Appearance of multidrug resistant strains of pathogenic bacteria (MDR = Multidrug resistant bacteria) is a serious medical issue in modern healthcare. Battle against infectious diseases caused by drug-resistant bacteria therefore inevitable involves searching for new bacterial
targets and the development of new antimicrobial agents. Peptidoglycan, an essential component of the bacterial cell wall which provides the structural integrity necessary for bacterial cells to resist internal osmotic pressure, remains one of essential and well validated targets for antibacterial therapy. Net-like structure of peptidoglycan consists of polysaccharide chains interconnected with short polypeptides. Interference in the synthesis or structure of peptidoglycan results in cell lysis and eventually bacterial death. Biosythesis of peptidoglycan is a complex process which involves around 20 reactions catalyzed by different enzymes that represent targets for development of new antibacterial agents. It occurs in the cythoplasm, on the inner side of bacterial cell membrane and on the outer cell membrane. In last decades more and more efforts have been made toward early cytoplasmic stages of biosynthesis, where Mur ligases play an important role. Mur ligases which consists of four enzymes (MurC-F), assemble the final intracellular peptidoglycan precursor UDP-MurNAc-pentapeptide by successive addition of L-Ala, D-Glu,
m-A2pm or L-Lys, and D-Ala-D-Ala to D-lactoyl part of peptidoglycan precursor UDP-Nacetylmuramic acid. Beside Mur ligases other important enzymes are involved in the synthesis of peptidoglycan. One of them is D-alanine:D-alanine (Ddl), which is responsible for supplying the MurF substrate, D-alanyl-D-alanine. Another ligase, murein peptide ligase (UDP-N-acetylmuramate:L-alanyl-γ-D-glutamyl-meso-diaminopimelate ligase or. Mpl), is responsible for constant remodeling of peptidoglycan during bacterial cell maturation and division. This enzyme is not essential for bacterial growth itself but since it is able to add tripeptide L-alanyl-γ-D-glutamyl-meso-diaminopimelate onto UDP-N-acetylmuramic acid in one step it is very important in recycling of peptidoglycan. In this thesis we focused our attention on MurC-F, DdlB and Mpl enzymes. There is little known in the literature about Mpl, whereas reaction mechanisms, specificity of enzymes and tridimensional structures of apoenzymes or complexes with substrates and products are known for other ligases. That will enable us to design new inhibitors of Mur ligases and DdlB with potential antibacterial activity. In the context of the research work, we first introduced in our laboratories in vitro assay with malachite green for the determination of enzyme activity. The assay is based on the determination of free phosphate released in the enzyme-catalyzed reaction and allows us to evaluate the inhibitory activity of potential Mur and DdlB inhibitors. First attempt in finding inhibitors was screening of inhouse bank of compounds from Faculty of chemistry. Diazenedicarboxamide inhibitors of DdlB were discovered in that manner. Later virtual screenig was performed on available crystal structures of MurD (E.coli), MurF (S. pneumonie) and DdlB (E. coli) enzymes using computational tools for highthroughput virtual screening eHits 6.0 and AutoDock 4.0 and NCI diversity set (National Cancer Institute bank
of compounds). Only high ranked compounds with strong predicted binding afinity were evaluated in vitro. New inhibitors with novel scaffolds of DdlB and MurD with IC50 in low micromolar range were discovered. Some inhibitors had also promising antibacterial activities against Gram-positive and Gram-negative bacteria. Kinetic experiments revealed that most of the DdlB inhibitors are ATP competitive. Compounds with hydroximethylcarbonyl fragment were designed and synthesized using peptidomimetic approach. Common reaction mechanisms of MurC-F and DdlB ligases via tetrahedral transition-state provided the starting point for new inhibitors. Since murein peptide ligase (Mpl) succesfuly adds tripeptide L-Ala-γ-D-Glu-meso-A2pm onto peptidoglycan precursor UDP-N-acetylmuramic acid we synthesized series of tripeptides with core structure L-Ala-γ-D-Glu-Xaa. Amino acid on the third position was altered using Lnorleucine, L-norvaline, L-diaminobutiric acid, L-2-minoheptanoic acid, L-alanine, Lglutamic acid etc. Although the enzyme is not essential for biosynthesis of peptidoglycan, incorporation of false tripeptides with Mpl would affect final composition and cross-linking of peptidoglycan which could lead to bacterial cell death. Obtained results reveal good starting point for optimization and further development of inhibitors, especially inhibitors of DdlB.
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