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Digital archive of theses discussed at the University of Pisa


Thesis etd-02242012-212847

Thesis type
Tesi di dottorato di ricerca
Thesis title
Enzymes and activation of intracellular signalling in cancer and neurodegenerative diseases
Academic discipline
Course of study
tutor Prof. Martini, Claudia
  • cancer
  • glia
  • neurodegenerative diseases
Graduation session start date
In recent years, many studies have shown evidence that glia play a central role in the central nervous system (CNS), creating many new questions. What is the normal function of glia and what is their role in disease of the CNS? Might glia be important drug targets? If so, this has the opportunity to open a new avenue of drug therapies for patients suffering from brain injuries and neurological disease.
In my thesis I described a number of studies of different compounds that exert effects on astroglial cells and on the associated neurological diseases, including inflammation and cancer.
In chapter 2, the biological characterization of a series of 2-phenylindol-3-ylglyoxylamides, and the test of their validity as new TSPO probes was reported. All of these compounds were characterized by the presence of a chemoreactive isothiocyanate group, able to bind the receptor protein irreversibly and covalently. Moreover, compound 18 featuring the NBD-fluorescent moiety was synthesized to develop an irreversible fluorescent probe. The presence of these two chemical groups (a chemoreactive group and a fluorescent chromophore) on a single molecule may offer numerous advantages, both in protein purification/characterization, and in protein cell visualization/density determination.
In chapter 3, I described the experiments performed using small molecule activators of Nrf2 mediated transcription on a reporter cell line. Two libraries were screened by the ‘fast track’ process and 6 hits were identified. In particular two of these molecules were able to induce the activation of the Nrf2-ARE pathway at a 10x times lower concentration than the reference compound. Further investigations, such as in silico assays, are necessary in order to determine chemical and physical characteristics of some of the selected molecules and to investigate their ability to activate the Nrf2-ARE pathway on human cells.
In chapters 4 and 5, the biological characterization of novel promising molecules acting on different interesting targets for glioma therapy is reported.
In chapter 4, the biological characterization of new MMP-inhibitors is reported. In the first part of this chapter, the attention has been directed towards a new class of MMP-2 inhibitors belonging to the recently identified family of N-O-isopropyl sulfonamide hydroxamates. Two of these MMP inhibitors (compound 1 and 2) were tested at nanomolar concentrations to investigate their potential efficacy in inhibiting the invasiveness and the proliferation of the human glioma cell line, U87MG. As a first step, the two compounds at nanomolar concentrations were assayed on their ability to inhibit MMP-2 cleavage of gelatin. Moreover, the MMP-2 expression was assessed following MMP inhibitor cell treatments. Of particular novelty was the effect of the inhibitors on the mRNA levels of MMP2 and in the future it would be interesting to look at the mechanism behind this observation. In order to help the development of multitarget therapies against GBM, the combined treatment of the U87MG glioma cells with the MMP inhibitors and the conventional chemotherapeutic drug TMZ was evaluated.
In the second half of chapter 4 the biological evaluation of a new series of 4-butylphenyl(ethynylthiophene)sulfonamido-based hydroxamates was described. As well, the effects of substitutions of different chemical groups on MMP-2 and MMP-9 activity and selectivity have been described. First, the reported compounds were examined in vitro by fluorometric assay on isolated enzymes. The compounds 5a-d, 6, 7 were then tested for their ability to inhibit in vitro invasion of U87MG glioma cells through matrigel and to inhibit cell growth.
Chapter 5, looked at glioma cell p53 reactivation by small molecule MDM2 inhibitors and their impact on cell viability and growth. The effect on p53 target genes was analysed at the mRNA level, as well as cell senescence and apoptosis. In particular, I investigated whether a novel small-molecule MDM2 inhibitor, named ISA27, affected the growth of the U87MG human GBM cell line and compared its efficacy with that of Nutlin-3. In this chapter I showed that ISA27 activated the p53 pathway in U87MG cells and elicited a dose- and time dependent inhibition of cell growth by the induction of permanent cell cycle arrest and apoptosis. Compared to Nutlin-3, ISA27 was effective at a lower dose and caused a faster antiproliferative response. If confirmed in vivo, the use of this MDM2 inhibitor could offer a novel therapy concept for the treatment of GBM patients by inducing tumor growth inhibition and regression.