Terpyridine Glycoconjugates and Their Metal Complexes: Antiproliferative Activity and Proteasome Inhibition.

Metal terpyridine complexes have gained substantial interest in many application fields, such as catalysis and supramolecular chemistry. In recent years, the biological activity of terpyridine and its metal complexes has aroused considerable regard. On this basis, we synthesised new terpyridine derivatives of trehalose and glucose to improve the water solubility of terpyridine ligands and target them in cancer cells through glucose transporters. Glucose derivative and its copper(II) and iron(II) complexes showed antiproliferative activity. Interestingly, trehalose residue reduced the cytotoxicity of terpyridine. Moreover, we tested the ability of parent terpyridine ligands and their copper complexes to inhibit proteasome activity as an antineoplastic mechanism.

Fraisinib: a calixpyrrole derivative reducing A549 cell-derived NSCLC tumor in vivo acts as a ligand of the glycine-tRNA synthase, a new molecular target in oncology.

Background and purpose: Lung cancer is the leading cause of death in both men and women, constituting a major public health problem worldwide. Non-small-cell lung cancer accounts for 85%-90% of all lung cancers. We propose a compound that successfully fights tumor growth in vivo by targeting the enzyme GARS1. Experimental approach: We present an in-depth investigation of the mechanism through which Fraisinib [meso-(p-acetamidophenyl)-calix(4)pyrrole] affects the human lung adenocarcinoma A549 cell line. In a xenografted model of non-small-cell lung cancer, Fraisinib was found to reduce tumor mass volume without affecting the vital parameters or body weight of mice. Through a computational approach, we uncovered that glycyl-tRNA synthetase is its molecular target. Differential proteomics analysis further confirmed that pathways regulated by Fraisinib are consistent with glycyl-tRNA synthetase inhibition. Key results: Fraisinib displays a strong anti-tumoral potential coupled with limited toxicity in mice. Glycyl-tRNA synthetase has been identified and validated as a protein target of this compound. By inhibiting GARS1, Fraisinib modulates different key biological processes involved in tumoral growth, aggressiveness, and invasiveness. Conclusion and implications: The overall results indicate that Fraisinib is a powerful inhibitor of non-small-cell lung cancer growth by exerting its action on the enzyme GARS1 while displaying marginal toxicity in animal models. Together with the proven ability of this compound to cross the blood-brain barrier, we can assess that Fraisinib can kill two birds with one stone: targeting the primary tumor and its metastases "in one shot." Taken together, we suggest that inhibiting GARS1 expression and/or GARS1 enzymatic activity may be innovative molecular targets for cancer treatment.

Dipyridamole for tracking amyloidogenic proteins aggregation and enhancing polyubiquitination.

Dipyridamole is currently used as a medication that inhibits blood clot formation and it is also investigated in the context of neurodegenerative and other amyloid related diseases. Here, we propose this molecule as a new diagnostic tool to follow the aggregation properties of three different amyloidogenic proteins tested (insulin, amylin and amyloid ß peptide 1-40). Results show that dipyridamole is sensitive to early stage amyloid formation undetected by thioflavin T, giving a different response for the aggregation of the three different proteins. In addition, we show that dipyridamole is also able to enhance ubiquitin chain growth, paving the way to its potential application as therapeutic agent in neurodegenerative diseases.

Pyrazolones Activate the Proteasome by Gating Mechanisms and Protect Neuronal Cells from ß-Amyloid Toxicity.

Proteasome malfunction parallels abnormal amyloid accumulation in Alzheimer's Disease (AD). Here we scrutinize a small library of pyrazolones by assaying their ability to enhance proteasome activity and protect neuronal cells from amyloid toxicity. Tube tests evidenced that aminopyrine and nifenazone behave as 20S proteasome activators. Enzyme assays carried out on an "open gate" mutant (α3ΔN) proteasome demonstrated that aminopyrine activates proteasome through binding the α-ring surfaces and influencing gating dynamics. Docking studies coupled with STD-NMR experiments showed that H-bonds and π-π stacking interactions between pyrazolones and the enzyme play a key role in bridging α1 to α2 and, alternatively, α5 to α6 subunits of the outer α-ring. Aminopyrine and nifenazone exhibit neurotrophic properties and protect differentiated human neuroblastoma SH-SY5Y cells from ß-amyloid (Aß) toxicity. ESI-MS studies confirmed that aminopyrine enhances Aß degradation by proteasome in a dose-dependent manner. Our results suggest that some pyrazolones and, in particular, aminopyrine are promising compounds for the development of proteasome activators for AD treatment.

