Development of new fluorescent phenanthridine-based probes for the site-specific detection and quantification of superoxide radical anion in biological systems – Vivo2

The objective of this project is to design and synthesize new HE derivatives that will push forwards the boundaries of the detection of superoxide in biological systems. To reach this goal we will combine physical chemistry, organic synthesis, biophysical studies and biological investigations, regrouped in 4 Tasks:

Task 1: mechanistic studies using theoretical calculations and synthesis of HE analogs, HPLC/MS studies, DFT Calculations, investigation of the oxidation process of HE by O2•– Kinetic studies of formation of 2-OH-E+, E+ and dimers Evaluation of the reactivity of HE analogs toward superoxide radical and other radical oxidants Modeling and designing HE analogs for mechanistic studies.

Task 2: Synthesis of improved HE analogs Intra-, extracellular targeting of HE Intracellular targeting of HE Extracellular targeting of HE Post-functionalization of 2-OH-E+ for a distinct fluorescence signature. Synthesis of Shift-HE to avoid the HPLC separation step and to detect directly the superoxide by fluorescence spectroscopy. Design and synthesis of HE derivatives with improved performances

Task 3: Modulation of the reactivity towards oxidizing proteins Binding and resistance studies by inclusion complexes Resistance studies using redox modulated HE derivatives (from Task 1) Deuteration of currently used probes

Task 4: Biological investigation. Evaluation of intracellular distribution and stability of the probes Evaluation of the performance of the probes for the detection of superoxide radical anion and other oxidants produced in model systems Investigation of the reactivity of the probes towards heme proteins and determination of the lifetime of the probes in cellular systems.

i) Synthesis of various HE model analogues have been prepared prouving that the substitution of the exocyclic by alkyl or carbamate groups is incompatible with the reaction with superoxide.Protocols to evaluate the performances of new synthesized HE derivatives is established.

ii) For site-specific measurements of extracellular and cytosolic superoxide, the synthesis of different esters of HE derivatives to specifically measure intracellular O2•– (e.g. AM-HE ester,HE ethyl ester) have been performed.

For intra mitochondrial superoxide detection, we prepared several analogs of Mito-HE differing in the length of the linking alkyl chain (n = 2-10). The capacity to react with superoxide in the presence of macrophage cells have been evaluated.
The study of these probes (ii) in biological systems are under investiguations.
iii) Typically, peroxidases and more generally heme proteins are responsible for the rapid consumption of HE leading to the formation of E+ and dimeric products.
We reached to covalently graft HE on mesoporous silica (SBA15-HE or silica-HE) and characterised it.

New generation of HE derivatives will be designed and synthesized. Kinetics of the reaction of the new HE analogs prepared will be performed.
Biological studies will be performed on the new synthesised HE derivatives

Resistance studies . Biophysical studies on new prepared HE derivatives will be investigated using the protocol established
Investigation of the reactivity of the Silica-HE towards heme proteins, superoxide.
Preparation of various analogues of Silica bearing HE by varying the nature of silica as well as the functional groups grafted on the external pores of the silica.

1. Zielonka J., Joseph J., Sikora A., Hardy M., Ouari O., Vasquez-Vivar J., Cheng G., Lopez M., Kalyanaraman B (2017). Mitochondria-Targeted Triphenylphosphonium-Based Compounds: Syntheses, Mechanisms of Action, and Therapeutic and Diagnostic Applications. Chem. Rev. 117, 10043-120. PMCID: PMC5611849
2. Zielonka J., Hardy M., Michalski R., Sikora A., Zielonka M., Cheng G., Ouari O., Podsiadly R., Kalyanaraman B. (2017). Recent Developments in the Probes and Assays for Measurement of the Activity of NADPH Oxidases. Cell Biochem Biophys. 75, 335-49. PMCID: PMC5693611
3. Hardy M, Zielonka J, Karoui H, Sikora A, Michalski R, Podsiadly R, Lopez M, Vasquez-Vivar J, Kalyanaraman B, Ouari O. Detection and Characterization of Reactive Oxygen and Nitrogen Species in Biological Systems by Monitoring Species-Specific Products. Antioxid Redox Signal 28: 1416-1432, 2018.
4. Cheng, G. Zielonka, M.; Dranka, B.; Kumar, S. N.; Myers, C. R.; Bennett, B.; Garces, A. M.; Dias Duarte Machado, L. G.; Thiebaut, D.; Ouari, O.; Hardy, M.; Zielonka, J.; Kalyanaraman, B. (2018). Detection of mitochondria-generated reactive oxygen species in cells using multiple probes and methods: Potentials, pitfalls, and the future. J. Biol. Chem. 293(26), 10363-10380

During the last decade, the development of new techniques for the detection of superoxide radical anion (O2•–) in biological systems has continued to receive increasing attention. Superoxide is positioned upstream in most radical cascades leading to Reactive Oxygen and Nitrogen Species (RONS) and irreversible oxidative damages to biomolecules. Therefore, the ability of rigorous detection of superoxide is of major importance.
However, currently, no method fulfills the requirements for the rigorous characterization of the role of O2•– in biological systems and the characterization of RONS in vitro and in vivo remains a challenge. Many probes have been criticized and discarded due to unreliable results, detection artefacts and/or poor reproducibility.
For the last decade hydroethidine (HE) has been shown to be a promising probe for the fluorescent detection of O2•–. Unfortunately, the use of HE in biological systems is limited by four main problems:

(i) limited understanding of the mechanism of HE reaction with O2•–,
(ii) lack of site-specific analogs of HE,
(iii) degradation of HE by heme proteins leading to misleading signals,
and (iv) requirement of HPLC-based products separation coupled with fluorescence, electrochemical or MS detection.

Our teams have been actively involved in developing techniques for the detection and the characterization of RONS (e. g. spin trapping, SOD mimics, fluorescence-based RONS probes) for the last 20 years and their significant contributions have been recognized in the field of free radical/redox biology. The major objectives of this proposal can be summarized as follows:

(i) determination of the reaction mechanism of HE with O2•– and other biologically-relevant oxidants, including experimental (kinetics, products characterization) and theoretical studies (DFT calculations), using HE analogs as model systems,
(ii) targeting HE type probes to intracellular and extracellular compartments for site-specific detection of O2•–,
(iii) supramolecular and other approaches to protect HE from non-specific oxidation by heme proteins,
(iv) the development of innovative probes for the direct and specific detection of O2•– by fluorescence,
(v) application of optimized probes for detection of O2•– in biological systems.

The molecular structure of the probe plays a key role in its performance, including selectivity and specificity. As the development of HE probe was not based on rational design, there is an opportunity to develop new more efficient HE-analogs for the detection and quantification of O2•–. This project involves one French partner (ICR) as well as two foreign partners, led by Dr. Kalyanaraman (Medical College of Wisconsin, Milwaukee, USA) and by Dr. Sikora (Lodz University of Technology, Poland). These partners have already funds to perform the work described in the proposal. Thus, no financial support is requested from ANR for these two groups. All collaborating groups have a strong experience and expertise and are among leaders in their field. Also, the project will capitalize and benefit from well-established and long-term collaborations between the three groups.

This project illustrates the need for joint and interdisciplinary effort to push forward the frontiers in the free radical and biomedical fields.