We report the results of a selection for single-stranded DNA oligonucleotide ligands to the serine protease thrombin using recently developed methods. This selection yielded a family of DNA sequences that conform to a consensus structure comprised of a unimolecular quadruplex motif and complementary flanking sequences capable of forming an additional Watson-Crick duplex motif. This novel quadruplex/duplex structure was not reported in a previous selection for DNA molecules which bind to thrombin [Bock et al. (1992) Nature 355, 564-566]. All quadruplex/duplex molecules tested bound to thrombin with higher affinity than quadruplex structures lacking the duplex structure. However, binding affinities did not always correlate with inhibitory potency since some molecules with high affinity were not potent inhibitors in vitro. 1H NMR spectroscopy studies demonstrated that the complementarity of bases in the duplex portion of a selected sequence allows it to form multimolecular structures. Constraining these molecules to the unimolecular quadruplex/duplex structure by bridging the 5' and 3' ends of the duplex motif with either triethylene glycol or disulfide bonds improved their thrombin inhibitory activity. All bridged quadruplex/duplex molecules were more potent inhibitors than molecules with only a quadruplex motif. Bridging the ends of these structures not only increased thrombin inhibition but also improved resistance to nucleases in serum more than 40-fold over the unbridged quadruplex. In addition, we have found that both the length and sequence of the duplex motif are important for inhibition.

Scientists have made tremendous efforts to gain understanding of the water molecules in proteins via indirect measurements such as molecular dynamic simulation and/or probing the polarity of the local environment. Here we present a tryptophan analogue that exhibits remarkable water catalysed proton-transfer properties. The resulting multiple emissions provide unique fingerprints that can be exploited for direct sensing of a site-specific water environment in a protein without disrupting its native structure. Replacing tryptophan with the newly developed tryptophan analogue we sense different water environments surrounding the five tryptophans in human thromboxane A 2 synthase. This development may lead to future research to probe how water molecules affect the folding, structures and activities of proteins.

Background: Adenosine monophosphate-activated protein kinase (AMPK) modulates cancer cell metabolism and survival. Results: The novel compound OSU-53 directly activates AMPK, inhibits multiple metabolic and oncogenic pathways, and induces apoptosis in triple-negative breast cancer cells. Conclusion: OSU-53 acts through a broad spectrum of antitumor activities downstream of AMPK activation. Significance: OSU-53 is a potent small molecule AMPK activator with translational potential for breast cancer therapy.

Indole-3-carbinol has emerged as a promising chemopreventive agent due to its in vivo efficacy in various animal models. However, indole-3-carbinol exhibits weak antiproliferative potency and is unstable in acidic milieu. Thus, this study was aimed at exploiting indole-3-carbinol to develop potent antitumor agents with improved chemical stability. This effort culminated in OSU-A9 {[1-(4-chloro-3-nitrobenzenesulfonyl)-1H-indol-3-yl]-methanol}, which is resistant to acid-catalyzed condensation, and exhibits 100-fold higher apoptosis-inducing activity than the parent compound. Relative to indole-3-carbinol, OSU-A9 displays a striking qualitative similarity in its effects on the phosphorylation or expression of multiple signaling targets, including Akt, mitogen-activated protein kinases, Bcl-2 family members, survivin, nuclear factor-KB, cyclin D1, p21, and p27. The ability of OSU-A9 to concurrently modulate this broad range of signaling targets underscores its in vitro and in vivo efficacy in prostate cancer cells. Nevertheless, despite this complex mode of mechanism, normal prostate epithelial cells were less susceptible to the antiproliferative effect of OSU-A9 than PC-3 and LNCaP prostate cancer cells. Treatment of athymic nude mice bearing established s.c. PC-3 xenograft tumors with OSU-A9 at 10 and 25 mg/kg i.p. for 42 days resulted in a 65% and 85%, respectively, suppression of tumor growth. Western blot analysis of representative biomarkers in tumor lysates revealed significant reductions in the intratumoral levels of phosphorylated (p-) Akt, Bcl-xL, and RelA, accompanied by robust increases in p-p38 levels. In conclusion, the ability of OSU-A9 to target multiple aspects of cancer cell survival with high potency suggests its clinical value in prostate cancer therapy. [Cancer Res 2007;67(16):7815-24]

Vitamin E is a fat-soluble vitamin that includes isomers of tocopherols and tocotrienols which are known for their antioxidant properties. Tocopherols are the predominant form encountered in the diet and through supplementation, and have garnered interest for their potential cancer therapeutic and chemopreventive effects, which include the dephosphorylation of Akt, a serine/threonine kinase that plays a pivotal role in important cellular processes, such as cell growth, survival, metabolism and motility. Full catalytic activation of Akt requires phosphorylation at both Thr308 and Ser473. Dephosphorylation of Ser473 drastically reduces Akt catalytic activity and the number of downstream substrates it can regulate. The mechanism by which α- and γ-tocopherol facilitate the selective dephosphorylation of the kinase Akt at Ser473 was investigated. We showed that this site-specific Akt dephosphorylation was mediated through the pleckstrin homology (PH) domain-dependent recruitment to the plasma membrane of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase, isoform 1), a phosphatase that dephosphorylates Akt at Ser473. The ability of α- and γ-tocopherol to induce PHLPP-mediated Akt inhibition established PHLPP as a “druggable” target. We structurally optimized these tocopherols to obtain derivatives with greater in vitro potency and in vivo tumor-suppressive activity in two prostate xenograft tumor models. Binding affinities for the PH domains of Akt and PHLPP1 were greater than for other PH domain-containing proteins, which may underlie the preferential membrane recruitment of these proteins. Molecular modeling revealed the structural determinants of the interaction with the PH domain of Akt that may inform strategies for continued structural optimization. These findings describe a mechanism by which tocopherols facilitate the dephosphorylation of Akt at Ser473, thereby providing insights into the mode of antitumor action of tocopherols and a rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors.