We have designed MI-219 as a potent, highly selective and orally active small-molecule inhibitor of the MDM2-p53 interaction. MI-219 binds to human MDM2 with a Ki value of 5 nM and is 10,000-fold selective for MDM2 over MDMX. It disrupts the MDM2-p53 interaction and activates the p53 pathway in cells with wild-type p53, which leads to induction of cell cycle arrest in all cells and selective apoptosis in tumor cells. MI-219 stimulates rapid but transient p53 activation in established tumor xenograft tissues, resulting in inhibition of cell proliferation, induction of apoptosis, and complete tumor growth inhibition. MI-219 activates p53 in normal tissues with minimal p53 accumulation and is not toxic to animals. MI-219 warrants clinical investigation as a new agent for cancer treatment.cancer therapy ͉ MDM2-p53 protein-protein interaction ͉ selective toxicity to tumors ͉ small-molecule inhibitor T he tumor suppressor p53 plays a central role in the regulation of cell cycle, apoptosis, DNA repair, and senescence (1-4). Because of the prominent role played by p53 in suppressing oncogenesis (5), it is not surprising that p53 function is impaired in all human cancers. Several distinct approaches have been pursued to restore p53 function as a new cancer therapeutic strategy (6-9). Three recent studies, using unique genetic mouse models, have demonstrated that the restoration of p53 leads universally to a rapid and robust regression of established sarcomas, lymphomas, and liver tumors (10)(11)(12)(13)(14). These studies provide strong evidence that established tumors remain persistently vulnerable to p53 tumorsuppressor function and that restoration of p53 function is therefore a powerful cancer therapeutic strategy (13).In Ϸ50% of human cancers, the gene encoding p53 is either deleted or mutated, rendering the p53 protein inactive (5, 15). In the remaining cancers, p53 retains its wild-type status but its function is effectively inhibited by its primary cellular inhibitor, the human MDM2 oncoprotein (mouse double minute 2, also termed HDM2 in humans) (5,16,17). One attractive pharmacological approach to p53 reactivation is to use a small molecule to block the MDM2-p53 interaction (6)(7)(8)18). The discovery of the Nutlins provided the important proof of the concept for this approach (7). Nutlins were shown to bind to MDM2, block the MDM2-p53 interaction, and activate wild-type p53 (7,(19)(20)(21). Nutlin-3a exhibits strong anti-tumor activity in multiple xenograft mouse models of human cancer (7,19). The discovery of the Nutlins has fueled enthusiasm for the development of small-molecule MDM2 inhibitors as a new class of anticancer therapy (6,8,22,23).One critical question in the development of MDM2 inhibitors for cancer treatment is their potential toxicity to normal tissues. This concern was heightened by a recent genetic study, which showed that p53 activation in the absence of the MDM2 gene causes severe toxicity to radiosensitive normal adult mouse tissues, leading to rapid animal death (24). Previous studies on ...

Signal transducer and activator of transcription 3 (STAT3) is an attractive cancer therapeutic target. Here we report the discovery of SD-36, a small-molecule degrader of STAT3. SD-36 potently induces the degradation of STAT3 protein in vitro and in vivo and demonstrates high selectivity over other STAT members. Induced degradation of STAT3 results in a strong suppression of its transcription network in leukemia and lymphoma cells. SD-36 inhibits the growth of a subset of acute myeloid leukemia and anaplastic large-cell lymphoma cell lines by inducing cell-cycle arrest and/or apoptosis. SD-36 achieves complete and long-lasting tumor regression in multiple xenograft mouse models at well-tolerated dose schedules. Degradation of STAT3 protein, therefore, is a promising cancer therapeutic strategy.

