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Combretastatin A-4 (CA-4), is a natural compound with a potent tubulin polymerization and cell growth inhibitor properties. For these reasons CA-4 is one of the most potent anti-vascular agents that shows strong cytotoxicity against a variety of human cancer cells, including multi-drug-resistant cancer cell lines. In order to complete the knowledge of metabolic fate of CA-4, the in vitro and in vivo phase II metabolism was investigated. Both in incubation with rat and human liver S9 preparation in the presence of 39-phosphoadenosine-5´-phosphosulfate (PAPS) as a cofactor the formation of a previously no reported sulphate metabolite was demonstrated through liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) data and comparison with a synthetic reference sample. In incubation of CA-4 using rat and human liver microsomes, the formation of CA-4 glucuronide was observed and chromatographic and mass spectral properties of the metabolite were achieved and compared with those of a synthetic reference sample. Incubation of CA-4 with rat and human liver S9 preparation in the presence of uridine-5´-diphosphoglucuronic acid trisodium salt (UDPGA) and an β-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system as cofactors resulted in the formation of glucuronides arising from phase I CA-4 metabolites. When CA-4 was administered intraperitoneally to rat at a dose of 30 mg kg-1, both glucuronide and sulphate metabolites were observed in LC-ESI-MS-MS chromatograms and their mass spectral data were identical to those obtained from synthetic standards. [ABSTRACT FROM AUTHOR]

Boronated carbohydrate derivatives have good potential for targeting malignant cells in Boron Neutron Capture Therapy (BNCT) due to their preferential glucose uptake. In particular, with the introduction of the ammonium trifluoroborate moiety, boronated sugars can function as both BNCT agents and Positron Emission Tomography (PET) tracers. Their 18 F radiolabeling allows real-time tracking of biodistribution. This study evaluates the chemical, metabolic, and plasma stability of ammonium trifluoroborates for pharmaceutical purposes using LC-HRMS, presenting stability data under various conditions -acidic, basic, pseudophysiological, and oxidative- and highlighting degradation products and mechanisms. The data are supported by 1 H NMR and 19 F NMR. Metabolic and plasma stabilities, along with preliminary toxicological data (MTT assays), are also provided to better predict the clinical applicability of these compounds., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Panza Luigi reports financial support was provided by Italian Ministry of Foreign Affairs and International Cooperation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Here, we report DNA-based synthetic nanostructures decorated with enzymes (hereafter referred to as DNA-enzyme swimmers) that self-propel by converting the enzymatic substrate to the product in solution. The DNA-enzyme swimmers are obtained from tubular DNA structures that self-assemble spontaneously by the hybridization of DNA tiles. We functionalize these DNA structures with two different enzymes, urease and catalase, and show that they exhibit concentration-dependent movement and enhanced diffusion upon addition of the enzymatic substrate (i.e., urea and H 2 O 2 ). To demonstrate the programmability of such DNA-based swimmers, we also engineer DNA strands that displace the enzyme from the DNA scaffold, thus acting as molecular "brakes" on the DNA swimmers. These results serve as a first proof of principle for the development of synthetic DNA-based enzyme-powered swimmers that can self-propel in fluids.

