Evaluation of adamantane hydroxamates as botulinum neurotoxin inhibitors: Synthesis,crystallography, modeling,kinetic and cellular based studies

Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based and thus have a limited window of treatment opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a therapeutic strategy for the treatment of botulism as it may provide an effective post exposure remedy. Using a combination of crystallographic and modeling studies a series of hydroxamates derived from 1-adamantylacetohydroxamic acid (3a) were prepared. From this group of compounds, an improved potency of about 17-fold was observed for two derivatives. Detailed mechanistic studies on these structures revealed a competitive inhibition model, with a K_i = 27 nM, which makes these compounds some of the most potent small molecule, non-peptidic BoNT/A LC inhibitors reported to date.     

The Aspartate-Less Receiver (ALR) Domains: Distribution,Structure and Function

Two-component signaling systems are ubiquitous in bacteria, Archaea and plants and play important roles in sensing and responding to environmental stimuli. To propagate a signaling response the typical system employs a sensory histidine kinase that phosphorylates a Receiver (REC) domain on a conserved aspartate (Asp) residue. Although it is known that some REC domains are missing this Asp residue, it remains unclear as to how many of these divergent REC domains exist, what their functional roles are and how they are regulated in the absence of the conserved Asp. Here we have compiled all deposited REC domains missing their phosphorylatable Asp residue, renamed here as the Aspartate-Less Receiver (ALR) domains. Our data show that ALRs are surprisingly common and are enriched for when attached to more rare effector outputs. Analysis of our informatics and the available ALR atomic structures, combined with structural, biochemical and genetic data of the ALR archetype RitR from Streptococcus pneumoniae presented here suggest that ALRs have reorganized their active pockets to instead take on a constitutive regulatory role or accommodate input signals other than Asp phosphorylation, while largely retaining the canonical post-phosphorylation mechanisms and dimeric interface. This work defines ALRs as an atypical REC subclass and provides insights into shared mechanisms of activation between ALR and REC domains.     

The crystal structure of phosphonate-inhibited D-Ala-D-Ala peptidase reveals an analogue of a tetrahedral transition state

D-Alanyl-D-alanine carboxypeptidase/transpeptidases (DD-peptidases) are beta-lactam-sensitive enzymes that are responsible for the final peptidoglycan cross-linking step in bacterial cell wall biosynthesis. A highly specific tripeptide phosphonate inhibitor was designed with a side chain corresponding to a portion of the Streptomyces R61 peptidoglycan. This compound was found to be a slow, irreversible inactivator of the DD-peptidase. Molecular modeling suggested that although a pentacoordinated intermediate of the phosphonylation reaction would not interact strongly with the enzyme, a tetracoordinated phosphonyl enzyme might be analogous to a transition state in the reaction with peptide substrates. To investigate this possibility, the crystal structure of the phosphonyl enzyme was determined. The 1.1 A resolution structure shows that the inhibitor has phosphonylated the catalytic serine (Ser62). One of the phosphonyl oxygens is noncovalently bound in the oxyanion hole, while the other is solvated by two water molecules. The conserved hydroxyl group of Tyr159 forms a strong hydrogen bond with the latter oxygen atom (2.77 A). This arrangement is interpreted as being analogous to the transition state for the formation of the tetrahedral intermediate in the deacylation step of the carboxypeptidase reaction. The proximity of Tyr159 to the solvated phosphonyl oxygen suggests that the tyrosine anion acts as a general base for deacylation. This transition state analogue structure is compared to the structures of noncovalent DD-peptidase reaction intermediates and phosphonylated beta-lactamases. These comparisons show that specific substrate binding to the peptidase induces a conformational change in the active site that places Ser62 in an optimal position for catalysis. This activated conformation relaxes as the reaction proceeds.     

Regulation of phenylacetic acid degradation genes of Burkholderia cenocepacia K56-2

Background  

Metabolically versatile soil bacteria Burkholderia cepacia complex (Bcc) have emerged as opportunistic pathogens, especially of cystic fibrosis (CF). Previously, we initiated the characterization of the phenylacetic acid (PA) degradation pathway in B. cenocepacia, a member of the Bcc, and demonstrated the necessity of a functional PA catabolic pathway for full virulence in Caenorhabditis elegans. In this study, we aimed to characterize regulatory elements and nutritional requirements that control the PA catabolic genes in B. cenocepacia K56-2.     

Inactivation of bacterial DD-peptidase by beta-sultams

N-Acyl-beta-sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to beta-sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the beta-sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located.     

Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators

PPARgamma coactivator 1alpha (PGC-1alpha) is a potent stimulator of mitochondrial biogenesis and respiration. Since the mitochondrial electron transport chain is the main producer of reactive oxygen species (ROS) in most cells, we examined the effect of PGC-1alpha on the metabolism of ROS. PGC-1alpha is coinduced with several key ROS-detoxifying enzymes upon treatment of cells with an oxidative stressor; studies with RNAi or null cells indicate that PGC-1alpha is required for the induction of many ROS-detoxifying enzymes, including GPx1 and SOD2. PGC-1alpha null mice are much more sensitive to the neurodegenerative effects of MPTP and kainic acid, oxidative stressors affecting the substantia nigra and hippocampus, respectively. Increasing PGC-1alpha levels dramatically protects neural cells in culture from oxidative-stressor-mediated death. These studies reveal that PGC-1alpha is a broad and powerful regulator of ROS metabolism, providing a potential target for the therapeutic manipulation of these important endogenous toxins.