Spectroscopic methods to analyze drug metabolites.

Drug metabolites have been monitored with various types of newly developed techniques and/or combination of common analytical methods, which could provide a great deal of information on metabolite profiling. Because it is not easy to analyze whole drug metabolites qualitatively and quantitatively, a single solution of analytical techniques is combined in a multilateral manner to cover the widest range of drug metabolites. Mass-based spectroscopic analysis of drug metabolites has been expanded with the help of other parameter-based methods. The current development of metabolism studies through contemporary pharmaceutical research are reviewed with an overview on conventionally used spectroscopic methods. Several technical approaches for conducting drug metabolic profiling through spectroscopic methods are discussed in depth.

Anti-leukemia activity of a Hsp70 inhibitor and its hybrid molecules.

In this study we examined the anti-leukemia activity of a small molecule inhibitor of Hsp70 proteins, apoptozole (Az), and hybrids in which it is linked to an inhibitor of either Hsp90 (geldanamycin) or Abl kinase (imatinib). The results of NMR studies revealed that Az associates with an ATPase domain of Hsc70 and thus blocks ATP binding to the protein. Observations made in the cell study indicated that Az treatment promotes leukemia cell death by activating caspase-dependent apoptosis without affecting the caspase-independent apoptotic pathway. Importantly, the hybrids composed of Az and geldanamycin, which have high inhibitory activities towards both Hsp70 and Hsp90, exhibit enhanced anti-leukemia activity relative to the individual inhibitors. However, the Az and imatinib hybrids have weak inhibitory activities towards Hsp70 and Abl, and display lower cytotoxicity against leukemia cells compared to those of the individual constituents. The results of a mechanistic study showed that the active hybrid molecules promote leukemia cell death through a caspase-dependent apoptotic pathway. Taken together, the findings suggest that Hsp70 inhibitors as well as their hybrids can serve as potential anti-leukemia agents.

Water-Floating Giant Nanosheets from Helical Peptide Pentamers.

One of the important challenges in the development of protein-mimetic materials is understanding the sequence-specific assembly behavior and dynamic folding change. Conventional strategies for constructing two-dimensional (2D) nanostructures from peptides have been limited to using ß-sheet forming sequences as building blocks due to their natural tendency to form sheet-like aggregations. We have identified a peptide sequence (YFCFY) that can form dimers via a disulfide bridge, fold into a helix, and assemble into macroscopic flat sheets at the air/water interface. Due to the large driving force for 2D assembly and high elastic modulus of the resulting sheet, the peptide assembly induces flattening of the initially round water droplet. Additionally, we found that stabilization of the helix by dimerization is a key determinant for maintaining macroscopic flatness over a few tens of centimeters even with a uniform thickness of <10 nm. Furthermore, the ability to transfer the sheets from a water droplet to another substrate allows for multiple stacking of 2D peptide nanostructures, suggesting possible applications in biomimetic catalysis, biosensors, and 2D related electronic devices.

Screening of Pre-miRNA-155 Binding Peptides for Apoptosis Inducing Activity Using Peptide Microarrays.

MicroRNA-155, one of the most potent miRNAs that suppress apoptosis in human cancer, is overexpressed in numerous cancers, and it displays oncogenic activity. Peptide microarrays, constructed by immobilizing 185 peptides containing the C-terminal hydrazide onto epoxide-derivatized glass slides, were employed to evaluate peptide binding properties of pre-miRNA-155 and to identify its binding peptides. Two peptides, which were identified based on the results of peptide microarray and in vitro Dicer inhibition studies, were found to inhibit generation of mature miRNA-155 catalyzed by Dicer and to enhance expression of miRNA-155 target genes in cells. In addition, the results of cell experiments indicate that peptide inhibitors promote apoptotic cell death via a caspase-dependent pathway. Finally, observations made in NMR and molecular modeling studies suggest that a peptide inhibitor preferentially binds to the upper bulge and apical stem-loop region of pre-miRNA-155, thereby suppressing Dicer-mediated miRNA-155 processing.

Solution structure of Rv0569, potent hypoxic signal transduction protein, from Mycobacterium tuberculosis.

The latent infection is unique characteristic of Mycobacterium tuberculosis to overcome human immune response for its survival. The M. tb develops adaptation to extreme stress conditions to increase the viability, thus easily acquires drug resistance than any other bacteria and maintains a long-term infection status without any symptoms. Rv0569 is a conserved hypothetical protein that overexpresses under dormant state induced by hypoxia, starvation, and medication. To study function and structure in detail, we determined the solution structure of Rv0569 by NMR. NOE and RDC restraints were used to calculate the structure, which was further refined with AMBER. Rv0569 is composed of five antiparallel ß-sheets and one α-helix. Rv0569 shows structural similarity with its homolog Rv2302, yet there is a big difference in the orientation of C-terminal α-helix between Rv0569 and Rv2302. According to previous studies, Rv0569 might comprise a hypoxia induced operon with the Rv0570 which is located 29 bp downstream of the Rv0569 and Rv0570 plays an important role in the latent infection. From our structure and bioinformatics research, we suggest that Rv0569 contributes to signaling transduction in hypoxic condition by binding with DNA for upregulation of Rv0570 or supporting Rv0570 for binding ATP during dormancy of tuberculosis.

Codon optimization enhances protein expression of human peptide deformylase in E. coli.

Human peptide deformylase (hPDF), located in the mitochondria, has recently become a promising target for anti-cancer therapy. However, the expression of the hPDF gene in Escherichia coli is not efficient likely due to extremely high levels of GC content as well as the presence of rare codons. We performed codon optimization of the hPDF gene in order to reduce GC content and to eliminate rare codons. Putative stable secondary structures of the optimized gene were also reduced. Codon optimization increased the expression of hPDF protein (residues 63-243) presumably by reducing the GC content. A large amount of soluble hPDF was obtained upon its fusion with thioredoxin (Trx-hPDF), although an insoluble fraction was still dominant. We confirmed that Co(2+) is an optimal metal for increasing the activity of purified Trx-hPDF, and that actinonin acts as an efficient inhibitor. Therefore, a large amount of purified hPDF protein would provide many benefits for the screening of various drug candidates.