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.

A constitutive equation for computing particle concentration and velocity fields in concentrated monomodal suspensions is proposed that consists of two parts: a Newtonian constitutive equation in which the viscosity depends on the local particle volume fraction and a diffusion equation that accounts for shear-induced particle migration. Particle flux expressions used to obtain the diffusion equation are derived by simple scaling arguments. Predictions are made for the particle volume fraction and velocity fields for steady Couette and Poiseuille flow, and for transient start-up of steady shear flow in a Couette apparatus. Particle concentrations for a monomodal suspension of polymethyl methacrylate spheres in a Newtonian solvent are measured by nuclear magnetic resonance (NMR) imaging in the Couette geometry for two particle sizes and volume fractions. The predictions agree remarkably well with the measurements for both transient and steady-state experiments as well as for different particle sizes.

Emerging evidence suggests that an amplifiable protease cascade consisting of multiple aspartatespecific cysteine proteases (ASCPs) Apoptosis is a fundamental biochemical cell-death pathway essential for normal tissue homeostasis, cellular differentiation, and development within a multicellular organism (for review, see refs. 1-3). Members of the growing family of aspartate-specific cysteine proteases (ASCPs) that include mammalian interleukin 13 converting enzyme (ICE) (4,5), Nedd2 (ICH-1) (6,7), CPP32 (8), Mch2 (9), Mch3 (10), TX (ICH-2, ICErel-II) (11)(12)(13), and ICErel-Ill (13) have been implicated as mediators of all apoptotic cell death (for review, see ref. 14).Cytotoxic T lymphocytes (CTL) induce apoptosis in their target cells possibly by activating members of the ASCP family (15). This hypothesis was supported by the observation that granzyme B, the CTL granule aspartate-specific serine protease, can cleave CPP32 (16). Recent observations suggested that the Fas-interacting protein FADD/MORT1 (17,18)

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor. (Blood. 2009; 114:2984-2992) IntroductionThe presence of genetic changes in cancer cells that lead to dysregulated activation of kinases frequently signals that the activated kinase is a contributing driver of disease, 1-4 and inhibitors of activated kinases can have a dramatic impact on disease progression in patients with these genetic alterations. 5,6 To clearly define the role of the dysregulated kinase, and to determine whether inhibition of the mutant kinase is sufficient to induce a therapeutic benefit, requires drugs capable of selectively, potently, and preferably sustainably inhibiting the activated kinase in patients.Activating mutations in the FLT3 receptor tyrosine kinase have been identified in up to 30% of acute myeloid leukemia (AML) patients. 7,8 The most common class of mutation is internal tandem duplications (ITDs) in the juxtamembrane domain 7,9 that lead to constitutive, ligand-independent activation of the kinase. 7,10 The prognosis for patients with FLT3-ITD mutations is significantly worse than that for patients with wild-type FLT3 when treated with standard therapy. [7][8][9][11][12][13][14][15][16] The presence of activating FLT3 mutations and the correlation of FLT3 activation with a poor prognosis strongly suggest that FLT3 is a driver of disease in AML, at least in patients with FLT3-ITD mutations. Several small molecule kinase inhibitors with activity against FLT3 have been evaluated in AML patients, including CEP-701 (lestaurtinib), PKC-412 (midostaurin), MLN-518 (tandutinib; previously known as CT-53518), sunitinib (SU-11248), sorafenib , and KW-2449. The compounds inhibit FLT3 in cellular assays and are efficacious in mouse models of FLT3-ITD AML. [17][18][19][20][21][22] In phase 1 and phase 2 clinical trials, conducted primarily in relapsed or refractory AML patients, responses were consistently observed with each of these drugs, 21,[23][24][25][26][27][28][29][30][31] however, responses generally were relatively limited and not durable. 21,[23][24][25]30 The studies did reveal a relationship between the likelihood of observing a clinical response and the pharmacokinetics/pharmacodynamics of FLT3 inhibition, and highlight...

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.

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Survivin is an anti-apoptotic protein that is overexpressed in most human cancers. We show that survivin forms complexes with a cellular protein, hepatitis B X-interacting protein (HBXIP), which was originally recognized for its association with the X protein of hepatitis B virus (HBX). Survivin±HBXIP complexes, but neither survivin nor HBXIP individually, bind pro-caspase-9, preventing its recruitment to Apaf1, and thereby selectively suppressing apoptosis initiated via the mitochondria/cytochrome c pathway. Viral HBX protein also interacts with the survivin± HBXIP complex and suppresses caspase activation in a survivin-dependent manner. Thus, HBXIP functions as a cofactor for survivin, and serves as a link between the cellular apoptosis machinery and a viral pathogen involved in hepatocellular carcinogenesis.

Heart failure is a common, lethal condition whose pathogenesis is poorly understood. Recent studies have identified low levels of myocyte apoptosis (80–250 myocytes per 105 nuclei) in failing human hearts. It remains unclear, however, whether this cell death is a coincidental finding, a protective process, or a causal component in pathogenesis. Using transgenic mice that express a conditionally active caspase exclusively in the myocardium, we demonstrate that very low levels of myocyte apoptosis (23 myocytes per 105 nuclei, compared with 1.5 myocytes per 105 nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy. Interestingly, these levels are four- to tenfold lower than those observed in failing human hearts. Conversely, inhibition of cardiac myocyte death in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure. To our knowledge, these data provide the first direct evidence that myocyte apoptosis may be a causal mechanism of heart failure, and they suggest that inhibition of this cell death process may constitute the basis for novel therapies