Exploration of ethylene glycol linked nitrofurantoin derivatives against Leishmania: Synthesis and in vitro activity.

Leishmaniasis is a neglected tropical disease that is caused by the Leishmania parasite. It is estimated that there are more than 350 million people at risk of infection annually. Current treatments that are in clinical use are expensive, have toxic side effects, and are facing parasitic resistance. Therefore, new drugs are urgently required. In the quest for new, safe, and cost-effective drugs, a series of novel ethylene glycol derivatives of nitrofurantoin was synthesised and the in vitro antileishmanial efficacy of the compounds tested against Leishmania donovani and Leishmania major strains. Arylated ethylene glycol derivatives were found to be the most potent, with submicromolar activity up to 294-fold greater than the parent compound nitrofurantoin. Analogues 2j and 2k had the best antipromastigote activities with submicromolar IC50 values against L. major IR-173 and antimonial-resistant L. donovani 9515 strains.

Exploring novel nitrofuranyl sulfonohydrazides as anti-Leishmania and anti-cancer agents: Synthesis, in vitro efficacy and hit identification.

Leishmaniasis and cancer are two deadly diseases that plague the human population. There are a limited number of drugs available for the treatment of these diseases; however, their overuse has resulted in pathogenic resistance. Recent studies have indicated the repurposing of nitro-containing compounds to be a new avenue into finding new treatments. In this study, new nitrofuranyl sulfonohydrazide derivatives were synthesized and evaluated for their in vitro antileishmanial and anticancer activities. The analogue 2h, featuring biphenyl moiety exhibited selective (SI > 10) submicromolar activity (IC50 0.97 µM) against acute promyelocytic leukemia cells hence was identified anticancer hit. This study revealed no antileishmanial hit. However, several promising analogues were uncovered and are worthy of further structural modifications to improve their toxicity and bioactivity profiles.

Chalcone-inspired rA1 /A2A adenosine receptor ligands: Ring closure as an alternative to a reactive substructure.

Over the past few years, great progress has been made in the development of high-affinity adenosine A1 and/or A2A receptor antagonists-promising agents for the potential treatment of Parkinson's disease. Unfortunately, many of these compounds raise structure-related concerns. The present study investigated the effect of ring closures on the rA1 /A2A affinity of compounds containing a highly reactive α,ß-unsaturated carbonyl system, hence providing insight into the potential of heterocycles to address these concerns. A total of 12 heterocyclic compounds were synthesised and evaluated in silico and in vitro. The test compounds performed well upon qualitative assessment of drug-likeness and were generally found to be free from potentially problematic fragments. Most also showed low/weak cytotoxicity. Results from radioligand binding experiments confirm that heterocycles (particularly 2-substituted 3-cyanopyridines) can replace the promiscuous α,ß-unsaturated ketone functional group without compromising A1 /A2A affinity. Structure-activity relationships highlighted the importance of hydrogen bonds in binding to the receptors of interest. Compounds 3c (rA1 Ki  = 16 nM; rA2A Ki  = 65 nM) and 8a (rA1 Ki  = 102 nM; rA2A Ki  = 37 nM), which both act as A1 antagonists, showed significant dual A1 /A2A affinity and may, therefore, inspire further investigation into heterocycles as potentially safe and potent adenosine receptor antagonists.

Synthesis and evaluation of methoxy substituted 2-benzoyl-1-benzofuran derivatives as lead compounds for the development adenosine A1 and/or A2A receptor antagonists.

A series of fourteen methoxy substituted 2-benzoyl-1-benzofuran derivatives were synthesised and their affinities determined for adenosine A1 and A2A receptors via radioligand binding assays to establish the structure activity relationships pertinent for A1 and A2A affinity. Compound 3j (6,7-dimethoxybenzofuran-2-yl)(3-methoxyphenyl)methanone exhibited A1 affinity (A1Ki (rat) = 6.880 µM) as well as A2A affinity (A2AKi (rat) = 0.5161 µM). Compounds 3a-b &3i-k exhibited selective affinity towards A1 with Ki values below 10 µM. The results indicate that C6,7-diOCH3 substitution on ring A in combination with meta (C3')-OCH3 substitution on ring B is beneficial for A1 and A2A affinity and activity. Compounds 3a-b &3j-k showed low cytotoxicity. Upon in vitro and in silico evaluation, compound 3j may be considered lead-like (i.e. a molecular entity suitable for optimization) and, thus, of value in the design of novel, potent and selective adenosine A1 and A2A receptor antagonists.

An update on derivatisation and repurposing of clinical nitrofuran drugs.

