Multivalent inhibition of the Aspergillus fumigatus KDNase

Aspergillus fumigatus is a saprophytic fungus and opportunistic pathogen often causing fatal infections in immunocompromised patients. Recently AfKDNAse, an exoglycosidase hydrolyzing 3-deoxy-D-galacto-D-glycero-nonulosonic acid (KDN), a rare sugar from the sialic acid family, was identified and characterized. The principal function of AfKDNAse is still unclear, but a study suggests a critical role in fungal cell wall morphology and virulence. Potent AfKDNAse inhibitors are required to better probe the enzyme's biological role and as potential antivirulence factors. In this work, we developed a set of AfKDNAse inhibitors based on enzymatically stable thio-KDN motifs. C2, C9-linked heterodi-KDN were designed to fit into unusually close KDN sugar binding pockets in the protein. A polymeric compound with an average of 54 KDN motifs was also designed by click chemistry. Inhibitory assays performed on recombinant AfKDNAse showed a moderate and strong enzymatic inhibition for the two classes of compounds, respectively. The poly-KDN showed more than a nine hundred fold improved inhibitory activity (IC₅₀ = 1.52 ± 0.37 μM, 17-fold in a KDN molar basis) compared to a monovalent KDN reference, and is to our knowledge, the best synthetic inhibitor described for a KDNase. Multivalency appears to be a relevant strategy for the design of potent KDNase inhibitors. Importantly, poly-KDN was shown to strongly decrease filamentation when co-cultured with A. fumigatus at micromolar concentrations, opening interesting perspectives in the development of antivirulence factors.