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Short hairpin RNAs (shRNAs) have been used to achieve stable target knockdown in a variety of biological systems. Here, we report the development of a tightly regulated tetracycline-responsive human U6 promoter for shRNA expression. By engineering two copies of the tet operators flanking the TATA box of the human U6 promoter, we created a U6 promoter derivative (2O2) that exhibited much lower basal transcriptional activity compared with recently reported inducible pol III dependent promoters. As a consequence of its tighter regulation, the 2O2 system greatly improved the success rate in making inducible knockdown cell lines. [Copyright &y& Elsevier]

p53 exerts its tumor suppressor activity through both transcription-dependent and transcription-independent processes. Although the transcription-dependent activity of p53 has been extensively studied, the mechanism for transcription-independent p53-mediated tumor suppression is less well known. Recently, it was reported that p53 can directly induce mitochondrial permeabilization and promote apoptosis. This occurs through complexation of the DNA-binding region of p53 with the anti-apoptotic proteins Bcl-xL and Bcl-2 (Mihara, M. et al. (2003) Mol. Cell 11, 577–590). Using nuclear magnetic resonance (NMR) spectroscopy we show that the interaction surface on p53 involves the same region that is used by the protein to contact DNA. The p53-binding site on Bcl-xL consists of the carboxy-terminus of the first α-helix, the loop between α3 and α4, and the loop between α5 and α6 of Bcl-xL. Furthermore, the interaction of p53 with Bcl-xL is blocked by the binding of a 25-residue peptide derived from the BH3 region of the pro-apoptotic protein referred to as Bad. [Copyright &y& Elsevier]

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Bcl-xL, a member of the Bcl-2 family, can inhibit many forms of programed cell death. The three-dimensional structure of Bcl-xL identified a 60 amino acid loop lacking defined structure. Although amino acid sequence within this region is not conserved among Bcl-2 family members, structural modeling suggested that Bcl-2 also contains a large unstructured region. Compared with the full-length protein, loop deletion mutants of Bcl-xL and Bcl-2 displayed an enhanced ability to inhibit apoptosis. Despite enhanced function, the deletion mutants did not have significant alterations in the ability to bind pro-apoptotic proteins such as Bax. The loop deletion mutant of Bcl-2 also displayed a qualitative difference in its ability to inhibit apoptosis. Full-length Bcl-2 was unable to prevent anti-IgM-induced cell death of the immature B cell line WEHI-231. In contrast, the Bcl-2 deletion mutant protected WEHI-231 cells from death. Substantial differences were observed in the ability of WEHI-231 cells to phosphorylate the deletion mutant of Bcl-2 compared with full-length Bcl-2. Bcl-2 phosphorylation was found to be dependent on the presence of an intact loop domain. These results suggest that the loop domain in Bcl-xL and Bcl-2 can suppress the anti-apoptotic function of these genes and may be a target for regulatory post-translational modifications. [ABSTRACT FROM AUTHOR]

The molecular chaperone HSP90 has been shown to facilitate cancer cell survival by stabilizing key proteins responsible for a malignant phenotype. We report here the results of parallel fragment-based drug design approaches in the design of novel HSP90 inhibitors. Initial aminopyrimidine leads were elaborated using high-throughput organic synthesis to yield nanomolar inhibitors of the enzyme. Second site leads were also identified which bound to HSP90 in two distinct conformations, an 'open' and 'closed' form. Intriguingly, linked fragment approaches targeting both of these conformations were successful in producing novel, micromolar inhibitors. Overall, this study shows that, with only a few fragment hits, multiple lead series can be generated for HSP90 due to the inherent flexibility of the active site. Thus, ample opportunities exist to use these lead series in the development of clinically useful HSP90 inhibitors for the treatment of cancers.

As part of a fully integrated and comprehensive strategy to discover novel antibacterial agents, NMR- and mass spectrometry-based affinity selection screens were performed to identify compounds that bind to protein targets uniquely found in bacteria and encoded by genes essential for microbial viability. A biphenyl acid lead series emerged from an NMR-based screen with the Haemophilus influenzae protein HI0065, a member of a family of probable ATP-binding proteins found exclusively in eubacteria. The structure-activity relationships developed around the NMR-derived biphenyl acid lead were consistent with on-target antibacterial activity as the Staphylococcus aureus antibacterial activity of the series correlated extremely well with binding affinity to HI0065, while the correlation of binding affinity with B-cell cytotoxicity was relatively poor. Although further studies are needed to conclusively establish the mode of action of the biphenyl series, these compounds represent novel leads that can serve as the basis for the development of novel antibacterial agents that appear to work via an unprecedented mechanism of action. Overall, these results support the genomics-driven hypothesis that targeting bacterial essential gene products that are not present in eukaryotic cells can identify novel antibacterial agents.