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PDF file - 1279K, Supplementary Figure 1. PANC-1 and Mia-PaCa cells are highly sensitive to RRD-251. Supplementary Figure 2. RRD-251 enhances the pro-apoptotic effects of gemcitabine. TABLE 1. Varying sensitivity of pancreatic cancer cells to RRD-251. Supplementary Table 2: RRD-251 enhances early and late apoptosis.

Although it is well established that cyclin-dependent kinases phosphorylate and inactivate Rb, the Raf-1 kinase physically interacts with Rb and initiates the phosphorylation cascade early in the cell cycle. We have identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251), that potently and selectively disrupts the Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E, and Rb/HDAC binding. The selective inhibition of Rb/Raf-1 binding suppressed the ability of Rb to recruit Raf-1 to proliferative promoters and inhibited E2F1-dependent transcriptional activity. RRD-251 inhibited anchorage-dependent and anchorage-independent growth of human cancer cells and knockdown of Rb with short hairpin RNA or forced expression of E2F1 rescued cells from RRD-251–mediated growth arrest. P.o. treatment of mice resulted in significant tumor growth suppression only in tumors with functional Rb, and this was accompanied by inhibition of angiogenesis, inhibition of proliferation, decreased phosphorylated Rb levels, and inhibition of Rb/Raf-1 but not Rb/E2F1 binding in vivo. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents. [Cancer Res 2008;68(10):3810–8]

Supplementary Figures 1-3 from A Small Molecule Disruptor of Rb/Raf-1 Interaction Inhibits Cell Proliferation, Angiogenesis, and Growth of Human Tumor Xenografts in Nude Mice

Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb–Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n = 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P = 0.0272), which correlated with reduced overall patient survival (P = 0.0186). To define the antitumor effects of RRD-251 (50 μmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50 mg/kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb–Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb–Raf-1 interaction might be a promising strategy targeting pancreatic cancer. Mol Cancer Ther; 12(12); 2722–34. ©2013 AACR.

Interferon-α2b (IFN-α2b) is used to treat melanoma but there is a need to improve its efficacy. IFN-α2b signaling requires STAT1/STAT2 tyrosine phosphorylation and is subject to negative regulation by phosphatases. In this study, we determined whether inhibition of the protein tyrosine phosphatase Shp2 could enhance IFN-α2b responses in human melanoma cells. Shp2 knockdown increased IFN-α2b-stimulated STAT1 Tyr-701 phosphorylation and ISRE-luciferase activity even though it did not affect STAT2 Tyr-690 phosphorylation in A375 cells. In A375 tumor xenografts, Shp2 knockdown enhanced the anti-melanoma effect of IFN-α2b. Furthermore, the Shp2 inhibitor SPI-112Me increased the IFN-α2b-induced STAT1 activation and anti-proliferative response in A375 and SK-MEL-2 cells. These results demonstrate that inhibition of Shp2 can enhance the anti-melanoma activity of IFN-α2b.

MDMX is a heterodimeric partner of MDM2 and a critical regulator of p53. The MDMX level is generally elevated in tumors with wild-type p53 and contributes to p53 inactivation. MDMX degradation is controlled in part by MDM2-mediated ubiquitination. Here, we show that MDMX turnover is highly responsive to changes in MDM2 level in non-transformed cells, but not in tumor cells. We found that loss of alternate reading frame (ARF) expression, which occurs in most tumors with wild-type p53, significantly reduces MDMX sensitivity to MDM2. Restoration of ARF expression in tumor cells enables MDM2 to degrade MDMX in a dose-dependent manner. ARF binds to MDM2 and stimulates a second-site interaction between the central region of MDM2 and MDMX, and thus increases MDMX-MDM2 binding and MDMX ubiquitination. These results reveal an important abnormality in the p53-regulatory pathway as a consequence of ARF deficiency. Loss of ARF during tumor development not only prevents p53 stabilization by proliferative stress but also causes accumulation of MDMX that compromises p53 activity. This phenomenon may reduce the clinical efficacy of MDM2-specific inhibitors by preventing MDMX downregulation.

