PDB ID : 1YSG Title : Solution Structure of the Anti-apoptotic Protein Bcl-xL in Complex with "SAR by NMR" Ligands Authors : Oltersdorf, T.; Elmore, S.W.; Shoemaker, A.R.; Armstrong, R.C.; Augeri, D.J.; Belli, B.A.; Bruncko, M.; Deckwerth, T.L.; Dinges, J.; Hajduk, P.J.; Joseph, M.K.; Kitada, S.; Korsmeyer, S.J.; Kunzer, A.R.; Letai, A.; Li, C.; Mitten, M.J.; Nettesheim, D.G.; Ng, S.; Nimmer, P.M.; O’Connor, J.M.; Oleksijew, A.; Petros, A.M.; Reed, J.C.; Shen, W.; Tahir, S.K.; Thompson, C.B.; Tomaselli, K.J.; Wang, B.; Wendt, M.D.; Zhang, H.; Fesik, S.W.; Rosenberg, S.H. Deposited on : 2005-02-08

Structural modifications were made to a previously described acyl dipeptide caspase inhibitor, leading to the oxamyl dipeptide series. Subsequent SAR studies directed toward the warhead, P2, and P4 regions of this novel peptidomimetic are described herein.

Inhibitor of apoptosis (IAP) proteins are overexpressed in many cancers and have been implicated in tumor growth, pathogenesis, and resistance to chemo- or radiotherapy. On the basis of the NMR structure of a SMAC peptide complexed with the BIR3 domain of X-linked IAP (XIAP), a novel series of XIAP antagonists was discovered. The most potent compounds in this series bind to the baculovirus IAP repeat 3 (BIR3) domain of XIAP with single-digit nanomolar affinity and promote cell death in several human cancer cell lines. In a MDA-MB-231 breast cancer mouse xenograft model, these XIAP antagonists inhibited the growth of tumors. Close structural analogues that showed only weak binding to the XIAP-BIR3 domain were inactive in the cellular assays and showed only marginal in vivo activity. Our results are consistent with a mechanism in which ligands for the BIR3 domain of XIAP induce apoptosis by freeing up caspases. The present study validates the BIR3 domain of XIAP as a target and supports the use of small molecule XIAP antagonists as a potential therapy for cancers that overexpress XIAP.

Cytosine arabinoside (ara-C) is a nucleoside analog used in the treatment of hematologic malignancies. One of the major side effects of ara-C chemotherapy is neurotoxicity. In this study, we have further characterized the cell death induced by ara-C in sympathetic neurons. Similar to neurons undergoing trophic factor deprivation-induced apoptosis, ara-C-exposed neurons became hypometabolic before death and upregulated c-myb, c-fos, and Bim. Bax deletion delayed, but did not prevent, ara-C toxicity. Neurons died by apoptosis, indicated by the release of mitochondrial cytochrome-c and caspase-3 activation. p53-deficient neurons demonstrated decreased sensitivity to ara-C, but neither p53 nor multiple p53-regulated genes were induced. Mature neurons showed increased ara-C resistance. These results demonstrate that molecular mechanisms underlying ara-C-induced death are similar to those responsible for trophic factor deprivation- induced apoptosis. However, substantial differences in neuronal death after these two distinct stress stimuli exist since ara-C toxicity, unlike the developmental death, can proceed in the absence of Bax.

Various aryloxy methyl ketones of the 1-naphthyloxyacetyl-Val-Asp backbone have been prepared. A systematic study of their structure-activity relationship (SAR) related to caspases 1, 3, 6, and 8 is reported. Highly potent irreversible broad-spectrum caspase inhibitors have been identified. Their efficacy in cellular models of cell death and inflammation are also discussed.

Studies dealing with transport of proteins from the oligodendrocyte cell body to the myelin sheath reveal the presence of different transport pathways. Proteolipid protein (PLP) is synthesized at the rough endoplasmic reticulum (ER) and then processed through the Golgi apparatus and transported to the myelin membranes. Myelin basic protein (MBP) on the other hand is synthesized locally at the ends of cell processes where its messenger RNA is translated on free ribosomes. Here we show that in rats that overexpress PLP, impairment of PLP transport from the cell body to the processes interferes with the translocation of other membrane proteins such as myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), but not with peripherally translated MBP. In addition, it also impedes the transport of non-myelin proteins, for example the amyloid precursor protein (APP). At the ultrastructural level, the ER of these metabolically disturbed oligodendrocytes revealed extreme swelling of the cisternae, and immunohistochemistry revealed intense expression of the ER chaperone molecule BiP/GRP78 and ER folding enzyme protein disulfide isomerase (PDI). These features suggest that these oligodendrocytes, which were found exclusively in gray matter areas of the spinal cord, started an unfolded protein response while suffering from ER stress. Some of these disturbed oligodendrocytes were seen to undergo programmed cell death. These results indicate that gray matter oligodendrocyte differ from white matter oligodendrocytes in their capacity to stabilize metabolic disturbances by an unfolded protein response.

