During the last 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 cancer (WP3).
The specific achievements are described below, according to each WP:
WP 1.1: Resolving the Ctr1-Atox1-ATP7B cycle
This WP was divided into three parts: the first one is understanding the effect of copper entry to the cell through Ctr1 and reduction mechanisms. Here, we resolved the Cu(II) and Cu(I) binding sites to hCtr1, and analyzed in detail the reduction mechanism of Cu(II) to Cu(I). We succeeded to express and purify the full Ctr1 using insect cell expression system. EPR and UV-Vis results showed that up to two Cu(II) ions and five Cu(I) ions can coordinate to Ctr1 monomer. We also showed that Ctr1 undergoes major conformational changes while binding to Cu(I) ions.
In the second part, we explored the copper transfer mechaism from Ctr1 to Atox1. Using EPR measurements and calculations we showed that Atox1 can accommodate various conformations, while it can accommodate a specific conformation depending on its interacting partner. We also revealed that while interacting with Ctr1, Atox1 is in its dimer form, while binding to MBDs in ATP7B, Atox1 breaks to monomers. 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. We also explored the Cu(I) binding site using EPR and MD calculations. In the last part, we looked in detail on the transfer mechasim from Atox1 to ATP7B .
WP 1.2: Targeting conditions that can affect or alter the Cu(I) cycle through Ctr1-Atox1-ATP7B transfer
In this WP, we explored factors that can interrupt with the copper cycle, such as:
(i) Competitive metal ions. We have recently summarized the results on the binding of diamagnetic metal ions to proteins in: Meron, S. et al.; Magnetochemistry, 2022, 8, 3.
(ii) Cu(II) chelators, to check their effect on the Cu(I) transfer and efflux rate. Herein, we showed that ATSM and Cu(II) does not bind to proteins that are involved in the copper cycle.
(iii) Methionine and cysteine based motifs which are identified as general binding sites for Cu(I) ions. Using in silico calculations, we designed two peptides that can interfere with copper metabolism in cancer.
WP2: Designing new biomarkers for hypoxic cells
The knowledge gained in WP1 assistes us in designing the 64Cu(II) radiotracer for diagnostic of hypoxic tissues. Our Cu(II) based complex is composed of three parts : Cu(II), ligand and peptide. Two lead compounds were chosen.
We performed stability tests, and explored the mechanisms of action of these compounds. 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. Moreover, western blot together with radioactive experiments showed that the uptake of the radiotracer by the cell is depndent on Ctr1 concentration. We isotope labelled our complexes with 64Cu(II) and evaluated its specific activity. We performed radioactive experiments in various cell types: Breast, liver, ovarian, prostate, bone. We also compared our uptake radiotracer with Cu-ATSM.
WP3: Developing new therapeutic approaches for copper dis-homeostasis disorders. In silico design of biomimetic peptides that can affect copper metabolism in cancer were designed and tested in various cancer types.