The protein tyrosine phosphatase (PTP) Shp2 (PTPN11) is an attractive target for anticancer drug discovery because it mediates growth factor signaling and its gain-of-function mutants are causally linked to leukemias. We previously synthesized SPI-112 from a lead compound of Shp2 inhibitor, NSC-117199. In this study, we demonstrated that SPI-112 bound to Shp2 by surface plasmon resonance (SPR) and displayed competitive inhibitor kinetics to Shp2. Like some other compounds in the PTP inhibitor discovery efforts, SPI-112 was not cell permeable, precluding its use in biological studies. To overcome the cell permeation issue, we prepared a methyl ester SPI-112 analog (SPI-112Me) that is predicted to be hydrolyzed to SPI-112 upon entry into cells. Fluorescence uptake assay and confocal imaging suggested that SPI-112Me was taken up by cells. Incubation of cells with SPI-112Me inhibited epidermal growth factor (EGF)-stimulated Shp2 PTP activity and Shp2-mediated paxillin dephosphorylation, Erk1/2 activation, and cell migration. SPI-112Me treatment also inhibited Erk1/2 activation by a Gab1-Shp2 chimera. Treatment of Shp2(E76K) mutant-transformed TF-1 myeloid cells with SPI-112Me resulted in inhibition of Shp2(E76K)-dependent cell survival, which is associated with inhibition of Shp2(E76K) PTP activity, Shp2(E76K)-induced Erk1/2 activation, and Bcl-XL expression. Furthermore, SPI-112Me enhanced interferon-gamma (IFN-gamma)-stimulated STAT1 tyrosine phosphorylation, ISRE-luciferase reporter activity, p21 expression, and the anti-proliferative effect. Thus, the SPI-112 methyl ester analog was able to inhibit cellular Shp2 PTP activity.

Clusterin (CLU), in its cytoplasmic form, is abundant in many advanced cancers and has been established to be cytoprotective against chemotherapeutic agents including docetaxel. However, little is known of the mechanism of its induction. Here, we provide evidence that AKT plays a critical role in upregulating cytoplasmic/secretory sCLU, which is responsible for docetaxel resistance. Western blot analysis indicated that docetaxel-resistant sublines derived from DU145 and PC3 prostate tumor cell lines displayed a markedly increased phospho-AKT level closely accompanied by heightened sCLU expression when compared with parental cells. To examine if AKT has a role in sCLU expression, AKT blockade was done by treatment with a specific inhibitor, API-2, or dominant-negative AKT transduction before analysis of sCLU gene expression. Loss of AKT function resulted in loss of sCLU and was accompanied by chemosensitization to docetaxel and increased cell death via a caspase-3–dependent pathway. To confirm that AKT affected resistance to docetaxel through sCLU and not through other mediators, tumor cells were first transfected with full-length CLU for overexpression and then treated with the AKT inhibitor API-2. We found that once sCLU was overexpressed, API-2 could not chemosensitize the tumor cells to docetaxel. Thus, the chemoresistance to docetaxel is mediated by sCLU and it can be induced by AKT. Lastly, AKT was found to mediate sCLU induction via signal transducer and activator of transcription 1 activation, which we have earlier shown to drive sCLU gene expression. These results identify a previously unrecognized pathway linking AKT to cytoprotection by sCLU in tumor cells. Mol Cancer Ther; 9(6); 1831–41. ©2010 AACR.

An efficient protocol for stereo- and regiocontrolled synthesis of small polyfunctional molecules is presented. The stereospecific addition of PhSeCl to 2,5-dihydro-2,5-dimethoxy-furan 1 in solvents, such as methylene chloride and methanol, gives cyclic and linear acetals 2 and 3, depending on the solvent used. Emphasis is given to the regiocontrolled hydrolysis of acetal groups for the preparation of stereodefined and highly functionalized C4 synthons, such as 8, 9 and 13.

[reaction: see text] The Sakurai-Hosomi-type allylation of aromatic and heteroaromatic aldehydes can be catalyzed by the new heterobidenate bipyridine monoxide PINDOX with high enantioselectivities. The sterochemical outcome is mainly controlled by the axial chirality in PINDOX, which in turn is determined by the annulated terpene units.