5-nitrofurans (NFs) have been in clinical use for over 60 years. These affordable drugs are used for the treatment of a broad spectrum of diseases ranging from urinary tract infections to cancer. The anti-pathogenic effect of clinical NFs occurs following a step-wise process involving activation by azoreduction, followed by nitroreduction catalysed by azoreductases and nitroreductases (NTRs), respectively. Azoreduction yields stable metabolites that have the ability to covalently bind to cellular proteins. Nitroreduction, on the other hand, occurs by type I or II reduction of the nitro group in the presence of parasitic NADPH-cytochrome P450 reductases. Type I NTRs catalyse, under anaerobic conditions, the reduction of NFs to produce anti-pathogenic hydroxylamine. Under aerobic conditions, nitroreduction catalysed by type II NTRs produces reactive oxygen and nitrogen species (ROS/RNS), causing oxidative stress to pathogens and ultimate death. This multi-activity nature of NFs thus allows the repurposing of these drugs from agricultural chemicals and basic antibiotics to efficient therapies against human life-threatening diseases. Cases of NF resistance in pathogens are also limited likely due to this multi-activity, as well as effectivity under both aerobic and anaerobic conditions. However, multi-activity of these drugs can also infer toxicity. Molecular derivatisation is an effective strategy to improve efficacy, lower toxicity, diversify activity and address pathogen resistance associated with the use of these drugs.

Sequence analysis of cell-free DNA derived from cultured human bone osteosarcoma (143B) cells.

The true importance of cell-free DNA in human biology, together with the potential scale of its clinical utility, is tarnished by a lack of understanding of its composition and origin. In investigating the cell-free DNA present in the growth medium of cultured 143B cells, we previously demonstrated that the majority of cell-free DNA is neither a product of apoptosis nor necrosis. In the present study, we investigated the composition and origin of this cell-free DNA population using next-generation sequencing. We found that the cell-free DNA comprises mainly of repetitive DNA, including α-satellite DNA, mini satellites, and transposons that are currently active or exhibit the capacity to become reactivated. A significant portion of these cell-free DNA fragments originates from specific chromosomes, especially chromosomes 1 and 9. In healthy adult somatic cells, the centromeric and pericentromeric regions of these chromosomes are normally densely methylated. However, in many cancer types, these regions are preferentially hypomethylated. This can lead to double-stranded DNA breaks or it can directly impair the formation of proper kinetochore structures. This type of chromosomal instability is a precursor to the formation of nuclear anomalies, including lagging chromosomes and anaphase bridges. DNA fragments derived from these structures can recruit their own nuclear envelope and form secondary nuclear structures known as micronuclei, which can localize to the nuclear periphery and bud out from the membrane. We postulate that the majority of cell-free DNA present in the growth medium of cultured 143B cells originates from these micronuclei.

The diverse origins of circulating cell-free DNA in the human body: a critical re-evaluation of the literature.

Since the detection of cell-free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non-invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle-induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno- or gene therapy. This review presents an extensive compilation of the putative origins of cfDNA and then contrasts the contributions of cellular breakdown processes with active mechanisms for the release of cfDNA into the extracellular environment. The involvement of cfDNA derived from both cellular breakdown and active release in lateral information transfer is also discussed. We hope to encourage researchers to adopt a more holistic view of cfDNA research, taking into account all the biological pathways in which cfDNA is involved, and to give serious consideration to the integration of in vitro and in vivo research. We also wish to encourage researchers not to limit their focus to the apoptotic or necrotic fraction of cfDNA, but to investigate the intercellular messaging capabilities of the actively released fraction of cfDNA and to study the role of cfDNA in pathogenesis.

Valproic acid alters the content and function of the cell-free DNA released by hepatocellular carcinoma (HepG2) cells in vitro.

BACKGROUND: It has long been believed that cell-free DNA (cfDNA) actively released into circulation can serve as intercellular messengers, and their involvement in processes such as the bystander effect strongly support this. However, this intercellular messaging function of cfDNA may have clinical implications that have not yet been considered. METHODS: CfDNA was isolated from the growth medium of HepG2 cells treated with valproic acid (VPA). This cfDNA was then administered to untreated cells and cellular metabolic activity was measured. RESULTS: VPA altered the characteristics of cfDNA released by treated HepG2 cells in vitro. When administered to untreated cells, the cfDNA from cells treated with VPA resulted in the dose-dependent induction of glycolytic activity within 36 min of administration, but little to no alterations in oxidative phosphorylation. The glycolytic activity lasted for 4-6 h, whereas changes in subsequent cfDNA release and characteristics were found to remain persistent after two 24 h treatments. Fragmented genomic DNA from VPA-treated cells did not induce the effects observed for cfDNA obtained VPA-treated cells. CONCLUSIONS: It is possible for cfDNA to, under in vitro conditions, transfer pharmaceutically-induced effects to untreated recipient cells. Further investigation regarding this occurrence under in vivo conditions is, therefore, strongly encouraged. GENERAL SIGNIFICANCE: The intercellular messaging functions of cfDNA present in donated biological fluids has potential clinical implications that require urgent attention. These implications may, however, also have potential as new forms of treatment that can circumvent pharmacological barriers.