Rasmussen's encephalitis is a progressive epileptic disorder characterized by unihemispheric lymphocytic infiltrates, microglial nodules, and neuronal loss leading to the destruction of the affected hemisphere. In this study, immunohistochemical evaluation of specimens from 11 patients revealed lymphocytic infiltrates that consisted mainly of CD3 + CD8 + T cells. Of these cells, 7.0% lay in direct apposition to MHC class I + neurons. Confocal laser microscopy revealed that these lymphocytes contained granzyme B in a polar orientation toward these perikarya. Single neurons underwent apoptosis. These findings indicate that a T‐cell–mediated cytotoxic reaction induces neuronal death in Rasmussen's encephalitis. This study directly shows, for what we believe is the first time, that a cytotoxic T‐cell mechanism contributes to loss of neurons in human brain disease.

Expression of apoptosis-associated proteins p53, bcl-2, bax, and caspase-3/CPP32, activation of caspase-3, and modification of proteins via poly(ADP-ribosyl)ation was studied in pontosubicular neuron necrosis (PSN), a form of perinatal brain damage revealing the morphological hallmarks of neuronal apoptosis, Immunoreactivity for p53 was completely absent. The majority of cells stained with the bax and procaspase-3 antibodies did not show morphological signs of apoptosis, In contrast, an antibody against activated caspase-3 almost exclusively stained cells with apoptotic morphology, Poly(ADP-ribosyl)ated proteins were only rarely detected in cells with apoptotic morphology. The expression patterns of bax, procaspase-3, bcl-2, and p53 in PSN were similar to that found in age-matched control brains, However, activated caspase-3 and poly-ADP-ribosylated proteins were exclusively found in apoptotic cells. These data indicate that detection of active caspase-3 is a reliable marker for apoptosis in formalin-fixed human tissue, and that neuronal apoptosis in pontosubicular neuron necrosis is accompanied by a pronounced activation of caspase-3.

Apoptotic cell death occurs in the injured and diseased central nervous system. It is mediated by a family of caspases, which are activated by the lethal stimulus and cleave multiple protein substrates that are critical for cell viability. Previous studies demonstrated that caspase‐mediated apoptotic cell death contributes to the loss of brain tissue after experimental cerebral ischemia and that peptidic caspase inhibitors can be efficacious in reducing infarct size after icv administration. Here we present the novel small molecule peptidomimetic caspase inhibitor IDN5370/CGP82630, which belongs to the structural class of oxoazepinoindoline caspase inhibitors. It is 10–100‐fold more potent than current peptidic inhibitors in inhibiting multiple caspases in vitro and promoting neuronal survival. IDN5370 and a derivative, IDN7866, were tested for their ability to reduce infarct size after permanent and transient cerebral ischemia. When administered icv to rats subjected to permanent middle cerebral artery occlusion (MCAO), IDN5370 significantly reduced cortical infarct as measured by magnetic resonance imaging at 2 days after artery occlusion. Protection of brain tissue persisted for 28 days after artery occlusion. To determine whether compounds of this structural class could reduce infarct size after peripheral administration, IDN7866, which penetrates the blood–brain barrier and inhibits caspase 3 in situ in the hippocampus after kainate‐induced seizures, was administered iv in both permanent and transient MCAO models. Infarct size was reduced significantly in both models 24 h after artery occlusion. These results demonstrate that peripherally administered peptidomimetic caspase inhibitors can attenuate brain injury after cerebral ischemia and confer a long‐lasting protective effect on the infarcted brain tissue. Drug Dev. Res. 52:579–586, 2001. © 2001 Wiley‐Liss, Inc.

Neuronal loss is prominent in Alzheimer's disease (AD), and its mechanisms remain unresolved. Apoptotic cell death has been implicated on the basis of studies demonstrating DNA fragmentation and an up-regulation of proapoptotic proteins in the AD brain. However, DNA fragmentation in neurons is too frequent to account for the continuous neuronal loss in a degenerative disease extending over many years. Furthermore, the typical apoptotic morphology has not been convincingly documented in AD neurons with fragmented DNA. We report the detection of the activated form of caspase-3, the central effector enzyme of the apoptotic cascade, in AD and Down's syndrome (DS) brain using an affinity-purified antiserum. In AD and DS, single neurons with apoptotic morphology showed cytoplasmic immunoreactivity for activated caspase-3, whereas no neurons were labeled in age-matched controls. Apoptotic neurons were identified at an approximate frequency of 1 in 1100 to 5000 neurons in the cases examined. Furthermore, caspase-3 immunoreactivity was detected in granules of granulovacuolar degeneration. Our results provide direct evidence for apoptotic neuronal death in AD with a frequency compatible with the progression of neuronal degeneration in this chronic disease and identify autophagic vacuoles of granulovacuolar degeneration as possible means for the protective segregation of early apoptotic alterations in the neuronal cytoplasm.

