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An inhibitor of the pleckstrin homology domain of CNK1 selectively blocks the growth of mutant KRAS cells and tumors Supplemental Information

Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain–containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild-type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial "addiction" to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane colocalization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild-type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS–driven cancer.Significance:These findings identify a therapeutic strategy to selectively block oncogenic KRas activity through the PH domain of Cnk1, which reduces its cell membrane binding, decreasing the efficiency of Ras signaling and tumor growth.

Supplementary Tables 1-2, Appendix materials, Figures 1-2 from In vitro and In vivo Activity of Novel Small-Molecule Inhibitors Targeting the Pleckstrin Homology Domain of Protein Kinase B/AKT

More than 1 million Americans yearly have a new or recurrent acute myocardial infarction (AMI). Although the mortality rate from AMI has decreased in recent years, post-MI congestive heart failure is increasing due to microvascular obstruction (MVO), ultimately limiting myocardial salvage. We aim to address this unmet need by devising an image-guided therapy called sonoreperfusion (SRP) to resolve MVO by ultrasound-targeted microbubble cavitation (UTMC). In this study, we hypothesized that fibrin-targeted phase shift microbubbles (FTPSMBs; ∼200 nm) (Microvascular Therapeutics, Inc) would improve SRP efficacy compared to fibrin-targeted microbubbles (FTMBs; 1–3 μm) to treat MVO owing to the smaller size and more efficient microthrombi penetration. A rat hindlimb model of MVO was created by directly injecting freshly prepared porcine microthrombi into the left femoral artery under contrast-enhanced ultrasound imaging (CEUS) guidance, and treated with UTMC (1 MHz, 1.5 MPa, 5 ms pulse duration, 5-sec pulse interval) with concomitant administration of FTPSMBs/FTMBs (3 mL/hr). The treatment effect was accessed with CEUS. UTMC with FTPSMBs caused more rapid and complete reperfusion of rat hindlimb following MVO compared with FTMB, likely owing to their small size and more effective thrombus penetration. Studies to explore the underlying molecular mechanisms associated with SRP treatments are underway.

Introduction: Microvascular obstruction (MVO) following coronary intervention is a common problem associated with worse clinical outcome, and has no consistent therapy. We are developing a new technique termed sonoreperfusion (SRP), to restore microvascular patency. This entails the use of ultrasound (US) to cause microbubble (MB) cavitation to dissolve microthrombi; this MB cavitation creates shear stress, activates nitric oxide release, and results in thrombolysis. In this pilot study, to further optimize SRP, we have used Phase Shift Microbubbles (PSMBs) (Microvascular Therapeutics, Inc). Owing to their much smaller size relative to standard MBs (~400 nm vs 3 μm) and greater microthrombus penetration, we will test the hypothesis that PSMBs attain successful reperfusion of MVO, and ultimately, will provide more rapid SRP, in our model of MVO. Methods: MVO of the rat ( n =3) hindlimb was created by direct injection of microthrombi into the left femoral artery, followed by infusion of therapeutic PSMBs. Definity MBs (Lantheus Medical Imaging) were infused through the jugular vein for contrast enhanced ultrasound imaging (CEUS). A transducer was positioned vertically above the hindlimb for therapeutic US pulses during concomitant administration of PSMBs. CEUS cine loops with burst replenishment were obtained at baseline (BL), 10 min post MVO, and after each of the two SRP treatment sessions (TX1, TX2) and analyzed (MATLAB). Results: Fig. 1 depicts a typical example demonstrating successful creation of MVO and complete reperfusion after SRP treatment. Data are expressed as mean ± standard error. Blood volume (dB) at BL (15.3 ± 1.1), MVO (5.2 ± 1.3), TX1 (19.8 ± 2.4), TX2 (17.9 ± 1.8) and flow rate (dB/sec) at BL (2.7 ± 0.4), MVO (0.2 ± 0.1) TX1 (2.1 ± 0.5), TX2 (2.7 ± 0.4). Conclusions: US targeted PSMB cavitation causes successful rapid reperfusion of rat hindlimb following MVO. Studies to explore the underlying molecular mechanisms associated with SRP of PSMBs are underway.

The Division of Cancer Prevention of the National Cancer Institute (NCI) and the Office of Disease Prevention of the National Institutes of Health co-sponsored the Translational Advances in Cancer Prevention Agent Development Meeting on August 27 to 28, 2020. The goals of this meeting were to foster the exchange of ideas and stimulate new collaborative interactions among leading cancer prevention researchers from basic and clinical research; highlight new and emerging trends in immunoprevention and chemoprevention as well as new information from clinical trials; and provide information to the extramural research community on the significant resources available from the NCI to promote prevention agent development and rapid translation to clinical trials. The meeting included two plenary talks and five sessions covering the range from pre-clinical studies with chemo/immunopreventive agents to ongoing cancer prevention clinical trials. In addition, two NCI informational sessions describing contract resources for the preclinical agent development and cooperative grants for the Cancer Prevention Clinical Trials Network were also presented.

OBJECTIVES: Early detection of Pancreatic Ductal Adenocarcinoma (PDAC) represents the most significant step towards the treatment of this aggressive lethal disease. Previously, we engineered a pre-clinical Thy1-targeted microbubble (MB(Thy1)) contrast agent that specifically recognizes Thy1 antigen overexpressed in the vasculature of murine PDAC tissues by ultrasound (US) imaging. In this study, we adopted a single-chain variable fragment (scFv) site-specific bioconjugation approach to construct clinically translatable MB(Thy1-scFv) and test for its efficacy in vivo in murine PDAC imaging and functionally evaluated the binding specificity of scFv ligand to human Thy1 in patient PDAC tissues ex vivo. MATERIALS AND METHODS: We recombinantly expressed the Thy1-scFv with a carboxy-terminus cysteine residue to facilitate its thioether conjugation to the PEGylated MBs presenting with maleimide functional groups. After the scFv-MB conjugations, we tested binding activity of the MB(Thy1-scFv) to MS1 cells over-expressing human Thy1 (MS1(Thy1)) under liquid shear stress conditions in vitro using a flow chamber set up at 0.6 mL/minute flow rate, corresponding to a wall shear stress rate of 100 seconds(−1), similar to that in tumor capillaries. For in vivo Thy1 US molecular imaging, MB(Thy1-scFv) were tested in the transgenic mouse model (C57BL/6J - Pdx1-Cre(tg/+); KRas(LSL-G12D/+;) Ink4a/Arf(−/−)) of PDAC, and in control mice (C57BL/6J) with L-arginine-induced pancreatitis or normal pancreas. To facilitate its clinical feasibility, we further produced Thy1-scFv without the bacterial fusion tags and confirmed its recognition of human Thy1 in cell lines by flow cytometry and patient PDAC frozen tissue sections of different clinical grades by immunofluorescence staining. RESULTS: Under shear stress flow conditions in vitro, MB(Thy1-scFv) bound to MS1(Thy1) cells at significantly higher numbers (3.0 ± 0.8 MB/cell; P

