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New Nuclear Medicine Imaging Radiotracer 64Cu(II) for diagnosing Hypoxia Conditions Based on the Cellular Copper Cycle

Periodic Reporting for period 2 - CuHypMECH (New Nuclear Medicine Imaging Radiotracer 64Cu(II) for diagnosing Hypoxia Conditions Based on the Cellular Copper Cycle)

Reporting period: 2019-03-01 to 2020-08-31

Imaging of hypoxia is important in many disease states in oncology, cardiology, and neurology. Hypoxia is a common condition encountered within the tumour microenvironment that drives proliferation, angiogenesis, and resistance to therapy. Despite on-going efforts to identify hypoxia, until now there is no clinically approved imaging biomarker, due to both low tumour uptake, and a low signal to background (S/B) ratio that affects the imaging quality. Nuclear Medicine is using labelled radio-isotopes for PET/CT and SPECT imaging. These radio-tracers diagnose the metabolic processes in the body. Among these tracers, 18F-FDG is the most routinely used as a marker of glucose metabolism. However, not all tumours consume glucose, and glucose consumption is not specific only for malignant tumours, which limits its application. Copper is a nutritional metal, recently examined as a radiotracer for hypoxia, owing to its to the oxidising environment. Clinical and in-vivo studies on various 64Cu(II)-PET radiotracers resulted in controversial reports on the specificity of the current tracers for hypoxia imaging due to non-selective bio-distribution & low S/B ratio. This multidisciplinary proposal focuses on the discovery of comprehensive signal pathways of the cellular copper cycle using advanced biophysical methods and a proprietary design of 64Cu(II) radiotracer. This radiotracer will be incorporated in the cellular copper cycle, and will enable to selectively target the oxidising environment in tumours. The design of the new radiotracer is based on systematic structural & functional mapping of the copper binding sites to the various copper proteins and the visualisation of the transfer mechanism. This new copper tracer should increase the selectivity of tumour uptake, stability, and improve bio-distribution. This project assimilates cold and hot chemistry and biology, while emphasising the clinical unmet need in metal based radiotracer that form stable complexes.

The specific objectives of CuHypMech are:
Objective no. 1: Map the cellular copper cycle at the molecular level. In order to design an effective biomarker, we will map in detail the copper cycle, and identify the various copper binding sites.
WP 1.1: Resolve the Ctr1-Atox1-ATP7B cycle
WP 1.2: Target conditions that can affect or alter the Cu(I) cycle through Ctr1-Atox1-ATP7B transfer.

Objective no. 2: Design new biomarkers for hypoxic cells. The designed biomarker will be incorporated in the cellular copper cycle and its mechanism will be resolved.
WP 2.1: Develop new radiotracer for PET/SPECT experiment based on 64Cu(II).
WP 2.2: Perform 64Cu(II) in cell experiments in hypoxic and normoxic conditions.

Objective no. 3: Develop new therapeutic approaches for copper dis-homeostasis disorders.
We will utilize the knowledge gained in this study on the copper cycle to develop biomimetic peptides to control the in-cell copper concentration.
WP 3.1: Design biomimetic peptides that can alter the cellular copper trafficking mechanism.
WP 3.2: Check our hypothesis for ALS therapy using in cell 64Cu experiments.
During the last 2.5 years, CuHypMech have gained a lot of knowledge on the copper transfer mechanism in the human cell, covered by WP1. The knowledge gained assisted us to design new radiotracer for diagnostic of hypoxic tumors (described in WP2) and peptides that can manipulate the copper metabolism, towards development of new therapeutic agents to neurological diseases (WP3).

The specific achievements are described below, according to each WP:

WP 1.1: Resolving the Ctr1-Atox1-ATP7B cycle
Task 1.1.1: Understanding the effect of copper entry to the cell through Ctr1 and reduction mechanisms. In this task, we would like to resolve all Cu(II) and Cu(I) binding sites to Ctr1, and to analyze in detail the reduction mechanism of Cu(II) to Cu(I). This task is essential, in order to design a proper radiotracer that will bind Ctr1. Using EPR and NMR experiments, we succeeded to resolve the first Cu(II) and Cu(I) sites in Ctr1 extracellular domain. We showed that H5 and H6 are essential residues for Cu(II) coordination, whereas the first three methionine residues M7, M9, and M12 form Cu(I) site. We reported our results in Shenberger et al. J. Coord. Chem. 2018, 71, 1985. We succeeded to express and purify Ctr1 using insect cell expression system. EPR results showed that up to six Cu(II) ions can coordinate to Ctr1 trimer. We also showed that Ctr1 undergoes some conformational changes while binding to Cu(I) ions. This work is currently in preparing for publication.

Task 1.1.2: Visualizing the interaction between Ctr1 and Atox1 and the effect of point mutations. To ensure that our radiotracer is properly incorporated in the copper cycle, the radiotracer’s design should not only reflect incorporation in Ctr1, but it must also be transferred to the Atox1 metallochaperone. Thus, herein, our mission is to analyze the Cu(I) transfer mechanism between Ctr1 and Atox1. Targeting key residues that are essential for the interaction between the two proteins as well as for Cu(I) coordination to these proteins. Using EPR measurements and calculations we recently showed that Atox1 can accommodate various conformations, while it can accommodate a specific conformation depending on its interacting partner (Levy et al. Protein Sci. 2017, 26, 1609, Levy et al. J. Phys Chem. B. 2016, 120, 12334). We also revealed that while interacting with Ctr1, Atox1 is in its dimer form, while binding to MBDs in ATP7B, Atox1 breaks to monomers (Qasem et al. Metallomics, 2019, 11, 1288, Magistrato et al. Curr. Opin. Struct, Biol. 2019, 58, 26). We also showed that cys to ser mutation in the copper binding site, greatly affect the interaction with the partner protein, and inhibit the copper transfer (Pavlin et al. Int. J. Mol. Biol. 2019, 20, 3462). We also explored the Cu(I) binding site using EPR and MD calculations, we revealed that C12 is critical residue for Cu(I) coordination, while C15 and K60 assist in preserving Atox1 dimerization (this work was submitted to publication).
Task 1.1.3: Exploring the interaction between Atox1 and ATP7B. In a recent paper we showed that MBD4 of ATP7B is critical for interaction with Atox1 metallocahperone, and proper copper transfer mechanism (Qasem et al. Metallomics, 2019, 11, 128).

