[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to design, synthesize, and evaluate novel antibacterial agents, derivatives of aryl-4-guanidinomethylbenzoate and N-aryl-4-guanidinomethylbenzamide.
A total of 44 derivatives of aryl-4-guanidin-omethylbenzoate (series A) and N-aryl-4-guanidinomethylbenzamide (series B) were synthesized and their antibacterial activities were assessed in vitro against a variety of Gram-positive and Gram-negative bacteria by an agar dilution method.
Twelve compounds showed potent bactericidal effects against a panel of Gram-positive germs, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), vancomycin-intermediate Staphylococcus aureus (VISA), and methicillin-resistant coagulase-negative staphylococci (MRCNS), with minimum inhibitory concentrations (MIC) ranging between 0.5 and 8 microg/mL, which were comparable to the MIC values of several marketed antibiotics. They exhibited weak or no activity on the Gram-negative bacteria tested. In addition, these compounds displayed high inhibitory activities towards oligopeptidase B of bacterial origin.
In comparison with the previously reported MIC values of several known antibiotics, the derivatives of aryl-4-guanidinomethylbenzoate and N-aryl-4-guanidinomethylbenzamide showed comparable in vitro bactericidal activities against VRE and VISA as linezolid. Their growth inhibitory effects on MRSA were similar to vancomycin, but were less potent than linezolid and vancomycin against MRCNS. This class of compounds may have the potential to be developed into narrow spectrum antibacterial agents against certain drug-resistant strains of bacteria.
[Show abstract][Hide abstract] ABSTRACT: Oligopeptidase B (OpdB) of Escherichia coli, previously called protease II, has a trypsin-like specificity, cleaving peptides at lysine and arginine residues and belongs to the prolyl oligopeptidase family of new serine peptidases. In this study, we report the fusion expression of E. coli oligopeptidase B with an N-terminal histidine tag using pET28a as the expression vector. Although most of the recombinant OpdB was produced as inclusion bodies, the solubility of the recombinant protease increased significantly when the expression temperature shifted from 37 to 30 degrees C. Recombinant OpdB (approximately 10 mg) could be purified from the soluble fraction of the crude extract of 1L log-phase E. coli culture containing 1.5 g wet bacterial cells. The purified OpdB has a molecular weight of approximately 80 kDa and a specific activity of 4.8 x 10(4) U/mg. OpdB could also be purified from the inclusion bodies with a lower yield. The recombinant enzyme was very stable under 40 degrees C. By comparison of the substrate specificity of the purified OpdB with that of OpdA, another trypsin-like protease in E. coli, we found that Boc-Glu-Lys-Lys-MCA is a specific substrate for E. coli OpdB. We also found that compared to OpdA, OpdB is much more sensitive to GMCHA-OPh(t)Bu, a synthetic trypsin inhibitor that can retard the growth of E. coli.
Protein Expression and Purification 07/2006; 47(2):645-50. · 1.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The antibacterial activities of NE-2001 were tested against 24 clinical isolates of Helicobacter pylori and compared with those of amoxicillin, clarithromycin, metronidazole, and furazolidone. The MIC(50) and MIC(90) of this synthetic compound on the isolates were 8 and 16 mug/ml, respectively. This action was highly selective against Helicobacter pylori; there was a >4-fold difference between the concentration of NE-2001 required to inhibit the growth of Helicobacter pylori and that required to inhibit the growth of common aerobic and anaerobic bacteria. Exposure of Helicobacter pylori (ATCC43504) to NE-2001 at the MIC (4 mug/ml), or at a greater concentration, resulted in an extensive loss of viability. The phenomenon was also observed at pH levels between 3.0 and 7.0. When two clinical Helicobacter pylori strains were successively cultured at subinhibitory concentrations of NE-2001, no significant changes in the bactericidal effects were found. The morphological alterations of Helicobacter pylori cells (ATCC43504), exposed to NE-2001 at various concentrations for 6 h, were observed using transmission electron microcopy. The bacterium displayed features such as swelling, vacuole-like structures in the cytoplasm, and cell destruction following exposure to NE-2001. The efficacy of NE-2001 was maintained when evaluated in eight clinical isolates resistant to metronidazole and five isolates resistant to both metronidazole and clarithromycin (MIC ranging between 4 and 16 mug/ml). The above-described results suggest that NE-2001 may have the potential to be developed as a candidate agent for the treatment of Helicobacter pylori infection.
