Identification of novel HSP90α/ß isoform selective inhibitors using structure-based drug design. demonstration of potential utility in treating CNS disorders such as Huntington's disease.

A structure-based drug design strategy was used to optimize a novel benzolactam series of HSP90α/ß inhibitors to achieve >1000-fold selectivity versus the HSP90 endoplasmic reticulum and mitochondrial isoforms (GRP94 and TRAP1, respectively). Selective HSP90α/ß inhibitors were found to be equipotent to pan-HSP90 inhibitors in promoting the clearance of mutant huntingtin protein (mHtt) in vitro, however with less cellular toxicity. Improved tolerability profiles may enable the use of HSP90α/ß selective inhibitors in treating chronic neurodegenerative indications such as Huntington's disease (HD). A potent, selective, orally available HSP90α/ß inhibitor was identified (compound 31) that crosses the blood-brain barrier. Compound 31 demonstrated proof of concept by successfully reducing brain Htt levels following oral dosing in rats.

Correlation between chemotype-dependent binding conformations of HSP90α/ß and isoform selectivity-Implications for the structure-based design of HSP90α/ß selective inhibitors for treating neurodegenerative diseases.

HSP90 continues to be a target of interest for neurodegeneration indications. Selective knockdown of the HSP90 cytosolic isoforms α and ß is sufficient to reduce mutant huntingtin protein levels in vitro. Chemotype-dependent binding conformations of HSP90α/ß appear to strongly influence isoform selectivity. The rational design of HSP90α/ß inhibitors selective versus the mitochondrial (TRAP1) and endoplasmic reticulum (GRP94) isoforms offers a potential mitigating strategy for mechanism-based toxicities. Better tolerated HSP90 inhibitors would be attractive for targeting chronic neurodegenerative diseases such as Huntington's disease.

Extracellular DNA-induced antimicrobial peptide resistance in Salmonella enterica serovar Typhimurium.

BACKGROUND: The Salmonella enterica serovar Typhimurium PhoPQ two component system (TCS) is activated by low Mg2+ levels, low pH and by antimicrobial peptides (AP). Under Mg2+ limitation, the PhoPQ system induces pmrD expression, which post-translationally activates the PmrAB TCS. PhoPQ and PmrAB control many genes required for intracellular survival and pathogenesis. These include the polymyxin resistance (pmr) operon, which is required for aminoarabinose modification of LPS and protecting the outer membrane from antimicrobial peptide disruption and killing. Extracellular DNA is a ubiquitous polymer in the matrix of biofilms and accumulates in some infection sites. Extracellular DNA chelates cations and thus activates the Pseudomonas aeruginosa PhoPQ/PmrAB systems, leading to expression of the orthologous arn (pmr) operon. RESULTS: Here we show that extracellular DNA induces expression of the S. Typhimurium pmr antimicrobial peptide resistance operon in a PhoPQ and PmrAB-dependent manner. Induction of the pmr genes by DNA was blocked when present with excess Mg2+. Exogenous DNA led to increased resistance of planktonic cultures to aminoglycosides, antimicrobial peptides (AP) and ciprofloxacin, but only AP resistance was PhoPQ/PmrAB-dependent. Extracellular DNA was shown to be a matrix component of S. Typhimurium biofilms cultivated in flow chambers and on glass surfaces. A pmrH-gfp fusion was highly expressed in flow chamber biofilms cultivated in medium with repressing levels of 10 mM Mg2+ and co-localized with eDNA. Expression of pmrH-lux was monitored in plastic peg biofilms and shown to require PhoPQ and PmrAB. Biofilms had higher levels of pmrH expression compared to planktonic cultures. We propose that DNA accumulation in biofilms contributes to the increased pmrH-lux expression in biofilms. CONCLUSIONS: The Salmonella PhoPQ/PmrAB systems and antimicrobial peptide resistance are activated by the cation chelating properties of extracellular DNA. DNA-induced AP resistance may allow immune evasion and increased survival of S. Typhimurium biofilms formed during extracellular growth stages of an infection or outside the host.

