Breaking the Walls: Development of Inhibitors of D,D- and L,D-Transpeptidases

Antimicrobial resistance remains one of the top ten global public health problems associated with an alarming increase in the number of bacterial infections that can no longer be treated easily with available chemotherapies. According to WHO, if no action is taken, deaths caused by resistant bacteria could reach 10 million annually by 2050. In order to overcome this problem, there is an indispensable need for identification and development of new antibacterial agents that bind to novel as well as already validated antibacterial drug targets. Since many large pharmaceutical companies have halted their efforts to discover new antibacterials, it is essential that smaller companies and particularly the academic community take on this task instead. Infections caused by resistant G- bacteria are particularly hard to treat because the impermeability of their outer membrane is increasingly restricting the access of many standard-of-care antibiotics. There is thus an urgent need to tackle the issue of impermeability outer membrane in G- pathogens with innovative therapeutic approaches. To this end, we want to combat resistant G- bacteria by destabilising their outer membrane; if successful, the envisaged compounds will act as potentiators of standard antibiotics, allowing them to penetrate outer membrane more effectively, ultimately leading to the death of bacterial cells. To assure a better outcome of the project, two approaches will be used in parallel to target bacterial cell wall biosynthesis. In the first approach, we will design and synthesize novel inhibitors of D,D-transpeptidases (also known as penicillin-binding proteins). D,D-transpeptidases are membrane-associated enzymes involved in the final steps of peptidoglycan biosynthesis as they catalyse the cross-linking of peptide chains. They are well validated targets for the discovery of novel antibacterial agents as they are inhibited by the most important antibiotic class, i.e. b-lactams. Here, we will use our extensive previous experiences in design and synthesis of monocyclic b-lactams to develop new super side-chain monobactams. These innovative monobactams will be prepared by optimized synthesis and will be decorated using sidechains of meropenem, ceftobiprole (and other similar structures) assuring high inhibitory potency against D,D-transpeptidases, including those from resistant bacteria, as well as potent antimicrobial activity. L,D-transpeptidases (LdtA, LdtB, and LdtC) from Escherichia coli catalyse other transpeptidation reactions. They attach the outer membrane-anchored lipoprotein (Lpp, Braun’s lipoprotein) to peptidoglycan, providing the only covalent connection between the outer membrane and the cell wall. In the second approach, we will develop selective inhibitors of LdtsA-C. As these enzymes utilise catalytic cysteine to catalyse transpeptidation, we will use our experiences in the development of cysteine-targeting inhibitors to develop the first inhibitors of Ldts from E. coli. To achieve this goal, a fragment-based drug discovery approach will be used. First, libraries of fragments will be screened on target enzymes, and then the binding modes of fragments will be determined using X-ray crystallography or NMR. In the next step, structure-based design will be used to generate the ideas allowing for improvement and growth of the fragments, and these improved compounds will be synthesized and evaluated pharmacologically. While novel inhibitors of D,D-transpeptidases will represent a source of important lead compounds with antibacterial activity, inhibitors of L,D-transpeptidases will sensitise G- bacteria to standard antibiotics by making their outer membrane more permeable. Such compounds will act as “potentiators” that will facilitate the entrance of newly developed (and existing) antibiotics to kill bacterial cells. This will be the first attempt to develop and evaluate inhibitors of LdtsA-C from E.coli as potentiators.