Cell-free DNA in a three-dimensional spheroid cell culture model: A preliminary study.

BACKGROUND: Investigating the biological functions of cell-free DNA (cfDNA) is limited by the interference of vast numbers of putative sources and causes of DNA release into circulation. Utilization of three-dimensional (3D) spheroid cell cultures, models with characteristics closer to the in vivo state, may be of significant benefit for cfDNA research. METHODS: CfDNA was isolated from the growth medium of C3A spheroid cultures in rotating bioreactors during both normal growth and treatment with acetaminophen. Spheroid growth was monitored via planimetry, lactate dehydrogenase activity and glucose consumption and was related to isolated cfDNA characteristics. RESULTS: Changes in spheroid growth and stability were effectively mirrored by cfDNA characteristics. CfDNA characteristics correlated with that of previous two-dimensional (2D) cell culture and human plasma research. CONCLUSIONS: 3D spheroid cultures can serve as effective, simplified in vivo-simulating "closed-circuit" models since putative sources of cfDNA are limited to only the targeted cells. In addition, cfDNA can also serve as an alternative or auxiliary marker for tracking spheroid growth, development and culture stability. BIOLOGICAL SIGNIFICANCE: 3D cell cultures can be used to translate "closed-circuit" in vitro model research into data that is relevant for in vivo studies and clinical applications. In turn, the utilization of cfDNA during 3D culture research can optimize sample collection without affecting the stability of the growth environment. Combining 3D culture and cfDNA research could, therefore, optimize both research fields.

Kinetic analysis, size profiling, and bioenergetic association of DNA released by selected cell lines in vitro.

Although circulating DNA (cirDNA) analysis shows great promise as a screening tool for a wide range of pathologies, numerous stumbling blocks hinder the rapid translation of research to clinical practice. This is related directly to the inherent complexity of the in vivo setting, wherein the influence of complex systems of interconnected cellular responses and putative DNA sources creates a seemingly arbitrary representation of the quantitative and qualitative properties of the cirDNA in the blood of any individual. Therefore, to evaluate the potential of in vitro cell cultures to circumvent the difficulties encountered in in vivo investigations, the purpose of this work was to elucidate the characteristics of the DNA released [cell-free DNA (cfDNA)] by eight different cell lines. This revealed three different forms of cfDNA release patterns and the presence of nucleosomal fragments as well as actively released forms of DNA, which are not only consistently observed in every tested cell line, but also in plasma samples. Correlations between cfDNA release and cellular origin, growth rate, and cancer status were also investigated by screening and comparing bioenergetics flux parameters. These results show statistically significant correlations between cfDNA levels and glycolysis, while no correlations between cfDNA levels and oxidative phosphorylation were observed. Furthermore, several correlations between growth rate, cancer status, and dependency on aerobic glycolysis were observed. Cell cultures can, therefore, successfully serve as closed-circuit models to either replace or be used in conjunction with biofluid samples, which will enable sharper focus on specific cell types or DNA origins.

An Enquiry Concerning the Characteristics of Cell-Free DNA Released by Cultured Cancer Cells.

Non-invasive screening that utilizes cell-free DNA (cfDNA) offers remarkable potential as a method for the early detection of genetic disorders and a wide variety of cancers. Unfortunately, one of the most prominent elements delaying the translation of cfDNA analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the origin of cfDNA is complicated by the apparently arbitrary variability of quantitative and qualitative characteristics of cfDNA in the blood of healthy as well as diseased individuals. These factors may contribute to false positive/negative results when applied to clinical pathology. Although many have acknowledged that this is a major problem, few have addressed it. We believe that many of the current difficulties encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. The results obtained in this study indicate that the release of cfDNA from 143B cells is not a consequence of apoptosis, necrosis or a product of DNA replication, but primarily the result of actively released DNA, perhaps in association with a protein complex. Moreover, this study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA.

A Quantitative Assessment of Cell-Free DNA Utilizing Several Housekeeping Genes: Measurements from Four Different Cell Lines.

Quantitative real-time PCR (qPCR) is regularly used to quantify cell-free nucleic acids (cfNAs) in order to identify biomarkers for various pathologies. However, studies have shown notable housekeeping gene expression variation between healthy and diseased tissues and treated versus untreated cell lines. The release of housekeeping genes by four cell lines was investigated and the housekeeping gene expression between cfNAs and mRNA of the cell lines was observed in order to elucidate their relationship.

Methodological Variables in the Analysis of Cell-Free DNA.