An efficient protocol for stereo- and regiocontrolled synthesis of small polyfunctional molecules is presented. The stereospecific addition of PhSeCl to 2,5-dihydro-2,5-dimethoxy-furan 1 in solvents, such as methylene chloride and methanol, gives cyclic and linear acetals 2 and 3, depending on the solvent used. Emphasis is given to the regiocontrolled hydrolysis of acetal groups for the preparation of stereodefined and highly functionalized C4 synthons, such as 8, 9 and 13.

Although it is well established that cyclin-dependent kinases phosphorylate and inactivate Rb, the Raf-1 kinase physically interacts with Rb and initiates the phosphorylation cascade early in the cell cycle. We have identified an orally active small molecule, Rb/Raf-1 disruptor 251 (RRD-251), that potently and selectively disrupts the Rb/Raf-1 but not Rb/E2F, Rb/prohibitin, Rb/cyclin E, and Rb/HDAC binding. The selective inhibition of Rb/Raf-1 binding suppressed the ability of Rb to recruit Raf-1 to proliferative promoters and inhibited E2F1-dependent transcriptional activity. RRD-251 inhibited anchorage-dependent and anchorage-independent growth of human cancer cells and knockdown of Rb with short hairpin RNA or forced expression of E2F1 rescued cells from RRD-251–mediated growth arrest. P.o. treatment of mice resulted in significant tumor growth suppression only in tumors with functional Rb, and this was accompanied by inhibition of angiogenesis, inhibition of proliferation, decreased phosphorylated Rb levels, and inhibition of Rb/Raf-1 but not Rb/E2F1 binding in vivo. Thus, selective targeting of Rb/Raf-1 interaction seems to be a promising approach for developing novel chemotherapeutic agents. [Cancer Res 2008;68(10):3810–8]

A series of modular bipyridine-type ligands 1 and 3-9 has been synthesized via a de novo construction of the pyridine nucleus. The chiral moieties of these ligands originate from the isoprenoid chiral pool, namely, beta-pinene (10 --1), 3-carene (14 --3 and 5), 2-carene (28 --4), alpha-pinene (43 --6-8), and dehydropregnenolone acetate (48 --9), respectively. Copper(I) complexes, derived from these ligands and (TfO)(2)Cu (1 mol %) upon an in situ reduction with phenylhydrazine, exhibit good enantioselectivity (up to 82% ee) and unusually high reaction rate (typicaly 30 min at room temperature) in allylic oxidation of cyclic olefins (52 --53). Copper-catalyzed cyclopropanation proceeded withor =76% enantioselectivity and approximately 3:1 to 99:1 trans/cis-diastereoselectivity (54 --55 + 56). The level of the asymmetric induction is discussed in terms of the ligand architecture that controls the stereochemical environment of the coordinated metal.

Allylation of aromatic and heteroaromatic aldehydes 1a-k with allyltrichlorosilane 2 can be catalyzed by the new heterobidentate, terpene-derived bipyridine N-monoxides 4, 6a,b, and 8-11 (/=10 mol %) to afford (S)-(-)-3 with high enantioselectivities (/=99% ee). The stereochemical outcome has been found to be controlled by the axial chirality of the catalyst, which in turn is determined by the central chirality of the annulated terpene units. Solvent effects on the conversion and the level of asymmetric induction have been elucidated, and MeCN has been identified as the optimal solvent for these catalysts.