// Gabor Somlyai 1 , T. Que Collins 2,3 , Emmanuelle J. Meuillet 4 , Patel Hitendra 5 , Dominic P. D’Agostino 6 and Laszlo G. Boros 7,8,9,10 1 HYD, LLC for Cancer Research & Drug Development, Budapest, Hungary, European Union 2 CignatureHealth Metabolic Clinic, Santa Monica, CA, USA 3 EPIGENIX Foundation, El Segundo, CA, USA 4 The University of Arizona Cancer Center, Tucson, AZ, USA 5 Moores Cancer Center, University of California San Diego School of Medicine, La Jolla, CA, USA 6 Department of Molecular Pharmacology and Physiology, Morsani College of Medicine, Hyperbaric Biomedical Research Laboratory, University of South Florida, Tampa, FL, USA 7 Department of Pediatrics, University of California Los Angeles School of Medicine, Westwood, CA, USA 8 Los Angeles Biomedical Research Institute (LABIOMED) at the Harbor-UCLA Medical Center, Torrance, CA, USA 9 SiDMAP, LLC, Culver City, CA, USA 10 UCLA Clinical and Translational Science Institute, Torrance, CA, USA Correspondence to: Laszlo G. Boros, email: // Keywords : imatinib, phenformin, deuterobolomics, lipitor, metformin Received : January 06, 2017 Accepted : February 24, 2017 Published : March 15, 2017 Abstract Phenformin’s recently demonstrated efficacy in melanoma and Gleevec’s demonstrated anti-proliferative action in chronic myeloid leukemia may lie within these drugs’ significant pharmacokinetics, pharmacodynamics and structural homologies, which are reviewed herein. Gleevec’s success in turning a fatal leukemia into a manageable chronic disease has been trumpeted in medical, economic, political and social circles because it is considered the first successful targeted therapy. Investments have been immense in omics analyses and while in some cases they greatly helped the management of patients, in others targeted therapies failed to achieve clinically stable recurrence-free disease course or to substantially extend survival. Nevertheless protein kinase controlling approaches have persisted despite early warnings that the targeted genomics narrative is overblown. Experimental and clinical observations with Phenformin suggest an alternative explanation for Gleevec’s mode of action. Using 13 C-guided precise flux measurements, a comparative multiple cell line study demonstrated the drug’s downstream impact on submolecular fatty acid processing metabolic events that occurred independent of Gleevec’s molecular target. Clinical observations that hyperlipidemia and diabetes are both reversed in mice and in patients taking Gleevec support the drugs’ primary metabolic targets by biguanides and statins. This is evident by structural data demonstrating that Gleevec shows pyridine- and phenyl-guanidine homology with Phenformin and identical phenylcarbamoyl structural and ligand binding homology with Lipitor. The misunderstood mechanism of action of Gleevec is emblematic of the pervasive flawed reasoning that genomic analysis will lead to targeted, personalized diagnosis and therapy. The alternative perspective for Gleevec’s mode of action may turn oncotargets towards metabolic channel reaction architectures in leukemia and melanoma, as well as in other cancers.

Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain-containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial “addiction” to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane co-localization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS-driven cancer.

Cnk1 (connector enhancer of kinase suppressor of Ras 1) is a pleckstrin homology (PH) domain–containing scaffold protein that increases the efficiency of Ras signaling pathways, imparting efficiency and specificity to the response of cell proliferation, survival, and migration. Mutated KRAS (mut-KRAS) is the most common proto-oncogenic event, occurring in approximately 25% of human cancers and has no effective treatment. In this study, we show that selective inhibition of Cnk1 blocks growth and Raf/Mek/Erk, Rho and RalA/B signaling in mut-KRAS lung and colon cancer cells with little effect on wild-type (wt)-KRAS cells. Cnk1 inhibition decreased anchorage-independent mut-KRas cell growth more so than growth on plastic, without the partial "addiction" to mut-KRAS seen on plastic. The PH domain of Cnk1 bound with greater affinity to PtdIns(4,5)P2 than PtdIns(3,4,5)P3, and Cnk1 localized to areas of the plasma membranes rich in PtdIns, suggesting a role for the PH domain in the biological activity of Cnk1. Through molecular modeling and structural modification, we identified a compound PHT-7.3 that bound selectively to the PH domain of Cnk1, preventing plasma membrane colocalization with mut-KRas. PHT-7.3 inhibited mut-KRas, but not wild-type KRas cancer cell and tumor growth and signaling. Thus, the PH domain of Cnk1 is a druggable target whose inhibition selectively blocks mutant KRas activation, making Cnk1 an attractive therapeutic target in patients with mut-KRAS–driven cancer. Significance: These findings identify a therapeutic strategy to selectively block oncogenic KRas activity through the PH domain of Cnk1, which reduces its cell membrane binding, decreasing the efficiency of Ras signaling and tumor growth.