In the last decade, the kynurenine pathway, which is the primary metabolic route for tryptophan (TRP) catabolism, has sparked great interest in the pharmaceutical sciences due to its role in immune regulation and cancer immunoediting. In this context, the development of cell-based assays might represent a tool to: i) characterize the cell secretome according to cell types; ii) gain more insight into the role of kynurenines in different disease scenarios; iii) screen hIDO1 (human indoleamine 2,3-dioxygenase) inhibitors and evaluate their effect on downstream TRP-catabolizing enzymes. This paper reports a validated Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method to simultaneously quantify TRP, L-kynurenine (KYN), xanthurenic acid (XA), 3-hydroxykynurenine (3OHKYN), kynurenic acid (KA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid (AA), 5-hydroxytryptamine (serotonin, 5HT) and tryptamine (TRYP) in Dulbecco's Modified Eagle and Eagle's Minimum Essential Media (DMEM and EMEM, respectively). The quantitative method was validated according to FDA, ICH and EMA guidelines, later applied: i) to assess the impact of selective inhibition of hIDO1 or hTDO (human tryptophan 2,3-dioxygenase) on the kynurenine pathway in A375 (melanoma), MDA-MB-231 (breast cancer), and U87 (glioblastoma) cell lines using multivariate analysis (MVA); ii) to determine the IC 50 values of both well-known (i.e., epacadostat, linrodostat) and the novel hIDO1 inhibitor (i.e., BL5) in the aforementioned cell lines. The proposed LC-MS/MS method is reliable and robust. Furthermore, it is highly versatile and suitable for applications in the preclinical drug research and in vitro assays., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Here we develop Lateral Flow Assays (LFAs) that employ as functional elements DNA-based structures decorated with reporter tags and recognition elements. We have rationally re-engineered tile-based DNA tubular structures that can act as scaffolds and can be decorated with recognition elements of different nature (i.e. antigens, aptamers or proteins) and with orthogonal fluorescent dyes. As a proof-of-principle we have developed sandwich and competitive multiplex lateral flow platforms for the detection of several targets, ranging from small molecules (digoxigenin, Dig and dinitrophenol, DNP), to antibodies (Anti-Dig, Anti-DNP and Anti-MUC1/EGFR bispecific antibodies) and proteins (thrombin). Coupling the advantages of functional DNA-based scaffolds together with the simplicity of LFAs, our approach offers the opportunity to detect a wide range of targets with nanomolar sensitivity and high specificity., (© 2023 Wiley-VCH GmbH.)

Inspired by naturally occurring regulatory mechanisms that allow complex temporal pulse features with programmable delays, we demonstrate here a strategy to achieve temporally programmed pulse output signals in DNA-based strand displacement reactions (SDRs). To achieve this, we rationally designed input strands that, once bound to their target duplex, can be gradually degraded, resulting in a pulse output signal. We also designed blocker strands that suppress strand displacement and determine the time at which the pulse reaction is generated. We show that by controlling the degradation rate of blocker and input strands, we can finely control the delayed pulse output over a range of 10 h. We also prove that it is possible to orthogonally delay two different pulse reactions in the same solution by taking advantage of the specificity of the degradation reactions for the input and blocker strands. Finally, we show here two possible applications of such delayed pulse SDRs: the time-programmed pulse decoration of DNA nanostructures and the sequentially appearing and self-erasing formation of DNA-based patterns.

We demonstrate here a strategy that allows the programmable and autonomous reorganization of self-assembled DNA polymers using redox chemistry. We have rationally designed different DNA monomers (tiles) that can co-assemble into tubular structures. The tiles can be orthogonally activated/deactivated with disulfide-linked DNA fuel strands that are degraded over time upon reduction because of the presence of a reducing agent in the system. The concentration of the disulfide fuels determines the activation kinetics of each DNA tile, which controls the degree of order/disorder in the formed co-polymer. The disulfide-reduction pathway can be employed together with enzymatic fuel-degradation pathways providing an additional level of control in the re-organization of DNA structures. Taking advantage of the different pH-sensitivities of disulfide-thiol and enzymatic reactions, we show that we can control the order in DNA-based co-polymers as a function of pH., (© 2023 Wiley-VCH GmbH.)

An approach to achieving dynamic and reversible decoration of DNA-based scaffolds is demonstrated here. To do this, rationally engineered DNA tiles containing enzyme-responsive strands covalently conjugated to different molecular labels are employed. These strands are designed to be recognized and degraded by specific enzymes (i.e., Ribonuclease H, RNase H, or Uracil DNA Glycosylase, UDG) inducing their spontaneous de-hybridization from the assembled tile and replacement by a new strand conjugated to a different label. Multiple enzyme-responsive strands that specifically respond to different enzymes allow for dynamic, orthogonal, and reversible decoration of the DNA structures. As a proof-of-principle of the strategy, the possibility to orthogonally control the distribution of different labels (i.e., fluorophores and small molecules) on the same scaffold without crosstalk is demonstrated. By doing so, DNA scaffolds that display different antibody recognition patterns are obtained. The approach offers the possibility to control the decoration of higher-order supramolecular assemblies (including origami) with several functional moieties to achieve functional biomaterials with improved adaptability, precision, and sensing capabilities., (© 2023 Wiley-VCH GmbH.)

