Hybrid Peptide-Alkoxyamine Drugs: A Strategy for the Development of a New Family of Antiplasmodial Drugs.

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.

Smart Alkoxyamines: A New Tool for Smart Applications.

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 = 42 000 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.

Shifting-Nitroxides to Investigate Enzymatic Hydrolysis of Fatty Acids by Lipases Using Electron Paramagnetic Resonance in Turbid Media.

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.

Selective On/Off-Nitroxides as Radical Probes to Investigate Non-radical Enzymatic Activity by Electron Paramagnetic Resonance.

A nitroxide carrying a peptide specific to the binding pocket of the serine proteases chymotrypsin and cathepsin G is prepared. This peptide is attached as an enol ester to the nitroxide. Upon enzymatic hydrolysis of the peptide, the enol ester moiety is transformed into a ketone moiety. This transformation affords a difference of 5 G in phosphorus hyperfine coupling constant between the electronic paramagnetic resonance (EPR) signals of each nitroxide. This property is used to monitor the enzymatic activity of chymotrypsin and cathepsin G by EPR. Michaelis constants were determined and match those reported for conventional optical probes.

The ß-phosphorus hyperfine coupling constant in nitroxide: part 3: titration of water by electron paramagnetic resonance.

Recently, we showed that the phosphorus hyperfine coupling constant aPß of persistent cyclic nitroxides decreased with the normalized polarity Reichardt's constant E. Thus, we investigated the changes in aPß in binary mixtures of solvents. The sensitivity of aPß to the solvent was high enough to allow us to perform water titration in THF, 1,4-dioxane, and acetonitrile by EPR. Accuracies of a few percent were achieved.

Enzymatically Shifting Nitroxides for EPR Spectroscopy and Overhauser-Enhanced Magnetic Resonance Imaging.

In vivo investigations of enzymatic processes using non-invasive approaches are a long-lasting challenge. Recently, we showed that Overhauser-enhanced MRI is suitable to such a purpose. A ß-phosphorylated nitroxide substrate prototype exhibiting keto-enol equilibrium upon enzymatic activity has been prepared. Upon enzymatic hydrolysis, a large variation of the phosphorus hyperfine coupling constant (Δa(P)=4 G) was observed. The enzymatic activities of several enzymes were conveniently monitored by electronic paramagnetic resonance (EPR). Using a 0.2 T MRI machine, in vitro and in vivo OMRI experiments were successfully performed, affording a 1200% enhanced MRI signal in vitro, and a 600% enhanced signal in vivo. These results highlight the enhanced imaging potential of these nitroxides upon specific enzymatic substrate-to-product conversion.

Alkoxyamines: toward a new family of theranostic agents against cancer.

Theranostics combines therapeutic and diagnostic or drug deposition monitoring abilities of suitable molecules. Here we describe the first steps of building an alkoxyamine-based theranostic agent against cancer. The labile alkoxyamine ALK-1 (t(1/2) = 50 min at 37 °C) cleaves spontaneously to generate (1) a highly reactive free alkyl radical used as therapeutic agents to induce cell damages leading to cell death and (2) a stable nitroxide used as contrast agent for Overhauser-enhanced magnetic resonance imaging (OMRI). The ALK-1 toxicity was studied extensively in vitro on the glioblastoma cell line U87-MG. Cell viability appeared to be dependent on ALK-1 concentration and on the time of the observation following alkoxyamine treatment. For instance, the LC50 at 72 h was 250 µM. Data showed that cell toxicity was specifically due to the in situ released alkyl radical. This radical induced oxidative stress, mitochondrial changes, and ultimately the U87 cell apoptosis. The nitroxide production, during the alkoxyamine homolysis, was monitored by OMRI, showing a progressive MRI signal enhancement to 6-fold concomitant to the ALK-1 homolysis. In conclusion, we have demonstrated for the first time that the alkoxyamines are promising molecules to build theranostic tools against solid tumors.

Alkoxyamines: a new family of pro-drugs against cancer. Concept for theranostics.

Development of anti-cancerous theranostic agents is a vivid field. This article describes a theranostic approach that relies on the triggering of cancer cell death by generation of alkyl radicals at the right place and at the right time using the presence of active proteases in the tumour environment. Alkoxyamines (R(1)R(2)NOR(3)) are labile molecules that homolyze into nitroxides (R(1)R(2)NO˙) and reactive alkyl radicals (R(3)˙). They are used as a source of active alkyl radicals for curing and nitroxides for monitoring by Overhauser-enhanced magnetic resonance imaging (OMRI). Herein, the requirements needed for applying alkoxyamines are described: (i) highly selective activation of the alkoxyamine by specific proteases; (ii) fast homolysis of the alkoxyamine C-ON bond at physiological temperature; (iii) activation of cell death processes through an increase of the local oxidative stress or potential re-activation of the immune system due to short-lived alkyl radicals; and (iv) imaging of the tumor and the drug release by sensing the nitroxide by OMRI.