Anti-apoptotic members of the Bcl-2 protein family, such as Bcl-2, Bcl-xL and Mcl-1, are among the most effective inhibitors of apoptosis and are frequently upregulated in various types of human cancer. A number of pro-apoptotic proteins share only the BH3 domain with other members of the Bcl-2 family, so called BH3-only proteins, and appear to function essentially as transdominant inhibitors by binding to anti-apoptotic Bcl-2 family proteins and neutralizing their cell-survival activity. The development of small molecules to mimic the BH3 proteins has been an intense area of research. However, to date available Bcl-2 family proteins inhibitors are either pan-inhibitors such as (−)-gossypol (which simultaneously inhibits Bcl-xl/BH3, Bcl-2/BH3 and Mcl-1/BH3 interactions) or are selective for the Bcl-xl/BH3 and Bcl-2/BH3 interactions. Hence a selective Mcl-1/BH3 inhibitor would greatly aid investigating the role of Mcl-1 in cancer and in drug resistance. Our current study is centered on the identification and the synthesis of small molecule selective inhibitors of the Mcl-1/BH3 interaction. To this aim we interrogated the hit-list from inhibitors of the MLSCN screen of the Mcl-1-NOXA interaction [(Emory, AID 1417) TR-FRET screen for Mcl-1-NOXA inhibitors]. Selected compounds were purchased and assessed for activity in a fluorescence polarization and ELISA assay, to reveal the hit HLM050001 (FP IC50 0.8 ± 0.1 µM; ELISA IC50 19.3 ± 2.5 µM). HLM050001binding to Mcl-1 is currently being assessed via co-crystallization and other biophysical techniques. We will report the synthesis and biological evaluation of focused libraries based on the initial hit and designed by modeling and docking to Mcl-1. Structure activity relationship studies around the hit will be disclosed as well as the outcomes of further rounds of chemical design and biological assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1354. doi:10.1158/1538-7445.AM2011-1354

MDM2 and MDMX function as key regulators of p53 by binding to its N terminus, inhibiting its transcriptional activity, and promoting its degradation. In particular, MDM2 is overexpressed in some of human tumors, and with MDMX contributes directly to loss of p53 function during the development of nearly 50% of human cancers. Due to p53 inactivation, MDM2 in many tumors confers tumor survival; therefore it is an important molecular target for anticancer therapy. Several studies showed that reactivation of wild type p53 in tumor cells can be obtained by disrupting the MDM2/p53 interaction with peptidic, peptidomimetic, and small molecule p53-mimetics. Specific successful examples include the Nutlins and spirooxindole analogs (MI-219 and MI-63). Amongst the peptidic and peptidomimetic inhibitors examined to date, none is nearly as effective as Nutlins and MI-219 in tumor killing in vitro. Hence, new inhibitors against MDM2 and/or MDMX are needed: as cell permeable chemical probes of the p53 pathway in cancer biology, and as templates for structure-based rational design of p53 activators for future therapeutic use. As part of our drug discovery program to identify antagonists of the p53/MDM2 and p53/MDMx protein-protein interactions, a high-throughput in-silico screen of a 3.2 millions virtual library of compounds (from Schrödinger, Inc.). A physical restraint was applied during the screen, in order to mimic binding to the hydrophobic cleft of MDM2 normally occupied by three p53 side chains (F19, W23, and L26) that are critical for MDM2/p53 binding. The top highest ranked 160 compounds were then assessed for their ability to block p53 interaction with MDM2 and MDMx in an ELISA assay. This resulted in the identification of E12/DP3-117, a small molecule disruptor of the p53/MDM2 protein-protein interaction with an IC50 value of 47 ± 14 μM. We will report the synthesis and biological evaluation of focused libraries based on the initial hit and on compounds showing improved activity. Structure activity relationship studies around the hits will be disclosed as well as the outcomes of further rounds of chemical design and biological assessment. Binding of E12/DP3-117 to MDM2 is currently being assessed via co-crystallization and other biophysical techniques. We will describe the use of the crystal structure of p53-like mutant peptides in complex with the N-terminal domains of Mdm2, as the basis for rational design of more potent MDM2 small-molecule/peptide hybrid inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3242. doi:10.1158/1538-7445.AM2011-3242

