susceptible to metal coordinating agents, the most useful of which incorporated the dipyridyl ketone hydrazone sub-structure. The impact of copper supplementation on the cytotoxic activity towards the melanoma line (MelRm) of metal coordinating agents when acting as ionophores is less predictable than the general improvement that has been seen in other cancer cells such as breast adenocarcinoma (MCF-7). The bimetallic nature of thiocarbohydrazone complexes with resultant loss of lipophilicity is a limiting factor in usage against MelRm. The cytotoxic activity of TSC against MelRm when used as copper ionophores could be markedly improved through combination with a partner drug capable of disrupting cellular defences to oxidative stress. In the absence of copper supplementation, both TSC and thiocarbohydrazones could be used to initiate cell cycle arrest and this could be employed to improve cytotoxicity profiles of other metallodrugs such as cisplatin.

Increased generation of intracellular reactive oxygen species initiates selective cytotoxicity against the MCF-7 cell line resultant from redox active combination therapy using copper-thiosemicarbazone complexes.

The combination of cytotoxic copper-thiosemicarbazone complexes with phenoxazines results in an up to 50-fold enhancement in the cytotoxic potential of the thiosemicarbazone against the MCF-7 human breast adenocarcinoma cell line over the effect attributable to drug additivity-allowing minimization of the more toxic copper-thiosemicarbazone component of the therapy. The combination of a benzophenoxazine with all classes of copper complex examined in this study proved more effective than combinations of the copper complexes with related isoelectronic azines. The combination approach results in rapid elevation of intracellular reactive oxygen levels followed by apoptotic cell death. Normal fibroblasts representative of non-cancerous cells (MRC-5) did not display a similar elevation of reactive oxygen levels when exposed to similar drug levels. The minimization of the copper-thiosemicarbazone component of the therapy results in an enhanced safety profile against normal fibroblasts.

Improved cytotoxicity of pyridyl-substituted thiosemicarbazones against MCF-7 when used as metal ionophores.

Zinc is the second most abundant transition metal in the human body, between 3 and 10% of human genes encoding for zinc binding proteins. We have investigated the interplay of reactive oxygen species and zinc homeostasis on the cytotoxicity of the thiosemicarbazone chelators against the MCF-7 cell line. The cytotoxicity of thiosemicarbazone chelators against MCF-7 can be improved through supplementation of ionic zinc provided the zinc ion is at a level exceeding the thiosemicarbazone concentration. Elimination of the entire cell population can be accomplished with this regime, unlike the plateau of cytotoxicity observed on thiosemicarbazone monotherapy. The cytotoxic effects of copper complexes of the thiosemicarbazone are not enhanced by zinc supplementation, displacement of copper from the complex being disfavoured. Treatment of MCF-7 with uncomplexed thiosemicarbazone initiates post G1 blockade alongside the induction of apoptosis, cell death being abrogated through subsequent supplementation with zinc ion after drug removal. This would implicate a metal depletion mechanism in the cytotoxic effect of the un-coordinated thiosemicarbazone. The metal complexes of the species, however, fail to initiate similar G1 blockade and apparently exert their cytotoxic effect through generation of reactive oxygen species, suggesting that multiple mechanisms of cytotoxicity can be associated with the thiosemicarbazones dependant on the level of metal ion association.

Selective induction of oxidative stress in cancer cells via synergistic combinations of agents targeting redox homeostasis.

Cancer cell resistance to chemotherapy is still a heavy burden that impairs the response of many cancer patients to conventional chemotherapy. Using drug combinations is one therapeutic approach to overcome the developing resistance to any one drug. Oxidative stress is now a generally regarded hallmark of cancer that can be one approach to selectively target cancer cells while sparing normal cells. With the aim of increasing oxidative stress in cancer cells to a lethal set point, we have generated and combined several series of redox active compounds that act at different points of the cellular oxidative cascade. The premise of such combinations is to deplete of endogenous antioxidant defence proteins (e.g., Glutathione) while concomitantly increasing the generation of ROS via metal redox recycling and Fenton chemistry which eventually leads to the disruption of cellular redox homeostasis and induction of cell death. Through this approach, we have identified highly synergistic combinations of two distinctive classes of compounds (Azines and Copper(II) complexes of 2-pyridyl ketone thiosemicarbazones) which are capable of eliminating cancer cells without concomitant increase in toxicity toward normal cells. In one of our most potent combinations, a combination index (CI) value of 0.056 was observed, representing a 17 fold enhancement in activity beyond additive effects. Such new combination regimen of redox active compounds can be one step closer to potentially safer low dose chemotherapy.

