Characterization of stable, constitutively expressed, chromosomal green and red fluorescent transcriptional fusions in the select agent bacterium, Francisella tularensis Schu S4 and the surrogate type B live vaccine strain (LVS).

Here, we constructed stable, constitutively expressed, chromosomal green (GFP) and red fluorescent (RFP) reporters in the genome of the surrogate strain, Francisella tularensis spp. holarctica LVS (herein LVS), and the select agent, F. tularensis Schu S4. A bioinformatic approach was used to identify constitutively expressed genes. Two promoter regions upstream of the FTT1794 and rpsF(FTT1062) genes were selected and fused with GFP and RFP reporter genes in pMP815, respectively. While the LVS strains with chromosomally integrated reporter fusions exhibited fluorescence, we were unable to deliver the same fusions into Schu S4. Neither a temperature-sensitive Francisella replicon nor a pBBR replicon in the modified pMP815 derivatives facilitated integration. However, a mini-Tn7 integration system was successful at integrating the reporter fusions into the Schu S4 genome. Finally, fluorescent F. tularensis LVS and a mutant lacking MglA were assessed for growth in monocyte-derived macrophages (MDMs). As expected, when compared to wild-type bacteria, replication of an mglA mutant was significantly diminished, and the overall level of fluorescence dramatically decreased with infection time. The utility of the fluorescent Schu S4 strain was also examined within infected MDMs treated with clarithromycin and enrofloxacin. Taken together, this study describes the development of an important reagent for F. tularensis research, especially since the likelihood of engineered antibiotic resistant strains will emerge with time. Such strains will be extremely useful in high-throughput screens for novel compounds that could interfere with critical virulence processes in this important bioweapons agent and during infection of alveolar macrophages.

Design of triazole-stapled BCL9 α-helical peptides to target the ß-catenin/B-cell CLL/lymphoma 9 (BCL9) protein-protein interaction.

The interaction between ß-catenin and B-cell CLL/lymphoma 9 (BCL9), critical for the transcriptional activity of ß-catenin, is mediated by a helical segment from BCL9 and a large binding groove in ß-catenin. Design of potent, metabolically stable BCL9 peptides represents an attractive approach to inhibit the activity of ß-catenin. In this study, we report the use of the Huisgen 1,3-dipolar cycloaddition reaction to generate triazole-stapled BCL9 α-helical peptides. The high efficiency and mild conditions of this "click" reaction combined with the ease of synthesis of the necessary unnatural amino acids allows for facile synthesis of triazole-stapled peptides. We have performed extensive optimization of this approach and identified the optimal combinations of azido and alkynyl linkers necessary for stapling BCL9 helices. The unsymmetrical nature of the triazole staple also allowed the synthesis of double-stapled BCL9 peptides, which show a marked increase in helical character and an improvement in binding affinity and metabolic stability relative to wild-type and linear BCL9 peptides. This study lays the foundation for further optimization of these triazole-stapled BCL9 peptides as potent, metabolically stable, and cell-permeable inhibitors to target the ß-catenin and BCL9 interaction.

Analysis of the interaction of BCL9 with beta-catenin and development of fluorescence polarization and surface plasmon resonance binding assays for this interaction.

The transcriptional activator beta-catenin is the primary mediator of the canonical Wnt signaling pathway and is frequently upregulated in many types of human cancer. Recent studies have suggested that the interaction of beta-catenin and its cofactor, B-cell lymphoma 9 (BCL9), is crucial for its transcriptional activity. Targeting this interaction using small molecules will improve our understanding of the beta-catenin/Wnt signaling pathway and may lead to the development of a new class of anticancer drugs. In this study, we developed a fluorescence polarization (FP)-based BCL9 binding assay. Using our initial FP assay, we performed extensive mutational analysis on four critical hydrophobic residues in the BCL9 peptide and determined the precise region in BCL9 responsible for binding to beta-catenin. These results led to further optimization of our FP assay, making it amenable for high-throughput screening (HTS). We also developed and validated a complementary surface plasmon resonance (SPR)-based binding assay and showed that our synthetic BCL9-based peptides are capable of fully inhibiting the binding of beta-catenin to wild-type BCL9 protein. Our studies provide not only further insight into the interaction between BCL9 and beta-catenin but also quantitative and reliable biochemical binding assays for the discovery of potent and specific small-molecule inhibitors of this interaction.

Studies on the mechanism of inhibition of bacterial ribonuclease P by aminoglycoside derivatives.

Ribonuclease P (RNase P) is a Mg2+-dependent endoribonuclease responsible for the 5'-maturation of transfer RNAs. It is a ribonucleoprotein complex containing an essential RNA and a varying number of protein subunits depending on the source: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Since bacterial RNase P is required for viability and differs in structure/subunit composition from its eukaryal counterpart, it is a potential antibacterial target. To elucidate the basis for our previous finding that the hexa-arginine derivative of neomycin B is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, we synthesized hexa-guanidinium and -lysyl conjugates of neomycin B and compared their inhibitory potential. Our studies indicate that side-chain length, flexibility and composition cumulatively account for the inhibitory potency of the aminoglycoside-arginine conjugates (AACs). We also demonstrate that AACs interfere with RNase P function by displacing Mg2+ ions. Moreover, our finding that an AAC can discriminate between a bacterial and archaeal (an experimental surrogate for eukaryal) RNase P holoenzyme lends promise to the design of aminoglycoside conjugates as selective inhibitors of bacterial RNase P, especially once the structural differences in RNase P from the three domains of life have been established.

Design and characterization of bivalent Smac-based peptides as antagonists of XIAP and development and validation of a fluorescence polarization assay for XIAP containing both BIR2 and BIR3 domains.

XIAP (X-chromosome-linked inhibitor of apoptosis protein) is an inhibitor of apoptosis by binding to and inhibition of caspase-3 and caspase-7 through its BIR2 domain and caspase-9 through its BIR3 domain. Smac (second mitochondria-derived activator of caspases) protein is an endogenous antagonist of XIAP. Smac forms a dimer and concurrently binds both the BIR2 and BIR3 domains in XIAP, functioning as a highly efficient and potent cellular inhibitor of XIAP. In this article, we have designed and synthesized a bivalent Smac-based ligand (Smac-1) and its fluorescent labeled analogue (Smac-1F) and characterized their interaction with different constructs of XIAP. Our study demonstrates that bivalent Smac-based ligands bind concurrently to both the BIR2 and BIR3 domains of XIAP and are more than 500 times more potent than the corresponding monovalent Smac-based ligands. Bivalent Smac-based ligands also function as much more potent antagonists of XIAP than do the corresponding monovalent Smac-based ligands in cell-free functional assays. Using Smac-1F and XIAP containing both BIR2 and BIR3 domains, we also developed and validated a new fluorescence polarization-based assay. Hence, our designed bivalent Smac-based peptides mimic the mode of dimeric Smac protein in their interaction with XIAP containing both BIR2 and BIR3 domains and achieve extremely high potency in binding and functional assays. Our study provides new insights into the mode of action of bivalent Smac ligands targeting XIAP and a basis for the design and development of cell-permeable, bivalent Smac mimetics.