6-Methoxyethylamino-numonafide inhibits hepatocellular carcinoma xenograft growth as a single agent and in combination with sorafenib.

Hepatocellular carcinoma (HCC) is the third leading form of cancer worldwide, and its incidence is increasing rapidly in the United States, tripling over the past 3 decades. The current chemotherapeutic strategies against localized and metastatic HCC are ineffective. Here we report that 6-methoxyethylamino-numonafide (MEAN) is a potent growth inhibitor of murine xenografts of 2 human HCC cell lines. At the same dose and with the same treatment strategies, MEAN was more efficacious in inhibiting tumor growth in mice than sorafenib, the only approved drug for HCC. Treatment by MEAN at an effective dose for 6 wk was well tolerated by animals. Combined therapy using both sorafenib and MEAN enhanced tumor growth inhibition over monotherapy with either agent. Additional experiments revealed that MEAN inhibited tumor growth through mechanisms distinct from those of either its parent compound, amonafide, or sorafenib. MEAN suppressed C-MYC expression and increased expression of several tumor suppressor genes, including Src homology region 2 domain-containing phosphatase-1 (SHP-1) and TXNIP (thioredoxin-interacting protein). As an encouraging feature for envisioned clinical application, the IC50 of MEAN was not significantly changed in several drug-resistant cell lines with activated P-glycoprotein drug efflux pumps compared to drug-sensitive parent cells, demonstrating the ability of MEAN to be effective in cells resistant to existing chemotherapy regimens. MEAN is a promising candidate for clinical development as a single-agent therapy or in combination with sorafenib for the management of HCC.-Liu, Y., Lou, G., Norton, J. T., Wang, C., Kandela, I., Tang, S., Shank, N. I., Gupta, P., Huang, M., Avram, M. J., Green, R., Mazar, A., Appella, D., Chen, Z., Huang, S. 6-Methoxyethylamino-numonafide inhibits hepatocellular carcinoma xenograft growth as a single agent and in combination with sorafenib.

Twisted cyanines: a non-planar fluorogenic dye with superior photostability and its use in a protein-based fluoromodule.

The cyanine dye thiazole orange (TO) is a well-known fluorogenic stain for DNA and RNA, but this property precludes its use as an intracellular fluorescent probe for non-nucleic acid biomolecules. Further, as is the case with many cyanines, the dye suffers from low photostability. Here, we report the synthesis of a bridge-substituted version of TO named α-CN-TO, where the central methine hydrogen of TO is replaced by an electron withdrawing cyano group, which was expected to decrease the susceptibility of the dye toward singlet oxygen-mediated degradation. An X-ray crystal structure shows that α-CN-TO is twisted drastically out of plane, in contrast to TO, which crystallizes in the planar conformation. α-CN-TO retains the fluorogenic behavior of the parent dye TO in viscous glycerol/water solvent, but direct irradiation and indirect bleaching studies showed that α-CN-TO is essentially inert to visible light and singlet oxygen. In addition, the twisted conformation of α-CN-TO mitigates nonspecific binding and fluorescence activation by DNA and a previously selected TO-binding protein and exhibits low background fluorescence in HeLa cell culture. α-CN-TO was then used to select a new protein that binds and activates fluorescence from the dye. The new α-CN-TO/protein fluoromodule exhibits superior photostability to an analogous TO/protein fluoromodule. These properties indicate that α-CN-TO will be a useful fluorogenic dye in combination with specific RNA and protein binding partners for both in vitro and cell-based applications. More broadly, structural features that promote nonplanar conformations can provide an effective method for reducing nonspecific binding of cationic dyes to nucleic acids and other biomolecules.

Fluorescent DNA nanotags featuring covalently attached intercalating dyes: synthesis, antibody conjugation, and intracellular imaging.

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.

Enhanced photostability of genetically encodable fluoromodules based on fluorogenic cyanine dyes and a promiscuous protein partner.

Fluoromodules are discrete complexes of biomolecules and fluorogenic dyes. Binding of the dyes to their cognate biomolecule partners results in enhanced dye fluorescence. We exploited a previously reported promiscuous binding interaction between a single-chain, variable fragment antibody protein and a family of cyanine dyes to create new protein-dye fluoromodules that exhibit enhanced photostability while retaining high affinity protein-dye binding. Modifications to the dye structure included electron-withdrawing groups that provide resistance to photo-oxidative damage. Low nanomolar equilibrium dissociation constants were found for the new dyes. Fluorescence microscopy illustrates how yeast can be surface-labeled with three different colors based on a single protein and appropriately chosen dyes.

A rainbow of fluoromodules: a promiscuous scFv protein binds to and activates a diverse set of fluorogenic cyanine dyes.

Combined magnetic and fluorescence cell sorting were used to select Fluorogen Activating Proteins (FAPs) from a yeast surface-displayed library for binding to the fluorogenic cyanine dye Dimethyl Indole Red (DIR). Several FAPs were selected that bind to the dye with low nanomolar Kd values and enhance fluorescence more than 100-fold. One of these FAPs also exhibits considerable promiscuity, binding with high affinity to several other fluorogenic cyanine dyes with emission wavelengths covering most of the visible and near-IR regions of the spectrum. This significantly expands the number and wavelength range of scFv-based fluoromodules.

Probing Mercaptobenzamides as HIV Inactivators via Nucleocapsid Protein†7.

Human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein 7 (NCp7), a zinc finger protein, plays critical roles in viral replication and maturation and is an attractive target for drug development. However, the development of drug-like molecules that inhibit NCp7 has been a significant challenge. In this study, a series of novel 2-mercaptobenzamide prodrugs were investigated for anti-HIV activity in the context of NCp7 inactivation. The molecules were synthesized from the corresponding thiosalicylic acids, and they are all crystalline solids and stable at room temperature. Derivatives with a range of amide side chains and aromatic substituents were synthesized and screened for anti-HIV activity. Wide ranges of antiviral activity were observed, with IC50 values ranging from 1 to 100 µm depending on subtle changes to the substituents on the aromatic ring and side chain. Results from these structure-activity relationships were fit to a probable mode of intracellular activation and interaction with NCp7 to explain variations in antiviral activity. Our strategy to make a series of mercaptobenzamide prodrugs represents a general new direction to make libraries that can be screened for anti-HIV activity.