Conidial NADase - a novel potential target to treat fungal infections

During the period Dec 2023-Sep 2024, we have consolidated the results from the biochemical and structural studies. These efforts have enabled us to establish several important and unique features of fungal NADases. Importantly, these enzymes belong to the class of TNT-domain proteins (from Tuberculosis necrotizing toxin). We showed that this class of enzymes is only present in bacteria and fungi and may therefore represent an attractive drug target. Within the class of fungal NADases, we identified a subdivision, based on the three-dimensional structures, that indicates the evolutionary independent development of three rather different mechanisms to stabilize a homodimeric state, which is critical for enzyme activity. One strategy is the introduction of the calcium binding site (described earlier in this project). Another, unexpected mechanism, which is found in the Fusarium enzyme, is the formation of intermolecular disulfide bonds which connect the C- and N-termini of the monomers. Yet another mechanism (based on partial X-ray structure and alpha-fold predictions) is the accumulation of ionic interactions in the dimer interface. These conclusions were based on the careful analyses of the obtained three-dimensional structures. As another important result, we noted that the catalytic centers of the three fungal NADases that we have been investigating are highly similar. Consequently, it can be assumed that inhibitors are likely to be effective on many different fungal NADases. Therefore, we wish to intensify our efforts to identify compounds from chemical libraries that exhibit inhibitory properties against fungal NADases.

Aspergilloses are among the most common human fungal infections and cause more than one million deaths every year. The airborne spores (conidia) of Aspergillus fumigatus are ubiquitously present in the environment and normally cleared from the lungs by the immune system. However, A. fumigatus infections represent a serious threat in immune-compromised patients with a lethality of up to 90%. The spectrum of antifungal drugs is rather limited and development of resistance has become a major challenge for the treatment of these infections. This proposal is based on our recent (unpublished) discovery of an NAD-cleaving enzyme (AfNADase) on the surface of conidia from Aspergillus fumigatus. Importantly, earlier virulence studies have indicated a prominent role of the encoding gene in the infection process; however, the function of the gene product has remained unknown. We hypothesize that the NADase activity may be critical for the adherence to the host cells or to evade the immune system. Our preliminary observations indicate that AfNADase represents a new class of enzymes with homologous representatives only in spore-forming fungi making it a promising drug target. In collaboration with internationally leading research groups, we will establish the molecular and catalytic properties of this new enzyme class including the X-ray structure. Moreover, we will use molecular genetics and a range of functional assays, including mouse models of A. fumigatus infection, to establish the biological function of AfNADase as well as its role in the infection process. Finally, we will produce a range of NAD analogs and test their inhibitory effect on AfNADase. Using effective analogs in biological assays, we intend to verify the suitability of AfNADase as a target for the treatment of aspergilloses and, potentially, other fungal infections.