Intrinsic Proteolytic Activities from Cancer Cells Are Sufficient to Activate Alkoxyamine Prodrugs and Induce Cell Death.

In search of better specificity and lower chances of resistance, protease-activatable alkoxyamine prodrugs to fight cancer have been proposed. These molecules are made of a peptide linked to an alkoxyamine. Proteolysis of the peptide converts the stable prodrug at 37 °C to a metastable alkoxyamine that spontaneously homolyzes into two free radicals: a stable nitroxide and a very reactive alkyl radical. The alkyl radical induces apoptosis in the surrounding cells by inducing random chemical alterations. Here, we show that varying the peptide moiety from succinyl-Ala-Ala-Pro-Val- to PyroGlu-Gly-Arg- or PyroGlu-Gly-Lys- is effective in switching the activating enzyme from elastase to urokinase. Furthermore, these prodrugs induce the death of HT-1080 cells, a cell line that secretes several active proteases in culture. This cytotoxic activity can be suppressed by protease inhibitors and does not affect cell lines devoid of active urokinase. We thus provide examples of alkoxyamine prodrugs that are efficiently activated by the limited intrinsic protease activity and that succeed in the destruction of cancer cell lines and cancer cells from tumor explants.

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.

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.

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.

Alkylverdazyls as a Source of Alkyl Radicals for Light-Triggered Cancer Cell Death.

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.

Design of a Targeting and Oxygen-Independent Platform to Improve Photodynamic Therapy: A Proof of Concept.

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.

Chemical modifications of imidazole-containing alkoxyamines increase C-ON bond homolysis rate: Effects on their cytotoxic properties in glioblastoma cells.

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.

Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins.

Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron-electron resonance (DEER) EPR measurements were performed for both the nitroxide-nitroxide and Gd(III)-nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.

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.

Dynamics of the intrinsically disordered protein CP12 in its association with GAPDH in the green alga Chlamydomonas reinhardtii: a fuzzy complex.

CP12 is a widespread regulatory protein of oxygenic photosynthetic organisms that contributes to the regulation of the Calvin cycle by forming a supra-molecular complex with at least two enzymes: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK). CP12 shares some similarities with intrinsically disordered proteins (IDPs) depending on its redox state. In this study, site-directed spin labeling (SDSL) combined with EPR spectroscopy was used to probe the dynamic behavior of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH, the first step towards ternary complex formation. The two N-terminal cysteine residues were labeled using the classical approach while the tyrosine located at the C-terminal end of CP12 was modified following an original procedure. The results show that the label grafted at the C-terminal extremity is in the vicinity of the interaction site whereas the N-terminal region remains fully disordered upon binding to GAPDH. In conclusion, GAPDH-CP12 is a fuzzy complex, in which the N-terminal region of CP12 keeps a conformational freedom in the bound form. This fuzziness could be one of the keys to facilitate binding of PRK to CP12-GAPDH and to form the ternary supra-molecular complex.

Enlarging the panoply of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR): sensitive and selective spin-labeling of tyrosine using an isoindoline-based nitroxide.

Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical spin labels are functionalized to be grafted on natural or site-directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based nitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.

New concepts in molecular imaging: non-invasive MRI spotting of proteolysis using an Overhauser effect switch.

BACKGROUND: Proteolysis, involved in many processes in living organisms, is tightly regulated in space and time under physiological conditions. However deregulation can occur with local persistent proteolytic activities, e.g. in inflammation, cystic fibrosis, tumors, or pancreatitis. Furthermore, little is known about the role of many proteases, hence there is a need of new imaging methods to visualize specifically normal or disease-related proteolysis in intact bodies. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, a new concept for non invasive proteolysis imaging is proposed. Overhauser-enhanced Magnetic Resonance Imaging (OMRI) at 0.2 Tesla was used to monitor the enzymatic hydrolysis of a nitroxide-labeled protein. In vitro, image intensity switched from 1 to 25 upon proteolysis due to the associated decrease in the motional correlation time of the substrate. The OMRI experimental device used in this study is consistent with protease imaging in mice at 0.2 T without significant heating. Simulations show that this enzymatic-driven OMRI signal switch can be obtained at lower frequencies suitable for larger animals or humans. CONCLUSIONS/SIGNIFICANCE: The method is highly sensitive and makes possible proteolysis imaging in three dimensions with a good spatial resolution. Any protease could be targeted specifically through the use of taylor-made cleavable macromolecules. At short term OMRI of proteolysis may be applied to basic research as well as to evaluate therapeutic treatments in small animal models of experimental diseases.