The discovery of new bioactivities is closely related to the generation of novel scaffolds, and in the past few years different strategies have been proposed to obtain unknown architectures from the manipulation of known compounds. In the present study, we exploited a vintage photochemical approach for the discovery of an unexpected pathway of reactivity related to Δ 1 -3-oxo-pentacyclic triterpenic acids gaining access to a new class of natural-unnatural 5(10→1) abeo -pentacyclic triterpenic acids.

Control over synthetic DNA-based nanodevices can be achieved with a variety of physical and chemical stimuli. Actuation with light, however, is as advantageous as difficult to implement without modifying DNA strands with photo-switchable groups. Herein, we show that DNA nanodevices can be controlled using visible light in photo-switchable aqueous buffer solutions in a reversible and highly programmable fashion. The strategy presented here is non-invasive and allows the remote control with visible light of complex operations of DNA-based nanodevices such as the reversible release/loading of cargo molecules.

In recent years, 4-phenylbutyric acid (4-PBA), an FDA-approved drug, has increasingly been used as a nonspecific chemical chaperone in vitro and in vitro, but its pharmacodynamics is still not clear. In this context, we developed and validated a Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) method to quantify 4-PBA in NeuroBasal-A and Dulbecco's Modified Eagle widely used cell culture media. Samples were injected on a Luna ® 3 µm PFP(2) 100 Å (100 × 2.0 mm) column maintained at 40 °C. Water and methanol both with 0.1% formic acid served as mobile phases in a step gradient mode. The mass acquisition was performed by selected ion monitoring (SIM) in negative mode for a total run time of 10.5 min at a flow rate of 0.300 mL/min. The analogue 4-(4-Nitrophenyl)-Butyric Acid served as internal standard. Validation parameters were verified according to FDA and EMA guidelines. The quantification ranges from 0.38-24 µM. Inter and intraday RSDs (Relative Standard Deviations) were within 15%. The developed LC-HRMS method allowed the estimation of 4-PBA absorption and adsorption kinetics in vitro in two experimental systems: (i) 4-PBA improvement of protein synthesis in an Alzheimer's disease astrocytic cell model; and (ii) 4-PBA reduction of endoplasmic reticulum stress in thapsigargin-treated melanoma cell lines.

Electrochemical aptamer-based (EAB) sensors utilize the binding-induced conformational change of an electrode-attached, redox-reporter-modified aptamer to transduce target recognition into an easily measurable electrochemical output. Because this signal transduction mechanism is single-step and rapidly reversible, EAB sensors support high-frequency, real-time molecular measurements, and because it recapitulates the reagentless, conformation-linked signaling seen in vivo among naturally occurring receptors, EAB sensors are selective enough to work in the complex, time-varying environments found in the living body. The fabrication of EAB sensors, however, requires that their target-recognizing aptamer be modified such that (1) it undergoes the necessary binding-induced conformational change and (2) that the thermodynamics of this "conformational switch" are tuned to ensure that they reflect an acceptable trade-off between affinity and signal gain. That is, even if an "as-selected" aptamer achieves useful affinity and specificity, it may fail when adapted to the EAB platform because it lacks the binding-induced conformational change required to support EAB signaling. In this paper we reveal the spectroscopy-guided approaches we use to modify aptamers such that they support the necessary binding-induced conformational change. Specifically, using newly reported aptamers, we demonstrate the systematic design of EAB sensors achieving clinically and physiologically relevant specificity, limits of detection, and dynamic range against the targets methotrexate and tryptophan.