Peptide-Alkoxyamine Drugs: An Innovative Approach to Fight Schistosomiasis: "Digging Their Graves with Their Forks".

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.

4-Amino-TEMPO loaded liposomes as sensitive EPR and OMRI probes for the detection of phospholipase A2 activity.

This work aims at developing a diagnostic method based on Electron Paramagnetic Resonance (EPR) measurements of stable nitroxide radicals released from "EPR silent" liposomes. The liposome destabilisation and consequent radical release is enzymatically triggered by the action of phospholipase A2 (PLA2) present in the biological sample of interest. PLA2 are involved in a broad range of processes, and changes in their activity may be considered as a unique valuable biomarker for early diagnoses. The minimum amount of PLA2 measured "in vitro" was 0.09 U/mL. Moreover, the liposomes were successfully used to perform Overhauser-enhanced Magnetic Resonance Imaging (OMRI) in vitro at 0.2 T. The amount of radicals released by PLA2 driven liposome destabilization was sufficient to generate a well detectable contrast enhancement in the corresponding OMRI image.

A system for in vivo on-demand ultra-low field Overhauser-enhanced 3D-Magnetic resonance imaging.

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.

Neutrophil Elastase-Activatable Prodrugs Based on an Alkoxyamine Platform to Deliver Alkyl Radicals Cytotoxic to Tumor Cells.

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.

Magnetic Resonance Imaging of Protease-Mediated Lung Tissue Inflammation and Injury.

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.

Neutrophil Elastase Activity Imaging: Recent Approaches in the Design and Applications of Activity-Based Probes and Substrate-Based Probes.

The last few decades of protease research has confirmed that a number of important biological processes are strictly dependent on proteolysis. Neutrophil elastase (NE) is a critical protease in immune response and host defense mechanisms in both physiological and disease-associated conditions. Particularly, NE has been identified as a promising biomarker for early diagnosis of lung inflammation. Recent studies have shown an increasing interest in developing methods for NE activity imaging both in vitro and in vivo. Unlike anatomical imaging modalities, functional molecular imaging, including enzymatic activities, enables disease detection at a very early stage and thus constitutes a much more accurate approach. When combined with advanced imaging technologies, opportunities arise for measuring imbalanced proteolytic activities with unprecedented details. Such technologies consist in building the highest resolved and sensitive instruments as well as the most specific probes based either on peptide substrates or on covalent inhibitors. This review outlines strengths and weaknesses of these technologies and discuss their applications to investigate NE activity as biomarker of pulmonary inflammatory diseases by imaging.

An elastase activity reporter for Electronic Paramagnetic Resonance (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI) as a line-shifting nitroxide.

Pulmonary inflammatory diseases are a major burden worldwide. They have in common an influx of neutrophils. Neutrophils secrete unchecked proteases at inflammation sites consequently leading to a protease/inhibitor imbalance. Among these proteases, neutrophil elastase is responsible for the degradation of the lung structure via elastin fragmentation. Therefore, monitoring the protease/inhibitor status in lungs non-invasively would be an important diagnostic tool. Herein we present the synthesis of a MeO-Suc-(Ala)2-Pro-Val-nitroxide, a line-shifting elastase activity probe suitable for Electron Paramagnetic Resonance spectroscopy (EPR) and Overhauser-enhanced Magnetic Resonance Imaging (OMRI). It is a fast and sensitive neutrophil elastase substrate with Km = 15 ±â€¯2.9 µM, kcat/Km = 930,000 s-1 M-1 and Km = 25 ±â€¯5.4 µM, kcat/Km = 640,000 s-1 M-1 for the R and S isomers, respectively. These properties are suitable to detect accurately concentrations of neutrophil elastase as low as 1 nM. The substrate was assessed with broncho-alveolar lavages samples derived from a mouse model of Pseudomonas pneumonia. Using EPR spectroscopy we observed a clear-cut difference between wild type animals and animals deficient in neutrophil elastase or deprived of neutrophil Elastase, Cathepsin G and Proteinase 3 or non-infected animals. These results provide new preclinical ex vivo and in vivo diagnostic methods. They can lead to clinical methods to promote in time lung protection.