The BCL2 family member BAX is required for the induction of apoptosis in neonatal sympathetic neurons after NGF withdrawal.Bax-deficient sympathetic neurons are NGF-independent for survival. To characterize the physiological state of neurons protected by BAX deficiency and to place BAX within the death pathway, we determine which of the molecular changes induced by NGF deprivation depend on BAX and compare the results with those for neurons protected by caspase inhibition. We find that neurons deficient in bothBaxandBcl2resist NGF-deprivation similar toBax-deficient neurons discounting a role for BCL2 in the mechanism by whichBaxdeficiency causes trophic factor independence. We identify two new molecular changes, phosphorylation of c-Jun on Ser63and α-spectrin proteolysis, which precede and accompany apoptosis, respectively. Early reversible changes induced by NGF withdrawal, such as decreased protein synthesis and glucose uptake, increased c-Jun phosphorylation, increased steady statec-junmRNA levels, and cellular atrophy, occur both in wild type andBax-deficient neurons and thus are BAX-independent. In contrast to neurons protected by caspase inhibition, noc-fosinduction occurs inBax-deficient neurons. Terminal irreversible events of apoptosis such as caspase-mediated α-spectrin proteolysis are prevented by bothBax-deficiency and caspase inhibition. This places BAX downstream or in a different pathway of the early changes and upstream of the terminal events such as those leading toc-fosinduction and caspase activation. This order indicates that the physiological state of NGF-deprived neurons protected byBaxdeficiency may be less perturbed than that of caspase inhibitor-saved neurons.

During development, sympathetic neurons are critically dependent on nerve growth factor (NGF) for survival. Neurons isolated from the superior cervical ganglia (SCG) of embryonic rodents and maintained for 1 week in vitro undergo programmed cell death in response to NGF deprivation. As the cells mature in vitro and in vivo, however, these neurons develop a resistance to NGF deprivation and become much less acutely dependent on NGF for survival. Using an in vitro model of neuronal maturation, we confirmed that SCG neurons maintained in culture for 3-4 weeks did not experience a dramatic loss in viability after NGF removal, yet they did undergo the initial biochemical and genetic changes elicited by NGF deprivation of young neurons. NGF deprivation of mature neurons produced rapid decreases in glucose uptake and protein and RNA synthesis rates, increased phosphorylation of c-Jun, and an increase in c-jun mRNA. Mature neurons, however, experienced a block in the cell death program before the final stages of the pathway activated in young neurons, which includes the induction of c-fos mRNA and characteristic apoptotic nuclear changes. This maturation-induced block was indistinguishable by these criteria from the block produced by Bax deficiency. Expression of Bax in mature neurons restored the apoptotic pathway, such that after NGF removal, Bax-overexpressing mature neurons resumed the apoptotic program, including the induction of c-Fos and passage through a caspase checkpoint. Thus, a block in the apoptotic program at or near the BAX checkpoint accounts for the decreased dependence of mature neurons on neurotrophic factor to maintain survival.

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.

Members of the BCL2-related family of proteins either promote or repress programmed cell death. BAX, a death-promoting member, heterodimerizes with multiple death-repressing molecules, suggesting that it could prove critical to cell death. We tested whether Bax is required for neuronal death by trophic factor deprivation and during development. Neonatal sympathetic neurons and facial motor neurons from Bax-deficient mice survived nerve growth factor deprivation and disconnection from their targets by axotomy, respectively. These salvaged neurons displayed remarkable soma atrophy and reduced elaboration of neurites; yet they responded to readdition of trophic factor with soma hypertrophy and enhanced neurite outgrowth. Bax-deficient superior cervical ganglia and facial nuclei possessed increased numbers of neurons. Our observations demonstrate that trophic factor deprivation–induced death of sympathetic and motor neurons depends on Bax.

Extensive neuronal death occurs in the developing nervous system. Death of neurons during this process is apoptotic and appears to utilize a pathway that is conserved in various mammalian cells and organisms. Recent evidence suggests that neuronal death during trauma, stroke, or neurode‐generative diseases may also occur by a similar mechanism. This review discusses the molecular mechanism of developmental neuronal death by examining the biochemical and molecular events associated with neuronal death after trophic factor withdrawal. The ability to inhibit neuronal death by manipulating the Bcl‐2 or the ICE‐family proteins demonstrates the importance of these proteins in the neuronal apoptotic pathway. The utility of inhibiting neuronal death by blocking the apoptotic pathway as therapy in neuropathological situations is discussed.

Degeneration of cholinergic neurons in the basal forebrain (CBF) is a prominent neuropathological feature of Alzheimer's disease and is thought responsible for some cognitive deficits seen in patients. An animal model of pure CBF degeneration would be valuable for analysis of the function of these neurons and testing therapeutic strategies. CBF neurons express receptors for nerve growth factor. In order to selectively destroy these neurons, we developed an immunotoxin using monoclonal antibody (192 IgG) to rat NGF receptor (p75NGFr) armed with the ribosome inactivating protein, saporin. In vitro 192-saporin was highly toxic to neurons expressing p75NGFr. Intraventricular injections of 192-saporin destroyed the CBF and impaired passive avoidance learning. These results indicate that 192-saporin treated rats can be used to model a key feature of Alzheimer's disease and that anti-neuronal immunotoxins are a powerful approach to selective neural lesioning.