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Tesi di Laurea Magistrale in Scienze Chimiche di Federico Pisanu

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

La presente invenzione attiene una nuova famiglia di composti isotiocianati e isoselencianati idonei ad essere utilizzati come: - farmaci innovativi con spiccato profilo di efficacia terapeutica, tali da consentire un miglior approccio alla patologia tumorale rispetto ai farmaci attuali, - teranostici per la contemporanea diagnosi e cura della patologia oncologica; - sonde fluorescenti per l'osservazione diretta di specifici organuli cellulari in applicazioni biomediche.

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Tesi di Laurea Triennale in Chimica di Federico Pisanu

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Tesi di laurea quinquiennale in Chimica e Tecnologie Farmaceutiche

Berberine is a natural isoquinoline alkaloid with significant antitumor activity against many types of cancer cells, including ovarian tumors. This study investigated the molecular mechanisms by which berberine differently affects cell growth of cisplatin (cDDP)-sensitive and -resistant and polyamine analogue cross-resistant human ovarian cancer cells. The results show that berberine suppresses the growth of cDDP-resistant cells more than the sensitive counterparts, by interfering with the expression of folate cycle enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS). In addition, the impairment of the folate cycle also seems partly ascribable to a reduced accumulation of folate, a vitamin which plays an essential role in the biosynthesis of nucleic acids and amino acids. This effect was observed in both lines, but especially in the resistant cells, correlating again with the reduced tolerance to this isoquinoline alkaloid. The data also indicate that berberine inhibits cellular growth by affecting polyamine metabolism, in particular through the upregulation of the key catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT). In this regard, berberine is shown to stimulate the SSAT induction by the spermine analogue N1, N12 bisethylspermine (BESpm), which alone was also able to downregulate DHFR mRNA more than TS mRNA. We report that the sensitivity of resistant cells to cisplatin or to BESpm is reverted to the levels of sensitive cells by the co-treatment with berberine. These data confirm the intimate inter-relationships between folate cycle and polyamine pathways and suggest that this isoquinoline plant alkaloid could be a useful adjuvant therapeutic agent in the treatment of ovarian carcinoma.

It is well known that Hsp90 chaperone protein contributes in stabilizing oncoproteins over-expressed in malignant cell lines, playing a key role in survival, proliferation, invasion, metastasis and angiogenesis, which represent the hallmark traits of the cancer.1 In the last decade Hsp90 has emerged as a possible therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. TRAP 1 (Tumor Necrosis Factor-Associated Protein ), the mitochondrial isoform of Hsp90, is a component of a mitochondrial pathway selectively up-regulated in tumor cells which antagonizes the proapoptotic activity of cyclophilin D, a mitochondrial permeability transition pore regulator, and is responsible together with Hsp90, for the maintenance of mitochondrial integrity, thus favoring cell survival. Interestingly, novel TRAP1 antagonists cause sudden collapse of mitochondrial function and selective tumor cell death, suggesting that this pathway may represent a novel molecular target to improve anticancer therapy. A number of derivatives from structures of known HSP90 inhibitors, previously synthesized by our research group, has been modified to direct them into mitochondria. The idea for a mitochondrial targeting of TRAP1 inhibitors is based on the assumption of the negative potential on the matrix face of the inner mitochondrial membrane. Thus, for a first synthetic approach we have decided to linking lipophilic cations to the Hsp90 molecule inhibitors, in order to give access to cationic drugs to internalize into the negative organelles: on the basis of literature data we selected different groups as the polyamines, which protonated at physiological pH, have the optimal characteristic to pass through the mitochondria membrane and internalize into the organelle. Alternatively a group of triphenylphosphonium salts derivatives as well as of pyridinium and guanidinium ones were also considered as cationic heads for targeting TRAP1 inhibitors into mitochondria. The preliminary biological results show that some Hsp90 inhibitors proved also of interest as TRAP1 ATPase inhibitors (IC50 0.4micromolar) and that some of these have the capacity to accumulate into mitochondrial compartment. A second synthetic approach, however, has been realized by acting directly on the pharmacophore, i.e. on the structure of resorcinol, making changes on some sites essential for the binding of Hsp90, in order to act, and then selectively inhibit TRAP1. In this case, starting from some biological activity of molecule inhibitors of cytosolic Hsp90, we have made changes to see if the interaction between them and the receptor site of TRAP1 can be improved. Biological assays of this second class of compounds are still in progress.

Dottorato di Ricerca in Biochimica Cellulare ed Attività dei Farmaci in Oncologia, XXVII Ciclo, a.a. 2014 Università della Calabria

A route for the preparation of carborane-containing glucuronic acid derivatives is described, starting from 1,2-O-isopropylidene-α-d- glucuronolactone. The obtained compounds are potential BNCT agents ready to be tested for their uptake in selected tumor cell lines. © 2004 Elsevier Ltd. All rights reserved.

