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The expansion of drug resistant parasites sheds a serious concern on several neglected parasitic diseases. Our recent results on cancer led us to envision the use of peptide-alkoxyamines as a highly selective and efficient new drug against schistosome adult worms, the etiological agents of schistosomiasis. Indeed, the peptide tag of the hybrid compounds can be hydrolyzed by worm's digestive enzymes to afford a highly labile alkoxyamine which homolyzes spontaneously and instantaneously into radicals-which are then used as a drug against Schistosome adult parasites. This approach is nicely summarized as digging their graves with their forks. Several hybrid peptide-alkoxyamines were prepared and clearly showed an activity: two of the tested compounds kill 50% of the parasites in two hours at a concentration of 100 µg/mL. Importantly, the peptide and alkoxyamine fragments that are unable to generate alkyl radicals display no activity. This strong evidence validates the proposed mechanism: a specific activation of the prodrugs by the parasite proteases leading to parasite death through in situ alkyl radical generation.
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The emergence and spread of drug-resistant Plasmodium falciparum parasites shed a serious concern on the worldwide control of malaria, the most important tropical disease in terms of mortality and morbidity. This situation has led us to consider the use of peptide-alkoxyamine derivatives as new antiplasmodial prodrugs that could potentially be efficient in the fight against resistant malaria parasites. Indeed, the peptide tag of the prodrug has been designed to be hydrolysed by parasite digestive proteases to afford highly labile alkoxyamines drugs, which spontaneously and instantaneously homolyse into two free radicals, one of which is expected to be active against P. falciparum. Since the parasite enzymes should trigger the production of the active drug in the parasite's food vacuoles, our approach is summarized as "to dig its grave with its fork". However, despite promising sub-micromolar IC50 values in the classical chemosensitivity assay, more in-depth tests evidenced that the anti-parasite activity of these compounds could be due to their cytostatic activity rather than a truly anti-parasitic profile, demonstrating that the antiplasmodial activity cannot be based only on measuring antiproliferative activity. It is therefore imperative to distinguish, with appropriate tests, a genuinely parasiticidal activity from a cytostatic activity.
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Antimaláricos , Citostáticos , Malária Falciparum , Malária , Humanos , Antimaláricos/química , Citostáticos/uso terapêutico , Malária/tratamento farmacológico , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum , Peptídeos/farmacologia , Peptídeos/uso terapêuticoRESUMO
Development of very-low field MRI is an active area of research. It aims at reducing operating costs and improve portability. However, the signal-to-noise issue becomes prominent at ultra-low field (<1 mT), especially for molecular imaging purposes that addresses specific biochemical events. In the context of preclinical molecular MRI of abnormal proteolysis the paper describes a MRI system able to produce Overhauser-enhanced MR images in living rats through in situ Dynamic Nuclear Polarization at 206 µT using stable and non-toxic nitroxides. In parallel conventional images are generated at 206 µT following pre-polarization at 20 mT. Results show that nitroxides are visualized in 3D within a few minutes in the lungs, kidneys and bladder post-administration. This system will be used for molecular imaging of inflammation using protease-specific nitroxide probes.
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Pulmão , Imageamento por Ressonância Magnética , Ratos , Animais , Espectroscopia de Ressonância Magnética , Imageamento por Ressonância Magnética/métodos , Óxidos de Nitrogênio/químicaRESUMO
The design of smart nanoplatforms presenting well-definite structures able to achieve controlled cascade action remotely triggered by external stimuli presents a great challenge. We report here a new nanosystem consisting of magnetic iron oxide nanoparticles covalently grafted with a thermosensitive radical initiator alkoxyamine, able to provide controlled and localized release of free radicals triggered by an alternating current (ac) magnetic field. These nanoparticles exhibit a high intrinsic loss power of 4.73 nHm2 kg-1 providing rapid heating of their surface under the action of an ac field, inducing the homolysis of alkoxyamine C-ON bond and then the oxygen-independent formation of radicals. This latter was demonstrated by electronic paramagnetic resonance spectroscopy, and the kinetics of homolysis has been investigated allowing a comparison of the temperature of alkoxyamine's homolysis with the one measured during the magnetothermia process.