WP 1.2: Targeting conditions that can affect or alter the Cu(I) cycle through Ctr1-Atox1-ATP7B transfer
In this WP, we plan to explore factors that can interrupt with the copper cycle:
Task 1.2.1: Competitive metal ions such as Ag(I), Zn(II), Hg2(II). We have previously shown that Ag(I) can block methionine based Cu(I) sites, and thus affects the Cu(I) transfer (Shenberger et al. J. Biol. Inorg. Chem. 2015, 20, 719). We do not have yet new results regarding other competitive metal ions, such as zinc or mercury.
Task 1.2.2: Cu(II) chelators, to check their effect on the Cu(I) transfer and efflux rate. We showed that ATSM and Cu(II) does not bind to proteins that are involved in the copper cycle, this work was recently published in Walke et al. ACS Omega, 2019, 4, 12278.
Task 1.2.3: Methionine and cysteine based motifs which are identified as general binding sites for Cu(I) ions. Our group has previously shown that the methionine motif can coordinate to the Ctr1 intracellular domain, and by this to facilitate the Cu(I) transfer. Using in silico calculations, two peptides that can interfere with proper copper transfer were designed. The peptides were synthesized with purity above 95%. MTT assay was performed in HEK293 cells. First, we checked copper toxicity, and saw that the cells are still viable when exposed to copper concentration of 10 microM. Then, cell experiments showed that these peptides interfere with copper metabolism.

WP2: Designing new biomarkers for hypoxic cells
The knowledge gained in WP1 assisted us in designing the 64Cu(II) radiotracer for diagnostic of hypoxic tissues. Our radiotracer composes 3 parts: (i) the ligand-which controls the incorporation of the radiotracer in the copper cycle according to the oxidation environment. (ii) The peptide- which guides the radiotracer to the Ctr1 extracellular domain, and (iii) the radioactive ion 64Cu(II).
WP 2.1: Developing new radiotracer for PET/SPECT experiment based on 64Cu(II).
Task 2.1.1: Preparation of Cu(II) complexes: Our Cu(II) derivative is composed of three parts : Cu(II), ligand and peptide. Based on our results, we found two peptides and two ligands that provided us with the best results, regarding incorporation in the copper cycle, stability of the complex, and sensitivity to hypoxic conditions.
Task 2.1.2: Resolving the Cu(II) complexes coordination to the Ctr1 protein, stability, and binding affinity parameters. We performed UV-Vis experiments, which showed that our complexes has high affinity to the Ctr1 extracellular part. EPR experiments confirmed their stability. (The data is described in Shenberger et al. PCT IL2018/051211)
Task 2.1.3: Sensitivity to the oxidation-reduction environment. We showed that our radiotracer is highly sensitive to the hypoxic conditions of the cell.
Task 2.1.4: Determination of transfer rate parameters in Ctr1-Atox1-ATP7B cycle. EPR experiments showed that the complexes are incorporated in the copper cycle, and once introducing inhibitor such as Ag(I), no penetration of the complex to the cell occurred. We are currently working in order to better resolve the transfer rate parameters of our complex.
Task 2.1.5 Isotope-labeling of the lead compound. We isotope labeled our complexes with 64Cu(II). We evaluated the specific activity of our complex.
We are currently conducting cell experiments using DA3 breast cancer cell line and HEK293 cell lines in hypoxic and normoxic conditions.

WP3: Developing new therapeutic approaches for copper dis-homeostasis disorders
WP 3.1 In silico design of biomimetic peptides that can increase the Cu(I) efflux rate.
On the basis of the structural information extracted from WP1, and as was mentioned in task 1.2.3 we designed two peptides that should interfere with the copper transfer mechanism. We are currently exploring their effect using in vitro experiments and cell experiments.
The mechanism of the copper cycle is explored using various biophysical, biochemical methods and all atoms simulations as well as MD simulations. We are mainly using magnetic resonance techniques, but we combines it with CD, various biochemical methods, and UV-Vis spectroscopy. Based on molecular level understanding, we designed a radioactive compound for PET imaging. We built a radioactive lab in our campus (including gamma counter (PerkinElmer)), we are allowed to perform cell experiments with 64Cu(II) till 600 microCi radiation. The synthesis of the radioactive complex is done in hot labs nearby – Isotopia. We are renting the facility per day, and our staff worked there for the synthesis of the complex. Then we transferred the complex in shielded syringes to BIU campus for cell experiments.
The design of the peptides (WP3) were done in silico using all atoms simulations.
Our project is highly multidisciplinary, where all experiments are performed by our group members and in house. From the molecular level understanding of the biological pathway, towards development of novel diagnostic and therapeutic compounds for cancer and neurological diseases. This is very novel and non-conventional for a research group to gain expertise in various biophysical and computational methods, through cell biology experiments, and nuclear medicine.
We are hoping that by the end of the project, we will be able to start animals experiments with our designed radioactive materials, and peptides that affect copper metabolism.