The naturally occurring isotope of hydrogen ((1)H), deuterium ((2)H), could have an important biological role. Deuterium depleted water delays tumor progression in mice, dogs, cats and humans. Hydratase enzymes of the tricarboxylic acid (TCA) cycle control cell growth and deplete deuterium from redox cofactors, fatty acids and DNA, which undergo hydride ion and hydrogen atom transfer reactions. A model is proposed that emphasizes the terminal complex of mitochondrial electron transport chain reducing molecular oxygen to deuterium depleted water (DDW); this affects gluconeogenesis as well as fatty acid oxidation. In the former, the DDW is thought to diminish the deuteration of sugar-phosphates in the DNA backbone, helping to preserve stability of hydrogen bond networks, possibly protecting against aneuploidy and resisting strand breaks, occurring upon exposure to radiation and certain anticancer chemotherapeutics. DDW is proposed here to link cancer prevention and treatment using natural ketogenic diets, low deuterium drinking water, as well as DDW production as the mitochondrial downstream mechanism of targeted anti-cancer drugs such as Avastin and Glivec. The role of (2)H in biology is a potential missing link to the elusive cancer puzzle seemingly correlated with cancer epidemiology in western populations as a result of excessive (2)H loading from processed carbohydrate intake in place of natural fat consumption.

Microsomal PGE2 synthase-1 (mPGES-1), the terminal enzyme in the formation of inducible PGE2, represents a potential target for cancer chemoprevention. We have previously shown that genetic abrogation of mPGES-1 significantly suppresses tumorigenesis in two preclinical models of intestinal cancer. In this study, we examined the role of mPGES-1 during colon tumorigenesis in the presence of dextran sulfate sodium (DSS)-induced inflammatory microenvironment. Using Apc (Δ14/+) in which the mPGES-1 gene is either wild-type (D14:WT) or deleted (D14:KO), we report that mPGES-1 deficiency enhances sensitivity to acute mucosal injury. As a result of the increased epithelial damage, protection against adenoma formation is unexpectedly compromised in the D14:KO mice. Examining the DSS-induced acute injury, cryptal structures are formed within inflamed areas of colonic mucosa of both genotypes that display the hallmarks of early neoplasia. When acute epithelial injury is balanced by titration of DSS exposures, however, these small cryptal lesions progress rapidly to adenomas in the D14:WT mice. Given that mPGES-1 is highly expressed within the intestinal stroma under the inflammatory conditions of DSS-induced ulceration, we propose a complex and dual role for inducible PGE2 synthesis within the colonic mucosa. Our data suggest that inducible PGE2 is critical for the maintenance of an intact colonic epithelial barrier, while promoting epithelial regeneration. This function is exploited during neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of the early initiated cryptal structures. Taken together, inducible PGE2 plays a complex role in inflammation-associated cancers that requires further analysis. Inducible PGE2 production by mPGES-1 is critical for the colonic mucosal homeostasis. This function is exploited in the presence of the neoplastic transformation in Apc (Δ14/+) mice as PGE2 contributes to the growth and expansion of the early cryptal structures.

The PGE2 pathway is important in inflammation-driven diseases and specific targeting of the inducible mPGES-1 is warranted due to the cardiovascular problems associated with the long-term use of COX-2 inhibitors. This review focuses on patents issued on methods of measuring mPGES-1 activity, on drugs targeting mPGES-1 and on other modulators of free extracellular PGE2 concentration. Perspectives and conclusions regarding the status of these drugs are also presented. Importantly, no selective inhibitors targeting mPGES-1 have been identified and, despite the high number of published patents, none of these drugs have yet made it to clinical trials.

This Letter reports the synthesis and biological evaluation of a collection of aminophthalazines as a novel class of compounds capable of reducing production of PGE2 in HCA-7 human adenocarcinoma cells. A total of 28 analogs were synthesized, assayed for PGE2 reduction, and selected active compounds were evaluated for inhibitory activity against COX-2 in a cell free assay. Compound 2xxiv (R1 = H, R2 = p-CH3O) exhibited the most potent activity in cells (EC50 = 0.02 μM) and minimal inhibition of COX-2 activity (3% at 5 μM). Furthermore, the anti-tumor activity of analog 2vii was analyzed in xenograft mouse models exhibiting good anti-cancer activity.

This research study sought to improve the treatment of pancreatic cancer by improving the drug delivery of a promising AKT/PDK1 inhibitor, PHT-427, in poly(lactic-co-glycolic) acid (PLGA) nanoparticles.PHT-427 was encapsulated in single-emulsion and double-emulsion PLGA nanoparticles (SE-PLGA-427 and DE-PLGA-427). The drug release rate was evaluated to assess the effect of the second PLGA layer of DE-PLGA-427. Ex vivo cryo-imaging and drug extraction from ex vivo organs was used to assess the whole-body biodistribution in an orthotopic model of MIA PaCa-2 pancreatic cancer. Anatomical magnetic resonance imaging (MRI) was used to noninvasively assess the effects of 4 weeks of nanoparticle drug treatment on tumor size, and diffusion-weighted MRI longitudinally assessed changes in tumor cellularity.DE-PLGA-427 showed delayed drug release and longer drug retention in the pancreas relative to SE-PLGA-427. Diffusion-weighted MRI indicated a consistent decrease in cellularity during drug treatment with both types of drug-loaded nanoparticles. Both SE- and DE-PLGA-427 showed a 6-fold and 4-fold reduction in tumor volume relative to untreated tumors and an elimination of primary pancreatic tumor in 68% of the mice.These results indicated that the PLGA nanoparticles improved drug delivery of PHT-427 to pancreatic tumors, which improved the treatment of MIA PaCa-2 pancreatic cancer.

Evaluations of tumor growth rates and molecular biomarkers are traditionally used to assess new mouse models of human breast cancers. This study investigated the utility of diffusion weighted (DW)-magnetic resonance imaging (MRI) for evaluating cellular proliferation of new tumor models of triple-negative breast cancer, which may augment traditional analysis methods. Eleven human breast cancer cell lines were used to develop xenograft tumors in severe combined immunodeficient mice, with two of these cell lines exhibiting sufficient growth to be serially passaged. DW-MRI was performed to measure the distributions of the apparent diffusion coefficient (ADC) in these two tumor xenograft models, which showed a correlation with tumor growth rates and doubling times during each passage. The distributions of the ADC values were also correlated with expression of Ki67, a biomarker of cell proliferation, and hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor receptor-2 (VEGFR2), which are essential proteins involved in regulating aerobic glycolysis and angiogenesis that support tumor cell proliferation. Although phosphatase and tensin homolog (PTEN) levels were different between the two xenograft models, AKT levels did not differ nor did they correlate with tumor growth. This last result demonstrates the complexity of signaling protein pathways and the difficulty in interpreting the effects of protein expression on tumor cell proliferation. In contrast, DW-MRI may be a more direct assessment of tumor growth and cancer cell proliferation.

CYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real-time PCR to establish the expression profile of CYR61 and integrin α(V)β5 in three non-small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of α(V)β5 integrin. Further, we investigated whether tumour cell-secreted CYR61 can facilitate cell migration by interacting with the α(V)β5 integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin α(V)β5 expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and α(V)β5 integrin as proteins that cooperate to mediate cancer cell migration.

This Letter presents the synthesis and biological evaluation of a collection of 2-aminothiazoles as a novel class of compounds with the capability to reduce the production of PGE 2 in HCA-7 human adenocarcinoma cells. A total of 36 analogs were synthesized and assayed for PGE 2 reduction, and those with potent cellular activity were counter screened for inhibitory activity against COX-2 in a cell free assay. In general, analogs bearing a 4-phenoxyphenyl substituent in the R 2 position were highly active in cells while maintaining negligible COX-2 inhibition. Specifically, compound 5l (R 1 = Me, R 2 = 4-OPh-Ph, R 3 = CH(OH)Me) exhibited the most potent cellular PGE 2 reducing activity of the entire series (EC 50 = 90 nM) with an IC 50 value for COX-2 inhibition of >5 μM in vitro. Furthermore, the anti-tumor activity of analog 1a was analyzed in xenograft mouse models exhibiting promising anti-cancer activity.

Chronic inflammation is associated with 25% of all cancers. In the inflammation-cancer axis, prostaglandin E(2) (PGE(2)) is one of the major players. PGE(2) synthases (PGES) are the enzymes downstream of the cyclooxygenases (COXs) in the PGE(2) biosynthesis pathway. Microsomal prostaglandin E(2) synthase 1 (mPGES-1) is inducible by pro-inflammatory stimuli and constitutively expressed in a variety of cancers. The potential role for this enzyme in tumorigenesis has been reported and mPGES-1 represents a novel therapeutic target for cancers. In order to identify novel small molecule inhibitors of mPGES-1, we screened the ChemBridge library and identified 13 compounds as potential hits. These compounds were tested for their ability to bind directly to the enzyme using surface plasmon resonance spectroscopy and to decrease cytokine-stimulated PGE(2) production in various cancer cell lines. We demonstrate that the compound PGE0001 (ChemBridge ID number 5654455) binds to human mPGES-1 recombinant protein with good affinity (K(D) = 21.3 ± 7.8 μM). PGE0001 reduces IL-1β-induced PGE(2) release in human HCA-7 colon and A549 lung cancer cell lines with EC(50) in the sub-micromolar range. Although PGE0001 may have alternative targets based on the results from in vitro assays, it shows promising effects in vivo. PGE0001 exhibits significant anti-tumor activity in SW837 rectum and A549 lung cancer xenografts in SCID mice. Single injection i.p. of PGE0001 at 100 mg/kg decreases serum PGE(2) levels in mice within 5 h. In summary, our data suggest that the identified compound PGE0001 exerts anti-tumor activity via the inhibition of the PGE(2) synthesis pathway.

Disruption of the phosphatidylinositol 3-kinase/AKT signaling pathway can lead to apoptosis in cancer cells. Previously we identified a lead sulfonamide that selectively bound to the pleckstrin homology (PH) domain of AKT and induced apoptosis when present at low micromolar concentrations. To examine the effects of structural modification, a set of sulfonamides related to the lead compound was designed, synthesized, and tested for binding to the expressed PH domain of AKT using a surface plasmon resonance-based competitive binding assay. Cellular activity was determined by means of an assay for pAKT production and a cell killing assay using BxPC-3 cells. The most active compounds in the set are lipophilic and possess an aliphatic chain of the proper length. Results were interpreted with the aid of computational modeling. This paper represents the first structure-activity relationship (SAR) study of a large family of AKT PH domain inhibitors. Information obtained will be used in the design of the next generation of inhibitors of AKT PH domain function.

Prostaglandin E2 (PGE2) is a bioactive lipid that can elicit a wide range of biological effects associated with inflammation and cancer. The physiological roles of PGE2 are diverse, mediated in part through activation of key downstream signaling cascades via transmembrane EP receptors located on the cell surface. Elevated levels of COX-2 and concomitant overproduction of PGE2 are often found in human cancers. These observations have led to the use of non-steroidal anti-inflammatory drugs (NSAIDs) as chemopreventive agents, particularly for colorectal cancer (CRC). Their long-term use, however, may be associated with gastrointestinal toxicity and increased risk of adverse cardiovascular events, prompting the development of other enzymatic targets in this pathway. This review will focus on recent efforts to target the terminal synthase, mPGES-1, for cancer chemoprevention. The role of mPGES-1 in the pathogenesis of various cancers is discussed. In addition, an overview of recent efforts to develop small molecule inhibitors that target the protein with high selectivity is also be reviewed.

Phosphatidylinositol 3-kinase/phosphatidylinositide-dependent protein kinase 1 (PDPK1)/Akt signaling plays a critical role in activating proliferation and survival pathways within cancer cells. We report the molecular pharmacology and antitumor activity of PHT-427, a compound designed to bind to the pleckstrin homology (PH) binding domain of signaling molecules important in cancer. Although originally designed to bind the PH domain of Akt, we now report that PHT-427 also binds to the PH domain of PDPK1. A series of PHT-427 analogues with variable C-4 to C-16 alkyl chain length were synthesized and tested. PHT-427 itself (C-12 chain) bound with the highest affinity to the PH domains of both PDPK1 and Akt. PHT-427 inhibited Akt and PDPK1 signaling and their downstream targets in sensitive but not resistant cells and tumor xenografts. When given orally, PHT-427 inhibited the growth of human tumor xenografts in immunodeficient mice, with up to 80% inhibition in the most sensitive tumors, and showed greater activity than analogues with C4, C6, or C8 alkyl chains. Inhibition of PDPK1 was more closely correlated to antitumor activity than Akt inhibition. Tumors with PIK3CA mutation were the most sensitive, and K-Ras mutant tumors were the least sensitive. Combination studies showed that PHT-427 has greater than additive antitumor activity with paclitaxel in breast cancer and with erlotinib in non–small cell lung cancer. When given >5 days, PHT-427 caused no weight loss or change in blood chemistry. Thus, we report a novel PH domain binding inhibitor of PDPK1/Akt signaling with significant in vivo antitumor activity and minimal toxicity. Mol Cancer Ther; 9(3); 706–17

