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.

Formation of Alginate/Chitosan Interpenetrated Networks Revealed by EPR Spectroscopy.

This study analyzes the physico-chemical properties of interpenetrated polymer networks (IPNs) and semi-IPN resulting from cross-linking chitosan with glutaraldehyde and alginate with Ca2+ cations, as a function of the order in which the cross-linking agents are added to the polymer mixture. Three physico-chemical methods were used to assess the differences between systems: rheology, IR spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. While rheology and IR spectroscopy are commonly used to characterize gel materials, EPR spectroscopy is rarely used, but has the advantage of providing local information about the dynamics of a system. The rheological parameters, which describe the global behavior of the samples, show that semi-IPN systems have a weaker gel behavior and the order of introducing the cross-linker in the polymer systems plays a role. The IR spectra of samples resulting by adding only Ca2+ or Ca2+ as the first cross-linker are similar to that of the alginate gel, while the spectra of samples in which glutaraldehyde is firstly added resemble the chitosan gel spectrum. Using spin-labeled alginate and spin-labeled chitosan, we monitored the changes occurring in the dynamic of the spin labels due to the formation of IPN and semi-IPN. The results show that the order of adding the cross-linking agents influences the dynamic of the IPN network, and that the formation of the alginate network determines the characteristics of the entire IPN system. The EPR data were correlated with the rheological parameters and IR spectra of the analyzed samples.

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.

Establishing plasmon contribution to chemical reactions: alkoxyamines as a thermal probe.

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.

Alkoxyamines Designed as Potential Drugs against Plasmodium and Schistosoma Parasites.

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.

Functionalized C-nucleosides as remarkable RNA binders: targeting of prokaryotic ribosomal A-site RNA.

RNA represents an extremely promising and yet challenging therapeutic target. Here, we report the design of a series of C-nucleosides as original RNA binders. Some of them bind strongly and selectively to A-site prokaryotic ribosomal RNA.

How intramolecular coordination bonding (ICB) controls the homolysis of the C-ON bond in alkoxyamines.

Because the C-ON bond homolysis rate constant k d is an essential parameter of alkoxyamine reactivity, it is especially important to tune k d without a major alteration of the structure of the molecule. Recently, several approaches have become known, e.g., protonation of functional groups and formation of metal complexes. In this paper, coordination reactions of [Zn(hfac)2(H2O)2] with a series of new SG1-based alkoxyamines affording complexes with different structures are presented. The k d values of the complexed forms of the alkoxyamines were compared to those of free and protonated ones to reveal the contribution of the electron-withdrawing property and structure stabilization. Together with previously published data, this work provides clues to the design of alkoxyamines that can be effectively activated upon coordination with metal ions. Furthermore, our results provide insight into the mechanism underlying the influence of complexation on the reactivity of alkoxyamines. This led us to describe different types of coordination: intramolecular in nitroxyl fragment, intramolecular in alkyl fragment, intramolecular between alkyl and nitroxyl fragment, and intermolecular one. All of them exhibit different trends which are dramatically altered by changes in conformation.

How intramolecular hydrogen bonding (IHB) controls the C-ON bond homolysis in alkoxyamines.

Recent amazing results (Nkolo et al., Org. Biomol. Chem., 2017, 6167) on the effect of solvents and polarity on the C-ON bond homolysis rate constants kd of alkoxyamine R1R2NOR3 led us to re-investigate the antagonistic effect of intramolecular hydrogen-bonding (IHB) on kd. Here, IHB is investigated both in the nitroxyl fragment R1R2NO and in the alkyl fragment R3, as well as between fragments, that is, the donating group on the alkyl fragment and the accepting group on the nitroxyl fragment, and conversely. It appears that IHB between fragments (inter IHB) strikingly decreases the homolysis rate constant kd, whereas IHB within the fragment (intra IHB) moderately increases kd. For one alkoxyamine, the simultaneous occurrence of IHB within the nitroxyl fragment and between fragments is reported. The protonation effect is weaker in the presence than in the absence of IHB. A moderate solvent effect is also observed.

Normal, Leveled, and Enhanced Steric Effects in Alkoxyamines Carrying a ß-Phosphorylated Nitroxyl Fragment.

The design of new R1R2NOR3 alkoxyamines for various applications relies on the accurate prediction of two kinetic parameters, the C-ON bond homolysis rate constant (kd) and its re-formation rate constant (kc). Relationships to describe the steric and polar effects of the R1R2NO fragment ruling kd have been developed. For all cyclic nitroxyl fragments, the steric effect is described as the sum of the bulkiness of the R1 and R2 groups (i.e., normal steric effect), while for the noncyclic nitroxyl fragment (except for one case), a leveled steric effect is assumed. In this work, we show that the normal steric effect also applies to noncyclic nitroxyl fragments and that for one case an enhanced steric effect is also observed, i.e., experimental kd >5-fold larger than the predicted value.

C-ON bond homolysis in alkoxyamines. Part 12: the effect of the para-substituent in the 1-phenylethyl fragment.

The application of alkoxyamines as initiators/controllers in nitroxide mediated polymerization and as agents for theranostics requires the development of switchable (from stable one to labile one) alkoxyamines. One way to achieve this is to tune the polarity of various groups carried by either the alkyl fragment or the nitroxyl fragment. Thus, the effect of protonation/deprotonation of the para-functionalized aryl moiety carried by the alkyl fragment in diethyl(2,2-dimethyl-1-((1,1-dimethylethyl)(1-para-subsitutedphenylethoxy)amino)propyl)phosphonate is investigated. An increase in kd is observed with increasing localized electrical effect, i.e., in the presence of electron withdrawing groups at the para position of the phenyl ring. A striking effect of the intimate ion pair on kd is also observed.

Artificial nucleobase-amino acid conjugates: a new class of TAR RNA binding agents.

The human immunodeficiency virus type-1 (HIV-1) Tat protein stimulates transcriptional elongation. Tat is involved in the transcription machinery by binding to the transactivation response region (TAR) RNA stem-loop structure, which is encoded by the 5' leader sequence found in all HIV-1 mRNAs. Herein, we report the rational design, synthesis, and in vitro evaluation of new RNA binding agents that were conceived in order to bind strongly and selectively to the stem-loop structure of TAR RNA and, thus, inhibit the Tat/TAR interaction. We have demonstrated that the conjugation of modified nucleobases, able to interact specifically with an RNA base pair, and various amino acids allows these motifs to bind the target RNA selectively and in a cooperative manner that leads to the inhibition of viral replication in HIV-infected cells.