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The synthesis of phosphorous indenoquinolines and their biological evaluation as topoisomerase 1 (TOP1) inhibitors and antiproliferative agents were performed. First, the preparation of new hybrid 5 H -indeno[2,1- c ]quinolines with a phosphine oxide group was performed by a two-step Povarov-type [4+2]-cycloaddition reaction between the corresponding phosphorated aldimines with indene in the presence of BF 3 ·Et 2 O. Subsequent oxidation of the methylene present in the structure resulted in the corresponding indeno[2,1- c ]quinolin-7-one phosphine oxides 10 . The synthesized derivatives were evaluated as TOP1 inhibitors showing higher inhibition values than CPT at prolonged incubation times (5 min). Inhibition of TOP1 was even observed after 30 min of incubation. The cytotoxic activities of these compounds were also studied against different cancer cell lines and a non-cancerous cell line. While some compounds showed cytotoxicity against some cancerous cells, none of the compounds showed any cytotoxicity against the non-cancerous cell line, MRC-5, in contrast to CPT, which exhibits high toxicity against this cell line. These results represent a very interesting advance since the heterocyclic phosphine oxide derivatives have important properties as TOP1 inhibitors and show an interesting cytotoxicity against different cell lines.

Malaria poses a serious global health problem, with half the world population being at risk. Regular screening is crucial for breaking the transmission cycle and combatting the disease spreading. However, current diagnostic tools relying on blood samples face challenges in many malaria-epidemic areas. In the present study, we demonstrate the detection of the malaria-causing Plasmodium parasite in non-invasive saliva samples (N = 61) from infected individuals by combining a DNA-based Rolling-circle-Enhanced-Enzyme-Activity-Detection (REEAD) sensor system with a chemiluminescence readout that could be detected with an in-house-developed affordable and battery-powered portable reader. We successfully transferred the technology to sub-Saharan Africa, where the malaria burden is high, and demonstrated a proof of concept in a small study (N = 40) showing significant differences ( p < 0.00001) between malaria-positive individuals (N = 33) and presumed asymptomatic negative individuals (N = 7) all collected in Gabon. This is the first successful application of the REEAD sensor system for the detection of malaria in saliva in a high-epidemic area and holds promise for the potential future use of REEAD for malaria diagnosis or surveillance based on non-invasive specimens in sub-Saharan Africa.

With the increasing need for effective compounds against cancer or pathogen-borne diseases, the development of new tools to investigate the enzymatic activity of biomarkers is necessary. Among these biomarkers are DNA topoisomerases, which are key enzymes that modify DNA and regulate DNA topology during cellular processes. Over the years, libraries of natural and synthetic small-molecule compounds have been extensively investigated as potential anti-cancer, anti-bacterial, or anti-parasitic drugs targeting topoisomerases. However, the current tools for measuring the potential inhibition of topoisomerase activity are time consuming and not easily adaptable outside specialized laboratories. Here, we present rolling circle amplification-based methods that provide fast and easy readouts for screening of compounds against type 1 topoisomerases. Specific assays for the investigation of the potential inhibition of eukaryotic, viral, or bacterial type 1 topoisomerase activity were developed, using human topoisomerase 1, Leishmania donovani topoisomerase 1, monkeypox virus topoisomerase 1, and Mycobacterium smegmatis topoisomerase 1 as model enzymes. The presented tools proved to be sensitive and directly quantitative, paving the way for new diagnostic and drug screening protocols in research and clinical settings.

Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.

The murine cell line GRX has been introduced as an experimental tool to study aspects of hepatic stellate cell biology. It was established from livers of C3H/HeN mice that were infected with cercariae of Schistosoma mansoni . Although these cells display a myofibroblast phenotype, they can accumulate intracellular lipids and acquire a fat-storing lipocyte phenotype when treated with retinol, insulin, and indomethacin. We have performed genetic characterization of GRX and established a multi-loci short tandem repeat (STR) signature for this cell line that includes 18 mouse STR markers. Karyotyping further revealed that this cell line has a complex genotype with various chromosomal aberrations. Transmission electron microscopy revealed that GRX cells produce large quantities of viral particles belonging to the gammaretroviral genus of the Retroviridae family as assessed by next generation mRNA sequencing and Western blot analysis. Rolling-circle-enhanced-enzyme-activity detection (REEAD) revealed the absence of retroviral integrase activity in cell culture supernatants, most likely as a result of tetherin-mediated trapping of viral particles at the cell surface. Furthermore, staining against schistosome gut-associated circulating anodic antigens and cercarial O- and GSL-glycans showed that the cell line lacks S. mansoni -specific glycostructures. Our findings will now help to fulfill the recommendations for cellular authentications required by many granting agencies and scientific journals when working with GRX cells. Moreover, the definition of a characteristic STR profile will increase the value of GRX cells in research and provides an important benchmark to identify intra-laboratory cell line heterogeneity, discriminate between different mouse cell lines, and to avoid misinterpretation of experimental findings by usage of misidentified or cross-contaminated cells.