In vivo Overhauser-enhanced MRI of proteolytic activity.

There is an increasing interest in developing novel imaging strategies for sensing proteolytic activities in intact organisms in vivo. Overhauser-enhanced MRI (OMRI) offers the possibility to reveal the proteolysis of nitroxide-labeled macromolecules thanks to a sharp decrease of the rotational correlation time of the nitroxide moiety upon cleavage. In this paper, this concept is illustrated in vivo at 0.2 T using nitroxide-labeled elastin orally administered in mice. In vitro, this elastin derivative was OMRI-visible and gave rise to high Overhauser enhancements (19-fold at 18 mm nitroxide) upon proteolysis by pancreatic porcine elastase. In vivo three-dimensional OMRI detection of proteolysis was carried out. A keyhole fully balanced steady-state free precession sequence was used, which allowed 3D OMRI acquisition within 20 s at 0.125 mm(3) resolution. About 30 min after mouse gavage, proteolysis was detected in the duodenum, where Overhauser enhancements were 7.2 ± 2.4 (n = 7) and was not observed in the stomach. Conversely, orally administered free nitroxides or pre-digested nitroxide-labeled elastin were detected in the mouse's stomach by OMRI. Combined with specific molecular probes, this Overhauser-enhanced MRI technique can be used to evaluate unregulated proteolytic activities in various models of experimental diseases and for drug testing.

Cellular density effect on RGD ligand internalization in glioblastoma for MRI application.

Cellular density is a parameter measured for glioma grade and invasiveness diagnosis. The characterization of the cellular density can be performed, non invasively, by magnetic resonance imaging (MRI), since, this technique displays a good resolution. Nevertheless MRI sensitivity is critical. Development of smart contrast agents appears useful to increase MRI signal to noise ratio (SNR). Tumor invasiveness is correlated with high expression of integrins that can be targeted by RGD motif. In this study, MRI contrast agents or fluorescent probes linked to RGD-peptides were used, in a glioma model, to assess the relation between RGD uptake/signal improvement/cell density and consequently tumor invasiveness. Experiments were performed in vitro with U87-MG glioma cells. Flow cytometry and microscopy experiments with RGD and iRGD-alexa488 demonstrated that cell internalization was dependent on cell density. The internalization involved a clathrin-dependent endocytosis. Cytoskeleton and particularly the microtubules were concerned. Actin filaments played a minor role. The internalization was also dependent on the glycolysis and the oxidative phosphorylations. The cellular density modulated the importance of the endocytosis pathways and of the metabolism but not the cytoskeleton contribution. The internalization of the RGD-peptide associated to gadolinium chelate increased the SNR of U87 cells. Moreover, following the cell density augmentation, the SNR increased with a low amplitude but a trend was clearly determined. In conclusion, RGD-peptide internalization appeared, in vitro, as a marker of cellular density. In perspective, the combination of these peptides with contrast agents associated to more sensitive MRI techniques could improve the MRI signal allowing the characterization of cellular density for tumor diagnosis.

Overhauser-enhanced MRI of elastase activity from in vitro human neutrophil degranulation.

BACKGROUND: Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research. METHODOLOGY/PRINCIPAL FINDINGS: We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10(4) cells per milliliter, well under the physiological neutrophils concentrations. CONCLUSIONS/SIGNIFICANCE: These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.

In vivo high-resolution 3D overhauser-enhanced MRI in mice at 0.2 T.

Overhauser-enhanced MRI (OMRI) offers the potentiality of detecting low-concentrated species generated by specific biological processes. However molecular imaging applications of OMRI need significant improvement in spatial localization. Here it is shown that 3D-OMRI of a free radical injected in tumor-bearing mice can be performed at high anatomical resolution at a constant field. A 30 mm cavity operating at 5.43 GHz was inserted in a C-shaped magnet for proton MRI at 0.194 T. Nude mice with or without brain-implanted C6 rat glioma were positioned in the cavity and injected with TOPCA (1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxylic acid). OMRI was performed in 3D within several minutes in the brain region without high overheating of the animals. Voxel size was 0.5 × 0.5 × 1 mm³ , providing good delineation of brain regions. Signal amplifications ranged from 2 in tumors to 10 in vessels several minutes after TOPCA injection. Time-course of signal enhancement could be measured by 2D OMRI at 15 s time intervals in a localized thin slice. The method opens the way for molecular imaging of biological activities able to generate OMRI-visible free radicals.