The formation of G-quadruplex structures within the nuclease hypersensitive element (NHE) III1 region of the c-myc promoter and the ability of these structures to repress c-myc transcription have been well established. However, just how these extremely stable DNA secondary structures are transformed to activate c-myc transcription is still unknown. NM23-H2/nucleoside diphosphate kinase B has been recognized as an activator of c-myc transcription via interactions with the NHE III1 region of the c-myc gene promoter. Through the use of RNA interference, we confirmed the transcriptional regulatory role of NM23-H2. In addition, we find that further purification of NM23-H2 results in loss of the previously identified DNA strand cleavage activity, but retention of its DNA binding activity. NM23-H2 binds to both single-stranded guanine- and cytosine-rich strands of the c-myc NHE III1 and, to a lesser extent, to a random single-stranded DNA template. However, it does not bind to or cleave the NHE III1 in duplex form. Significantly, potassium ions and compounds that stabilize the G-quadruplex and i-motif structures have an inhibitory effect on NM23-H2 DNA-binding activity. Mutation of Arg88 to Ala88 (R88A) reduced both DNA and nucleotide binding but had minimal effect on the NM23-H2 crystal structure. On the basis of these data and molecular modeling studies, we have proposed a stepwise trapping-out of the NHE III1 region in a single-stranded form, thus allowing single-stranded transcription factors to bind and activate c-myc transcription. Furthermore, this model provides a rationale for how the stabilization of the G-quadruplex or i-motif structures formed within the c-myc gene promoter region can inhibit NM23-H2 from activating c-myc gene expression. [Mol Cancer Ther 2009;8(5):1363–77]

LAMMER kinases (also known as CDC-2-like or CLKs) are a family of dual specificity serine/threonine protein kinases that are found in all sequenced eukaryotic genomes. In the fission yeast, Schizosaccharomyces pombe, the LAMMER kinase gene, Lkh1, positively regulates the expression of the antioxidant defense genes, superoxide dismutase 1 (sod1+, CuZn-SOD) and catalase (ctt1+, CAT). We have shown that mutations in the Drosophila LAMMER kinase gene, Darkener of apricot (Doa), protect against the decrease in life span caused by the reactive oxygen species (ROS) generator paraquat, and at the same time show an increase in cytoplasmic (CuZn-Sod or SOD1) and mitochondrial superoxide dismutase (Mn-Sod or SOD2) protein levels and activity. The siRNA-mediated knock down of the human LAMMER kinase gene, CLK-1, in HeLa and MCF-7 human cell lines leads to an increase in both SOD1 activity and mRNA transcript levels. These data suggest that SOD1 is negatively regulated by LAMMER kinases in Drosophila and human cell lines and that this regulation may be conserved during evolution.

Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies.

Measuring intracellular metabolism has increasingly led to important insights in biomedical research. (13)C tracer analysis, although less information-rich than quantitative (13)C flux analysis that requires computational data integration, has been established as a time-efficient method to unravel relative pathway activities, qualitative changes in pathway contributions, and nutrient contributions. Here, we review selected key issues in interpreting (13)C metabolite labeling patterns, with the goal of drawing accurate conclusions from steady state and dynamic stable isotopic tracer experiments. ispartof: Current Opinion in Biotechnology vol:34 pages:189-201 ispartof: location:England status: published

Secondary bile acids have long been postulated to be tumor promoters in the colon; however, their mechanism of action remains unclear. In this study, we examined the actions of bile acids at the cell membrane and found that they can perturb membrane structure by alteration of membrane microdomains. Depletion of membrane cholesterol by treating with methyl-beta-cyclodextrin suppressed deoxycholic acid (DCA)-induced apoptosis, and staining for cholesterol with filipin showed that DCA caused a marked rearrangement of this lipid in the membrane. Likewise, DCA was found to affect membrane distribution of caveolin-1, a marker protein that is enriched in caveolae membrane microdomains. Additionally, fluorescence anisotropy revealed that DCA causes a decrease in membrane fluidity consistent with the increase in membrane cholesterol content observed after 4 h of DCA treatment of HCT116 cells. Significantly, by using radiolabeled bile acids, we found that bile acids are able to interact with and localize to microdomains differently depending on their physicochemical properties. DCA was also found to induce tyrosine phosphorylation and activate the receptor tyrosine kinase epidermal growth factor receptor in a ligand-independent manner. In contrast, ursodeoxycholic acid did not exhibit any of these effects even though it interacted significantly with the microdomains. Collectively, these data suggest that bile acid-induced signaling is initiated through alterations of the plasma membrane structure and the redistribution of cholesterol.

Objective Vascular endothelial growth factor (VEGF) interacts with two high-affinity receptor tyrosine kinases (RTK) on vascular endothelium to initiate complementary but disparate biologic responses. We previously reported that acute myeloid leukemia (AML) cells express VEGF and one or both VEGF-A receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). To evaluate receptor-selective trophic response to VEGF-A in AML cells, we investigated receptor-specific ligand activation responsible for VEGF-initiated clonogenic response. Materials and methods Using KG1 (VEGFR-1 + /VEGFR-2 + ) and HL60 (VEGFR-1 + ) cells with differential VEGF receptor display, we investigated ligand-induced clonogenic response and receptor-initiated signaling after stimulation with VEGF-A, the VEGFR-1 selective ligand placental growth factor (PlGF), or receptor-specific antibody agonists. Results Recombinant human (rhu)-VEGF increased S-phase fraction and stimulated colony formation in both KG1 and HL60 cells. Ligation of VEGFR-1 or VEGFR-2 with receptor-specific antibody agonists triggered equivalent and concentration-dependent stimulation of colony recovery in KG1 cells, whereas clonogenic response in HL60 cells was restricted to VEGFR-1 activation by antibody or PlGF. In serum-deprived KG1 and HL60 cells, rhu-VEGF stimulated rapid and sustained phosphorylation of Akt/PKB that was inhibited by the phosphatidyl inositol 3-kinase (PI3-K) kinase inhibitor wortmannin. Preincubation with wortmannin inhibited VEGF-induced colony formation in a concentration-dependent fashion. rhu-VEGF–induced clonogenic response and Akt phosphorylation was abolished by the VEGF-RTK inhibitor SU-5416 at concentrations greater than 10 μM, whereas MEK inhibition by PD98059 (1 and 10 μM) was ineffective. In vivo suppression of Akt phosphorylation was confirmed in myeloblast lysates from three patients with advanced myeloid malignancies treated with SU5416. Conclusion These data indicate that VEGF interaction with either VEGFR-1 or VEGFR-2 initiates a clonogenic response in AML cells that is PI3-kinase dependent. RTK inhibitors with broad specificity for angiogenic receptors represent novel therapeutics that merit further clinical investigation in AML.