Escherichia coli sequence type 131 as a prominent cause of antibiotic resistance among urinary Escherichia coli isolates from reproductive-age women.

The recent emergence of multidrug-resistant Escherichia coli sequence type 131 (ST131) has coincided with an increase in general antibiotic resistance of E. coli, suggesting that ST131 has a contributing role in resistance. However, there is little information about the contribution of ST131 to different clinical syndromes or the basis for its impressive emergence and epidemic spread. To investigate this, we studied 953 E. coli isolates from women of reproductive age in the central west region of New South Wales, Australia, including 623 urinary isolates from patients with cystitis (cystitis isolates) (n = 322) or pyelonephritis (pyelonephritis isolates) (n = 301) and 330 fecal isolates from healthy controls. The characteristics studied included ST131 clonal group status, resistance to different antibiotics, presence of virulence factor (VF) genes, and biofilm production. As expected, fecal isolates differed significantly from urinary (cystitis and pyelonephritis) isolates in most of the studied characteristics. Antibiotic resistance was significantly more common in ST131 than in non-ST131 isolates. Both antibiotic resistance and ST131 were more common in pyelonephritis than cystitis isolates and least so among fecal isolates. Within each source group, individual VF genes were more prevalent and VF scores were higher for ST131 than for non-ST131 isolates. For ST131 only, the prevalences of most individual VF genes and VF scores were the lowest in the fecal isolates, higher in the cystitis isolates, and highest in the pyelonephritis isolates. Biofilm production was strongly associated with ST131 status and antibiotic resistance. These results clarify the distribution of the ST131 clonal group and its epidemiological associations in our region and suggest that it exhibits both enhanced virulence and increased antibiotic resistance compared with those of other urinary tract infection (UTI) and fecal E. coli isolates from women of reproductive age.

Genotypic and phenotypic characterization of Escherichia coli isolates from children with urinary tract infection and from healthy carriers.

BACKGROUND: Urinary tract infections (UTI) are common in children and contribute significantly to morbidity and mortality. The major cause is Escherichia coli, carrying multiple virulence-associated factors (VFs). However, the specific traits that distinguish childhood uropathogenic E. coli from fecal commensals of healthy children are incompletely defined. METHODS: We used a multiplex polymerase chain reaction-based reverse line blot assay, several additional polymerase chain reactions and phenotypic methods to compare distributions of virulence traits (22 VF genes, UTI-associated O types, phylogenetic groups, sequence type 131 and expression of selected VFs), between 212 E. coli isolates from children ≤ 5 years with UTI (109 cystitis and 103 pyelonephritis) and 115 fecal isolates from healthy children of similar age, collected during the same time period. RESULTS: The studied traits were most prevalent among pyelonephritis, followed closely by cystitis isolates and were uncommon among fecal isolates. Eight VF genes differentiated pyelonephritis from cystitis isolates, but aggregate VF scores in these 2 UTI groups were similar. Most of the studied phenotypic characteristics showed a similar descending prevalence gradient from pyelonephritis, through cystitis, to fecal isolates. Coexpression of biofilm components, curli and cellulose, was strongly associated with pyelonephritis, phylogenetic group B2, individual VF genes and higher VF scores. Two-thirds (67%) of clinical isolates belonged to phylogenetic group B2 and, of these, 12% belonged to the sequence type 131 clonal group, compared with 14% and 1%, respectively, of fecal isolates. CONCLUSIONS: These findings identify specific virulence factors, O types and a virulent clonal group (sequence type 131), as potential targets for UTI prevention strategies in children.

Multiplex PCR-based reverse line blot assay for simultaneous detection of 22 virulence genes in uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are among the most common bacterial infections and are responsible for significant morbidity and health care costs worldwide. The main bacterial cause of uncomplicated UTI is Escherichia coli, which possesses numerous virulence factors (VFs). Many studies of the pathogenesis of E. coli UTI have centered on VF genes. Hence, the development of better molecular assays to study VF genes would facilitate these studies. We developed a highly sensitive and specific multiplex PCR-based reverse line blot (mPCR/RLB) assay to simultaneously detect 22 VF genes of uropathogenic E. coli and then used it to characterize 180 isolates from nonpregnant women of child-bearing age with cystitis and 153 fecal isolates from similar-age healthy women, in regional New South Wales, Australia. The assay accurately identified all VF genes (of the 22 under study) known to be present in 30 previously characterized control strains. The detection limits were 28 ng of DNA from E. coli isolates and 50 CFU/ml in mock-infected urine specimens containing known concentrations of E. coli. Cystitis isolates (compared to the fecal isolates) showed a significantly higher prevalence of 18 individual VF genes and contained significantly more VF genes per isolate (median number, 18.5 versus 6.5 [P = 0.001]). Discordance between paired probes for a given VF gene occurred in several clinical test isolates but no reference strains and among the test isolates was associated with fecal source (10% of VF genes versus 2% for cystitis isolates [P < 0.001]). This novel mPCR/RLB method is a potentially powerful tool for investigating the prevalence and distribution of VFs in E. coli.

