ABSTRACT
As a model system for designing new inhibitors of bacterial cell division, we studied the essential and highly conserved FtsZ GTPase from Pseudomonas aeruginosa. A collection of GTP analogues were prepared using the solid-phase parallel synthesis approach. The synthesized GTP analogues inhibited the GTPase activity of FtsZ with IC(50) values between 450microM and 2.6mM, and 5 compounds inhibited Staphylococcus aureus growth in a biological assay. The FtsZ spectrophotometric assay developed for screening of synthesized compounds is the first step in identification of antibacterials targeting the bacterial cell division essential proteins.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Bacterial Proteins/antagonists & inhibitors , Cytoskeletal Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Pseudomonas aeruginosa/enzymology , Staphylococcus aureus/drug effects , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Cell Division/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , GTP Phosphohydrolases/antagonists & inhibitors , Guanosine Triphosphate/analogs & derivatives , Guanosine Triphosphate/chemical synthesis , Guanosine Triphosphate/pharmacology , Microbial Sensitivity Tests , Staphylococcus aureus/growth & developmentABSTRACT
The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates D-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-L-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC50 value was estimated at 4 microM for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-L-alanine:D-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC50 value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein-protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein-protein interaction domains can be identified by phage display.
Subject(s)
Ligases/antagonists & inhibitors , Ligases/chemistry , Pseudomonas aeruginosa/metabolism , Amino Acid Motifs , Amino Acid Sequence , Binding Sites , Computational Biology/methods , Dose-Response Relationship, Drug , Inhibitory Concentration 50 , Models, Chemical , Molecular Sequence Data , Peptide Library , Peptides/chemistry , Protein Binding , Protein Conformation , Sequence Analysis, DNA , Sequence Homology, Amino Acid , SpectrophotometryABSTRACT
We have developed a screening assay by thin-layer chromatography (TLC) to identify inhibitors for the bacterial essential enzymes MurA, -B, and -C. Libraries of compounds were synthesized using the mix-and-split combinatorial chemistry approach. Screening of the pooled compounds using the developed assay revealed the presence of many pools active in vitro. Pools of interest were tested for antibacterial activity. Individual molecules in the active pools were synthesized and retested with the TLC assay and with bacteria. We focused on the best five compounds for further analysis. They were tested for inhibition on each of the three enzymes separately, and showed no inhibition of MurA or MurB activity but were all inhibitors of MurC enzyme. This approach yielded interesting lead compounds for the development of novel antibacterial agents.
