Inhibitor of Apoptosis Proteins (IAPs) are negative regulators of apoptosis and their overexpression is observed in many cancer cells, correlating with the inhibition of caspases. IAPs inhibitory function is exploited by the BIR domains, which were firstly identified in baculovirus. Among the IAP family, XIAP (X chromosome-linked IAP) directly inhibits caspases preventing proteolytic cleavage through its BIR2 and BIR3 domains; furthermore, XIAP-BIR1 in a dimeric form recognizes TAB1, a kinase activator, regulating pro-survival pathways. In the last years, cIAPs have become crucial players of the extrinsic pathway; in fact, through the recognition of TRAFs (TNF Receptor Associated Factors) by the cIAP2-BIR1 domain, they are recruited to the TNF- Receptor Signaling Complex and act as E3 ubiquitin ligases. One of the most promising approaches that have been proposed to inhibit these proteins is represented by the structure-based design of small molecules, named Smac-mimetics, that mimic Smac/DIABLO (Second mitochondria-derived activator of caspases/Direct IAp Binding protein with Low pI), an endogenous antagonist of IAPs. Initially designed in 2001 against the BIR3 domain of XIAP, Smac-mimetics have shown to prevent the inhibitory action of XIAP on initiator and executioner caspases, but also to bind to the BIR3 of cIAPs, inducing their autoubiquitylation and degradation. This work focuses on the cloning, expression and purification of the IAPs constructs of interest and their biochemical and biophysical characterization alone and in the presence of some of the Smac-mimetics from our library. The X-ray technique on crystals and protein solutions allowed a structural study of the protein-ligand complexes at atomic level, favoring the process of drug lead optimization. Furthermore, the screening of libraries of pharmacologically active compounds through in silico docking searches on new targets, such as XIAP- and cIAP2-BIR1, resulted in the discovery of potential new pro-apoptotic leads, whose clinical properties are known. Since in the last months new macromolecular protein complexes have been identified as involved in apoptosis and pro-survival pathways, novel protocols for the expression and purification of cIAP1 and TRAFs full length constructs have been optimized to obtain pure and homogeneous samples ready for the structural characterization.

STRUCTURAL INSIGHTS INTO INHIBITOR OF APOPTOSIS PROTEINS RECOGNITION BY PRO-APOPTOTIC COMPOUNDS / F. Cossu ; tutor: M. Bolognesi ; coordinatore: P. Plevani. - : . Universita' degli Studi di Milano, 2012 Jan 23. ((24. ciclo, Anno Accademico 2011. [10.13130/cossu-federica_phd2012-01-23].

STRUCTURAL INSIGHTS INTO INHIBITOR OF APOPTOSIS PROTEINS RECOGNITION BY PRO-APOPTOTIC COMPOUNDS

F. Cossu
2012-01-23

Abstract

Inhibitor of Apoptosis Proteins (IAPs) are negative regulators of apoptosis and their overexpression is observed in many cancer cells, correlating with the inhibition of caspases. IAPs inhibitory function is exploited by the BIR domains, which were firstly identified in baculovirus. Among the IAP family, XIAP (X chromosome-linked IAP) directly inhibits caspases preventing proteolytic cleavage through its BIR2 and BIR3 domains; furthermore, XIAP-BIR1 in a dimeric form recognizes TAB1, a kinase activator, regulating pro-survival pathways. In the last years, cIAPs have become crucial players of the extrinsic pathway; in fact, through the recognition of TRAFs (TNF Receptor Associated Factors) by the cIAP2-BIR1 domain, they are recruited to the TNF- Receptor Signaling Complex and act as E3 ubiquitin ligases. One of the most promising approaches that have been proposed to inhibit these proteins is represented by the structure-based design of small molecules, named Smac-mimetics, that mimic Smac/DIABLO (Second mitochondria-derived activator of caspases/Direct IAp Binding protein with Low pI), an endogenous antagonist of IAPs. Initially designed in 2001 against the BIR3 domain of XIAP, Smac-mimetics have shown to prevent the inhibitory action of XIAP on initiator and executioner caspases, but also to bind to the BIR3 of cIAPs, inducing their autoubiquitylation and degradation. This work focuses on the cloning, expression and purification of the IAPs constructs of interest and their biochemical and biophysical characterization alone and in the presence of some of the Smac-mimetics from our library. The X-ray technique on crystals and protein solutions allowed a structural study of the protein-ligand complexes at atomic level, favoring the process of drug lead optimization. Furthermore, the screening of libraries of pharmacologically active compounds through in silico docking searches on new targets, such as XIAP- and cIAP2-BIR1, resulted in the discovery of potential new pro-apoptotic leads, whose clinical properties are known. Since in the last months new macromolecular protein complexes have been identified as involved in apoptosis and pro-survival pathways, novel protocols for the expression and purification of cIAP1 and TRAFs full length constructs have been optimized to obtain pure and homogeneous samples ready for the structural characterization.
BOLOGNESI, MARTINO
PLEVANI, PAOLO
apoptosis ; IAPs ; Smac/DIABLO ; Smac-mimetics ; virtual docking ; crystallography ; thermal stability
Settore BIO/11 - Biologia Molecolare
STRUCTURAL INSIGHTS INTO INHIBITOR OF APOPTOSIS PROTEINS RECOGNITION BY PRO-APOPTOTIC COMPOUNDS / F. Cossu ; tutor: M. Bolognesi ; coordinatore: P. Plevani. - : . Universita' degli Studi di Milano, 2012 Jan 23. ((24. ciclo, Anno Accademico 2011. [10.13130/cossu-federica_phd2012-01-23].
Doctoral Thesis
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