BCL-2 proteins are critical for cell survival and are overexpressed in many tumors. ABT-737 is a small-molecule BH3 mimetic that exhibits single-agent activity against lymphoma and small-cell lung cancer in preclinical studies. We here report that ABT-737 effectively kills acute myeloid leukemia blast, progenitor, and stem cells without affecting normal hematopoietic cells. ABT-737 induced the disruption of the BCL-2/BAX complex and BAK-dependent but BIM-independent activation of the intrinsic apoptotic pathway. In cells with phosphorylated BCL-2 or increased MCL-1, ABT-737 was inactive. Inhibition of BCL-2 phosphorylation and reduction of MCL-1 expression restored sensitivity to ABT-737. These data suggest that ABT-737 could be a highly effective antileukemia agent when the mechanisms of resistance identified here are considered.

The protooncogene Bcl-2 functions as a suppressor of apoptosis in growth factor-dependent cells, but a postreceptor signaling mechanism is not known. We recently reported that interleukin 3 (IL-3) and erythropoietin, or the protein kinase C activator bryostatin-1 (Bryo), not only suppresses apoptosis but also stimulates the phosphorylation of Bcl-2 (May, W. S., Tyler, P. G., Ito, T., Armstrong, D. K., Qatsha, K. A., and Davidson, N. E. (1994) J. Biol. Chem. 269, 26865-26870). To test whether phosphorylation is required for Bcl-2 function, conservative serine 3 alanine mutations were produced at the seven putative protein kinase C phosphorylation sites in Bcl-2. Results indicate that the S70A Bcl-2 mutant fails to be phosphorylated after IL-3 or Bryo stimulation and is unable to support prolonged cell survival either upon IL-3 deprivation or etoposide treatment when compared with wild-type Bcl-2. In contrast, a Ser 3 Glu mutant, S70E, which may mimic a potential phosphate charge, more potently suppressed the etoposide-induced apoptosis than wild type in the absence of IL-3. Since the loss of function S70A mutant can heterodimerize with its partner protein and death effector Bax, these findings demonstrate that Bcl-2:Bax heterodimerization is not sufficient and Bcl-2 phosphorylation is required for full Bcl-2 death suppressor signaling activity.

Malaria is one of the most significant causes of childhood mortality but disease control efforts are threatened by resistance of the Plasmodium parasite to current therapies. Continued progress in combating malaria requires development of new, easy to administer drug combinations with broad ranging activity against all manifestations of the disease. DSM265, a triazolopyrimidine-based inhibitor of the pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH), is the first DHODH inhibitor to reach clinical development for treatment of malaria. We describe studies profiling the biological activity, pharmacological and pharmacokinetic properties, and safety of DSM265, which supported its advancement to human trials. DSM265 is highly selective towards DHODH of the malaria parasite Plasmodium, efficacious against both blood and liver stages of P. falciparum, and active against drug-resistant parasite isolates. Favorable pharmacokinetic properties of DSM265 are predicted to provide therapeutic concentrations for more than 8 days after a single oral dose in the range of 200–400 mg. DSM265 was well tolerated in repeat dose and cardiovascular safety studies in mice and dogs, was not mutagenic, and was inactive against panels of human enzymes/receptors. The excellent safety profile, blood and liver-stage activity, and predicted long human half-life position DSM265 as a new potential drug combination partner for either single-dose treatment or once weekly chemoprevention. DSM265 has advantages over current treatment options that are dosed daily or are inactive on the parasite liver-stage

Phosphorylation of Bcl2 at serine 70 may result from activation of a classic protein kinase C (PKC) isoform and is required for functional suppression of apoptosis by Bcl2 in murine growth factor-dependent cell lines (Ito, T., Deng, X., Carr, B., and May, W. S. (1997) J. Biol. Chem. 272, 11671-11673). Human pre-B REH cells express high levels of Bcl2 yet remain sensitive to the chemotherapeutic agents etoposide, cytosine arabinoside, and Adriamycin. In contrast, myeloid leukemiaderived HL60 cells express less than half the level of Bcl-2 but are >10-fold more resistant to apoptosis induced by these drugs. The mechanism responsible for this apparent dichotomy appears to involve a deficiency of mitochondrial PKC␣ since 1) HL60 but not REH cells contain highly phosphorylated Bcl2; 2) PKC␣ is the only classical isoform co-localized with Bcl2 in HL60 but not REH mitochondrial membranes; 3) the natural product and potent PKC activator bryostatin-1 induces mitochondrial localization of PKC␣ in association with Bcl2 phosphorylation and increased REH cell resistance to drug-induced apoptosis; 4) PKC␣ can directly phosphorylate wild-type but not phosphorylation-negative and loss of function S70A Bcl2 in vitro; 5) stable, forced expression of exogenous PKC␣ induces mitochondrial localization of PKC␣, increased Bcl2 phosphorylation and a >10-fold increase in resistance to drug-induced cell death; and (6) PKC␣-transduced cells remain highly sensitive to staurosporine, a potent PKC inhibitor. Furthermore, treatment of the PKC␣ transformants with bryostatin-1 leads to even higher levels of mitochondrial PKC␣, Bcl2 phosphorylation, and REH cell survival following chemotherapy. While these findings strongly support a role for PKC␣ as a functional Bcl2 kinase that can enhance cell resistance to antileukemic chemotherapy, they do not exclude the possibility that another Bcl2 kinase(s) may also exist. Collectively, these findings identify a functional role for PKC␣ in Bcl2 phosphorylation and in resistance to chemotherapy and suggest a novel target for antileukemic strategies.

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential anti-malarials we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound towards clinical candidate status.

No abstract

Cell division is finely controlled by various molecules including small G proteins and kinases/phosphatases. Among these, Aurora B, RhoA, and the GAP MgcRacGAP have been implicated in cytokinesis, but their underlying mechanisms of action have remained unclear. Here, we show that MgcRacGAP colocalizes with Aurora B and RhoA, but not Rac1/Cdc42, at the midbody. We also report that Aurora B phosphorylates MgcRacGAP on serine residues and that this modification induces latent GAP activity toward RhoA in vitro. Expression of a kinase-defective mutant of Aurora B disrupts cytokinesis and inhibits phosphorylation of MgcRacGAP at Ser387, but not its localization to the midbody. Overexpression of a phosphorylation-deficient MgcRacGAP-S387A mutant, but not phosphorylation-mimic MgcRacGAP-S387D mutant, arrests cytokinesis at a late stage and induces polyploidy. Together, these findings indicate that during cytokinesis, MgcRacGAP, previously known as a GAP for Rac/Cdc42, is functionally converted to a RhoGAP through phosphorylation by Aurora B.

). The potent apoptotic agent ceramide can activate a PP2A, suggesting that one potential component of the ceramide-induced death signal may involve the inactivation of Bcl2. Results indicate that C2-ceramide but not inactive C2-dihydroceramide, was found to specifically activate a mitochondrial PP2A, which rapidly and completely induced Bcl2 dephosphorylation and correlated closely with ceramide-induced cell death. Using a genetic approach, the gain-of-function S70E Bcl2 mutation, which mimics phosphorylation, fails to undergo apoptosis even with the addition of high doses of ceramide (IC 50 > 50 M). In contrast, cells overexpressing exogenous wild-type Bcl2 were sensitive to ceramide at dosages where PP2A is fully active and Bcl2 would be expected to be dephosphorylated (IC 50 ‫؍‬ 14 M). These findings indicate that in cells expressing functional Bcl2, the mechanism of death action for ceramide may involve, at least in part, a mitochondrial PP2A that dephosphorylates and inactivates Bcl2.