Anti-cancer chemotherapeutics in target therapy: from advanced preclinical studies on promising Au(III) peptidomimetics toward the design of new receptor-recognizable metal-based agents

At present, though there is no clinically available antineoplastic drug that acts selectively on the tumor tissue, chemotherapy is still the main form of treatment for cancer. To date, platinum-based drugs, whose forerunner is cisplatin, are among the most employed chemotherapeutics in spite of their treatment-related toxicity. Starting from the chemical properties of platinum and the severe toxicological profile of the drug cisplatin, novel Au(III)-dithiocarbamato complexes have been recently designed and biologically studied, showing particularly promising antitumor activity (both in vitro and in vivo), lack of cross-resistance, and reduced toxic and nephrotoxic side-effects compared to cisplatin, accounting for a different mechanism of action. Following these encouraging results, we aimed to exploit the peculiar features of the tumor microenvironment in order to obtain a targeted delivery of our potential drugs into cancer tissues, resulting in further effectiveness increase and side-effects reduction. Accordingly, for a site-specific delivery strategy, we planned to functionalize the dithiocarbamato ligand moiety by means of biomolecules that are internalized into the pathological cells upon binding to a specific cell membrane transporter or a receptor, upregulated compared to healthy cells. With respect to human transporters, Au(III)-dithiocarbamato complexes functionalized with di- and tripeptides (our second-generation compounds), designed to exploit the peptide transporters PEPT1 and PEPT2, have been recently synthetized and studied, turning out the filing of an international patent, just extended worldwide. On the other hand, in this work the receptor-mediated strategies have been taken into account as well (third-generation compounds). Concerning the second-generation complexes, preliminary screening tests on different human tumor cell lines indicated AuD6 and AuD8 as suitable candidates for advanced preclinical studies. We report here on their synthesis, chemical characterization and the biological data collected throughout a 6-month stay as a visiting PhD student at the Barbara Ann Karmanos Cancer Institute (Detroit, U.S.A.) in collaboration with Prof. Q. Ping Dou. Several advanced preclinical studies were carried out evaluating the anticancer activity both in vitro and in vivo, the acute toxicity on animal models and providing insights into the mechanism of action in order to enter phase I clinical trials. The triple-negative breast cancer cell line MDA-MB-231 was chosen as a model since these cells are highly metastatic and invasive. Notably, AuD6 and AuD8 overcame the in vitro resistance of cisplatin against this cell line, displayed outstanding anticancer activity on xenografts (subcutaneous administration) and very negligible acute toxicity in mice upon intravenous and per os administrations. In addition to showing a good chemotherapeutic index in terms of toxicity-activity ratio, these two compounds proved strong inhibitors of proteasome – a significant intracellular protein degradation machinery – both in vitro and in vivo. Therefore, a crystallographic study, aimed at investigating the mechanism of inhibition of 20S proteasome by AuD8 (chosen as a model compound), was performed by means of biotechnological methods. With respect to the pharmacokinetic profile of the considered compounds, serum albumin was taken into account as it is the most abundant plasma protein and it is a well-known carrier of drugs and endogenous substances such as vitamins and lipids. An investigation of the possible interaction between AuD8 and the serum protein was carried out by circular dichroism and fluorescence spectrophotometry, replacing the human serum albumin with the bovine counterpart since they have an almost identical sequence. Concerning our third-generation of complexes – designed to work as targeted bullets that could go straight to the cancer cells, bypassing normal ones – we focused on the cholecystokinin (CCK) receptors which belong to the G-protein-coupled receptor superfamily and are overexpressed in many neoplasms. The targeted drug delivery has been achieved by complexation of the metal center with the CCK8 peptide. Thus, the CCK8 peptide was first synthesized, purified and characterized by mass spectrometry, elemental analysis, NMR and FT-IR spectroscopies. The corresponding Au(III) dithiocarbamato derivative was then synthesized and partially characterized by 1H-NMR and UV-Vis spectrophotometry. Afterwards, we addressed our attention also to the “click chemistry” field, designing and developing a versatile gold(III)-based building block to be functionalized with different targeting peptides. The related overexpressed receptors can specifically recognize and bind the corresponding conjugates, thus intaking them into cytoplasm by mediated internalization. An international patent application is going to be filed about this innovative strategy. More recently, as a natural extension of our previous studies, we have also taken into account Cu(II), being an endogenous metal characterized by very interesting biochemical properties. Concerning the relationship between copper and cancer, it is worth highlighting that copper plays a significant role in angiogenesis and therefore is more abundant in cancer cells than in healthy ones, leading to a selectivity of copper-based anticancer agents toward neoplastic tissues. In the present work, two different classes of Cu(II) dithiocarbamato complexes were designed to gain insights into structure-activity relationships and to get the know-how with respect to the chemical behavior of this transition metal in terms of reaction conditions and stability in different solvents. Their synthesis, wide chemical characterization and preliminary biological data are herein reported. Overall, these findings will be useful for the future development of the more sophisticated copper-based receptor compounds