Wnts are important for various developmental and oncogenic processes. Here we show that Wnt signaling functions at synapses in hippocampal neurons. Tetanic stimulations induce N-methyl-Daspartate receptor-dependent synaptic Wnt3a release, nuclear ␤-catenin accumulations, and the activation of Wnt target genes. Suppression of Wnt signaling impairs long term potentiation. Conversely, activation of Wnt signaling facilitates long term potentiation. These findings suggest that Wnt signaling plays a critical role in regulating synaptic plasticity.Wnts are a family of secreted, lipid-modified signaling proteins that act as short range ligands to locally activate receptor-mediated signaling cascades. Wnt signaling is critical for a variety of developmental processes; its dysregulation is the causal factor for many diseases, especially cancer (1, 2). Wnt ligands bind to Frizzled receptors to activate intracellular signaling cascades, including the Wnt/␤-catenin, Wnt/Ca 2ϩ , and Wnt/planar cell polarity pathways (3). In the canonical Wnt/␤-catenin-signaling pathway, Frizzled receptors signal through Dishevelled to inhibit the kinase activity of glycogen synthase kinase 3␤ in a protein degradation complex containing Axin, ␤-catenin, and other proteins. When Wnt signaling is inactive, ␤-catenin is phosphorylated by glycogen synthase kinase 3␤ and thus rapidly degraded via the proteosome pathway; the activation of Wnt signaling stabilizes ␤-catenin by inhibiting glycogen synthase kinase 3␤. Stabilized ␤-catenin translocates to the nuclei and binds the TCF/LEF family of transcription factors to regulate the expression of Wnt target genes (1, 2). In addition, ␤-catenin is a component of the cadherin complex and plays an important role in regulating cell-cell adhesion (4). Among many Wnt-regulated developmental processes are neural patterning and differentiation (5), including hippocampal formation (6 -8), dendritic morphogenesis (9, 10), axon guidance (11-16), and synapse formation (11,17,18).Wnts are expressed in the brain (19 -21). Dysregulation of Wnt signaling has been suggested as an etiological cause for specific mental disorders. For example, Wnt signaling is up-regulated in schizophrenic brains (22)(23)(24). Several studies suggested an association of Frizzled-3 with schizophrenic susceptibility (25-27). On the other hand, downregulation of the Wnt/␤-catenin pathway is implicated in Alzheimer disease etiology (28 -34). A recent study indicated that cocaine exposures affect the expression of many Wnt signal-related genes (35). In addition, mutation of Dishevelled-1 in mice produces behavioral impairments (36), whereas mutation of a Drosophila Wnt receptor, Derailed (13), causes memory deficits (37, 38). Collectively, available data point to the role of Wnt signaling in the modulation of brain functions. However, the mechanism by which Wnt signaling regulates brain function is completely unknown.Here we report the synaptic localization of Wnt signaling proteins in mouse hippocampal neurons, the activity-induced Wnt3a rel...

Rationale-Identification of behaviors specifically mediated by the dopamine D2 and D3 receptors would allow for the determination of in vivo receptor selectivity and aide the development of novel therapeutics for dopamine-related diseases.Objectives-These studies were aimed at evaluating the specific receptors involved in the mediation of D2/D3 agonist-induced yawning and hypothermia.Correspondence to: James H. Woods. NIH Public AccessAuthor Manuscript Psychopharmacology (Berl) Methods-The relative potencies of a series of D2-like agonists to produce yawning and hypothermia were determined. The ability of D3-and D2-selective antagonists to inhibit the induction of yawning and hypothermia were assessed, and a series of D2/D3 antagonists were characterized with respect to their ability to alter yawning induced by a low and high dose of PD-128,907 as well as sumanirole-induced hypothermia.Results-D3-preferring agonists induced yawning at lower doses than those required to induce hypothermia, and the D2-preferring agonist, sumanirole, induced hypothermia at lower doses than were necessary to induce yawning. The rank order of D3 selectivity was pramipexole > PD-128,907 = 7-OH-DPAT = quinpirole = quinelorane > apomorphine = U91356A. Sumanirole had only D2 agonist effects. PG01037, SB-277011A and U99194 were all D3-selective antagonists, whereas haloperidol and L-741,626 were D2-selective antagonists and nafadotride's profile of action was more similar to the D2 antagonists than to the D3 antagonists.Conclusions-D3 and D2 receptors have specific roles in the mediation of yawning and hypothermia, respectively, and the analysis of these effects allow inferences to be made regarding the selectivity of D2/D3 agonists and antagonists with respect to their actions at D2 and D3 receptors.