Akt, a serine/threonine kinase that promotes cell survival, is activated by binding of its pleckstrin homology (PH) domain to membrane phosphatidylinositol (PtdIns)-3-phosphates formed by PtdIns-3-kinase. D-3-Deoxy-phosphatidyl-myo-inositols that cannot be phosphorylated on the 3-position of the myo-inositol group are inhibitors of the Akt PH domain. The most active compound is D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (PX-316). PX-316 administered intraperitoneally to mice at 150 mg/kg inhibits Akt activation in HT-29 human tumor xenografts up to 78% at 10 h with recovery to 34% at 48 h. Phosphorylation of GSK-3beta, a downstream target of Akt, is also inhibited. There is no decrease in PtdIns(3,4,5)-trisphosphate levels by PX-316, showing it is not an inhibitor of PtdIns-3-K in vivo. Gene expression profiling of HT-29 tumor xenografts shows many similarities between the effects of PX-316 and the PtdIns-3-K inhibitor wortmannin, with downregulation of several ribosomal-related genes, while PX-316 uniquely increases the expression of a group of mitochondrial-related genes. PX-316 has antitumor activity against early human MCF-7 breast cancer and HT-29 colon cancer xenografts in mice. PX-316 formulated in 20% hydroxypropyl-beta-cyclodextrin for intravenous administration is well tolerated in mice and rats with no hemolysis and no hematological toxicity. Thus, PX-316 is the lead compound of a new class of potential agents that inhibit Akt survival signaling.

Prostate cancer is the most common cancer diagnosed and the second leading cause of cancer-related deaths in men in the United States. The etiological factors that give rise to prostate cancer are not known. Therefore, it is not possible to develop primary intervention strategies to remove the causative agents from the environment. However, secondary intervention strategies with selenium (Se) compounds and other agents represent a viable option to reduce the morbidity and mortality of prostate cancer. In this review, we discuss ongoing clinical trials. In addition, we discuss preclinical mechanistic studies that provide insights into the biochemical and molecular basis for the anti-carcinogenic activity of both inorganic and organic forms of Se. © 2003 Wiley-Liss, Inc.

The Grb2-associated binding protein 1 (GAB1) integrates signals from different signaling pathways and is over-expressed in many cancers, therefore representing a new therapeutic target. In the present study, we aim to target the pleckstrin homology (PH) domain of GAB1 for cancer treatment. Using homology models we derived, high-throughput virtual screening of five million compounds resulted in five hits which exhibited strong binding affinities to GAB1 PH domain. Our prediction of ligand binding affinities is also in agreement with the experimental K D values. Furthermore, molecular dynamics studies showed that GAB1 PH domain underwent large conformational changes upon ligand binding. Moreover, these hits inhibited the phosphorylation of GAB1 and demonstrated potent, tumor-specific cytotoxicity against MDA-MB-231 and T47D breast cancer cell lines. This effort represents the discovery of first-in-class GAB1 PH domain inhibitors with potential for targeted breast cancer therapy and provides novel insights into structure-based approaches to targeting this protein., Author Summary In this paper, we described the identification and evaluation of a set of first-in-class potent inhibitors targeting a new cancer target, Grb2-associated binder-1 (GAB1), which integrates signals from different signaling pathways, and is frequently over-expressed in cancer cells. To achieve our goals, we have employed intensive computational modeling to understand the structure of the GAB1 pleckstrin homology (PH) domain and screened five million compounds. Upon biological evaluation, we found that several inhibitors that induced large conformational changes of the target structure exhibited strong selective binding to GAB1 PH domain. Particularly, these inhibitors demonstrated potent and tumor-specific cytotoxicity in breast cancer cells. This represents a groundbreaking discovery in targeting GAB1 signaling which may be used for cancer therapy, especially for triple negative breast cancer patients.

Metabolomics technologies continue to develop not only to study endpoint steady-state concentrations of numerous metabolites in normal and cancer cells but also to examine metabolic flux and networks. These techniques are of importance for understanding tumor cell metabolism and for the development of new drugs and treatment strategies. The choice of tracer substrates is central as 13C-labeled substrates readily improve real-time reaction visibility by increasing metabolic network transparencies in cancer metabolomics. In this chapter, targeted [1,2- 13C2]-d-glucose single tracer fate associations are compared with the external [U- 13C18]-stearate oxidation model for thiazolidinedione efficacy testing in primary liver tumor cells. Although the externally supplied [U- 13C18]-stearate tracer readily labels multiple products by acetyl-CoA exchange, parallel stearate synthesis and mobilization from unlabeled intracellular pools disrupt its uptake after drug treatment. This can be overcome by using cross-labeled 13C-stearate from [1,2- 13C2]-d-glucose as the internal tracer and the independent explanatory variable to study associations among markers of rosiglitazone-induced stearate breakdown in a single [1,2- 13C2]-d-glucose tracer experiment during drug efficacy testing in cultured cells.

Two 3-hydroxy(phosphono)methyl-bearing phosphatidylinositol ether lipid analogues were synthesized and shown to be inhibitors of Akt and PI3-K. These compounds were also shown to inhibit the growth of HT-29 human colon cancer cells and MCF-7 human breast cancer cells.