Transcriptional regulation of sdiA by cAMP-receptor protein, LeuO, and environmental signals in Salmonella enterica serovar Typhimurium.

The sdiA gene encodes for a LuxR-type transcription factor, which is active when bound to N-acyl homoserine lactones (AHLs). Because Salmonella enterica serovar Typhimurium does not produce AHLs, SdiA senses signals produced by other organisms. SdiA is not expressed constitutively, and response is limited to conditions in which elevated expression occurs, but little is known about the regulation of sdiA expression. Here we map the sdiA promoter and define several regulators that directly or indirectly act on the promoter. The major activator of sdiA expression is cAMP-receptor protein (CRP), and we define the CRP operator in the sdiA promoter using promoter and crp mutants. LeuO activates sdiA expression to a lesser extent than does CRP. We demonstrate that LeuO directly binds the sdiA promoter and the Rcs phosphorelay represses sdiA expression. In this study, NhaR, IlvY, and Fur affected sdiA expression indirectly and weakly. Expression in late-stationary phase depended on RpoS. AHL-dependent expression of the SdiA-regulated gene rck correlated to the observed sdiA transcriptional changes in regulator mutants. The data demonstrate that regulation of sdiA involves integration of multiple environmental and metabolic signals.

Cysteine biosynthesis, oxidative stress and antibiotic resistance in Salmonella typhimurium.

The efficacy of antibiotics varies under different growth conditions due to the induction of specific or more general defense pathways, but the mechanisms are not completely understood. Actively swarming Salmonella show elevated resistance to many types of antibiotics. Previously, we had shown that cysteine biosynthesis was important for the induced antibiotic resistance phenotype of swarm cells. Here we examine the connection of cysteine to oxidative stress and demonstrate that the antioxidant properties of cysteine or cysteine-derived metabolites contribute to the antibiotic resistance in both vegetative and swarm cell populations. We observed that cys auxotrophs were oxidatively stressed, and in wild-type cells expression of the cys regulon was induced during periods of oxidative stress. In swarm cells, we found a 6-fold increase in reduced glutathione compared to swim cells and a corresponding increased resistance to oxidants. Wild-type and cys auxotrophs exhibited the same sensitivities to gentamicin, polymyxin and ciprofloxacin when grown anaerobically, suggesting that induced oxidative stress defense was contributing to elevated antibiotic resistance in swarm cells aerobically. Induction of the CysB regulon by addition of exogenous inducer resulted in elevated antibiotic resistance independently of swarming.

L-Cysteine is required for induced antibiotic resistance in actively swarming Salmonella enterica serovar Typhimurium.

Swarm-cell differentiation in Salmonella enterica serovar Typhimurium (S. typhimurium) results in a biosynthetic mode of growth, despite growing on a rich medium, and cells that have elevated antibiotic resistance. These phenotypes are not a prerequisite for swarm motility. By blocking the switch to anabolic growth using amino acid auxotrophs and screening for the presence of elevated antibiotic resistance in the swarm state, we found that cysteine biosynthesis is crucial for complete swarm-cell differentiation. Mutants were made in each cys biosynthetic operon and all had decreased antibiotic resistance in the swarm state, while swim-cell resistance remained the same as that of wild-type cells. This swarm-state-specific decreased resistance in Deltacys strains could be restored to wild-type levels by the addition of cysteine to swarm medium. Two regulatory mutants, DeltacysB and DeltacysE, failed to swarm unless cysteine was supplemented to the medium. We show that all CysB-responsive operons involved in cysteine biosynthesis are upregulated in the swarm state, even though swarm cells are cultivated on a medium that represses cysteine biosynthesis in the swim state. While swarm medium has sufficient cysteine for growth of S. typhimurium, it does not contain enough for swarm-cell differentiation. We hypothesize that in these cells, the additional cysteine requirement is for use in pathways not directly related to cell growth.