Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

Progressive cerebral deposition of the 39-43-amino-acid amyloid beta-protein (A beta) is an invariant feature of Alzheimer's disease which precedes symptoms of dementia by years or decades. The only specific molecular defects that cause Alzheimer's disease which have been identified so far are missense mutations in the gene encoding the beta-amyloid precursor protein (beta-APP) in certain families with an autosomal dominant form of the disease (familial Alzheimer's disease, or FAD). These mutations are located within or immediately flanking the A beta region of beta-APP, but the mechanism by which they cause the pathological phenotype of early and accelerated A beta deposition is unknown. Here we report that cultured cells which express a beta-APP complementary DNA bearing a double mutation (Lys to Asn at residue 595 plus Met to Leu at position 596) found in a Swedish FAD family produce approximately 6-8-fold more A beta than cells expressing normal beta-APP. The Met 596 to Leu mutation is principally responsible for the increase. These data establish a direct link between a FAD genotype and the clinicopathological phenotype. Further, they confirm the relevance of the continuous A beta production by cultured cells for elucidating the fundamental mechanism of Alzheimer's disease.

The amyloid beta peptide (A beta P) is a small fragment of the much larger, broadly distributed amyloid precursor protein (APP). Abundant A beta P deposition in the brains of patients with Alzheimer's disease suggests that altered APP processing may represent a key pathogenic event. Direct protein structural analyses showed that constitutive processing in human embryonic kidney 293 cells cleaves APP in the interior of the A beta P, thus preventing A beta P deposition. A deficiency of this processing event may ultimately prove to be the etiological event in Alzheimer's disease that gives rise to senile plaque formation.

Studies of Drosophila and mammals have revealed the importance of insulin signaling through phosphatidylinositol 3-kinase and the serine/threonine kinase Akt/protein kinase B for the regulation of cell, organ, and organismal growth. In mammals, three highly conserved proteins, Akt1, Akt2, and Akt3, comprise the Akt family, of which the first two are required for normal growth and metabolism, respectively. Here we address the function of Akt3. Like Akt1, Akt3 is not required for the maintenance of normal carbohydrate metabolism but is essential for the attainment of normal organ size. However, in contrast to Akt1 ؊/؊ mice, which display a proportional decrease in the sizes of all organs, Akt3 ؊/؊ mice present a selective 20% decrease in brain size. Moreover, although Akt1-and Akt3-deficient brains are reduced in size to approximately the same degree, the absence of Akt1 leads to a reduction in cell number, whereas the lack of Akt3 results in smaller and fewer cells. Finally, mammalian target of rapamycin signaling is attenuated in the brains of Akt3؊/؊ but not Akt1 ؊/؊ mice, suggesting that differential regulation of this pathway contributes to an isoform-specific regulation of cell growth.While complex organisms grow toward determinate final sizes, there must be precise regulation within each tissue as well as coordination among organs to reach these final sizes (18,24). The regulation of both cell number and size contributes to the establishment of organ size, whereas cell number appears to be predominant in determining differences between species. Several factors, including circulating hormones and metabolites as well as cell-autonomous signaling cascades, control these processes (31). One of the key extracellular effectors determining organismal size is insulin-like growth factor 1 (IGF1). As demonstrated by genetic studies with mice, IGF1 is required for normal embryonic and postnatal growth (4,43,44,59). In addition, IGF1 controls the sizes of individual organs (43, 59). For example, normal brain growth requires IGF1 (6, 43), as IGF1-deficient brains are reduced in size secondary to a decrease in both cell number and cell size (6,15). Similarly, humans with IGF1 deficiency display severe growth retardation and suffer from mental retardation (75).In addition to extracellular factors, the intracellular signaling pathways determining growth are being uncovered. IGF1 acts through the type 1 IGF receptor to modulate an evolutionarily conserved pathway of molecules involved in the regulation of growth and metabolism (38,53). For many hormones, including IGF1 and insulin, binding to a receptor stimulates its protein tyrosine kinase activity, leading to the phosphorylation of scaffold proteins of the insulin receptor substrate (IRS) family. IRS proteins assemble complexes that include a number of potential signaling proteins, of which the lipid kinase phosphatidylinositol 3-kinase (PI3K) appears to be the most critical for the maintenance of cell size and proliferation (10). PI3K catalyzes the generation of phospha...

