Inhibitors of anti-apoptotic proteins for cancer therapy

Abstract

Apoptosis, or programmed cell death, is the principal mechanism through which unwanted or damaged cells are safely eliminated. Just as diverse growth stimuli ultimately induce cellular proliferation through common pathways in the cell cycle, a set of evolutionarily conserved genes regulate the final aspects of the cell-death pathway. Balance between these proliferative and apoptotic processes is essential for normal tissue homeostasis. Although cancer has historically been considered a disease of uncontrolled cell division, abnormal resistance to apoptosis is now understood to contribute to tumor initiation, progression and resistance to chemotherapy [1]. A family of aspartate specific cysteine proteases called caspases drives apoptotic cell death. Members of this protein family normally exist as pro-enzymes that are activated by proteolytic cleavage and can be functionally subdivided into a hierarchy of 'initiator' and 'executioner' caspases. Initiator caspases (caspases 6, 8, 9, 10, 12) are activated during early apoptosis signaling and serve to propagate the death signal by cleaving and activating executioner caspases (caspases 2, 3, 7). The resulting proteolytic cascade leads to cleavage of numerous intracellular targets, and ultimately to cell death and the formation of apoptotic bodies that are rapidly engulfed and cleared by other cells [2,3]. Two apoptosis signaling pathways exist that differ in the origin of their death signal, but converge upon a common pathway (Fig. 1). The extrinsic or death receptor pathway is initiated by an extracellular stimulus of a membrane bound receptor such as the action of Fas ligand on the Fas receptor. Upon surface activation, the cytoplasmic side of the receptor recruits and activates initiator caspases (e.g. caspase-8) that in turn activate executioner caspases (e.g. caspase-3). The intrinsic or mitochondrial apoptotic pathway responds to signals of stress or cell damage such as hypoxia, detachment, disregulated cell cycle, DNA damage or chemotherapy treatment. This results in the release of cytochrome c from the mitochondria into the cytosol where if forms a complex with Apaf-1 (apoptotic protease activating factor-1), dATP (or ATP) and the inactive initiator caspase procaspase-9. Within this complex, known as the apoptosome, caspase-9 is activated. Once activated, caspase-9 cleaves and activates executioner caspases (e.g. caspase-7) [4]. Programmed cell death is a highly conserved and tightly regulated process that is governed by the delicate checks and balances between families of pro-apoptotic and anti-apoptotic proteins. Upsetting this balance leads to deficient apoptotic signaling and is a common mechanism by which tumor cells can develop a survival benefit or resistance to chemotherapy. Two groups of proteins, members of the B-Cell Lymphoma (Bcl-2) and inhibitors of apoptosis protein (IAP) families, are endogenous inhibitors of apoptosis that are overexpressed in many tumor cells. Members of the Bcl-2 and IAP families are non-enzymatic proteins that exert their inhibitory function through direct protein-protein interactions with their proapoptotic counterparts. Anti-apoptotic Bcl-2 family proteins act directly at the mitochondria and function to block cytochrome c release and can therefore inhibit only the intrinsic cell death pathway [5]. IAP proteins act further downstream by directly binding to and inhibiting both initiator and effector caspases and can therefore block both the extrinsic and the intrinsic pathways [6,7]. Small molecule antagonism of these endogenous inhibitors of apoptosis requires the disruption of high affinity protein-protein interactions. Affinities of small molecule inhibitors are derived from competition binding studies using peptides that mimic one of the protein binding partners. Fluorescent polarization assays (FPA) detect the displacement of fluorescently labeled peptide [8] while biosensor assays typically detect displacement of a protein from an