2011 - 2012, The cell membrane functions as a platform for the assembly of many signal transduction pathways and provides an additional level of regulation in cell signaling networks. The complex dynamic structure of the plasma membrane allows lipid–lipid and lipid–protein interactions, as well as the interaction of lipid–protein complexes with the submembrane cytoskeleton. The existence of membrane microdomains adds further complexity to such interactions, as well as the messages propagated in cells through G proteins and other non-permanent (extrinsic) membrane proteins. Each G protein can simultaneously bear a myristoyl, palmitoyl and isoprenyl moiety and therefore, many lipid molecules associated with G proteins may arise in G protein coupled receptor (GPCR)-rich membrane microdomains. These lipids can regulate the biophysical properties of membranes, which in turn modulate the interaction and activity of G proteins. The aim of the first part of my work was to understand the effect of these moieties on membrane structure and G protein-membrane interactions. Although recent studies found that the Gβγ dimer drives the interaction of G-proteins with nonlamellar-prone membranes, little is known about the molecular basis of this interaction. For this reason, I also investigated the interaction of the C-terminus of the Gγ protein with model membranes with or without the isoprenyl moieties. From the very beginning of my work, my studies have focused on the interaction of saturated and unsaturated fatty acids with cell membranes. Many of these molecules, in fact, have proven to be active against certain types of cancers and other diseases. Among all these molecules it is important to remember the Minerval, an analogue of oleic acid developed in the laboratory of Prof. Escribà, which has led to a reduction of up to 80% in the development of lung cancer and glioma cells. A promising molecule with high anti-inflammatory activity and synthesized by the group of Prof. Escribà like the previous one, is a modification of arachidonic acid: the 2-hydroxy arachidonic acid (AAOH). Due to the similarity with arachidonic acid (AA), that is the natural substrate of cyclooxygenase (COX), the second part of my work consisted in the investigation of the interaction of AAOH with COX-1 and COX-2. The results, in terms of free energy of binding and the Fukui function, demonstrated the potential of AAOH as non-toxic non-steroidal anti-inflammatory drug (NSAID). Recent findings pointed unambiguously to membranes as additional cellular sensors in activating a stress protein response, from prokaryotes to mammalian cells, at the beginning of temperature rise or other stresses. Aging or pathophysiological conditions can also be linked to the development of subtle membrane changes or “membrane-defects”, responsible for a dysregulated expression of heat shock proteins (HSPs). Chaperone co-inducers, among which can be mentioned the hydroxylamines such as BGP-15 and NG-094, are substances that cannot induce HSPs by itself, but can enhance HSP induction in combination with other mild stresses. A chaperone co-inducer also has the ability to lower the temperature threshold of the heat shock response and may provide suitable therapeutic candidates for many disease states since they are capable of affecting stressed rather than unstressed cells. The third part of my work consisted in molecular dynamics investigations of BGP-15 and NG-094 on membranes made of sphingomyelin and cholesterol at different composition to understand some aspects of membrane functioning. BGP-15 and NG-094 can induce an alteration in membrane’s fluidity similar to those induced by heat and a strong reorganization of sphingomyelin headgroups with an increased penetration of water. Taken together, all the results suggested that hydroxylamines have a strong effect on microdomains reorganization showing a great potential to become a new class of pharmaceuticals to combat most various protein-misfolding diseases and aging. All these investigations are relevant in the context of the physiology of cells, whose alterations may lead to pathologies whose treatment could be addressed by modifying membrane lipid composition and structure through so-called “Membrane-lipid therapy”. [edited by author], XI n.s.

VEICOLAZIONE DI ANTITUMORALI PER IL TRATTAMENTO DI TUMORI EPATICI

Presso il Centro di Eccellenza in Biocristallografia dell'Università di Trieste si stanno sviluppando diversi tipi di bioconiugati della vitamina B12 che permettono l'utilizzo della proteina naturale transcobalammina come "cavallo di Troia" per trasportare all'interno della cellula farmaci antitumorali o diagnostici.

The synthesis of new substituted E-3-(3-indolylmethylene)1,3-dihydroindol-2-ones is reported. The antitumor activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. Structure-activity relationships are discussed. The action of selected compounds was investigated in MCF-7 breast cancer cells. The ability of these derivatives to inhibit cellular proliferation was accompanied by increased level of p53 and its transcriptional targets p21 and Bax, interference in the cell cycle progression with cell accumulation in the G2/M phase, and lastly activation of apoptosis.

