Riassunto analitico
Protein-protein interactions (PPIs) play pivotal roles in nearly all biological processes including cell proliferation, growth, differentiation, cellular signaling and programmed cell death or apoptosis.
Selective modulation of specific PPIs by small molecules has emerged as an important approach to investigate biological processes, for validating new drug targets, and for the development of new therapeutics. Major research efforts have been lately directed towards the development of new therapeutics reactivating the apoptotic program in tumor cells by specifically targeting PPIs.
The induction of apoptosis is in fact one of the most widespread chemotherapeutic strategies because a hallmark of most cancer cells is their ability to escape programmed cell death and to resist to traditional anticancer drug treatments.
The aim of the research proposal was to combine several research approaches for the early-stage discovery of novel small molecular-weight compounds able to interfere with PPI of anti-apoptotic proteins, in particular MDM2/p53 and Bcl-2/Bak, Bax.
The main objective of the work was to obtain an experimental validation of the PPI inhibitory activity of a restricted number of commercial organic molecules that were selected by means of in silico techniques by a collaborative partner (BioChemoInformatic laboratory, University of Bologna).
The experiments of this work have been performed on HL-60 cells, HCT116 cells and two derived isogenic cell lines, HCT116 p53-/- and HCT116 Bax-/-.
First of all, we tested a set of virtual screening compounds supposed to inhibit Bcl-2. We used HL-60 cells which express high levels of Bcl-2 protein. In order to study the cellular and molecular activities of these compounds we compared them to biological profiles of ABT-737, a potent small-molecule chemical that acts like a BH3-only protein to antagonize anti-apoptotic Bcl-2 family members.
Some of the hit compounds determined a substantial increase in the percentage of cells undergoing SubG1 and the activation of the pro-apoptotic protein Bax was observed.
We then explored the possibility to identify compounds able to dual-target Bcl-2 and MDM2, i.e. simultaneous inhibition. To this purpose, another set of virtual screening compounds was tested on human colorectal carcinoma HCT116 cells and compared to ABT-737 and Nutlin 3-a (a cis-imidazoline selective inhibitor of MDM2).
One compound showed the ability to activate the principal targets of p53. In addition, cell cycle analysis on HCT116 wt, p53-/- and Bax-/- suggested the reactivation of both p53 and Bax/Bak pro-apoptotic proteins indicating the possible use of this compound for further lead optimization purpose towards the discovery of effective anti-apoptotic multi-target drugs.
Overall, the results shown in this thesis provide evidence that the selected compounds may offer lead structures for drug development of new and effective anticancer therapeutic agents able to reactivate the apoptotic process in malignant cells.
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