In recent years, cell-free DNA (cfDNA) analysis has received increasing amounts of attention as a potential non-invasive screening tool for the early detection of genetic aberrations and a wide variety of diseases, especially cancer. However, except for some prenatal tests and BEAMing, a technique used to detect mutations in various genes of cancer patients, cfDNA analysis is not yet routinely applied in clinical practice. Although some confusing biological factors inherent to the in vivo setting play a key part, it is becoming increasingly clear that this struggle is mainly due to the lack of an analytical consensus, especially as regards quantitative analyses of cfDNA. In order to use quantitative analysis of cfDNA with confidence, process optimization and standardization are crucial. In this work we aim to elucidate the most confounding variables of each preanalytical step that must be considered for process optimization and equivalence of procedures.

Reference gene selection for in vitro cell-free DNA analysis and gene expression profiling.

OBJECTIVES: (i) To optimize cell-free DNA (cfDNA) and mRNA quantification using eight housekeeping genes (HKGs), (ii) to determine if there is a difference in the occurrence of HKGs in the cfDNA and mRNA of normal cells and cancer cells, and (iii) to investigate whether there is some selectivity involved in the release of cfDNA. DESIGN AND METHODS: cfDNA was isolated directly from the growth medium of 3 cultured cancer cell lines and one non-malignant, primary cell line. At the same time interval, mRNA was isolated from these cells and cDNA was synthesized. CfDNA and cDNA were then amplified with real-time PCR utilizing eight different HKGs. RESULTS: For all cell lines tested, Beta-actin (ACTB) is the most appropriate HKG to use as a control for cfDNA and mRNA quantification. There was no clear difference in the occurrence of HKGs between cancer cells and healthy cells. Lastly, there is a consistent and distinct difference between the mRNA expression and cfDNA of all cell lines. CONCLUSIONS: This study reveals a new candidate HKG for a robust control in cfDNA analysis and gene expression profiling, and should be considered for optimal analysis. Furthermore, results indicate that cfDNA is selectively released from cells into culture medium.

Adjustments to the preanalytical phase of quantitative cell-free DNA analysis.

Evaluating the kinetics of cell-free DNA (cfDNA) in the blood of cancer patients could be a strong auxiliary component to the molecular characterization of cfDNA, but its potential clinical significance is obscured by the absence of an analytical consensus. To utilize quantitative cfDNA assessment with confidence, it is crucial that the preanalytical phase is standardized. In a previous publication, several preanalytical variables that may affect quantitative measurements of cfDNA were identified, and the most confounding variables were assessed further using the growth medium of cultured cancer cells as a source of cfDNA ("Cell-free DNA: Preanalytical variables" [1]). The data accompanying this report relates to these experiments, which includes numerous changes to the sample handling and isolation protocols, and can be used for the interpretation of these results and other similar experiments by different researchers.

Characterization of the cell-free DNA released by cultured cancer cells.

The most prominent factor that delays the translation of cell-free DNA (cfDNA) analyses to clinical practice is the lack of knowledge regarding its origin and composition. The elucidation of the former is complicated by the seemingly random fluctuation of quantitative and qualitative characteristics of cfDNA in the blood of healthy and diseased individuals. Besides methodological discrepancies, this could be ascribed to a web of cellular responses to various environmental cues and stressors. Since all cells release cfDNA, it follows that the cfDNA in the blood of cancer patients is not only representative of tumor derived DNA, but also of DNA released by healthy cells under different conditions. Additionally, cfDNA released by malignant cells is not necessarily just aberrant, but likely includes non-mutated chromosomal DNA fragments. This may cause false positive/negative results. Although many have acknowledged that this is a major problem, few have addressed it. We propose that many of the current stumbling blocks encountered in in vivo cfDNA studies can be partially circumvented by in vitro models. Accordingly, the purpose of this work was to evaluate the release of cfDNA from cultured cells and to gauge its potential use for elucidating the nature of cfDNA. Results suggest that the occurrence of cfDNA is not a consequence of apoptosis or necrosis, but primarily a result of actively secreted DNA, perhaps in association with a protein complex. This study demonstrates the potential of in vitro cell culture models to obtain useful information about the phenomenon of cfDNA.

Cell-free DNA: Preanalytical variables.

Since the discovery of cell-free DNA (cfDNA) in human blood, most studies have focused on diagnostic and prognostic uses of these markers for solid tumors. Except for some prenatal tests and BEAMing, cfDNA analysis has not yet been translated to clinical practice and routine application appears distant. This can be attributed to overlapping factors: (i) a lack of knowledge regarding the origin and function of cfDNA, (ii) insufficient molecular characterization, and (iii) the absence of an analytical consensus. In this review, we address the latter determinant and focus specifically on quantitative analysis of cfDNA. While the literature reports limited value for a single quantitative assessment, cfDNA kinetic assessment will be an essential component to qualitative characterization. In order to establish quantitative analysis for accurate kinetic assessments, process optimization and standardization are crucial. This report elucidates the most confounding variables of each preanalytic step that must be considered for optimal analysis.