A small-molecule mimetic of Smac/Diablo that specifically counters the apoptosis-inhibiting activity of IAP proteins has been shown to enhance apoptosis induced by cell surface death receptors as well as chemotherapeutic drugs. Survey of a panel of 50 human non-small-cell lung cancer cell lines has revealed, surprisingly, that roughly one-quarter of these lines are sensitive to the treatment of Smac mimetic alone, suggesting that an apoptotic signal has been turned on in these cells and is held in check by IAP proteins. This signal has now been identified as the autocrine-secreted cytokine tumor necrosis factor alpha (TNFalpha). In response to autocrine TNFalpha signaling, the Smac mimetic promotes formation of a RIPK1-dependent caspase-8-activating complex, leading to apoptosis.

We describe the synthesis and properties of a small molecule mimic of Smac, a pro-apoptotic protein that functions by relieving inhibitor-of-apoptosis protein (IAP)-mediated suppression of caspase activity. The compound binds to X chromosome- encoded IAP (XIAP), cellular IAP 1 (cIAP-1), and cellular IAP 2 (cIAP-2) and synergizes with both tumor necrosis factor alpha (TNFalpha) and TNF-related apoptosis-inducing ligand (TRAIL) to potently induce caspase activation and apoptosis in human cancer cells. The molecule has allowed a temporal, unbiased evaluation of the roles that IAP proteins play during signaling from TRAIL and TNF receptors. The compound is also a lead structure for the development of IAP antagonists potentially useful as therapy for cancer and inflammatory diseases.

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We have studied a naturally occurring small-molecule antimitotic called diazonamide A. Diazonamide A is highly effective at blocking spindle assembly in mammalian cell culture and does so through a unique mechanism. A biotinylated form of diazonamide A affinity purifies ornithine ␦-amino transferase (OAT), a mitochondrial enzyme, from HeLa cell and Xenopus egg extracts. In the latter system, the interaction between diazonamide A and OAT is regulated by RanGTP. We find that specific OAT knockdown in human cervical carcinoma and osteosarcoma cells by RNA interference blocks cell division and causes cell death, the effects largely phenocopying diazonamide A treatment in these cell lines. Our experiments reveal an unanticipated, paradoxical role for OAT in mitotic cell division and identify the protein as a target for chemotherapeutic drug development.A variety of small molecules block progression through M phase of the cell cycle. The most common are tubulin ligands. Tubulin-binding toxins have helped elucidate the structure and organization of the mitotic spindle, and certain of these toxins are clinically effective as cancer chemotherapy. However, systemic disruption of the tubulin cytoskeleton has drawbacks. Microtubule poisons disturb nonmitotic functions of the cytoskeleton in both replicating cells and differentiated nondividing cells. Wasting, neutropenia, and peripheral neuropathy are severe dose-limiting toxicities common to this family of drugs in vivo (1). As a result, considerable effort has been made to identify alternative antineoplastics that target mitotic regulatory factors or components of the spindle other than tubulin (2). The development of specific inhibitors of the aurora kinases (3) and the kinesin motor protein Eg5 (2) are notable recent examples.

A poptosis in mammalian cells is executed by the proteolytic activities of caspases that are activated in response to apoptotic stimuli via two distinct signaling pathways 1 . These pathways have different initiator caspases that cleave and activate a common set of executioner caspases, including caspases 3, 6 and 7. The intrinsic (or mitochondrial) pathway of apoptosis is activated in response to DNA damage and other stress signals within the cell. Cytochrome c is consequently released from mitochondria into the cytosol and binds its adaptor protein, Apaf-1, to form the large 'apoptosome' complex, which recruits and activates caspase-9 (ref. 2). The extrinsic pathway is triggered from the exterior of the cell by members of the TNF superfamily, which bind and activate their corresponding death receptors [3][4][5] . For example, binding of TRAIL to the extracellular domain (ECD) of the death receptors DR4 (TRAIL-Receptor 1 (TRAIL-R1)) and DR5 (TRAIL-R2) promotes clustering of these receptors. The ligand-receptor complex in turn engages the adaptor protein Fas-associated death domain (FADD) via its cytoplasmic death domains. FADD then recruits the initiator caspase-8 through its N-terminal death-effector domain to form a death-inducing signaling complex (DISC) [6][7][8][9] .Caspase activation is tightly regulated by members of the inhibitor of apoptosis protein (IAP) family, such as XIAP, cIAP1 and cIAP2 (refs. 10-12). In response to death signals, a second mitochondriaderived activator of apoptosis (Smac; also called Diablo) is released from the mitochondria into the cytosol, where it binds the Bir domains of XIAP to relieve its inhibition of caspases and the Bir domains of cIAPs to induce their degradation. The four N-terminal residues (alanine, valine, proline and isoleucine) of Smac are sufficient for this interaction and have been mimicked using a peptidomimetic approach to generate cell-permeable small molecules that function similarly to the Smac protein [13][14][15] . Previously, we demonstrated that Smac mimetics not only effectively kill cancer cell lines with an autocrine TNFα signal but also substantially increase the cell-killing efficiency of TRAIL [13][14][15] .Selective activation of the apoptotic pathway provides tremendous therapeutic potential for cancer treatment 16 . Various strategies are currently under active clinical investigation, including the use of Bcl-2 inhibitors, Smac mimetics or IAP antagonists and deathreceptor agonists 17 . Among the death receptor-targeted agents, the therapeutic use of TNFα and agonistic CD95-specific antibodies has been hampered by their toxic side effects, particularly a severe inflammatory response through NF-κB activation 3,18 . However, mounting evidence from clinical trials has indicated that targeting DR4 and DR5 with agonist antibodies or recombinant TRAIL selectively eliminates tumor cells while sparing normal cells 19,20 . Although targeting TRAIL pathways may be promising as a safe anticancer therapy, the clinical use of TRAIL itself is limited by it...

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with K i ‫؍‬ 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8-170 ng͞ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.

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