Improved Synthesis of Biotinol-5'-AMP: Implications for Antibacterial Discovery.

An improved synthesis of biotinol-5'-AMP, an acyl-AMP mimic of the natural reaction intermediate of biotin protein ligase (BPL), is reported. This compound was shown to be a pan inhibitor of BPLs from a series of clinically important bacteria, particularly Staphylococcus aureus and Mycobacterium tuberculosis, and kinetic analysis revealed it to be competitive against the substrate biotin. Biotinol-5'-AMP also exhibits antibacterial activity against a panel of clinical isolates of S. aureus and M. tuberculosis with MIC values of 1-8 and 0.5-2.5 µg/mL, respectively, while being devoid of cytotoxicity to human HepG2 cells.

A nucleotide signature for identification of Aglaia stellatopilosa Pannell.

Members of the genus Aglaia have been reported to contain bioactive phytochemicals. The genus, belonging to the Meliaceae family, is represented by at least 120 known species of woody trees or shrubs in the tropical rain forest. As some of these species are very similar in their morphology, taxonomic identification can be difficult. A reliable and definitive molecular method which can identify Aglaia to the level of the species will hence be useful in comparing the content of specific bioactive compounds between the species of this genus. Here, we report the analysis of DNA sequences in the internal transcribed spacer (ITS) of the nuclear ribosomal DNA and the observation of a unique nucleotide signature in the ITS that can be used for the identification of Aglaia stellatopilosa. The nucleotide signature consists of nine bases over the length of the ITS sequence (654 bp). This uniqueness was validated in 37 samples identified as Aglaia stellatopilosa by an expert taxonomist, whereas the nucleotide signature was lacking in a selection of other Aglaia species and non-Aglaia genera. This finding suggests that molecular typing could be utilized in the identification of Aglaia stellatopilosa.

Escherichia coli biotin protein ligase: characterization and development of a high-throughput assay.

The rapid rise in pathogenic bacteria resistant to current treatments, coupled with the paucity of new therapeutic agents in the pipeline, has resulted in a significant need for new antibiotics. One strategy to overcome resistance requires new chemical entities that inhibit key enzymes in essential metabolic processes that have not been previously targeted and for which there is no preexisting drug resistance. Biotin protein ligase (BPL), required to complete acetyl CoA carboxylase's capability for fatty acid biosynthesis, is one target that has not yet been fully explored. However, its application in large-scale compound screens has been limited due to the lack of a truly high-throughput assay for enzyme activity. Here we report a novel assay system for BPL from Escherichia coli (BirA). This assay employs fluorescence polarization technology together with a unique peptide substrate for BirA. Additionally, the multiple handling steps and requirement for radiolabeled ligands associated with previous assays have been eliminated. Kinetic analysis of MgATP (K(m) 0.25+/-0.01 mM) and biotin (K(m) 1.45+/-0.15 microM) binding produced results consistent with published data. Inhibition studies with end products of the BPL reaction, AMP and pyrophosphate, further validated the assay. Statistical analysis, performed upon both intraassay and interassay results (n = 30), showed the coefficient of variance to be <10% across all data sets. Furthermore, Z' factors between 0.5 and 0.8 demonstrated the utility of this technology in high-throughput applications.

Senile systemic amyloidosis presenting with heart failure: a comparison with light chain-associated amyloidosis.

BACKGROUND: Small deposits of amyloid are often found in the hearts of elderly patients. However, extensive deposition of transthyretin-derived amyloid fibrils in the heart (senile systemic amyloidosis [SSA]) can cause heart failure. The clinical features of SSA that involve the heart are ill defined, and the condition may be overlooked as a cause of heart failure. We sought to better define the clinical, echocardiographic, and electrocardiographic features of cardiac involvement in SSA and to compare them with the findings in patients with light chain-associated (AL) amyloidosis that affects the heart. METHODS: Eighteen consecutive patients with SSA and heart failure evaluated at a tertiary referral center for the diagnosis and treatment of amyloidosis were compared with 18 randomly selected patients with AL amyloidosis that involved the heart. All patients underwent a complete clinical and biochemical evaluation. Echocardiograms and electrocardiograms were interpreted by blinded investigators. RESULTS: Patients with SSA were older than those with AL amyloidosis and were all male. Proteinuria (protein output of >1 g per 24 hours) was common in AL amyloidosis but was not present in SSA. Left ventricular wall thickness was greater in patients with SSA than those with AL amyloidosis, but despite thicker walls and older age, the severity of heart failure was less in the SSA group and the median survival was much longer (75 vs 11 months; P = .003). CONCLUSIONS: Senile systemic amyloidosis is a disorder of elderly men and is characterized by amyloidosis clinically limited to the heart. In contrast to the rapid progression of heart failure in AL amyloidosis, SSA results in slowly progressive heart failure. The difference in survival, despite evidence of more myocardial disease in the senile group, suggests that heart failure in AL amyloidosis may have a toxic component, possibly related to the circulating monoclonal light chain.