## 3 Results and Discussion
### Gene Essential Assessment: A Tool for Drug Target Discovery? A set of _C. albicans_ essential genes [69], were used to evaluate the model's ability to predict essentiality. For each gene a simulation was performed, on the same environmental conditions, described in the reference [69] (YNB medium), eliminating the corresponding reactions for that gene. Only protein-coding genes present in the model were considered. The model was able to correctly predict 78% (84 out of 108) of the identified essential genes (File S5). It is important to highlight that, in this type of models, the regulatory network is not considered, so it is expected that some predictions are not close to reality. To evaluate whether the assessment of gene essentiality could be a promising tool in drug target discovery, each one of the identified essential enzymes in the RPMI medium was searched in the DrugBank database [70], as a possible drug target of known antimicrobial agents. RPMI medium simulates human serum, thus allowing to simulate the natural environment faced by _Candida albicans_ in systemic infections. Interestingly, 11 ERG genes, including the well-known azide drug target _ERG11_, were predicted by the model to be essential in RPMI medium. Although most ERG genes are not essential, the inhibition of the activity of this pathway has a fungistatic effect indeed. They encode the enzymes that guide the last steps of ergosterol biosynthesis. This pathway is the main target of azide drugs, one of the most common antifungal agents to treat _Candida_ infections [71]. These drugs act by blocking ergosterol biosynthesis inhibiting the Erg11 encoded by the _ERG11_ gene. When an azide drug binds to this enzyme, ergosterol synthesis is inhibited, leading to lower concentrations of this metabolite in the plasma membrane [72]. Given that ergosterol is part of the _C. albicans_' biomass, it is acceptable to consider that enzymes that participate in its synthesis pathway can be essential, making most ERG genes attractive alternatives as new drug targets [73]. Many additional proteins stand out as promising new drug targets, including some for which there are already predicted inhibitory drugs, based on the results for homologous proteins in other organisms. For example, Atovaquone is a drug used as a fixed-dose combination with Malarone for treating uncomplicated malaria cases or as chemoprophylaxis in travelers. This drug is an analogue of ubiquinone and targets enzyme 1.3.5.2 encoded by _URA9_ in _Plasmodium falciparum_. Atovaquone acts as a competitive inhibitor of ubiquinol inhibiting the mitochondrial electron transport chain at the bc1 complex, resulting in a loss of mitochondrial function [74]. It would be interesting to check if these drugs are also active against _C. albicans_ by targeting CaUra9. Another promising example of a predicted _C. albicans_ drug target is Fol1, which corresponds to enzyme 2.5.1.15. Fol1 is the target of the sulfa drugs (sulfonamides and sulfones), a very well-known class of drugs, used to treat infectious diseases [75]. The effect of sulfa drugs on _C. albicans_ has not been sufficiently investigated. However, it seems that sulfa-fluconazole combination results in increased antifungal activity against _C. albicans_, leading to the reversal of azide resistance in previously resistant strains [76]. Since a reaction can be catalyzed by a protein encoded by more than one gene, and genes may encode more than one protein, we decided to analyze the model's critical reactions. This analysis allowed to increase the number of confirmed drug targets predicted as essential enzymes. As an example, the FKS genes stand out. They are not considered essential as the enzyme beta-1,3-glucan synthase can be encoded by more than one FKS/GSC/GSL genes [77]. However, the model predicts the reaction in which this enzyme participates as essential. The beta-1,3-glucan synthase is the target of the echinocandin class of antifungal drugs.