It is well accepted that escape of apoptosis is a major mechanism for cancer progression and drug resistance. Anti-apoptotic members of the Bcl-2 protein family, such as Bcl-2, Bcl-xL and Mcl-1, are among the most effective inhibitors of apoptosis and are frequently upregulated in various types of human cancer. A number of pro-apoptotic proteins share only the BH3 domain with other members of the Bcl-2 family, so called BH3-only proteins, and appear to function essentially as transdominant inhibitors by binding to anti-apoptotic Bcl-2 family proteins and neutralizing their cell-survival activity. The development of small molecules to mimic the BH3 proteins has been an intense area of research. It is clear from the development of ABT737 (and its orally available relative ABT-263) that targeting of Bcl-2/Bcl-xL by small molecule BH3-mimetics is a promising strategy for the development of a new class of anticancer drugs. However the clinical potential of ABT737 may be limited since it does not inhibit the pro-survival Bcl-2 family protein Mcl-1. To date available Bcl-2 family proteins inhibitors are either pan-inhibitors such as (-)-gossypol (which simultaneously inhibits Bcl-xl/BH3, Bcl-2/BH3 and Mcl-1/BH3 interactions) or are selective for the Bcl-xl/BH3 and Bcl-2/BH3 interactions. Hence a selective Mcl-1/BH3 inhibitor would greatly aid investigating the role of Mcl-1 in cancer and in drug resistance and complement the activity of ABT737. Our current study is centered on the identification and the synthesis of small molecule selective inhibitors of the Mcl-1/BH3 interaction. To this aim we interrogated the hit-list from inhibitors of the MLSCN screen of the Mcl-1-NOXA interaction [(Emory, AID 1022) TR-FRET screen for Mcl-1-NOXA inhibitors]. We docked 2100 of the experimental 2100 MLSCN hits with Mcl-1 protein structure derived from the Mcl-1-NOXA X-ray crystal structure. We then examined the top 375 compounds. We then ranked them by docking score, the number of MLSCN biological assays tested and number of hits in these assays. We then selected compounds that docked well and had been tested in many MLSCN assays for which they were not hits. Selected compounds were purchased and assessed for activity in an Alphascreen and ELISA assay, to reveal the hit HL5-026 (IC50 = 46 µM). HL5-026 is an inhibitor of the Mcl-1/Bim-BH3 interaction that has four-fold selectivity over Bcl-xl/Bim-BH3. HL5-026 binding to Mcl-1 is currently being assessed via co-crystallization and other biophysical techniques. We will report the synthesis and biological evaluation of focused libraries based on the initial hit and designed by modeling and docking to Mcl-1. First generation libraries have revealed compounds of improved activity. Structure activity relationships around the hits will be disclosed as well as the outcomes of further rounds of chemical design and biological assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 732.

Protein tyrosine phosphorylation affects cellular activities that control tumor growth and progression. Protein tyrosine phosphorylation, a key regulatory process of signal transduction pathways, is controlled by the action protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Kinases are now a clinically proven anticancer drug target. The development of PTP inhibitors is, by comparison, a relatively new field. Since PTPs do not require a small molecule cofactor they generally show a low propensity to bind a small molecule inhibitor. Nevertheless there are now examples of highly potent PTP inhibitors that provide promising proof-of-principle that PTPs are druggable. Most PTP inhibitors have a charged or highly polar functional group that mimics the phosphotyrosine residue, that often leads to poor ADME and cell permeability properties. We will report our efforts to address the problems of cell permeability we have encountered in our Shp2 PTP inhibitor program. We have previously reported a series of isatin based Shp2 inhibitors based on a compound (NSC-117199) identified from screen of the NCI Diversity Set. We synthesized >100 analogs of NSC-117199 in our lead optimization effort, which yielded several compounds with approximately 50-fold increase in potency and > 10-fold increase in selectivity (Shp2 versus Shp1). However, these Shp2 PTP inhibitors have either a polar nitro or carboxyl group and have no detectable cellular activity. We will report three approaches to address this problem. The first approach was to prepare neutral ester prodrugs. Several compounds have clearly demonstrable activity against Shp2 in cells. A series of esters has also been designed to also improve the water solubility of the PTP inhibitor. A second approach taken was to further modify the isatin scaffold that does not necessitate the use of a prodrug strategy. We will report the discovery of new isatins related to NSC-117199 that have new modifications that result in cell active Shp2 inhibitors. Finally we will report the discovery and preliminary optimization of new classes of cell active Shp2 inhibitors based on alternative scaffolds. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 761.