Armando Lucero-Acuña,1 Justin J Jeffery,2 Edward R Abril,3,4 Raymond B Nagle,3,4 Roberto Guzman,1 Mark D Pagel,2,3,5,6 Emmanuelle J Meuillet3,7,8 1Department of Chemical and Environmental Engineering, University of Arizona, 2Department of Biomedical Engineering, University of Arizona, 3University of Arizona Cancer Center, 4Department of Pathology, University of Arizona, 5Department of Chemistry and Biochemistry, University of Arizona, 6Department of Medical Imaging, University of Arizona, 7Department of Molecular and Cell Biology, University of Arizona, 8Department of Nutritional Sciences, University of Arizona, Tucson, AZ, USA Abstract: The K-ras mutation in pancreatic cancer can inhibit drug delivery and increase drug resistance. This is exemplified by the therapeutic effect of PH-427, a small molecule inhibitor of AKT/PDK1, which has shown a good therapeutic effect against a BxPC3 pancreatic cancer model that has K-ras, but has a poor therapeutic effect against a MiaPaCa-2 pancreatic cancer model with mutant K-ras. To increase the therapeutic effect of PH-427 against the MiaPaCa-2 pancreatic cancer model with mutant K-ras, we encapsulated PH-427 into poly(lactic-co-glycolic acid) nanoparticles (PNP) to form drug-loaded PH-427-PNP. PH-427 showed a biphasic release from PH-427-PNP over 30 days during studies in sodium phosphate buffer, and in vitro studies revealed that the PNP was rapidly internalized into MiaPaCa-2 tumor cells, suggesting that PNP can improve PH-427 delivery into cells harboring mutant K-ras. In vivo studies of an orthotopic MiaPaCa-2 pancreatic cancer model showed reduced tumor load with PH-427-PNP as compared with treatment using PH-427 alone or with no treatment. Ex vivo studies confirmed the in vivo results, suggesting that PNP can improve drug delivery to pancreatic cancer harboring mutant K-ras. Keywords: nanoparticles, pancreatic cancer, AKT, bioluminescence imaging, drug delivery

Numerous epidemiological studies have reported that metformin, a well-known and widely used anti-diabetic drug, may provide protective benefits in decreasing pancreatic cancer risk among the diabetic population. Following a brief introduction regarding metformin’s history and pharmacological properties, this book chapter presents epidemiological findings showing how metformin is associated with protection against pancreatic cancer. We also introduce the anti-cancer effects of metformin through AMPK-independent and AMPK-dependent manners [1-6]. These mechanisms include its inhibitory effects on the insulin growth factor-1 (IGF-1), G protein-coupled receptor (GPCR) and mTORC1 signaling pathways [3-10]. We then discuss the metabolic effects of metformin in cancer. For example, metformin has been shown to inhibit glycolysis in various cancer cell lines [11-13]. Metformin is a known inhibitor of complex I of the electron transport chain [14-18], potentially limiting the intact oxidative respiration capabilities of the cancer cell. We also discuss in depth the anti-cancer mechanisms of action of metformin in the context of lipid metabolism as reported in numerous models. These include metformin’s ability to increase fatty acid β-oxidation in adipocytes [19] and its ability to inhibit hepatic lipogenesis [2]. As shown by numerous studies [20-23], metformin also possesses antilipogenic properties, potentially limiting this critical metabolic pathway that confers cancer pancreatic cell survival advantage.

The serine-threonine protein kinase Akt is a direct downstream target of phosphatidylinositol 3-kinase (PI3-K). The PI3-K-generated phospholipids regulate Akt activity via directly binding to the Akt PH domain. The binding of PI3-K-generated phospholipids is critical to the relocalization of Akt to the plasma membrane, which plays an important role in the process of Akt activation. Activation of the PI3-K/Akt signaling pathway promotes cell survival. To elucidate the structural basis of the interaction of PI3-K-generated phospholipids with the Akt PH domain with the objective of carrying out structure-based drug design, we modeled the three-dimensional structure of the Akt PH domain. Comparative modeling-based methods were employed, and the modeled Akt structure was used in turn to construct structural models of Akt in complex with selected PI3-K-generated phospholipids using the computational docking approach. The model of the Akt PH domain consists of seven beta-strands forming two antiparallel beta-sheets capped by a C-terminal alpha-helix. The beta1-beta2, beta3-beta4, and beta6-beta7 loops form a positively charged pocket that can accommodate the PI3-K-generated phospholipids in a complementary fashion through specific hydrogen-bonding interactions. The residues Lys14, Arg25, Tyr38, Arg48, and Arg86 form the bottom of the binding pocket and specifically interact with the 3- and 4-phophate groups of the phospholipids, while residues Thr21 and Arg23 are situated at the wall of the binding pocket and bind to the 1-phosphate group. The predicted binding mode is consistent with known site-directed mutagenesis data, which reveal that mutation of these crucial residues leads to the loss of Akt activity. Moreover, our model can be used to predict the binding affinity of PI3-K-generated phospholipids and rationalize the specificity of the Akt PH domain for PI(3,4)P2, as opposed to other phospholipids such as PI(3)P and PI(3,4,5)P3. Taken together, our modeling studies provide an improved understanding of the molecular interactions present between the Akt PH domain and the PI3-K-generated phospholipids, thereby providing a solid structural basis for the design of novel, high-affinity ligands useful in modulating the activity of Akt.

3-Modified d - myo -inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibitors of PI3-K and Akt. These data are presented along with IC 50 values for the inhibition of the growth of three cancer cell lines.

1d-3,4-Dideoxyphosphatidylinositol ether lipid 2, a PI analog, was synthesized through a sequence of protection/deprotection protocols and two Barton deoxygenation reactions, starting from l-(−)-quebrachitol. DDPIEL is 18-fold more potent than its monodeoxy counterpart DPIEL in the inhibition of PI3-K.