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Current chemotherapies suffer low specificity and sometimes drug resistance. Neutrophil elastase activity in cancer is associated with poor prognosis and metastasis settlement. More generally, tumors harbor various and persistent protease activities unseen in healthy tissues. In an attempt to be more specific, we designed prodrugs that are activatable by neutrophil elastase. Upon activation, these alkoxyamine-based drugs release cytotoxic alkyl radicals that act randomly to prevent drug resistance. As a result, U87 glioblastoma cells displayed high level caspase 3/7 activation during the first hour of exposure in the presence of human neutrophil elastase and the prodrug in vitro. The apoptosis process and cell death occurred between 24 and 48 h after exposure with a half lethal concentration of 150 µM. These prodrugs are versatile and easy to synthetize and can be adapted to many enzymes.
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Antineoplásicos , Glioblastoma , Pró-Fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Glioblastoma/patologia , Humanos , Elastase de Leucócito/metabolismo , Neutrófilos/metabolismo , Pró-Fármacos/metabolismoRESUMO
Two alkylated verdazyl radicals (AlkVZs) were investigated as active compounds for photoinitiated controlled MCF-7 cell death. Observed results unambiguously showed that AlkVZ could be a potential structural moiety for the design of a novel family of photodynamic therapy agents. The main advantage of the proposed substances is an oxygen-independent generation of active radicals, which play a pivotal role in the treatment of oxygen-deficient tumors.
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Morte Celular/efeitos dos fármacos , Células MCF-7/efeitos dos fármacos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Radicais Livres/metabolismo , HumanosRESUMO
Frataxin (FXN) is a highly conserved mitochondrial protein whose deficiency causes Friedreich's ataxia, a neurodegenerative disease. The precise physiological function of FXN is still unclear; however, there is experimental evidence that the protein is involved in biosynthetic iron-sulfur cluster machinery, redox imbalance, and iron homeostasis. FXN is synthesized in the cytosol and imported into the mitochondria, where it is proteolytically cleaved to the mature form. Its involvement in the redox imbalance suggests that FXN could interact with mitochondrial superoxide dismutase (SOD2), a key enzyme in antioxidant cellular defense. In this work, we use site-directed spin labelling coupled to electron paramagnetic resonance spectroscopy (SDSL-EPR) and fluorescence quenching experiments to investigate the interaction between human FXN and SOD2 in vitro. Spectroscopic data are combined with rigid body protein-protein docking to assess the potential structure of the FXN-SOD2 complex, which leaves the metal binding region of FXN accessible to the solvent. We provide evidence that human FXN interacts with human SOD2 in vitro and that the complex is in fast exchange. This interaction could be relevant during the assembly of iron-sulfur (FeS) clusters and/or their incorporation in proteins when FeS clusters are potentially susceptible to attacks by reactive oxygen species.
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Pulmonary inflammation usually involves strong neutrophil recruitment with a marked release of proteases such as neutrophil elastase (NE). Noninvasive in vivo assessment of unregulated elastase activity in the lungs would provide a valuable diagnostic tool. Here, it is proposed to use Overhauser-enhanced magnetic resonance imaging (OMRI) in mice where inflammation was induced by the instillation of lipopolysaccharide (LPS). OMRI contrast in the lungs was generated by a dedicated NE free radical substrate. The free radical decayed more rapidly in LPS-treated mouse lungs than in control mice, indicating the occurrence of increased proteolysis under inflammation. Preclinical detection of abnormal proteolysis opens the way for new diagnosis modality and antiprotease testing in vivo.
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The nature of plasmon interaction with organic molecules is a subject of fierce discussion about thermal and non-thermal effects. Despite the abundance of physical methods for evaluating the plasmonic effects, chemical insight has not been reported yet. In this contribution, we propose a chemical insight into the plasmon effect on reaction kinetics using alkoxyamines as an organic probe through their homolysis, leading to the generation of nitroxide radicals. Alkoxyamines (TEMPO- and SG1-substituted) with well-studied homolysis behavior are covalently attached to spherical Au nanoparticles. We evaluate the kinetic parameters of homolysis of alkoxyamines attached on a plasmon-active surface under heating and irradiation at a wavelength of plasmon resonance. The estimation of kinetic parameters from experiments with different probes (Au-TEMPO, Au-SG1, Au-SG1-TEMPO) allows revealing the apparent differences associated with the non-thermal contribution of plasmon activation. Moreover, our findings underline the dependency of kinetic parameters on the structure of organic molecules, which highlights the necessity to consider the nature of organic transformations and molecular structure in plasmon catalysis.
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Photodynamic therapy (PDT) is a promising technique to treat different kinds of disease especially cancer. PDT requires three elements: molecular oxygen, a photoactivatable molecule called the photosensitizer (PS), and appropriate light. Under illumination, the PSs generate, in the presence of oxygen, the formation of reactive oxygen species including singlet oxygen, toxic, which then destroys the surrounding tissues. Even if PDT is used with success to treat actinic keratosis or prostate cancer for example, PDT suffers from two major drawbacks: the lack of selectivity of most of the PSs currently used clinically as well as the need for oxygen to be effective. To remedy the lack of selectivity, targeting the tumor neovessels is a promising approach to destroy the vascularization and cause asphyxia of the tumor. KDKPPR peptide affinity for the neuropilin-1 (NRP-1) receptor overexpressed on endothelial cells has already been proven. To compensate for the lack of oxygen, we focused on photoactivatable alkoxyamines (Alks), molecules capable of generating toxic radicals by light activation. In this article, we describe the synthesis of a multifunctional platform combining three units: a PS for an oxygen-dependent PDT, a peptide to target tumor neovessels, and an Alk for an oxygen-independent activity. The synthesis of the compound was successfully carried out, and the study of its photophysical properties showed that the PS retained its capacity to form singlet oxygen and the affinity tests confirmed the affinity of the compound for NRP-1. Thanks to the electron paramagnetic resonance spectroscopy, a technique of choice for radical investigation, the radicals generated by the illumination of the Alk could be detected. The proof of concept was thus successfully established.
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Sistemas de Liberação de Medicamentos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/uso terapêutico , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Estrutura Molecular , Oxigênio , Peptídeos , FotoquímicaRESUMO
Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich's ataxia. Frataxin's actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.
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Dicroísmo Circular/métodos , Compostos Férricos/química , Compostos Ferrosos/química , Proteínas de Ligação ao Ferro/química , Ferro/química , Marcadores de Spin , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Humanos , Concentração de Íons de Hidrogênio , Agregação Patológica de Proteínas , Ligação Proteica , Conformação Proteica , Espectrometria de Fluorescência/métodos , FrataxinaRESUMO
In 1986, Rizzardo et al. discovered the nitroxide-mediated polymerization which relies on the reversibility of homolysis of the C-ON bond of alkoxyamine R1R2NOR3, a unique property of these molecules. This discovery has generated a tremendous endeavor in the field of polymer chemistry. Alkoxyamines have been used as initiators/controllers for nitroxide-mediated polymerization. Moreover, photoexcitable alkoxyamines that dissociate under light at different wavelengths have also been developed for polymer chemistry. Over the past few years, alkoxyamines have started to be used in materials sciences. In many cases (e.g., self-healing polymers), the development of smart materials requires the use of smart building blocks, that is, molecules or systems whose properties and/or structures change upon external stimuli. Alkoxyamines exhibit a unique property: reversible homolysis (i.e., homolysis of the C-ON bond into alkyl R3⢠and nitroxyl R1R2NO⢠radicals and