Due to increasing resistance to antibacterial agents the design and synthesis of new antibacterial agents is becoming a new focal point of the field. Among the most validated targets for new antibacterial compounds are enzymes which are a part of peptidoglycan biosynthetic pathway constituting an essential part of the bacterial cell wall and thus any anomaly in this macromolecule can lead to cell lysis. Some of the initial stages of the peptidoglycan biosynthesis are catalyzed by enzymes from the Mur ligase family (MurC, MurD, MurE and MurF), which were chosen as targets in our research. We evaluated a hypothesis stating that sulfonohydrazides could be designed to inhibit Mur ligases. Various structurally diverse new sulphonohydraside inhibitors of Mur ligases were designed, synthesized and biochemically evaluated using modern approaches in medicinal chemistry. Initially, we focused mainly on MurC enzyme for which only few inhibitors had been previously known. However, during our continuous research inhibitors of other Mur ligazes were also designed. One of the approaches was to use benzylidenerodanines as lead compounds which are well-documented MurC inhibitors. Leads were then further modified on the
thioxothiazolidin-4-one ring which was first replaced by structurally similar non-cyclic sulfonohydrazone moiety. All other parts of molecule were then modified in an attempt to increase the inhibitory activity on MurC. p-chlorobenzil substituent in the lead compound was thus replaced by various arylsulfonates, aryamides, and arylesters. We have also acquired the right to the Slovenian patent by the The Slovenian Intellectual Property Office. However, despite the manifested inhibitory activity in the micromolar region it has been afterwards discovered that this inhibitory activity was most probably the result of a nonspecific promiscuous binding activity. On a parallel line a more rational design based on the structure of the enzyme’s active site to new MurC inhibitors was attempted. Using published X-ray crystal structures of MurC complexed with the reaction product, computer based virtual screening and de novo design we prepared new sulfonohydrazide moiety based inhibitors, which, according to our model, were designed to bind into diphosphate binding pocket with sulfonohydrazide moiety. Basic structural requirements for good inhibitory activity against MurC using critical evaluation of of biochemical testing results were depicted. The initial hits with leucine as a
building block were having the inhibitory activity in the middle micromolar range and were used in further studies. One of the first inhibitors with IC50 of 240 μM was also found to posses weak antibacterial activity with MIC of 128 μg/mL against S. aureus. Further modifications were directed towards increased solubility of compounds as well as towards their increased inhibitory activity. Leucine was therefore replaced by threonine and in some compounds sulfonohydrazide was also replaced by hydrazide. With the aim of increasing the number of H-bonds between the compounds and the enzyme, differently substituted arenes were used. Rigidisation of compounds where proline was used instead of leucine was also one of the attempts in increasing the potency. However, despite promising docking results no compound was found active.
In a search for new compounds with the potential to inhibit MurB, MurC, MurD and MurE, we also designed a series of compounds which consisted of peptide mimetic, diphosphate mimetic and phenyl substitute for sugar residue, which orients both mimetics towards the proper conformation and, to some level restricts the molecule’s degrees of freedom. Sulfonylcarbamates, sulfonamidothreonine and sulfonamidothreoninohydrazine were used as pyrophosphate mimetics. The hydroxamic acid functionality, a well-known metal complex forming group, was also introduced into some of the mimetics, in order to form strong interactions with the Mg2+ in the enzymes’ active sites. This series of compounds was tested and one was found to be active against MurC in middle μM range. With the procedures described above we managed to synthesize several inhibitors with their inhibitory activities in the middle μM range. In the course of our scientific research we found sulfonohydrazide to be a suitable fragment in the design of novel inhibitors of Mur ligases. Using structure-based design the compunds were efficiently docked into the vicinity of diphosphate binding pocket. Furthermore, using eHITS we were able to predict inhibitory activity with over 50% accuracy and therefore this docking software proved to be a suitable tool for the design of new inhibitors. According to the results of the eHITS and of biochemical testing it was concluded that sulfonohydrazide fragment could be replaced with structurally similar hydrazide fragment, however, a risk of lower solubility of designed compounds was present. In addition, leucine was found to be the most suitable building block for the inhibitory activity. It has also been established that both phenyl fragments enable several modifications which can be used to tune up the inhibitory activity towards MurC as well as MurD. These important discoveries, which have been made in the disertation, contribute significantly to the understanding of the structure activity relationship in the design of novel inhibitors of Mur ligases, which participate in bacterial cell wall biosynthesis.
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