This paper reports the synthesis of compounds formed by two indole systems separated by a heterocycle (pyridine or piperazine). As a primary screening, the new compounds were submitted to the National Cancer Institute for evaluation of antitumor activity in the human cell line screen. The pyridine derivatives were far more active than the piperazine derivatives. For the study of the mechanism of action, the most active compounds were subjected to COMPARE analysis and to further biological tests including proteasome inhibition and inhibition of plasma membrane electron transport. The compound bearing the 5-methoxy-2-indolinone moiety was subjected to the first in vivo experiment (hollow fiber assay) and was active. It was therefore selected for the second in vivo experiment (human tumor xenograft in mice). In conclusion we demonstrated that this approach was successful, since some of the compounds described are much more active than the numerous, so far prepared and tested 3-indolylmethylene-2-indolinones.

The synthesis of new 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones and 3-(5-imidazo[2,1-b]thiadiazolylmethylene)-2-indolinones is reported. The antitumor activity was evaluated according to the protocols available at the National Cancer Institute (NCI), Bethesda, MD. To investigate the mechanism of action of the most potent antitumor agent of this series, its effect on growth of HT-29 colon carcinoma cells was studied. Its ability to inhibit cellular proliferation was mediated by cell cycle arrest at the G2/M phase, accompanied by inhibition of ornithine decarboxylase (ODC), the limiting enzyme of polyamine synthesis, and followed by induction of apoptosis.

Le convolutamidine A-E sono alcaloidi isolati dal briozoo marino Amathia convoluta nel 1995. Questi composti hanno uno scheletro comune costituito da un 4,6-dibromo-3-idrossioxindolo e contengono un stereocentro quaternario sul C-3, ma differiscono per la catena laterale legata al C-3. Qui di seguito è riportata la sintesi della (R)-convolutamidina A, ottenuta per addizione enantioselettiva di acetone alla 4,6 dibromoisatina. Questa metodologia sintetica è stata messa a punto per l’addizione enantioselettiva di acetone all’isatina e costituisce il primo esempio di reazione di questo tipo.

cis-2-Deoxy-2-acetamido glycosyl isocyanides are shown to be excellent precursors for the rapid preparation of bicyclic pyranoid glycosyl ureas, exploiting, in this case, an anomalous nucleophilic reactivity of the acetamido group. © Georg Thieme Verlag Stuttgart.

Il DNA è uno dei target più studiati verso il quale possono venire indirizzati agenti dotati di attività antitumorale. Per il metabolismo cellulare e la stabilità del DNA, risulta importante l’omeostasi di poliammine naturali come putrescina e spermina. Applicando la teoria del “templato universale”1 alla struttura di MDL 276952, composto a struttura poliamminica dotato di attività antiproliferativa, abbiamo progettato poliammine a potenziale attività antitumorale, sostituendo la catena centrale diamminoalchilica con nuclei aromatici presenti in numerosi agenti intercalatori, nell’intento di ottenere derivati dotati di buona affinità per il DNA e capaci inoltre di intercalarsi tra le sue due eliche. Una valutazione preliminare dei derivati sintetizzati è stata effettuata in vitro in linee cellulari di carcinoma mammario (SKBR3), della prostata (PC3) e di leucemia (CEM).

The design and synthesis of anticancer E-3-(3,4,5-trimethoxybenzylidene)-1,3-dihydroindol-2-ones is reported. Strong COMPARE correlations among the cell line responses suggest that these compounds may be acting similarly through a combination of different mechanisms of action. The 5-methoxy derivative (2h) was the most active compound with a mean pGI50 of 6.34, and it is now under review by Biological Evaluation Committee of the National Cancer Institute for possible further studies.

The new water soluble ruthenium complexes [(C5R5)RuCl(PTA)2] (R = H, Me; PTA = 1,3,5-triaza-7-phosphaadamantane) have been synthesised and applied to regioselective catalytic hydrogenation of unsaturated ketones in aqueous biphasic conditions and in studies of citotoxity towards selected tumor cell strains.

The simplified synthesis of a portion of Globo-H hexasaccharide, a saccharide antigen overexpressed by breast cancer cells, and its conjugation to two different immunogenic proteins are described. The conjugated vaccines, evaluated in a preclinical model, showed good immunogenic potential.

sono stati sviluppati sistemi nanoparticellari in grado di intrappolare elevate quantità di farmaci e di legare materiale genico. I sistemi preparati sono stati caratterizzati da un punto di vista chimico-fisico e tecnologico farmaceutico. Sono stati infine utilizzati per prove in vitro e i risultati ottenuti hanno mostrato che essi possiedono le caratteristiche per potere essere considerati dei validi carrier per farmaci e materiale genico nel trattamento oncologico.