Antimicrobial Agents and Chemotherapy 09/2005; 49(8):3468-73. · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study describes the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) from Helicobacter pylori. A gene encoding LAP was cloned from H. pylori and the expressed 55 kDa protein displayed homology to aminopeptidases from Gram-negative bacteria, plants and mammals. This LAP demonstrated amidolytic activity against L-leucine-p-nitroanilide. Optimal activity was observed at pH 8.0 and 45 degrees C, with V(max) of 232 mumol min(-1) (mg protein)(-1) and S(0.5) of 0.65 mM. The data suggest that LAP could be allosteric (n(H)=2.27), with regulatory homohexamers, and its activity was inhibited by ion chelators and enhanced by divalent manganese, cobalt and nickel cations. Bestatin inhibited both LAP activity (IC(50)=49.9 nM) and H. pylori growth in vitro. The results point to the potential use of LAP as a drug target to develop novel anti-H. pylori agents.
[Show abstract][Hide abstract] ABSTRACT: The synthesis and anti-Helicobacter pylori activity of a novel agent NE2001, 4-(4-methylbenzyl)-4'-[guanidinomethylbenzoyloxy] biphenyl-4-carboxylate hydrochloride, are described. NE2001 had a specific inhibitory effect on the growth of H. pylori preceded by the suppression DNA synthesis in the cell. The effects of NE2001 on RNA and protein syntheses in H. pylori were also examined.
[Show abstract][Hide abstract] ABSTRACT: 1.1. Proteinase inhibitor B from the root of the arrowhead vegetable legume has been cross-linked with DMA, DMS and EGS. The cross-linked AI-B with DMA resulted only in the monomer inhibitor, but with DMS and EGS, the dimer, trimer and a small amount of tetramer were obtained besides the monomer.2.2. Difficulty in the isolation and separation of monomer, dimer, trimer and tetramers of AI-B by classical column (TSK-2000-SW) on HPLC was encountered. The successful separation and isolation of monomer and dimer was obtained using an advanced column (Shim-Pack-Diol-300) for exclusion HPLC.3.3. The assay of inhibitory activity showed that the monomer and dimer had a much lower activity than the native AI-B. Furthermore, the monomeric activity was decreased more drastically than those of the dimeric. The circular dichroism (CD) spectra showed that the monomeric conformation had been changed, but the dimeric conformation had not.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry. 01/1988; 91(3):497-502.
[Show abstract][Hide abstract] ABSTRACT: 1.1. Isolation of CSF-1 from human urine was performed through five purification steps. These include concentration by dialysis, silica gel absorption, hydrophobic chromatography and phenyl-Sepharose CL-6B, Fast Protein Liquid Chromatography (FPLC) and finally preparative electrophoresis on polyacrylamide gels. These methods have been reported in a previous paper (Tao et al., 1987).2.2. The isolated CSF-1 which exhibits a one band pattern on SDS-PAGE under non-reducing conditions after Coosmassie Blue and silver stainings, CSF-1 was purified 100,000-fold and has a specific activity of 2.16 x 107 units/mg protein. Its apparent Mr is 57,000 with an isoelectric point pI = 5.8–6.0 CSF-l is a glycoprotein with 40% of carbohydrate (w/w).3.3. An almost complete removal of the carbohydrate moiety from CSF-1 was obtained after treatment with trifluoromethanesulfonic (TFMS) acid followed by gel filtration on Sephadex G-25 (Fine). The deglycosylated (DG) CSF-1 possesses an apparent Mr of 38,000 and an isoelectric point, pI: 6.2 as compared to native-CSF-1 (N-CSF-1), Mr = 57,000 and pI = 5.8 respectively.4.4. The TFMS treatment did not alter the activities of CSF-1 as shown by biological assay and receptor binding assay. The thermostability experiment revealed that DG-CSF-1 was less stable than N-CSF-1. The circular dichroism spectra (CD) of N-CSF-1 and DG-CSF-1 were different.5.5. The features of interaction of iodinated-N-CSF-1 and iodinated-DG-CSF-1 with single cell suspensions from human peritoneal macrophage were studied. The binding activity of peritoneal macrophage was the highest among all cells examined. The carbohydrates compositions of DG-CSF, the N-acetylglucosamine and galactosamine are established.
Comparative Biochemistry and Physiology Part B: Comparative Biochemistry.