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.

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...

Overexpression of the antiapototic proteins Bcl-2 and Bcl-xL provides a common mechanism through which cancer cells gain a survival advantage and become resistant to conventional chemotherapy. Inhibition of these prosurvival proteins is an attractive strategy for cancer therapy. We recently described the discovery of a selective Bcl-xL antagonist that potentiates the antitumor activity of chemotherapy and radiation. Here we describe the use of structure-guided design to exploit a deep hydrophobic binding pocket on the surface of these proteins to develop the first dual, subnanomolar inhibitors of Bcl-xL and Bcl-2. This study culminated in the identification of 2, which exhibited EC50 values of 8 nM and 30 nM in Bcl-2 and Bcl-xL dependent cells, respectively. Compound 2 demonstrated single agent efficacy against human follicular lymphoma cell lines that overexpress Bcl-2, and efficacy in a murine xenograft model of lymphoma when given both as a single agent and in combination with etoposide.

Development of a rationally designed potentiator of cancer chemotherapy, via inhibition of Bcl-X(L) function, is described. Lead compounds generated by NMR screening and directed parallel synthesis displayed sub-microM binding but were strongly deactivated in the presence of serum. The dominant component of serum deactivation was identified as domain III of human serum albumin (HSA); NMR solution structures of inhibitors bound to both Bcl-X(L) and HSA domain III indicated two potential optimization sites for separation of affinities. Modifications at both sites resulted in compounds with improved Bcl-X(L) binding and greatly increased activity in the presence of human serum, culminating in 73R, which bound to Bcl-X(L) with a K(i) of 0.8 nM. In a cellular assay 73R reversed the protection afforded by Bcl-X(L) overexpression against cytokine deprivation in FL5.12 cells with an EC(50) of 0.47 microM. 73R showed little effect on the viability of the human non small cell lung cancer cell line A549. However, consistent with the proposed mechanism, 73R potentiated the activity of paclitaxel and UV irradiation in vitro and potentiated the antitumor efficacy of paclitaxel in a mouse xenograft model.

Alphavirus vectors, derived principally from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus, are widely used for gene expression studies in vitro and are being developed for both vaccine and gene therapy applications (25). Typically, these vectors are constructed in a format known as a replicon, due to the selfamplifying nature of the vector RNA (30). Replicons contain both the cis and trans alphavirus genetic elements required for RNA replication, as well as heterologous gene expression via the native subgenomic promoter. Upon introduction into cells, replicon RNA is translated to produce four nonstructural proteins (nsPs), which together comprise the alphaviral replicase. Replication proceeds through a minus-strand RNA intermediate and subsequently generates two distinct positive-strand RNA species, corresponding to a genomic-length vector RNA and an abundant subgenomic RNA encoding the heterologous gene (27). The replicon RNA can be packaged into virion-like particles by providing the structural proteins in trans, from in vitro-transcribed defective helper RNA (4, 15-17) or using packaging cell lines (16). Alternatively, the replicon RNA can be introduced directly into cells as plasmid DNA (2,6,8,13).In most mammalian cells, host macromolecular synthesis is inhibited following the introduction of alphavirus replicons, leading to eventual cell death by an apoptotic mechanism (11, 25). Thus, application of these vectors for some gene therapy applications and extended gene expression studies in cultured cells is limited. Given the many other attractive features of the alphavirus replicon system, it would be useful to extend the utility of these vectors to include long-term expression and reduced cytopathogenicity options.Under appropriate conditions, alphaviruses and alphavirusderived vectors can establish persistence in cultured cells (14,26,29) or exhibit delayed onset of cytopathic effects (9). The establishment of SIN replicon persistence in BHK cells has been associated with mutation of the protease domain of nsP2 (7, 10), and studies have suggested that the use of such mutants for long-term expression may be possible (1, 3). It remains to be determined whether mutation of other alphavirus nsPs or nsP2 domains can provide a noncytopathic phenotype by a similar or alternative mechanism.To expand the utility of the noncytopathic replicon and further explore how persistence is established, we isolated additional SIN replicons with this phenotype, as well as SFV replicons with a similar phenotype. Mutations that conferred the establishment of persistent replication were mapped to several regions of nsP2 for both SIN and SFV replicons, in addition to the same residue 726 mutation identified previously (7, 10). These mutations had various effects on the levels of genomic and subgenomic replicon RNA and, in some cases, processing of the nonstructural polyprotein.Selection of replicons that establish persistent replication. To select alphavirus replicon variants capable of...