The principle of Targeted Alpha Therapy (TAT) is to destroy cancer cells with alpha-emitting- radionuclides bound to cancer selective carrier molecules, such as antibodies or peptides. Alpha particle-emitting radionuclides are promising for two reasons. On the one hand, the high toxicity of alpha particles related to the high linear energy transfer (LET ˜ 100 keV/µm) confer them the possibility to destroy cells not anymore sensitive to chemotherapy or external beam radiation. On the other hand, they are particularly interesting for the treatment of small size tumors (residual disease, micro metastases) due to the short range (< 100 µm) of alpha particles in human tissue. The challenge is to deliver the radioactive atoms at the target and to find the right balance between toxicity and anti-tumour effect. The efficacy and safety of targeted alpha therapy has been shown in a large number of pre-clinical studies and has been successfully translated to clinical trials. Among the different potential alpha-emitters, At-211 is considered to be one of the most promising. It was used in two clinical trials in the US and in Sweden, and is the subject of a wide research program in Nantes. This project is coordinated by the “Centre de Recherches en Cancérologie Nantes Angers” and is motivated by the arrival of the cyclotron ARRONAX in Nantes, which will produce At-211 by the middle of 2010. A clinical trial of prostate cancer metastases is scheduled in the frame of the project Alpharit financed by Oseo. This will correspond to the first trial of TAT in France. At-211 binding to carrier molecules remains however a difficult task On the one hand, it is a rare element since it has only short half-life radioactive isotopes; they have to be produced by nuclear reactions, such as in cyclotron for At-211. On the other hand, it is an invisible element. The amount of astatine produced allows working at ultra trace concentrations (typically 10-11 to 10-15 M) and no spectroscopic tools can be used to evaluate astatine chemistry at the microscopic level. As a result, Astatine chemistry is not well understood. By analogy with labelling protocols of iodide disease-targeting carrier molecules, the studies focused on formation of astatine-carbon bounds. While some approaches provide “adequate” in vivo stability to move into clinical studies, notably when the labelled molecule is administrated to a compartmental space (e.g. intraperitoneal, tumour resection etc.), additional studies still need to be conducted to develop improved labelling approaches and particularly for systemically administration. A team of researchers in Poland have proposed a mode of fixation with At- based on the high in vivo stability of the trans-[RhCl2(cis/trans-16S4-diol)]+ complex. Thus, some AtMCl+ (M=Rh(III) or Ir(III)) entities are synthesized and complexed with chelating agents. This promising way must be optimised and validated. Another approach is to develop some ligands for a direct chelation of astatine. In this case, a metallic form of astatine is prepared and a coordination bound is created. Considering the complexity to evaluate astatine chemistry, only a few studies have been conducted on this subject. To date, there have been no examples of chelates that are stable to in vivo deastatination. The exploration of the metallic character of astatine is at the center of this project. In a recent study, we have defined a robust pourbaix diagram of astatine in non-complexing aqueous medium showing the existence of two stable metallic forms of At, i.e. At+ and AtO+. The aim of this project is to explore the reactivity of AtO+ with respect to ligands (what are the atoms interacting with AtO+? What is the nature of the bounds formed) with the objective to synthesize some ligands that could be used for TAT. As a result, we would propose alternative labelling methods with respect to those used or under development using the “halogen” character of astatine.

KinBioFRET proposal is based on an original combination of multidisciplinary partners: technological and methodological development team (Marc Tramier) directly concerned by the development of FRET biosensor methodologies and three biological partners (Jean-Pierre Tassan, Claude Prigent and Roland Le Borgne) directly interested into dynamics of kinase activities in different fields of biology. We propose to develop new FRET biosensors to measure the activity of two essential mitotic kinases, MELK and Aurora A, in the frame of spatio-temporal regulation of these kinases during cell division in living genetically tractable model organisms. First, to be able to increase sensitivity, speed of time-lapse acquisition and then biological relevance of these bioprobes, KinBioFRET will develop new technological and methodological approaches by using fastFLIM for quantitative heteroFRET measurement and by introducing homoFRET bioprobes by fluorescence anisotropy measurement. Second, one of the methodological objectives is to be able to follow in the same time two (or more) FRET bioprobes using the homoFRET approach. This will be achieved by measuring MELK and Vinculin tension sensors using multicolor homoFRET biosensors simultaneously in Xenopus embryos. Third, we will engineer new FRET biosensors to study spatio-temporal regulation of the mitotic kinase Aurora A in Drosophila. The choice of Aurora A is intimately linked to the biological questions addressed by four independent groups of biologists at IGDR. In addition to the FRET change from C- and N-ter fluorescent protein tags within Aurora A, we propose to develop an Aurora A FRET biosensor constituted of a phospho-binding domain recognizing an Aurora A substrate peptide sequence and sandwiched between two fluorescent proteins. This latter FRET biosensor will contain various target sequences for specific subcellular localizations such as centromers, centrosomes, and plasma membrane. Finally, the methodological approaches developed here will be directly used to investigate two biological questions: (i) the comparison of blastula and gastrula dividing cells to simultaneously follow spatio-temporal dynamics of MELK kinase activities and mechanical tension in Xenopus embryos, and (ii) the spatio-temporal regulation of Aurora A activity following symmetric and asymmetric cellular division in the context of Drosophila live pupae. We believe that this proposal is novel and ambitious for several reasons: multidisciplinary approaches, development of quantitative fluorescence microscopy methods dedicated to FRET bioprobes, development of new Aurora A kinase FRET biosensors, development of homoFRET biosensors by anisotropy measurements, development of simultaneous spatio-temporal dynamics of FRET biosensors, novelty of expected biological results in the field of kinase regulation during cell division in developing living organism.