Comparative genomics allowed the identification of drug targets against human fungal pathogens

Abstract

Background: The prevalence of invasive fungal infections (IFIs) has increased steadily worldwide in the last few decades. Particularly, there has been a global rise in the number of infections among immunosuppressed people. These patients present severe clinical forms of the infections, which are commonly fatal, and they are more susceptible to opportunistic fungal infections than non-immunocompromised people. IFIs have historically been associated with high morbidity and mortality, partly because of the limitations of available antifungal therapies, including side effects, toxicities, drug interactions and antifungal resistance. Thus, the search for alternative therapies and/or the development of more specific drugs is a challenge that needs to be met. Genomics has created new ways of examining genes, which open new strategies for drug development and control of human diseases.

Results: In silico analyses and manual mining selected initially 57 potential drug targets, based on 55 genes experimentally confirmed as essential for Candida albicans or Aspergillus fumigatus and other 2 genes (kre2 and erg6) relevant for fungal survival within the host. Orthologs for those 57 potential targets were also identified in eight human fungal pathogens (C. albicans, A. fumigatus, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Paracoccidioides lutzii, Coccidioides immitis, Cryptococcus neoformans and Histoplasma capsulatum). Of those, 10 genes were present in all pathogenic fungi analyzed and absent in the human genome. We focused on four candidates: trr1 that encodes for thioredoxin reductase, rim8 that encodes for a protein involved in the proteolytic activation of a transcriptional factor in response to alkaline pH, kre2 that encodes for α-1,2-mannosyltransferase and erg6 that encodes for Δ(24)-sterol C-methyltransferase.

Conclusions: Our data show that the comparative genomics analysis of eight fungal pathogens enabled the identification of four new potential drug targets. The preferred profile for fungal targets includes proteins conserved among fungi, but absent in the human genome. These characteristics potentially minimize toxic side effects exerted by pharmacological inhibition of the cellular targets. From this first step of post-genomic analysis, we obtained information relevant to future new drug development.

Figure 1

Phylogenetic analysis between human pathogenic fungi performed by Bayesian analysis. Phylogenetic trees generated from Bayesian analysis of the genes trr1 (a), rim8 (b), kre2 (c) and erg6 (d). The length of the vertical lines linking one protein is proportional to the estimated distance between their sequences. The posterior probability values were added to the phylogenetic branches. Af: Aspergillus fumigatus, Bd: Blastomyces dermatitidis, Ca: Candida albicans, Ci: Coccidioides immitis, Cn: Cryptococcus neoformans, Hc: Histoplasma capsulatum, Pl: Paracoccidioides lutzii, Pb3: P. brasiliensis isolate 3, Pb18: P. brasiliensis isolate 18.

Figure 2

The predicted tridimensional structure of TRR1 and KRE2 proteins obtained by homology modeling. The structures of TRR1 (a) and KRE2 (b) proteins. The α-helix is represented by the color red, the β-sheet is represented by yellow and loops are represented by green. (a - left panel) The KRE2 active site presents the conserved residues His292, His357, Asp330 and Glu298. (Mutation of these residues abolished the protein activity in C. albicans.) Additional residues found in the KRE2 active site include Glu216, which interacts with the metal ion Mn²⁺ and creates the reactive nucleophylic center for the glycosyltransferase reaction, and Tyr189, which coordinates the donor and acceptor binding that allows the transfer of the mannose to the growing oligosaccharide. (b - right panel) The TRR1 protein is composed of two domains that comprise the binding sites of NADPH and FAD. The NADPH binding domain contains the active Cys145 and Cys148 residues. Other important residues of the TRR1 active site are Ala151, Val152, Pro153 and Ile154 that form a hydrophobic region in the NADPH binding domain.