Amino- and chloro-8-hydroxyquinolines and their copper complexes as proteasome inhibitors and antiproliferative agents.

Proliferation and programmed cell death are tightly correlated with the ubiquitin-proteasome system (UPS). Alterations in the UPS may be implicated in pathological conditions such as the proteasome over-activity in cancer cells. Mounting evidence indicates that many types of actively proliferating malignant cells are more sensitive to proteasome inhibition than normal cells, and therefore UPS inhibitors are actively pursued as anticancer agents. The approval of the proteasome inhibitor drug bortezomib for the treatment of myeloma and lymphoma further highlights the need for UPS inhibitors. Recent studies have suggested that clioquinol and 5-amino-8-hydroxyquinoline can inhibit proteasome activity and induce apoptosis in human cancer cells. As for clioquinol, a copper-dependent and -independent mechanism has been proposed to explain the inhibition of the proteasome whereas the activity of 5-amino-8-hydroxyquinoline has not been explored in the presence of copper(ii) ions. Herein, we investigated the biological activity of some 8-hydroxyquinolines by using human ovarian (A2780) and lung (A549) cancer cells. The effect of copper(ii) on the activity of these compounds was also evaluated. The investigated systems inhibit the chymotrypsin-like activity of the proteasome and induce growth inhibition and apoptosis in a concentration-dependent manner. Copper(ii) ions increase the activity of 8-hydroxyquinoline derivatives except in the case of 5-amino-8-hydroxyquinoline. This study suggests the great potential of amino- and chloro-8-hydroxyquinolines as anticancer agents. Furthermore, it clarifies some aspects concerning the activity of 5-amino-8-hydroxyquinoline, which has been previously proposed as a proteasome inhibitor capable of overcoming resistance to bortezomib.

New comprehensive studies of a gold(III) Dithiocarbamate complex with proven anticancer properties: Aqueous dissolution with cyclodextrins, pharmacokinetics and upstream inhibition of the ubiquitin-proteasome pathway.

The gold(III)-dithiocarbamate complex AuL12 (dibromo [ethyl-N-(dithiocarboxy-kS,kS')-N-methylglycinate] gold(III)), is endowed with promising in vitro/in vivo antitumor activity and toxicological profile. Here, we report our recent strategies to improve its water solubility and stability under physiological conditions along with our efforts for unravelling its tangled mechanism of action. We used three types of α-cyclodextrins (CDs), namely ß-CD, Me-ß-CD and HP-ß-CD to prepare aqueous solutions of AuL12. The ability of these natural oligosaccharide carriers to enhance water solubility of hydrophobic compounds, allowed drug stability of AuL12 to be investigated. Moreover, pharmacokinetic experiments were first carried out for a gold(III) coordination compound, after i.v. injection of the nanoformulation AuL12/HP-ß-CD to female mice. The gold content in the blood samples was detected at scheduled times by AAS (atomic absorption spectrometry) analysis, highlighting a fast biodistribution with a tß1/2 of few minutes and a slow escretion (tα1/2 of 14.3 h). The in vitro cytotoxic activity of AuL12 was compared with the AuL12/HP-ß-CD mixture against a panel of three human tumor cell lines (i.e., HeLa, KB and MCF7). Concerning the mechanism of action, we previously reported the proteasome-inhibitory activity of some our gold(III)-based compounds. In this work, we moved from the proteasome target to upstream of the important ubiquitin-proteasome pathway, testing the effects of AuL12 on the polyubiquitination reactions involving the Ub-activating (E1) and -conjugating (E2) enzymes.

Ubiquitin Associates with the N-Terminal Domain of Nerve Growth Factor: The Role of Copper(II) Ions.

