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New Inhibitors of Bacterial and Fungal Cell Wall Biosynthesis

Final Activity Report Summary - NEW ANTIBIOTICS (New inhibitors of bacterial and fungal cell wall biosynthesis)

The fungal cell wall consists of large sections of oligosaccharide materials, the most abundant being beta-glucan, a polymer made up of beta-(1-3)glucose with a variable degree of beta-(1-6)glucose branching. Chitin, a polymer of beta(1-4)N-acetylglucosamine (GlcNAc), forms another main part of the fungal cell wall. The present study focused on the attempted inhibition of both of these non-mammalian oligosaccharides' biosynthesis. Chitin is assembled stepwise by the enzyme chitin synthase, which transfers single GlcNAc residues to a growing oligomeric chain, the donor substrate for the enzyme being uridine diphosphate-GlcNAc (UDP-GlcNAc). The elongation step in glucan biosynthesis is catalysed by the glucan synthase, the donor substrate for the enzyme being uridine diphosphate glucose UDPG.

This research program proposes a new strategy to inhibit this biosynthetic pathway, namely a chain termination approach. This strategy involves the use of a monomeric carbohydrate building block that has been modified at the hydroxyl group at which further carbohydrate units would be added after this unit is incorporated into the growing oligosaccharide chain. Potential chain terminators of chitin and glucan biosyntheses are therefore GlcNAc residues in which the 4-hydroxyl has been modified and glucose derivatives in which the 3-hydroxyl has been replaced (in both cases, groups such as: H, OMe, F, NHAc and N3 have been used to replace OH group). If such materials are processed by enzymes, then their transfer to the terminus of the growing chitin and glucan chains would result in a chain termination step since the required 4-hydroxyl of GlcNAc and 3-hydroxyl of glucose, at which subsequent units would be added, will now be lacking. The most obvious approach was that of synthesising and testing the modified UDP donors themselves, since these are the actual substrates processed by the enzymes. In parallel, selected intermediates from the reaction sequence were tested in view of their potential as pro-drugs (they may be converted to the active UDP donor once inside the cell).

From previous experience on the use of oxazolines as transition-state mimics that are readily processed by hexosaminidases, it was reasoned that such oxazolines may themselves serve as activated substrates for chitin synthase. Therefore, after the synthesis and the antifungal testing of the 4-modified UDP-GlcNAc derivatives, another target was the synthesis and testing of a selection of GlcNAc derived oxazolines in which the key 4-hydroxyl was replaced with groups such as: H, OMe, F, NHAc and N3.

The antifungal action of the putative chain termination compounds was assessed using two assays on microconidia and germlings of the dermatophyte Trichophyton rubrum. The assays measured the effects of the modified compounds on adhesion and germination of fungal spores/germlings versus the control compounds. None of the UDP derivatives or the precursors tested displayed any significant anti-fungal activity in cell adhesion or germination assays, while putative chain-terminating benzoylated oxazolines and their precursors (anomeric acetates) all significantly inhibited T. rubrum germination at concentrations of 1 mM, whereas the control had no effect. One rationalisation of these results is the better compound polarity profile of the benzoate esters, which are probably more able to penetrate into cells and, therefore, probably act as prodrugs. Direct proof of the precise mode of action will require substantial further investigation. However, whatever the precise molecular mode of action, this work has demonstrated that novel antifungal carbohydrates can be rationally designed by using a chain-termination approach.