We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59CJ-OH (API; 1-5 Mmol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G 1 cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the ;-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.

Purpose: It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA doublestrand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RAS mt ) cells. Experimental Design: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RAS mt (A549) and K-RAS wt (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser 139 ), ATM (Ser 1981 ), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr 2609 ) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gH2AX foci and frequency of micronuclei. Results: BIBX reduced clonogenic survival of K-RAS mt -A549 cells, but not of K-RAS wt -FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS mt -A549 and MDA-MB-231 cells, but not in K-RAS wt -FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr 2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RAS mt nor in K-RAS wt cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation. Conclusion: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.

We report herein the design of potent and orally active small-molecule inhibitors of the MDM2-p53 interaction. Compound 5 binds to MDM2 with a K i value of 0.6 nM, activates p53 at concentrations as low as 40 nM, and potently and selectively inhibits cell growth in tumor cells with wild-type p53 over tumor cells with mutated/deleted p53. Compound 5 has a good oral bioavailability and effectively inhibits tumor growth in the SJSA-1 xenograft model. The p53 tumor suppressor is an attractive cancer therapeutic target because its tumor suppressor activity can be stimulated to eradicate tumor cells. [1][2][3] In tumor cells with wild-type p53, the p53 activity is effectively inhibited by its endogenous inhibitor, the human MDM2 protein, through direct binding to p53. 4,5 The interaction site between MDM2 and p53 proteins is mediated by a well-defined pocket in MDM2 and a short helix from p53, making this site attractive for the design of small-molecule inhibitors to block the MDM2-p53 protein-protein interaction. 6,7 Reactivation of p53 by blocking the MDM2-p53 interaction using a smallmolecule inhibitor is being pursued as an exciting, new cancer therapeutic strategy. [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22] We have recently reported the design of spiro-oxindoles as a new class of potent, selective, cell permeable, non-peptidic, small-molecule inhibitors of the MDM2-p53 interaction. 9-11 Using a structure-based approach, we have obtained compound 1 (MI-63, Figure 1) as a potent and cell-permeable MDM2 inhibitor. Compound 1 binds to MDM2 protein with a low nanomolar affinity in our fluorescence-polarization (FP) based, competitive, biochemical binding assay. 10 Consistent with its mode of action, compound 1 potently inhibits cell growth in cancer cells with wild-type p53 and is selective over cancer cells with mutated/deleted p53. In our subsequent pharmacokinetic (PK) evaluations, compound 1 was found to have a poor PK profile and a modest oral bioavailability ( Analysis of the predicted binding model for 1 showed that the morpholinyl group is partially exposed to solvent. 10 This suggested that the morpholinyl group in 1 may be replaced by other groups without a detrimental effect on binding to MDM2 and cellular activity. We have therefore carried out chemical modifications of this region to investigate the structure-activity relationship on binding, cellular activity and PK parameters.We first designed and synthesized compounds 2 and 3 (Figure 1), in which the morpholinyl group in compound 1 is replaced by a methylpiperazinyl group or a methylpiperidinyl group, respectively. Our binding experiments showed that these two compounds bind to MDM2 with K i values of 1.5 and 2.0 nM, respectively (Table 1). Consistent with their high binding affinities to MDM2, they potently inhibit cell growth in the cancer cell lines with wild-type p53, and display excellent selectivity over cancer cell lines with deleted p53 (Table 1 and Supporting Information). However, PK testing showed that while compounds...

Organ preservation protocols in head and neck squamous cell carcinoma (HNSCC) are limited by tumors that fail to respond. We observed that larynx preservation and response to chemotherapy is significantly associated with p53 overexpression, and that most HNSCC cell lines with mutant p53 are more sensitive to cisplatin than those with wild-type p53. To investigate cisplatin resistance, we studied two HNSCC cell lines, UM-SCC-5 and UM-SCC-10B, and two resistant sublines developed by cultivation in gradually increasing concentrations of cisplatin. The cisplatin-selected cell lines, UM-SCC-5PT and UM-SCC-