Many biochemical pathways involving nerve growth factor (NGF), a neurotrophin with copper(II) binding abilities, are regulated by the ubiquitin (Ub) proteasome system. However, whether NGF binds Ub and the role played by copper(II) ions in modulating their interactions have not yet been investigated. Herein NMR spectroscopy, circular dichroism, ESI-MS, and titration calorimetry are employed to characterize the interactions of NGF with Ub. NGF1-14 , which is a short model peptide encompassing the first 14 N-terminal residues of NGF, binds the copper-binding regions of Ub (KD =8.6 10-5 m). Moreover, the peptide undergoes a random coil-polyproline type II helix structural conversion upon binding to Ub. Notably, copper(II) ions inhibit NGF1-14 /Ub interactions. Further experiments performed with the full-length NGF confirmed the existence of a copper(II)-dependent association between Ub and NGF and indicated that the N-terminal domain of NGF was a valuable paradigm that recapitulated many traits of the full-length protein.

Copper(II) ions affect the gating dynamics of the 20S proteasome: a molecular and in cell study.

Due to their altered metabolism cancer cells are more sensitive to proteasome inhibition or changes of copper levels than normal cells. Thus, the development of copper complexes endowed with proteasome inhibition features has emerged as a promising anticancer strategy. However, limited information is available about the exact mechanism by which copper inhibits proteasome. Here we show that Cu(II) ions simultaneously inhibit the three peptidase activities of isolated 20S proteasomes with potencies (IC50) in the micromolar range. Cu(II) ions, in cell-free conditions, neither catalyze red-ox reactions nor disrupt the assembly of the 20S proteasome but, rather, promote conformational changes associated to impaired channel gating. Notably, HeLa cells grown in a Cu(II)-supplemented medium exhibit decreased proteasome activity. This effect, however, was attenuated in the presence of an antioxidant. Our results suggest that if, on one hand, Cu(II)-inhibited 20S activities may be associated to conformational changes that favor the closed state of the core particle, on the other hand the complex effect induced by Cu(II) ions in cancer cells is the result of several concurring events including ROS-mediated proteasome flooding, and disassembly of the 26S proteasome into its 20S and 19S components.

Semax, an ACTH4-10 peptide analog with high affinity for copper(II) ion and protective ability against metal induced cell toxicity.

Heptapeptide Semax, encompassing the sequence 4-7 of N-terminal domain of the adrenocorticotropic hormone (ACTH) and a C-terminal Pro-Gly-Pro tripeptide, belongs to a short regulatory peptides family. This compound has been found to affect learning processes and to exert marked neuroprotective activities on cognitive brain functions. Dys-homeostasis of metal ions is involved in several neurodegenerative disorders and growing evidences have showed that brain is a specialized organ able to concentrate metal ions. In this work, the metal binding ability and protective activity of Semax and its metal complexes were studied. The equilibrium study clearly demonstrated the presence of three complex species. Two minor species [CuL] and [CuLH-1]- co-exist together with the [CuLH-2]2- in the pH range from 3.6 to 5. From pH5 the [CuLH-2]2- species becomes predominant with the donor atoms around copper arranged in a 4N planar coordination mode. Noteworthy, a reduced copper induced cytotoxicity was observed in the presence of Semax by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay on a SHSY5Y neuroblastoma and RBE4 endothelial cell lines.

New conjugates of superoxide dismutase/catalase mimetics with cyclodestrins.

Mimetics of antioxidant enzymes such as superoxide dismutases (SOD) or catalases are reported as potential new drugs able to reduce oxidative stress damage. In particular, manganese(III) complexes of salen-type ligands have been studied as both SOD and catalase mimetics. In this paper, we report the synthesis of two novel conjugates of salen-type ligands with the beta-cyclodextrin, the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N'-bis(salicylidene))]-beta-cyclodextrin and the 6-deoxy-6-[(S-cysteamidopropyl(1,2-diamino)N,N'-bis(3-methoxysalicylidene))]-beta-cyclodextrin, their spectroscopic characterization, and the synthesis and the characterization of their manganese(III) complexes. The SOD-like activity of the metal complexes was investigated by the indirect Fridovich method. The catalase like activity was tested using a Clark-type oxygen electrode. The peroxidase activity was tested using the ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)) assay. The glycoconjugation of salen-manganese(III) complexes yields compounds with enhanced SOD activity. These complexes also show catalase and peroxidase activities higher than the simple salen complexes (EUK 113 and EUK 108).