Inhibition of anthrax lethal factor by ssDNA aptamers.

Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a Kd value of 11.0 ±â€¯2.7 nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC50 value of 15 ±â€¯1.5 µM and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF.

Inhibition of Bacillus anthracis metallo-ß-lactamase by compounds with hydroxamic acid functionality.

Metallo-ß-lactamases (MBLs) that catalyze hydrolysis of ß-lactam antibiotics are an emerging threat due to their rapid spread. A strain of the bacterium Bacillus anthracis has its ability to produce and secrete a MBL, referred to Bla2. To address this challenge, novel hydroxamic acid-containing compounds such as 3-(heptyloxy)-N-hydroxybenzamide (compound 4) and N-hydroxy-3-((6-(hydroxyamino)-6-oxohexyl)oxy)benzamide (compound 7) were synthesized. Kinetic analysis of microbial inhibition indicated that the both sides of hydroxamic acids containing compound 7 revealed a reversible, competitive inhibition with a Ki value of 0.18 ± 0.06 µM. The result has reflected that the both sides of dihydroxamic acids in a molecule play a crucial role in the binding affinity rather than monohydroxamic containing compound 4 which was unable to inhibit Bla2. In addition, in silico analysis suggested that compound 7 was coordinated with a zinc ion in the active site of enzyme. These observations suggest that the dihydroxamic acid-containing compound may be a promising drug candidate, and a further implication for designing new inhibitors of Bla2.

Kinetic characterization of a slow-binding inhibitor of Bla2: thiomaltol.

The increasing prevalence of drug resistant bacteria is a pandemic problem. Metallo-ß-lactamases (MBLs) are one of the main causes of drug resistance due to hydrolysis of ß-lactam antibiotics. Thus, the development of effective inhibitors of MBLs remains urgent. The compound thiomaltol was used as a lead compound to investigate its ability to inhibit metallo-ß-lactamase from Bacillus anthracis (Bla2), which causes anthrax. Kinetic evaluation with nitrocefin as a substrate indicates that thiomaltol inhibits Bla2 in a time-dependent manner with an IC(50) value of 290 µM after 20 min preincubation. Progress curve analysis and reversibility tests suggest that thiomaltol is a reversible, slow-binding inhibitor with a K(i) of 85 ± 30 µM. Furthermore, studies on the modality of inhibition and in silico analysis indicate thiomaltol to be a competitive inhibitor. The results demonstrate that thiomaltol is a promising lead compound for slow binding inhibitor design of Bla2.

Purification development and characterization of the zinc-dependent metallo-ß-lactamase from Bacillus anthracis.

Metallo-ß-lactamase from Bacillus anthracis (Bla2) catalyzes the hydrolysis of ß-lactam antibiotics which are commonly prescribed to combat bacterial infections. Bla2 contributes to the antibiotic resistance of this bacterium. An understanding of it is necessary to design potential inhibitors that can be introduced with current antibiotics for effective eradication of anthrax infections. We have purified Bla2 using Ni(2+)-affinity chromatography with over 140-fold increase in activity with a yield of 3.5%. The final specific activity was 19,000 units/mg. Purified Bla2 displays different K ( m ), V ( max ), and (k ( cat ) /K (M)) with penicillin G and cephalexin as substrates and is also sensitive to pH, with maximum activity between pH 7.0-9.0. The IC(50) (50% inhibition concentration) value of EDTA against Bla2 is 630 nM, which can be understood by observing its three-dimensional interaction with the enzyme.