reformation via the coupling of these two species). Until now, this property has been controlled only by changes in temperatures or by light irradiation. Chemical and/or biochemical control of the homolysis event would open new gates for the application of these molecules in different fields such as biology and medicine. Thus, the concept of smart alkoxyamines is discussed and exemplified via the activation of alkoxyamines using chemical or/and biochemical changes amplifying the polar, steric, and stabilization effects. In situ activation is also discussed. It is shown that (i) increasing the electron-withdrawing properties of the alkyl fragment weakens the C-ON bond and thus favors homolysis but is opposite for the nitroxyl fragment; (ii) increasing the steric hindrance on the nonactive site affords dramatic conformation changes which weaken the C-ON bond; and (iii) increasing the stabilization of the released alkyl radical weakens the C-ON bond. Solvent effects and intramolecular hydrogen bonding are also discussed. Reactions used to highlight our purpose are either reversible or nonreversible and used under conditions that are as mild as possible (temperatures below 40 °C and atmospheric pressure). For example, a several (thousands of millions of) millions of orders of magnitude enhancement of the homolysis rate constant is observed upon enzymatic hydrolysis at 37 °C, meaning that a shift from a stable alkoxyamine (t1/2 = 42â¯000 milleniums) to a highly labile alkoxyamine (tmax = 1500 s for 35% conversion) is achieved. Applications of this concept are discussed for safe NMP initiators and for theranostic agents.
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The on-demand generation of stable organic radicals from the precursors can be considered as an essential challenge for the plethora of applications in various fields of science. In this contribution, we prepared a range of N-(methyl)benzyl derivatives of 6-oxoverdazyl via atom transfer radical addition from moderate to high yields and studied their thermal- and photo-initiated homolysis. The kinetics of homolysis was measured, and the dissociating rate constant kd, activation energy Ea and frequency factor A were estimated. Variation of the substituent at the C3-position of the verdazyl ring was successfully applied for fine-tuning the homolysis rate: the value of kd was higher for alkylverdazyls with electron-withdrawing groups, e.g., the para nitro group afforded a 6-fold increase in kd. In contrast to thermal homolysis, the rate of photoinduced decomposition depends on both the extinction coefficient and the value of activation energy. Thus, nitro-containing alkylated verdazyls show the highest homolysis rate in both types of initiations. The achieved results afford a novel opportunity in the controlled generation of verdazyls and further application of these compounds in medicine and chemical industry.
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Malaria and schistosomiasis are major infectious causes of morbidity and mortality in the tropical and sub-tropical areas. Due to the widespread drug resistance of the parasites, the availability of new efficient and affordable drugs for these endemic pathologies is now a critical public health issue. In this study, we report the design, the synthesis and the preliminary biological evaluation of a series of alkoxyamine derivatives as potential drugs against Plasmodium and Schistosoma parasites. The compounds (RS/SR)-2F, (RR/SS)-2F, and 8F, having IC50 values in nanomolar range against drug-resistant P. falciparum strains, but also five other alkoxyamines, inducing the death of all adult worms of S. mansoni in only 1 h, can be considered as interesting chemical starting points of the series for improvement of the activity, and further structure activity, relationship studies. Moreover, investigation of the mode of action and the rate constants kd for C-ON bond homolysis of new alkoxyamines is reported, showing a possible alkyl radical mediated biological activity. A theoretical chemistry study allowed us to design new structures of alkoxyamines in order to improve the selectivity index of these drugs.
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Anti-Helmínticos , Antimaláricos , Plasmodium falciparum/crescimento & desenvolvimento , Schistosoma mansoni/crescimento & desenvolvimento , Animais , Anti-Helmínticos/síntese química , Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Antimaláricos/síntese química , Antimaláricos/química , Antimaláricos/farmacologia , HumanosRESUMO
Nitroxide-mediated polymerization is now a mature technique, at 35 years of age. During this time, several variants have been developed: electron spin capture polymerization (ESCP), photoNMP (NMP2), chemically initiated NMP (CI-NMP), spin label NMP (SL-NMP), and plasmon-initiated NMP (PI-NMP). This mini-review is devoted to the features and applications of these variants.
