Isolation and Structure Determination of Two Novel Phenazines from a Streptomyces with Inhibitory Activity against Metallo-enzymes, Including Metallo-.BETA.-lactamase.

Abstract

Two novel metabolites, SB 212021 and SB 212305, have been isolated from a Streptomyces and shown to have molecular formulae of C15H10N2O5 and C20H17N3O8S, respectively. The structures were deduced by a combination of NMR techniques and mass spectral fragmentation patterns and shown to be novel membersof the phenazine group of antibiotics. In the absence of added zinc, both compounds had IC50's of 1^75^for the Bacteroides fragilis 262 CfiA and Xanthomonas maltophilia h-l metallo-/Mactamases. The compounds also inhibited ACEwith IC50's of 55 and 68 fiM, respectively. Mode of action studies illustrate that the compounds inihibit some metalloenzymes by chelation of the active site metal ion. They exhibit poor antibacterial activity.Metallo/Mactamases, whilst not as prevalent in nature as their serine counterparts, do constitiute a possible threat to /Mactam chemotherapy due to the unavailability of effective inhibitors.Although only produced by a limited number of organisms they are potentially transferable by plasmids and could therefore pose a wider threat1*. The search for inhibitors of this enzyme is thus aimed at identifying structural types from natural product screening that could provide leads for chemical modification/synthesis.We report on the isolation of two novel phenazines, designated SB 212021 (1) and SB 212305 (2), produced by an unidentified Streptomyces sp. and found to inhibit zinc-dependent metallo-/Mactamase from Bacillus cereus. This paper describes the isolation, physico-chemical properties, structure determination and biological activity of SB 212021 and SB 212305. Materials and Methods Fermentation ConditionsThe unidentified Streptomyces sp. was maintained as a vegetative cell suspension stored in glycerol 10% under liquid nitrogen. Vegetative cell and spore suspension (1 ml) was used to inoculate seed medium (100ml) containing 1.5% agar. The seed stage medium consisted of yeast extract (Oxoid) 0.5%, malt extract (Oxoid) 1.0%, glycerol 1.0% and peptone soya (Oxoid) 0.5% dissolved in distilled water and adjusted to pH 6.5 before sterilisation in an autoclave at 121°C for 15 minutes. Four spiked 500 ml flasks, each containing 60 ml of seed stage mediumas defined above, were inoculated with 3 plugs from a culture plate. The inoculated flasks were incubated on a gyratory shaking table at 240 rpm for 60 hours at 28°C and after this time were used to inoculate 70 spiked 500ml flasks, each containing 60ml of seed stage medium as defined above (inoculum level ca. 5%, 3ml/flask).These inoculated flasks were shaken at 240rpm for 4.5 days at 28°C. The harvested broth was

Succinic Acids as Potent Inhibitors of Plasmid-borne IMP-1 Metallo-β-lactamase

“…17 and 18)) including phenazines (19), trifluoromethyl alcohol and ketone derivatives of L-and D-alanine (20), thioesters (18,(21)(22)(23), thiols (24 -28), biphenyl tetrazoles (29,30), and amino acid-derived hydroxamates (31). Biphenyl tetrazoles have been shown to reverse imipenem resistance in a clinical isolate of Bacteroides fragilis (29), and thioesters have been shown to reverse resistance to the carbapenem L-742,728 in a laboratory strain of Escherichia coli expressing IMP-1 (32).…”

Probing substrate binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis

“…A similar grouping scheme (B1, B2, and B3) based on structural properties of the metallo-β-lactamases has recently been offered [41]. The diversity of the group 3 β-lactamases is best exemplified by the enzymes' vastly differing efficacies towards non-clinical inhibitors; these differences predict that one inhibitor may not inhibit all metallo-β-lactamases [18,[20][21][22][23][24][25][26][27][28][29]. To combat this problem, we are characterizing a metallo-β-lactamase from each of the subgroups in an effort to identify a common structural or mechanistic aspect of the enzymes that can be targeted for the generation of an inhibitor.…”

The 1.5-Å Structure of Chryseobacterium meningosepticum Zinc β-Lactamase in Complex with the Inhibitor, D-Captopril

“…Because of the great interest in MBL inhibition, several classes of inhibitors have been described (17,18); these include phenazines (19), ketone derivatives of L-and D-alanine and trifluoromethyl alcohol (20), thioesters (21,22), biphenyl tetrazoles (23,24), amino acid-derived hydroxamates (25), thiols (26 -30), and tricyclic natural products (31). However, despite many of these having good inhibitory properties, only a few thiols have a broad spectrum of inhibition for MBLs.…”