Synthesis and characterization of poly-phosphane coinage metals complexes and study on the protein ligation and catalysis

A series of coinage metals centered poly-phosphane complexes has been synthesized and characterized under photophysical, chemical and biochemical aspects also in combination with a protein. Poly-phosphane metal complexes possess many properties in the field of luminescence,[1] catalysis, [2] chemo sensing, [3] and of anticancer activity [4]. In this study phosphane ligands containing the carboxylic functional group in ortho or para position of PPh3 have been used. The introduction of this polar group has the double aim either to make more hydrophilic the complexes and to tune the binding ability of the phosphane. In the case of gold(I) complexes, the poly-phosphine compound have been studied in comparison with the corresponding [bis-triphenylphosphine-gold(I)chloride], where the carboxylic group is absent, to evaluate the influence of its presence in the photopysical properties as well as on the interaction with dihydrofolate reductase, a protein involved in cell proliferation, DNA duplication and many other biological functions [5]. Affinity constants have been estimated through quenching of fluorescence studies and inhibition constants have been evaluated through rate constant determination of the reduction of dihydrofolate (H2F) to tetrahydrofolate (H4F) with reduced nicotinamide adenide dinucleotide phosphate (NADPH) as hydride donor. The tests highlighted a catalytic activity of the gold(I) compounds versus the H2F, which is the substrate of the enzyme. A strong effect of the enzyme on the luminescence properties of the gold(I) complexes have been observed. A coinage metals homolog series have been also evaluated as antiproliferative agent by in vitro MTT tests. Scheme. Schematic view of a homolog series of coinage metals complexes under study. References 1. R. Edward, T. Tiekink, J. G. Kang, Coord. Chem. Rev. 2009, 253, 1627-1648 2. David J. Gorin, Benjamin D. Sherry, and F. Dean Toste, Chem. Rev. 2008, 108, 3351–3378 3. X. He, V. W. Yam, Coord. Chem. Rev. 2011, 2111-2123 4. R. Galassi, A.Burini, O. Camille Simon, A. Dolmella, D. Micozzi, S. Vincenzetti, S. Pucciarelli Dalton Trans., 2015, 44, 3043-3056 DOI: 10.1039/C4DT01542H 5

Azolate gold(I) phosphane complexes as innovative therapies for the treatment of HER2-driven breast cancer

Gold(I) compounds have been known as cytotoxic agents since 30 years ago1. Lastly, the inhibition activity studies on compounds (such as LAuL’, where L is a phosphane and L’ a co-ligand) led to the individuation of a likely molecular target2, renewing the interest on the field of these metallodrugs. In the design of active gold compounds, the proper hydro / lipophilic balancing provides the lowering of the overall toxicity, maintaining both a good cellular uptake and anticancer properties. Imidazoles and pyrazoles as co-ligands afford to gold(I) phosphane compounds having cytotoxic activity, but enough polarity to be soluble in physiological media. Different azolate gold(I)phosphane complexes have been synthesized. They contain substituents on imidazole or pyrazole ligands such as R = NO2, CF3, CN, Cl, CH2OH) or substituents such as COOH or COONHEt3 in the phosphane moiety. Some of them have been already tested as antitumoral in some panels of cancer cells, resulting active3. In this work we present the study of the cytotoxic effects of several gold(I) compounds and a natural compound on an in vitro model of HER2-overexpressing breast cancer. We tested the effectiveness of these compounds as potential anticancer agents on SKBR-3 cell line, a human breast cancer cell line that overexpresses the HER2 (Neu/ErbB-2) gene product4. These cells display an epithelial morphology in tissue culture and are a useful preclinical model to screen for new therapeutic agents which could overcome the drawback of resistance to HER2-targeted therapies5. In order to screen the cytotoxic activity of these new compounds on SKBR-3 cells we performed different cell viability assays. As conclusion we observed a detrimental effect on the cytotoxicity for those compounds having an ionic structure or highly hydrophilic polar substituents on the azolate or phosphane ligands and a remarkable activity for those compounds having the Ph3PAu+ moiety and substituted imidazolate as co-ligands. 1) Benoît Bertrand, and Angela Casini. Dalton Trans., 2014, 43, 4209. DOI: 10.1039/c3dt52524d 2) a) Peter J. Barnard, Susan J. Berners-Price. Coord. Chem. Rev. 2007, 251, 1889–1902. DOI:10.1016/j.ccr.2007.04.006. b) A. Bindoli, M. P. Rigobello, G. Scutari, C. Gabbiani, A. Casini, L. Messori, Coord. Chem. Rev., 2009, 253, 1692–1707. DOI: 10.1016/j.ccr.2009.02.026. 3) a) R. Galassi, A. Burini, S. Ricci, M. Pellei, M. P. Rigobello, A. Citta, A. Dolmella, V. Gandin, C Marzano. Dalton Trans., 2012, 41, 5307. DOI: 10.1039/c2dt11781a b) 4) Fogh J, Fogh JM, Orfeo T, 1977, One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst. , 59(1):221-6. DOI: 10.1016/j.bmcl.2013.11.058. 5) Saturnino C, Sirignano E, Botta A, Sinicropi MS, Caruso A, Pisano A, Lappano R, Maggiolini M, Longo P, 2014, New titanocene derivatives with high antiproliferative activity against breast cancer cells. Bioorg Med Chem Lett., 1;24(1):136-40. DOI: 10.1016/j.bmcl.2013.11.058

