Inhibition of ß-carbonic anhydrases from Brucella suis with C-cinnamoyl glycosides incorporating the phenol moiety.

A small series of C-glycosides containing the phenol moiety was tested for the inhibition of the ß-class carbonic anhydrases (ßCAs, EC 4.2.1.1) from Brucella suis. Many compounds showed activities in the micromolar or submicromolar range and excellent selectivity for pathogen CAs over human isozymes. Glycosides incorporating the 3-hydroxyphenyl moiety showed the best inhibition profile, and therefore this functionality represents lead for the development of novel anti-infectives with a new mechanism of action.

C-glycosides incorporating the 6-methoxy-2-naphthyl moiety are selective inhibitors of fungal and bacterial carbonic anhydrases.

A small series of C-glycosides containing the methoxyaryl moieties was tested for the inhibition of the ß-class carbonic anhydrases (CAs, EC 4.2.1.1) from Cryptococcus neoformans and Brucella suis. Many compounds showed activities in the micromolar or submicromolar range and excellent selectivity for pathogen CAs over human isozymes. The deprotected glycosides incorporating the 6-methoxy-2-naphthyl moiety showed the best inhibition profile and therefore represent leads for the development of novel anti-infectives with a new mechanism of action.

Molecular modeling studies on nucleoside hydrolase from the biological warfare agent Brucella suis.

Brucella suis is a dangerous biological warfare agent already used for military purposes. This bacteria cause brucellosis, a zoonosis highly infective and difficult to fight. An important selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. We present here the first three-dimensional structure of B. suis NH (BsNH) and propose this enzyme as a molecular target to the drug design in the fight against brucellosis. In addition, we performed molecular docking studies, aiming to analyze the three-dimensional positioning of nine known inhibitors of Chritidia fasciculata NH (CfNH) in the active sites of BsNH and CfNH. We also analyzed the main interactions of some of these compounds inside the active site of BsNH and the relevant factors to biological activity. These results, together with further molecular dynamics (MD) simulations, pointed out to the most promising compound as lead for the design of potential inhibitors of BsNH. Most of the docking and MD results corroborated to each other and the docking results also suggested a good correlation with experimental data.

Enzymatic, immunological and phylogenetic characterization of Brucella suis urease.

BACKGROUND: The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined. RESULTS: Urease encoded by the ure-1 operon of Brucella suis strain 1330 was purified to homogeneity using ion exchange and hydrophobic interaction chromatographies. The urease was purified 51-fold with a recovery of 12% of the enzyme activity and 0.24% of the total protein. The enzyme had an isoelectric point of 5, and showed optimal activity at pH 7.0 and 28-35 degrees C. The purified enzyme exhibited a Michaelis-Menten saturation kinetics with a Km of 5.60 +/- 0.69 mM. Hydroxyurea and thiourea are competitive inhibitors of the enzyme with Ki of 1.04 +/- 0.31 mM and 26.12 +/- 2.30 mM, respectively. Acetohydroxamic acid also inhibits the enzyme in a competitive way. The molecular weight estimated for the native enzyme was between 130-135 kDa by gel filtration chromatography and 157 +/- 7 kDa using 5-10% polyacrylamide gradient non-denaturing gel. Only three subunits in SDS-PAGE were identified: two small subunits of 14,000 Da and 15,500 Da, and a major subunit of 66,000 Da. The amino terminal sequence of the purified large subunit corresponded to the predicted amino acid sequence encoded by ureC1. The UreC1 subunit was recognized by sera from patients with acute and chronic brucellosis. By phylogenetic and cluster structure analyses, ureC1 was related to the ureC typically present in the Rhizobiales; in contrast, the ureC2 encoded in the ure-2 operon is more related to distant species. CONCLUSION: We have for the first time purified and characterized an active urease from B. suis. The enzyme was characterized at the kinetic, immunological and phylogenetic levels. Our results confirm that the active urease of B. suis is a product of ure-1 operon.