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Previously, we described alkoxyamines bearing a pyridine ring as new pro-drugs with low molecular weights and theranostic activity. Upon chemical stimulus, alkoxyamines undergo homolysis and release free radicals, which can, reportedly, enhance magnetic resonance imaging and trigger cancer cell death. In the present study, we describe the synthesis and the anti-cancer activity of sixteen novel alkoxyamines that contain an imidazole ring. Activation of the homolysis was conducted by protonation and/or methylation. These new molecules displayed cytotoxic activities towards human glioblastoma cell lines, including the U251-MG cells that are highly resistant to the conventional chemotherapeutic agent Temozolomide. We further showed that the biological activities of the alkoxyamines were not only related to their half-life times of homolysis. We lastly identified the alkoxyamine (RS/SR)-4a, with both a high antitumour activity and favourable logD7.4 and pKa values, which make it a robust candidate for blood-brain barrier penetrating therapeutics against brain neoplasia.
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Aminas/química , Antineoplásicos/química , Imidazóis/química , Pró-Fármacos/química , Aminas/metabolismo , Aminas/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Carbono/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Glioblastoma/patologia , Meia-Vida , Humanos , Nitrogênio/química , Oxigênio/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , EstereoisomerismoRESUMO
While optical methods are not efficient enough for the easy, fast, and efficient detection of enzymatic activity in turbid media, the properties of the electron paramagnetic resonance (EPR) technique make it suitable for use in such media. Nitroxides which exhibit a change in their EPR hyperfine coupling constants upon enzymatic activity and are selective to lipases were developed under the name of shifting-nitroxides. Several fatty acids, exhibiting saturated and unsaturated chains of various lengths, were coupled with the shifting-nitroxide via an enol ester link and tested against several lipases. As the solubility of fatty acids is low in HEPES buffer, experiments were performed in turbid aqueous solution. Almost all labeled fatty acids were hydrolyzed by Candida rugosa lipase, and more selectivity is observed with Porcine Pancreas lipase type II. No activity was observed for lipase AK Amano 20, Candida antartica lipase B, and Mucor miehei lipase.
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Espectroscopia de Ressonância de Spin Eletrônica , Ácidos Graxos/metabolismo , Lipase/metabolismo , Óxidos de Nitrogênio/química , Animais , Candida/enzimologia , HidróliseRESUMO
Because alkoxyamines are employed in a number of important applications, such as nitroxide-mediated polymerization, radical chemistry, redox chemistry, and catalysis, research into their reactivity is especially important. Typically, the rate of alkoxyamine homolysis is strongly dependent on temperature. Nonetheless, thermal regulation of such reactions is not always optimal. This review describes various ways to reversibly change the rate of Câ»ON bond homolysis of alkoxyamines at constant temperature. The major methods influencing Câ»ON bond homolysis without alteration of temperature are protonation of functional groups in an alkoxyamine, formation of metalâ»alkoxyamine complexes, and chemical transformation of alkoxyamines. Depending on the structure of an alkoxyamine, these approaches can have a significant effect on the homolysis rate constant, by a factor of up to 30, and can shorten the half-lifetime from days to seconds. These methods open new prospects for the application of alkoxyamines in biology and increase the safety of (and control over) the nitroxide-mediated polymerization method.
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After quantum calculations of absolute free energies of polyatomic sulfur derivatives or hydrocarbon oligomer compounds, we have studied the variations of G n as a function of the number of repeating units n (sulfur atoms or hydrocarbon units, n = 2 to about 60), and observed that these values can be correlated by (a) a nearly perfect linear relationship, G n = An + B (A and B, constants), with a high accuracy which enables an extrapolation for higher values of n, and (b) a power law:, δ G = G n/ n - G( n-1) /( n - 1) = C/ n d ( C and d, constants). From comparison of variations of the C-C bond lengths, we show that the conjugation of trans-polyenes (up to C60H62) is more important than for polyynes (up to C60H2).