Azolate/phosphane Gold(I) compounds in antiproliferative therapy: a new frontier for the azolate gold(I) chemistry

Azolate/phosphane Gold(I) compounds in antiproliferative therapy: a new frontier for the azolate gold(I) chemistry Azolate gold(I) phosphane compounds have become good candidate for anticancer applications.[1] It was highlighted that azolate gold(I) phosphane compounds were mostly very active in the regards of many panel of cancer cells, in addition to cis-platin resistant cells. Moreover, inhibition studies of pivotal enzymes, such as the seleno dependent ThioredoxinaReductase (TrxR), and an enzyme involved in DNA synthesis such as DeHydroFolateReductase, were carried out highlighting in both cases IC50 ranging from nano- to micromolar scale, respectively.[1][2] In order to study the effectiveness of these new azolate gold(I) phosphane compounds as potential anticancer agents, and to understand in depth the Structure Activity Relationship (SAR) relationship, different cell viability assays (MTT assays) were performed on a human in vitro model of HER2-overexpressing breast cancer: SKBR-3 cells.[3] After this preliminary screening, the most promising and effective compounds were selected to extend the study on A17 cell line, a murine preclinical model of Basal Like Breast Cancer (BLBC).[4] Hence, their efficacy in suppressing BLBC growth in vivo was tested and IHC analysis on explanted tumors were carried on. Overall, in vitro assays demonstrated a remarkable activity for those compounds having the Ph3PAu+ moiety and substituted imidazolate as co-ligands. Concerning the in vivo study the compounds act significantly delaying tumor growth. Accordingly, IHC analysis revealed a remarkable anti-angiogenic activity associated with a lower expression of proliferative markers and a higher level of apoptotic markers in treated tumours in comparison with controls. Moreover, respect to cisplatin these compounds displayed a lower nephrotoxicity, although their liver toxicity was higher. These promising results open the way to further investigations in order to understand the mechanism of action of these new azolate gold (I) posphane complexes. Leave one line blank [1] R. Galassi et al., Dalton Trans., 2012, (41), pp 5307-5318. [2] R. Galassi et al., Dalton Trans., 2015, (44), pp 3043-3056. [3] Fogh, J., Fogh J. M., Orfeo T, J Natl Cancer Inst. , 1977, (59), pp 221-6; [4] M. Galiè et al., Carcinogenesis , 2005, (11), pp 1868-187

Personalized liposome–protein corona in the blood of breast, gastric and pancreatic cancer patients

When nanoparticles (NPs) are dispersed in a biofluid, they are covered by a protein corona the composition of which strongly depends on the protein source. Recent studies demonstrated that the type of disease has a crucial role in the protein composition of the NP corona with relevant implications on personalized medicine. Proteomic variations frequently occur in cancer with the consequence that the bio-identity of NPs in the blood of cancer patients may differ from that acquired after administration to healthy volunteers. In this study we investigated the correlation between alterations of plasma proteins in breast, gastric and pancreatic cancer and the biological identity of clinically approved AmBisome-like liposomes as determined by a combination of dynamic light scattering, zeta potential analysis, one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) and semi-quantitative densitometry. While size of liposome–protein complexes was not significantly different between cancer groups, the hard corona from pancreatic cancer patients was significantly less negatively charged. Of note, the hard corona from pancreatic cancer patients was more enriched than those of other cancer types this enrichment being most likely due to IgA and IgG with possible correlations with the autoantibodies productions in cancer. Given the strict relationship between tumor antigen-specific autoantibodies and early cancer detection, our results could be the basis for the development of novel nanoparticle-corona-based screening tests of cancer

IN VITRO AND IN VIVO STUDIES FOR THE TREATMENT OF BASAL LIKE BREAST CANCER (BLBC) WITH AZOLATE/PHOSPHANE GOLD(I) COMPOUNDS

