Iminosugars as Antibacterial Agents? Corbohydrate Derived Analogues of the Anti-Tuberculosis Agent Ethambutol as Potential Inhibitors of Bacterial Cell Wall Metabolising Enzymes

Recently, information has become available that iminosugar related compounds might emerge as novel anti- bacterial agents. Due to inhibitory effects on glycosyl hydrolases and glycosyl transferases which are unique to the bacterial world, highly efficient anti-bacterial principles could possibly be at hand which are benign to the human/animal host who/which is lacking the enzymes addressed by selective inhibition. The cell wall of mycobacteria, the causative agents of such diseases as tuberculosis, leprosy, or severe skin lesions, consists of two major polysacchride containing sub-structures, which are an arabinogalactan (AG) and a lipoarabinomannan (LAM). Ethambutol [(S,S)-2,2`-(ethylenediimino)di-l-butanol], a drug long known as one of the few agents with bacteriostatic effects on mycobacteria, was recently discovered to prevent the correct biosynthesis of these polysaccharides. From the biological effects of ethambutol it can be concluded that this comparably simple compound can be regarded an enzyme inhibiting sugar mimetic. Based on this hypothesis, novel interesting analogues could well be, unlike ethambutol, sugar derivatives containing structural Motifs of D-arabinofuranose or D-galactose. Targeted structures will initially be new, asymmetrical analogues of ethambutol in order to establish a simple screening model as well as viable structure- activity relationships. Subsequently, several five-membered iminoalditols will be examined. Independently, a series of potential inhibitors of AG biosynthesis will be prepared. Another part of the proposed project involves the synthesis and biological evaluation of bulgecin related compounds as potential inhibitors of Soluble Lytic Transglycosylase (SLT), an enzyme vital for the survival and growing of bacteria. Synthetic approaches will be based on sound in-house methodology previously established in the course of projects P8415 and 10805 CHE. Biochemical evaluation of products will be conducted by international experts in their fields.

The main goal of the project was the attempt to discover novel antibacterial substances which highly selectively disturb metabolic processes which only occur in bacteria. This would minimise toxic side-effects of antibacterial chemotherapeutic compounds which could be a significant benefit for long-term treatment as necessary with certain bacterial infections. As the target, mycobacteria, the causative agents for tuberculosis (3-4 mill. people die per year), leprosy, as well as a range of animal and life stock diseases, were chosen. Emphasis was laid in particular on the search for new compounds with activity against Mycobacterium tuberculosis. The importance of novel antibacterial agents for the presence and the future has been increasingly recognised and emphasised, such as, very recently, in the starting 6th European Framework Programme. Based on previous extended research in the area of iminosugars, a family of natural products and non-natural derivatives thereof, in which a nitrogen atom replaces the ring oxygen, we have tried to prepare inhibitors of a particular enzyme which is responsible for a vital step in the cell wall synthesis of Mycobacterium tuberculosis. The aim of research conducted in parallel was to derivatise the important anti-tuberculosis drug ethambutol, which was found to be a strong inhibitor of the particular enzyme under consideration here. With the aid of international collaborations such as the department of chemistry of the University of British Columbia, Vancouver, as well as AgResearch, a science institution in New Zealand devoted to, amongst others, antibacterial research, biologcal activities of the new compounds prepared were investigated. Unfortunately, it turned out that ethambutol is not a viable lead structure for anti-tuberculosis drugs, as any even minute deviation from the parent structure resulted in reduced biological activity against mycobacteria. Despite the not convincing anti-tuberculosis activities of numerous tested compounds, some of these products turned out to be highly efficient inhibitors of carbohydrate metabolising enzymes. A particularily useful effect was observed when the parent structures were combined with fluorescent molecules.The resulting novel inhibitors not only bind extremely powerfully to the active site of the respective enzyme but also carry a "molecular torch" which can be employed for diagnostic purposes. With a view to analytical methods employed in biochemistry and medicine, such inhibitors are valuable diagnostic compounds. The limited availability of novel and highly specific diagnostic substances which are based on non-radioactive technologies and are high-throughput compatible, makes the new fluorescent products highly interesting candidates for new biochemical-analytical methodologies, in particular for applications in proteomics research.