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A critical role for reactive oxygen species (ROS) in photoreceptor apoptosis has been established. However, the exact molecular mechanisms triggered by oxidative stress in photoreceptor cell death remain undefined. This study delineates the molecular events that occur after treatment of the photoreceptor cell line 661W with the nitric oxide donor sodium nitroprusside (SNP). Cytosolic calcium levels increased during photoreceptor apoptosis, leading to activation of the calcium-dependent proteases calpains. Furthermore, caspase activation also occurred following SNP insult. However, although treatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone inhibited caspase activity per se in SNP-treated 661W cells, it did not prevent apoptosis. On the other hand, CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran) acted as a scavenger of ROS and reduced 661W photoreceptor apoptosis induced by SNP by preventing the activation of a pathway in which calpains have a key role. In summary, we report for the first time that both caspases and calpains are involved in 661W photoreceptor apoptosis and that calpain activation can be prevented by the ROS scavenger CR-6.The cell death process of apoptosis is characterized by a series of morphological and biochemical changes, including membrane blebbing, loss of plasma membrane asymmetry, chromatin cleavage, and DNA fragmentation (1, 2). Apoptosis plays a central role in tissue modeling during development and, together with the cell-generating process of mitosis, is responsible for the maintenance of cell numbers in multicellular organisms. Deregulation of apoptosis has been well documented in several human pathologies, including cancer, neurodegenerative diseases, and AIDS (3,4). Apoptosis also appears to be responsible for the cell loss seen in several disorders of the retina, including retinitis pigmentosa (a heterogeneous group of inherited disorders), glaucoma, and macular degeneration (5-8). Experiments aimed at unraveling the signaling pathways of apoptosis have identified several distinct mechanisms, and it has largely been accepted that caspases play a key role in both the initiation and execution pathways of apoptosis. However, the involvement of caspases does not seem to be clear-cut in some tissue systems. For example, there is still considerable controversy as to whether caspases play a role in retinal cell death (9 -12). There is also some uncertainty about the role of caspases in neurodegenerative conditions (13-15). Recent work from our laboratory has indicated that photoreceptor death in animal models of retinitis pigmentosa proceeds in the absence of caspase activity, suggesting a caspase-independent mechanism of cell destruction (11,12,16). The exact mechanisms operating in photoreceptor death are still unclear but may involve calpains rather than caspases as the executing enzymes. These studies also suggested a key role for reactive oxygen species (ROS) 1 and reactive nitrogen intermediates (RNI) since inhibitors of...

Apoptosis is a biological process relevant to human disease states that is strongly regulated through protein-protein complex formation. These complexes represent interesting points of chemical intervention for the development of molecules that could modulate cellular apoptosis. The apoptosome is a holoenzyme multiprotein complex formed by cytochrome c-activated Apaf-1 (apoptotic proteaseactivating factor), dATP and procaspase-9 that link mitochondria disfunction with activation of the effector caspases and in turn is of interest for the development of apoptotic modulators. In the present study we describe the identification of compounds that inhibit the apoptosome-mediated activation of procaspase-9 from the screening of a diversityoriented chemical library. The active compounds rescued from the library were chemically optimised to obtain molecules that bind to both recombinant and human endogenous Apaf-1 in a cytochrome c-noncompetitive mechanism that inhibits the recruitment of procaspase-9 by the apoptosome. These newly identified Apaf-1 ligands decrease the apoptotic phenotype in mitochondrial-mediated models of cellular apoptosis. Keywords: apoptosis; apoptosome; Apaf-1; caspasa-3; caspasa-9; combinatorial libraries; inhibitor; molecular recognition; peptoid; protein-protein interactions; small molecule Abbreviations: Apaf-1, apoptotic protease-activating factor; DEVDase, hydrolysis of Ac-DEVD-afc; DTT, dithiothreitol; FCS, fetal-calf serum; MEFs, mouse embryo fibroblasts; MMP, mitochondrial membrane potential; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Ni-NTA, (Ni 2 þ -nitrilotriacetate)-agarose; peptoid f1a, 5 0 -6 0 carboxyfluorescein-labelled peptoid 1a; PBS, phosphate-buffered saline; PGA, poly-(L-glutamic acid); rApaf-1, recombinant Apaf-1

Abstract:The identification and cloning of the vanilloid receptor 1 (TRPV1) represented a significant step for the understanding of the molecular mechanisms underlying the transduction of noxious chemical and thermal stimuli by peripheral nociceptors. TRPV1 is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons. TRPV1 channel activity is remarkably potentiated by pro-inflammatory agents, a phenomenon that is thought to underlie the peripheral sensitisation of nociceptors that leads to thermal hyperalgesia. Cumulative evidence is building a strong case for the involvement of this receptor in the etiology of both peripheral and visceral inflammatory pain, such as inflammatory bowel disease, bladder inflammation and cancer pain. The validation of TRPV1 receptor as a key therapeutic target for pain management has thrust intensive drug discovery programs aimed at developing orally active antagonists of the receptor protein. Nonetheless, the real challenge of these drug discovery platforms is to develop antagonists that preserve the physiological activity of TRPV1 receptors while correcting over-active channels. This is a condition to ensure normal pro-prioceptive and nociceptive responses that represent a safety mechanism to prevent tissue injury. Recent and exciting advances in the function, dysfunction and modulation of this receptor will be the focus of this review.

Despite the tremendous academic and industrial investment to develop therapeutic modulators of thermoTRPs, it apparently seems that we are still far from the first successful product, although hope is maintained high for all compounds currently in clinical trials. A major concern has been the appearance of side effects. A better knowledge of the thermosensory protein networks (signal-plexes), along with the application of system biology approaches may provide novel strategies to modulate thermoTRPs activity with improved therapeutic index. A case in point is TRPV1, where acting on interacting proteins is providing new therapeutic opportunities.

A novel inhibitor of interactions between signaling proteins in T cells demonstrates promising preventive and therapeutic effects in several models of autoimmune disease.

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