IN VITRO AND IN VIVO STUDIES FOR THE TREATMENT OF BASAL LIKE BREAST CANCER (BLBC) WITH AZOLATE/PHOSPHANE GOLD(I) COMPOUNDS Rossana Galassia, Alfredo Burinia, Oumarou Camille Simona, Anna Teresa Ramadoria, Stefania Pucciarelli,b Albana Hisy,c Manuela Iezzi,c Valentina Gambini b, Martina Tiliob, Cristina Marchini b, Augusto Amicib a School of Science and Technology, Chemistry Division, Camerino University, Via Sant’ Agostino, I-62032 Italy. b Department of Biosciences and Veterinary, University of Camerino, Via Gentile III da Varano, I-62032, Italy c Aging Research Centre, G. d’Annunzio University, Chieti, 66100, Italy e-mail: [email protected] Breast cancer is a heterogeneous disease classified into molecular subtypes with distinctive gene expression signatures. Of all the molecular subtypes, BLBC has the worst negative outcome and prognosis. BLBCs are generally estrogen receptor (ER-) and progesterone receptor (PR)-negative and also lack high expression/amplification of HER2, limiting targeted therapeutic options. Thus, to date, Cisplatin remains the only possible therapeutic choice in the adjuvant or metastatic setting in the BLBC. Considering also several and serious side effects, new therapies are therefore an urgent unmet medical need for this patient population. Azolate gold(I) phosphane compounds have become good candidate for anticancer applications.[1] It was highlighted that azolate gold(I) phosphane compounds were mostly very active in the regards of many panel of cancer cells, in addition to cis-platin resistant cells. They show a mechanism of action involving the inhibition of seleno dependent ThioredoxinaReductase (TrxR), but they inhibit with IC50 in the micromolar scale also many other enzymes such as DeHydroFolateReductase.[1][2] In order to study the effectiveness of these new azolate gold(I) phosphane compounds as potential anticancer agents, different cell viability assays (MTT assays) were performed on a human in vitro model of HER2-overexpressing breast cancer: SKBR-3 cells.[3] After this preliminary screening, the most promising and effective compounds were selected to extend the study on A17 cell line, a murine preclinical model of Basal Like Breast Cancer (BLBC).[4] Hence, their efficacy in suppressing BLBC growth in vivo was tested and IHC analysis on explanted tumors were carried on. Overall, in vitro assays demonstrated a remarkable activity for those compounds having the Ph3PAu+ moiety and substituted imidazolate as co-ligands. Concerning the in vivo study the compounds act significantly delaying tumor growth. Accordingly, IHC analysis revealed a remarkable anti-angiogenic activity associated with a lower expression of proliferative markers and a higher level of apoptotic markers in treated tumours in comparison with controls. Moreover, respect to cisplatin these compounds displayed a lower nephrotoxicity, although their liver toxicity was higher. These promising results open the way to further investigations in order to understand the mechanism of action of these new azolate gold (I) posphane complexes. References [1] R. Galassi et al., Dalton Trans., 2012, (41), pp 5307-5318. [2] R. Galassi et al., Dalton Trans., 2015, (44), pp 3043-3056. [3] Fogh, J., Fogh J. M., Orfeo T, J Natl Cancer Inst. , 1977, (59), pp 221-6; [4] M. Galiè et al., Carcinogenesis , 2005, (11), pp 1868-187

The water soluble ruthenium(II) organometallic compound[Ru(p-cymene)(bis(3,5 dimethylpyrazol-1-yl)methane)Cl]Clsuppresses triple negative breast cancer growth by inhibiting tumorinfiltration of regulatory T cells

Ruthenium compounds have become promising alternatives to platinum drugs by displaying specific activities against different cancers and favorable toxicity and clearance properties. Here, we show that the ruthenium(II) complex [Ru(p-cymene)(bis(3,5-dimethylpyrazol-1-yl)methane)Cl]Cl (UNICAM-1) exhibits potent in vivo antitumor effects. When administered as four-dose course, by repeating a single dose (52.4 mg kg-1) every three days, UNICAM-1 significantly reduces the growth of A17 triple negative breast cancer cells transplanted into FVB syngeneic mice. Pharmacokinetic studies indicate that UNICAM-1 is rapidly eliminated from kidney, liver and bloodstream thanks to its high hydrosolubility, exerting excellent therapeutic activity with minimal side effects. Immunohistological analysis revealed that the efficacy of UNICAM-1, mainly relies on its capacity to reverse tumor-associated immune suppression by significantly reducing the number of tumor-infiltrating regulatory T cells. Therefore, UNICAM-1 appears very promising for the treatment of TNBC. © 2016 Elsevier Ltd. All rights reserve

