Invasive fungal infections represent a public health problem that worsens over the years with the increasing resistance to current antimycotic agents. Therefore, there is a compelling medical need of widening the antifungal drug repertoire, following different methods such as drug repositioning, identification and validation of new molecular targets and developing new inhibitors against these targets. In this work we developed a structure-based strategy for drug repositioning and new drug design, which can be applied to infectious fungi and other pathogens. Instead of applying the commonly accepted off-target criterion to discard fungal proteins with close homologues in humans, the core of our approach consists in identifying fungal proteins with active sites that are structurally similar, but preferably not identical to binding sites of proteins from the so-called “human pharmacolome”. Using structural information from thousands of human protein target-inhibitor complexes, we identified dozens of proteins in fungal species of the genera Histoplasma, Candida, Cryptococcus, Aspergillus and Fusarium, which might be exploited for drug repositioning and, more importantly, also for the design of new fungus-specific inhibitors. As a case study, we present the in vitro experiments performed with a set of selected inhibitors of the human mitogen-activated protein kinases 1/2 (MEK1/2), several of which showed a marked cytotoxic activity in different fungal species.

Background Invasive fungal infections account for a high burden of morbidity and mortality. This is aggravated because of the toxicity and resistance problems associated to current antifungal drugs, which in whole target only a handful of fungal molecules. In this scenario, new target identification and drug design, together with drug repurposing, represent promising strategies. Methods We aim to identify and test in vitro potential new therapeutic targets in fungi. Our strategy consists in identifying fungal proteins with active sites (meaning the set of residues lining the binding pocket) that are similar, but not identical!, to sites of proteins from the human pharmacolome. A high structural similarity with a human counterpart allows validation of the fungal target using cross-reactive inhibitors of the human protein. On the other hand, a few amino acid differences in the binding pocket produce local topological and chemical changes that create a "design space" for new specific inhibitors of the fungal target. Results Applying our own bioinformatics approach and taking advantage of the >200 available crystal structures of proteins of the human pharmacolome in complex with inhibitors, we have identified ca. 30 proteins in several fungal species of the genera Histoplasma, Candida, Criptococcus, Aspergillus and Fusarium, whose binding sites share at least 70% amino acid identity with their similar binding pockets in human pharmaceutical targets. So far we have assayed in vitro, in seven different fungal species, ca. 60 known inhibitors of around twenty of the orthologous human proteins. Some of the tested inhibitors have been previously assayed in different species in drug repurposing screenings, while others, to our knowledge, have not been yet tested. Over a dozen of these compounds, targeting eight different protein targets, showed IC50 values in the micromolar order in one or across several species. In general, yeasts were more significantly affected than molds. Conclusion Our results point to new potential fungal targets that can be exploited for the design of new antifungal agents. Ongoing work by our group aims to identify, by virtual screening, specific inhibitors for several of these potential targets. Disclosures All Authors: No reported disclosures