RET-mediated gene expression pattern is affected by isoform but not oncogenic mutation.

The inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2) is caused by mutations of the RET receptor tyrosine kinase and is characterized by medullary thyroid carcinoma. MEN 2 subtypes have distinct mutational spectrums and vary in severity. The most severe disease subtype, MEN 2B, is associated with a specific RET mutation (M918T) that has been predicted to alter downstream signaling and target gene expression patterns. We used gene expression microarray analysis to identify target genes modulated by RET. We compared two oncogenic RET mutants, associated with MEN 2A (2ARET) or MEN 2B (2BRET) disease subtypes, that are predicted to have distinct downstream target genes. We showed that overall, 2ARET and 2BRET modulated genes with similar functional ontologies. Further, when we validated our microarray data by quantitative real time PCR, we did not detect major differences in gene expression associated with these mutants when differences in receptor activity levels were considered. We did, however, detect differences in gene expression induced by two RET COOH-terminal isoforms, RET9 and RET51, irrespective of the RET form present (wildtype, 2ARET, or 2BRET). Our data suggest that similar transcriptional programs contribute to all forms of MEN 2 but that differences in target gene expression may contribute to developmental pattern differences observed between RET isoforms.

A model for GFR alpha 4 function and a potential modifying role in multiple endocrine neoplasia 2.

Mutations of the RET proto-oncogene are found in the majority of patients with the inherited cancer syndrome multiple endocrine neoplasia type 2 (MEN 2). A minority of cases, however, have no detectable RET mutation and there is considerable phenotypic variation within and among MEN 2 families with the same RET mutation, suggesting a role for other loci in this disease. A candidate for such a gene is glial cell line-derived neurotrophic factor receptor alpha 4 (GFRA4), which encodes a cell surface-bound co-receptor (GFR alpha 4) required for interaction of RET with its ligand persephin. The GFRA4 gene has multiple alternative splices leading to three distinct protein isoforms that are prominently expressed in thyroid. We postulated that mutations of GFRA4 contribute to MEN 2 in the absence of RET mutations or modify the RET mutation phenotype. We screened patients with MEN 2 or MEN 2-like phenotypes, with and without RET mutations, for variants of GFRA4. We identified 10 variants, one of which was over represented in, and two of which were found exclusively in, our patient populations. One of these was a single-base substitution upstream of the GFR alpha 4 coding region, where it may alter gene expression. The second was a 7 bp insertion, which results in a change in reading frame for all three GFR alpha 4 isoforms. This would cause a relative shift in membrane bound and soluble forms of GFR alpha 4, which would significantly alter the formation of RET signalling complexes. Our data suggest a model of wild-type GFR alpha 4 isoform expression that includes both activating and inhibiting co-receptors for RET.

Transcriptional repression of the RET proto-oncogene by a mitogen activated protein kinase-dependent signalling pathway.

Transcription factors play important roles in regulating cell growth and differentiation. In this study, treatment of the MTC cell line, TT, with phorbol 12-myristate 13-acetate (PMA) was shown to reduce neurite outgrowth which may be associated with de-differentiation and loss of the transformed phenotype. Northern blotting revealed that PMA transiently induced early growth response gene 1 (Egr-1) expression and decreased RET expression. Transient transfection analyses using 5'-deletion constructs of the basal RET promoter, demonstrated the requirement of a region between -70 and -33 bp for PMA-inducible expression. Gel shift and supershift studies demonstrated that PMA induced Egr-1 formed part of a complex capable of binding to the RET minimal promoter. Overexpression of Egr-1 displaced both sephacryl and phosphocellulose protein 1 (Sp1) and Sp3 from a GC-box element previously found to be important for RET basal expression. Furthermore, use of a raf-1 inducible TT cell line, that has been previously shown to downregulate RET expression, revealed that this downregulation may be linked to the induction of Egr-1. Our data suggest that regulation of RET expression during development and in medullary thyroid carcinoma may be determined, at least in part, by this complex of Sp and Egr-1 proteins.