The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with "70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk-cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensinrat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.Corticotropin-releasing factor (CRF), a 41-amino acid peptide, regulates the secretion of adrenocorticotropin and other proopiomelanocortin products from the anterior pituitary. CRF also coordinates the endocrine, behavioral, and autonomic responses to stress. Within the past few years, substantial evidence has accumulated from both laboratory and clinical studies implicating CRF as a physiological mediator of stress responses and stress-induced disorders (1-6). Immunocytochemical studies have shown that CRF is found within the paraventricular nucleus of the hypothalamus as well as limbic areas such as the central and medial nuclei of the amygdala, the bed nucleus of the stria terminalis, substantia inominata, septum, preoptic area, the lateral hypothalamus, and brain stem nuclei involved in stress responses and regulation of autonomic function, such as the locus coeruleus, the parabrachial nucleus, and the dorsal vagal complex (see ref. 7). CRF, when administered intracerebroventricularly, results in behavioral, physiological, and autonomic responses that are similar to those observed when animals are exposed to a stressful environment (4-6).

The structures of two isoforms of Bcl-2 that differ by two amino acids have been determined by NMR spectroscopy. Because wildtype Bcl-2 behaved poorly in solution, the structures were determined by using Bcl-2͞Bcl-x L chimeras in which part of the putative unstructured loop of Bcl-2 was replaced with a shortened loop from Bcl-xL. These chimeric proteins have a low pI compared with the wild-type protein and are soluble. The structures of the two Bcl-2 isoforms consist of 6 ␣-helices with a hydrophobic groove on the surface similar to that observed for the homologous protein, Bcl-xL. Comparison of the Bcl-2 structures to that of Bcl-xL shows that although the overall fold is the same, there are differences in the structural topology and electrostatic potential of the binding groove. Although the structures of the two isoforms of Bcl-2 are virtually identical, differences were observed in the ability of the proteins to bind to a 25-residue peptide from the proapoptotic Bad protein and a 16-residue peptide from the proapoptotic Bak protein. These results suggest that there are subtle differences in the hydrophobic binding groove in Bcl-2 that may translate into differences in antiapoptotic activity for the two isoforms.

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.

Progressive cerebral deposition of extracellu- (15,16). A related issue concerns the mechanism whereby the normal, age-related deposition of (AP in selected brain regions and vessels is augmented in AD. The local proteolytic processing of the precursor and the role of amyloidassociated proteins, such as the serine protease inhibitor a1-antichymotrypsin (24), require elucidation.Although molecular studies of 8APP have advanced rapidly, the native precursor protein in human brain and other tissues has not been detected and characterized. To begin to address some of the questions posed above, we produced antibodies to synthetic peptides with sequences predicted from amyloidogenic and nonamyloidogenic regions of J3APP and have identified forms of the precursor molecule in brain, nonneural tissues, and cultured cells of several species. METHODSPreparation of Antibodies to Synthetic Peptides. Several peptides were synthesized according to the sequence deduced from a P3APP cDNA (14). Those used in this study were: amino acid residues 597-624 (peptide (31-28), comprising the first 28 residues of PAP; residues 676-695 (peptide C1), comprising the 20 C-terminal amino acids of PAPP; and residues 681-695, a second C-terminal peptide (C2), comprising the last 15 residues. The latter peptide and an antiserum to it were the gifts of Tsuyoshi Ishii (Psychiatric Research Institute, Tokyo). Peptide C1 was HPLC-purified and coupled to edestin. Peptide C2 had been coupled to keyhole limpet hemocyanin. Peptide p1-28 was injected uncoupled. Antisera were assayed by serial dilutions on dot blots of unconjugated peptide. Previously characterized antibodies used in this study included two antisera (A and C) to the -4-kDa P3AP purified from amyloid-rich fractions of AD cortex (12,25), two antisera (Fl and Ph) to the -4-kDa ,8AP purified from AD meningovascular amyloid (10), a paired helical filament-specific antiserum having no reaction with amyloid deposits (26), and an antiserum to heat-stable microtubule-associated proteins (principally X and MAP 2) purified from fetal human brain (25).Abbreviations: AD, Alzheimer disease; flAP, f8-amyloid protein; f3APP, 13-amyloid precursor polypeptide. 7341The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.