Background : Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods : Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results : We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion : REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

This work describes, for the first time, the synthesis of dialkyl (2-arylquinolin-8-yl)phosphonate derivatives. The preparation was carried out through a direct and simple process as a multicomponent Povarov reaction of aminophenylphosphonates, aldehydes, and styrenes and subsequent oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or, alternatively, by a cycloaddition reaction between phosphonate aldimines and acetylenes. Based on phosphonate group structural characteristics, considered as phosphorous isosteres of carboxylic heterocycles, they may present interesting biological properties related to cell proliferation. In the current report, a new series of dialkyl (2-arylquinolin-8-yl)phosphonates have been synthesized and their antiproliferative effect evaluated on different human cancer and embryonic cells, as well as on Leishmania infantum parasites, a eukaryotic protist responsible for visceral leishmaniasis. Thereby, the antitumor effect was assessed in human lung adenocarcinoma cells (A549), human ovarian carcinoma cells (SKOV3), and human embryonic kidney cells (HEK293) versus the non-cancerous lung fibroblasts cell line (MRC5). On the other hand, the antileishmanial activity was tested against both stages of L. infantum cell cycle, namely free-living promastigotes and intramacrophage amastigotes, using a primary culture of Balb/c splenocytes to calculate the selectivity index. Besides the antiproliferative and antileishmanial capacities, their behavior as topoisomerase 1B inhibitors has been evaluated as a possible mechanism of action.

DNA sensors can be used as robust tools for high-throughput drug screening of small molecules with the potential to inhibit specific enzymes. As enzymes work in complex biological pathways, it is important to screen for both desired and undesired inhibitory effects. We here report a screening system utilizing specific sensors for tyrosyl-DNA phosphodiesterase 1 (TDP1) and topoisomerase 1 (TOP1) activity to screen in vitro for drugs inhibiting TDP1 without affecting TOP1. As the main function of TDP1 is repair of TOP1 cleavage-induced DNA damage, inhibition of TOP1 cleavage could thus reduce the biological effect of the TDP1 drugs. We identified three new drug candidates of the 1,5-naphthyridine and 1,2,3,4-tetrahydroquinolinylphosphine sulfide families. All three TDP1 inhibitors had no effect on TOP1 activity and acted synergistically with the TOP1 poison SN-38 to increase the amount of TOP1 cleavage-induced DNA damage. Further, they promoted cell death even with low dose SN-38, thereby establishing two new classes of TDP1 inhibitors with clinical potential. Thus, we here report a dual-sensor screening approach for in vitro selection of TDP1 drugs and three new TDP1 drug candidates that act synergistically with TOP1 poisons.

The heterogeneity of tumor cells and the potential existence of rare cells with reduced chemotherapeutic response is expected to play a pivotal role in the development of drug resistant cancers. Herein, we utilized the colon cancer cell lines, Caco2 and DLD1, to investigate heterogeneity of topoisomerase 1 (TOP1) activity in different cell subpopulations, and the consequences for the chemotherapeutic response towards the TOP1 targeting drug, camptothecin. The cell lines consisted of two subpopulations: one (the stem-cell-like cells) divided asymmetrically, was camptothecin resistant, had a differently phosphorylated TOP1 and a lower Casein Kinase II (CKII) activity than the camptothecin sensitive non-stem-cell-like cells. The tumor suppressor p14ARF had a different effect in the two cell subpopulations. In the stem-cell-like cells, p14ARF suppressed TOP1 activity and downregulation of this factor increased the sensitivity towards camptothecin. It had the opposite effect in non-stem-cell-like cells. Since it is only the stem-cell-like cells that have tumorigenic activity our results point towards new considerations for future cancer therapy. Moreover, the data underscore the importance of considering cell-to-cell variations in the analysis of molecular processes in cell lines.

With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution.

The so-called Rolling Circle Amplification allows for amplification of circular DNA structures in a manner that can be detected in real-time using nucleotide-based molecular beacons that unfold upon recognition of the DNA product, which is being produced during the amplification process. The unfolding of the molecular beacons results in a fluorescence increase as the Rolling Circle Amplification proceeds. This can be measured in a fluorometer. In the current study, we have investigated the possibility of using two different molecular beacons to detect two distinct Rolling Circle Amplification reactions proceeding simultaneously and in the same reaction tube by measurement of fluorescence over time. We demonstrate the application of this fluorometric readout method, for automated and specific detection of the activity of the type IB topoisomerase from the malaria parasite Plasmodium falciparum in the presence of human cell extract containing the related topoisomerase I from humans. The obtained results point towards a future use of the presented assay setup for malaria diagnostics or drug screening purposes. In longer terms the method may be applied more broadly for real-time sensing of various Rolling Circle Amplification reactions., Competing Interests: The authors declare no conflict of interest.

Human topoisomerase I (hTopI) is an essential cellular enzyme. The enzyme is often upregulated in cancer cells, and it is a target for chemotherapeutic drugs of the camptothecin (CPT) family. Response to CPT-based treatment is dependent on hTopI activity, and reduction in activity, and mutations in hTopI have been reported to result in CPT resistance. Therefore, hTOPI gene copy number, mRNA level, protein amount, and enzyme activity have been studied to explain differences in cellular response to CPT. We show that Rolling Circle Enhanced Enzyme Activity Detection (REEAD), allowing measurement of hTopI cleavage-religation activity at the single molecule level, may be used to detect posttranslational enzymatic differences influencing CPT response. These differences cannot be detected by analysis of hTopI gene copy number, mRNA amount, or protein amount, and only become apparent upon measuring the activity of hTopI in the presence of CPT. Furthermore, we detected differences in the activity of the repair enzyme tyrosyl-DNA phosphodiesterase 1, which is involved in repair of hTopI-induced DNA damage. Since increased TDP1 activity can reduce cellular CPT sensitivity we suggest that a combined measurement of TDP1 activity and hTopI activity in presence of CPT will be the best determinant for CPT response.

Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I. The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family. The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented DNA sensor, suggesting a potential application of the sensor for first line screening for potential topoisomerase I targeting anti-cancer drugs.