The effects of the biguanide anti-hyperglycemic agent, metformin ( N , N ′-dimethyl-biguanide), on insulin signaling was studied in a human hepatoma cell line (HepG2). Cells were cultured in the absence (control cells) or in the presence of 100 μM of a cholesterol derivative, hemisuccinate of cholesterol. Cholesterol hemisuccinate-treatment alters cholesterol and lipid content of HepG2 and modulates membrane fluidity. Cholesterol hemisuccinate-treatment induces a decrease in insulin responsiveness and creates an "insulin-resistant" state in these cells. Exposure to 100 μM of metformin resulted in a significant enhancement of insulin-stimulated lipogenesis in control and cholesterol hemisuccinate-treated cells. In control cells, metformin altered glycogenesis in a biphasic manner. In cholesterol hemisuccinate-treated cells, metformin inhibited basal glycogenesis but restored insulin-stimulated glycogenesis. Hence, to understand the mechanism of metformin action, we analyzed early steps in the insulin signaling pathway, including insulin receptor autophosphorylation, mitogen-activated-protein kinase and phosphatidylinositol 3-kinase activities, in both control and cholesterol hemisuccinate-treated cells. Overall, the results suggest that metformin may interact with the insulin receptor and/or a component involved in the early steps of insulin signal transduction.

The PGE2 pathway is important in inflammation-driven diseases and specific targeting of the inducible mPGES-1 is warranted due to the cardiovascular problems associated with the long-term use of COX-2 inhibitors. This review focuses on patents issued on methods of measuring mPGES-1 activity, on drugs targeting mPGES-1 and on other modulators of free extracellular PGE2 concentration. Perspectives and conclusions regarding the status of these drugs are also presented. Importantly, no selective inhibitors targeting mPGES-1 have been identified and, despite the high number of published patents, none of these drugs have yet made it to clinical trials.

CYR61 is one of the six proteins of the CCN family of proteins known to play diverse roles in angiogenesis, cellular proliferation, survival, migration and wound healing. However, the specific function of CYR61 in cancer is unclear, and the literature remains controversial. We used quantitative real-time PCR to establish the expression profile of CYR61 and integrin α(V)β5 in three non-small cell lung cancer, five colorectal cancer, one breast cancer and one oesophageal squamous carcinoma cell lines. We showed that the levels of CYR61 were significantly increased in oesophageal squamous carcinoma cell line along with the enhanced levels of α(V)β5 integrin. Further, we investigated whether tumour cell-secreted CYR61 can facilitate cell migration by interacting with the α(V)β5 integrin. Using tumour cell lines with low, intermediate and high CYR61 expression and their isogenic variants as a cellular model, we determined that integrin α(V)β5 expressed on these tumour cells is required for cell migration. Moreover, we showed that the modulation of expression levels of CYR61 in these cancer cells affected their capacity for migration. These results represent an advance to the understanding of the role of CYR61 and α(V)β5 integrin as proteins that cooperate to mediate cancer cell migration.

Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal synthase responsible for the synthesis of the pro-tumorigenic prostaglandin E2 (PGE2). mPGES-1 is overexpressed in a wide variety of cancers. Since its discovery in 1997 by Bengt Samuelsson and collaborators, the enzyme has been the object of over 200 peer-reviewed articles. Although today mPGES-1 is considered a validated and promising therapeutic target for anticancer drug discovery, challenges in inhibitor design and selectivity are such that up to this date there are only a few published records of small-molecule inhibitors targeting the enzyme and exhibiting some in vivo anticancer activity. This review summarizes the structures, and the in vitro and in vivo activities of these novel mPGES-1 inhibitors. Challenges that have been encountered are also discussed.

The serine/threonine kinase Akt has proven to be a significant signaling target, involved in various biological functions. Because of its cardinal role in numerous cellular responses, Akt has been implicated in many human diseases, particularly cancer. It has been established that Akt is a viable and feasible target for anticancer therapeutics. Analysis of all Akt kinases reveals conserved homology for an N-terminal regulatory domain, which contains a pleckstrin-homology (PH) domain for cellular translocation, a kinase domain with serine/threonine specificity, and a C-terminal extension domain. These well defined regions have been targeted, and various approaches, including in silico methods, have been implemented to develop Akt inhibitors. In spite of unique techniques and a prolific body of knowledge surrounding Akt, no targeted Akt therapeutics have reached the market yet. Here we will highlight successes and challenges to date on the development of anticancer agents modulating the Akt pathway in recent patents as well as discuss the methods employed for this task. Special attention will be given to patents with focus on those discoveries using computer-aided drug design approaches.

Selective blockade of hypoxia-inducible gene expression by designed small molecules would prove valuable in suppressing tumor angiogenesis, metastasis and altered energy metabolism. We report the design, synthesis, and biological evaluation of a dimeric epidithiodiketopiperazine (ETP) small molecule transcriptional antagonist targeting the interaction of the p300/CBP coactivator with the transcription factor HIF-1alpha. Our results indicate that disrupting this interaction results in rapid downregulation of hypoxia-inducible genes critical for cancer progression. The observed effects are compound-specific and dose-dependent. Controlling gene expression with designed small molecules targeting the transcription factor-coactivator interface may represent a new approach for arresting tumor growth.

The phosphatidylinositol 3-kinase/AKT signaling pathway plays a critical role in activating survival and antiapoptotic pathways within cancer cells. Several studies have shown that this pathway is constitutively activated in many different cancer types. The goal of this study was to discover novel compounds that bind to the pleckstrin homology (PH) domain of AKT, thereby inhibiting AKT activation. Using proprietary docking software, 22 potential PH domain inhibitors were identified. Surface plasmon resonance spectroscopy was used to measure the binding of the compounds to the expressed PH domain of AKT followed by an in vitro activity screen in Panc-1 and MiaPaCa-2 pancreatic cancer cell lines. We identified a novel chemical scaffold in several of the compounds that binds selectively to the PH domain of AKT, inducing a decrease in AKT activation and causing apoptosis at low micromolar concentrations. Structural modifications of the scaffold led to compounds with enhanced inhibitory activity in cells. One compound, 4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide, inhibited AKT and its downstream targets in cells as well as in pancreatic cancer cell xenografts in immunocompromised mice; it also exhibited good antitumor activity. In summary, a pharmacophore for PH domain inhibitors targeting AKT function was developed. Computer-aided modeling, synthesis, and testing produced novel AKT PH domain inhibitors that exhibit promising preclinical properties. [Cancer Res 2009;69(12):5073–81]