In vivo protein corona patterns of lipid nanoparticles

ct In physiological environments (e.g. the blood), nanoparticles (NPs) are surrounded by a layer of biomolecules referred to as a 'protein corona' (PC). The most tightly NP-bound proteins form the so-called hard corona (HC), the key bio-entity that determines the NP's biological identity and physiological response. To date, NP-HC has been almost exclusively characterized in vitro, while NP-protein interactions under realistic in vivo conditions remain largely unexplored. In this study, we thoroughly characterized the in vivo HC of a NP formulation that forms around lipid nanoparticles with a lipid composition equal to that of clinically used liposomal amphotericin B (AmBisome®) after the recovery of the NPs from the blood circulation of FVB/N mice 10 minutes post intravenous administration. In vitro HC formed by 10 minutes incubation of NPs in FVB/N mouse plasma was used for comparison. Here we show that the biological identity (i.e. size, zeta-potential and aggregation state) of NPs in vivo is significantly different from that acquired in vitro. Furthermore, the variety of protein species in the in vivo HC was considerably larger. The present work has demonstrated that characterization of the in vivo HC is essential to provide an accurate molecular description of the biological identity of NPs in physiological environments

Phage-Based Anti-HER2 Vaccination Can Circumvent Immune Tolerance against Breast Cancer

D16HER2 is a splice variant of HER2 and defined as the transforming isoform in HER2-positive breast cancer. It has been shown that D16HER2 promotes breast cancer aggressiveness and drug resistance. In the present work, we used in silico modeling to identify structural differences between D16HER2 and the wild-type HER2 proteins. We then developed DNA vaccines specifically against the D16HER2 isoform and showed that these immunotherapies hampered carcinogenesis in a breast cancer transplantable model. However, the vaccines failed to elicit immune protection in D16HER2 transgenic mice because of tolerogenic mechanisms toward the human HER2 self-antigen, a scenario commonly seen in HER2þ patients. Thus, we engineered bacteriophages with immunogenic epitopes of D16HER2 exposed on their coat for use as anticancer vaccines. These phage-based vaccines were able to break immune tolerance, triggering a protective anti-D16HER2 humoral response. These findings provide a rationale for the use of phage-based anti-HER2/D16HER2 vaccination as a safe and efficacious immunotherapy against HER2- positive breast cancers. Cancer Immunol Res; 6(12); 1486–98

Irreversible inhibition of Δ16HER2 is necessary to suppress Δ16HER2- positive breast carcinomas resistant to Lapatinib

HER2 tyrosine kinase receptor is a validated target in breast cancer therapy. However, increasing evidence points to a major role of Δ16HER2 splice variant commonly coexpressed with HER2 and identified as a clinically important HER2 molecular alteration promoting aggressive metastatic breast cancer. Consistently, mice transgenic for the human Δ16HER2 isoform (Δ16HER2 mice) develop invasive mammary carcinomas with early onset and 100% penetrance. The present study provides preclinical evidence that Δ16HER2 expression confers de novo resistance to standard anti-HER2-therapies such as Lapatinib and acquired resistance to the selective Src inhibitor Saracatinib in breast cancer. Of note, Dacomitinib, an irreversible small molecule pan-HER inhibitor, was able to completely suppress Δ16HER2-driven breast carcinogenesis. Thus, only Dacomitinib may offer benefit in this molecularly defined patient subset by irreversibly inhibiting Δ16HER2 activation

Irreversible inhibition of Δ16HER2 is necessary to suppress Δ16HER2-positive breast carcinomas resistant to Lapatinib

HER2 tyrosine kinase receptor is a validated target in breast cancer therapy. However, increasing evidence points to a major role of Δ16HER2 splice variant commonly coexpressed with HER2 and identified as a clinically important HER2 molecular alteration promoting aggressive metastatic breast cancer. Consistently, mice transgenic for the human Δ16HER2 isoform (Δ16HER2 mice) develop invasive mammary carcinomas with early onset and 100% penetrance. The present study provides preclinical evidence that Δ16HER2 expression confers de novo resistance to standard anti-HER2-therapies such as Lapatinib and acquired resistance to the selective Src inhibitor Saracatinib in breast cancer. Of note, Dacomitinib, an irreversible small molecule pan-HER inhibitor, was able to completely suppress Δ16HER2-driven breast carcinogenesis. Thus, only Dacomitinib may offer benefit in this molecularly defined patient subset by irreversibly inhibiting Δ16HER2 activation