Histone deacetylases: Targets for antifungal drug development

Abstract

The interaction of pathogens and its hosts causes a drastic change in the transcriptional landscape in both cells. Among the several mechanisms of gene regulation, transcriptional initiation is probably the main point. In such scenario, the access of transcriptional machinery to promoter is highly regulated by post-translational modification of histones, such as acetylation, phosphorylation and others. Inhibition of histone deacetylases is able to reduce fungal pathogens fitness during infection and, therefore, is currently being considered for the development of new antifungal therapy strategies.

Post-translational modification of histones represents an important mechanism of gene expression regulation in eukaryotic cells. The impact of such modifications, either singly or in combination, is to form a language that could be deciphered by a set of proteins that regulate downstream functions in chromatin. Among such modifications, acetylation of lysine lying at N- and C-terminal domains that protrude from the nucleosome core particle plays an important role in gene expression regulation. This occurs mainly at transcriptional level by altering DNA-histone and histone-histone interactions and by the function of other proteins that can alter chromatin dynamics and functions.¹ Histone acetylation is a dynamic process regulated by the activity of 2 groups of enzymes conserved from yeast to humans: histone acetyltransferases (HAT), generally associated with the positive regulation of transcription, and histone deacetylases (HDAC), whose function is linked to negative regulation of transcription.² HDACs constitute a family of enzymes that are able to remove the acetyl group from histones and other cellular proteins. These enzymes can be classified by sequence homology into the Rpd3/Hda1 (classical HDAC) and into the sirtuin family.³ A recent genomic analysis revealed that the number of genes that codes for HDACs in fungal genomes varies from 2 to 11². In Ascomycota, classical HDACs gene numbers range from 2 to 5, and the number of sirtuin family genes ranges from 2 to 9². A similar number of genes of both families are found in Basidiomycota (2 to 7 for classical HDACs and 3 to 8 for sirtuin HDACs, respectively) and other Phyla (Blastocladiomycota, Chytridiomycota, Microsporidia, and Zygomycota).²

The pathogenic yeast Cryptococcus neoformans, together with its sibling species Cryptococcus gattii, causes cryptococcosis, a life-threatening disease with over 1 million new cases and 600.000 deaths every year.⁴ This disease is normally characterized by an initial pneumonia that could evolve to meningitis, which is normally the death cause. Cryptococcosis is generally treated with antifungal drugs, as fluconazole, flucytosine and amphotericin B.⁵ However, resistance to fluconazole was already observed in this pathogenic yeast.⁶ In this way, new targets for the development of antifungals are needed. In this issue of Virulence, Brandão and coworkers described the effects of pharmacological inhibition of HDACs in the human fungal pathogen C. neoformans.⁷ Employing the HDAC inhibitors sodium butyrate (SB) and Trichostatin A (TSA), the authors found that pivotal virulence factors, such as growth at 37°C, melanin synthesis, phospholipase and capsule polysaccharide production are affected in a dose dependent fashion. In addition, they found alterations in morphogenetic traits (filamentation and mating) and in cell cycle (leading to arrest at G2/M). However, they could not found differences in the infectious potential of pre-treated fungal cells in a non-mammalian model of cryptococcosis. The results presented by Brandão and coworkers show that the effects of HDAC inhibition by SB were more pronounced, if not unique, than those obtained with inhibition by TSA. HDACs are currently considered as targets for the development of new antifungal drugs, since such enzymes have been described as regulators of key virulence aspects in important pathogenic fungal species. Gene inactivation experiments led to association of individual HDAC genes with morphological transitions, virulence and expression and regulation of important drug resistance associated proteins, as the chaperone Hsp90 protein and drug efflux pumps.^8-11 Interestingly, inactivation of C. albicans HDAC coding genes HDA1 and RPD3 led to reduced trailing growth and reduced capability to evolve azole resistance, possibly due to a effect of these HDACs on the expression regulation of efflux pumps.¹² In the pathogenic mold Aspergillus fumigatus, HDAC gene inactivation led to defects in germination and in secondary metabolite production.¹³ HDACs coding genes from plant fungal pathogens have also been characterized and associated with key events in virulence of Fusarium graminearum and Magnaporthe oryzae.² Recent evidences show that HDACs inhibition in pathogenic fungi constitutes a promising therapeutic strategy, resulting in altered expression of genes necessary for virulence or drug resistance. In line with this, treatment of C. albicans and other pathogenic Candida species with TSA lowered the expression of ERG genes (the products are targets of the azole drugs) and CDR/MDR1 genes (code for multidrug transporters).¹⁴ One of the pivotal work that supports this employment of HDACs inhibitors as antifungal drugs describes the synergistic effect of MGCD290, a HDAC inhibitor, with different azoles (fluconazole, posaconazole or voriconazole) in opportunistic fungal isolates from genera Candida, Cryptococcus, Aspergillus, Rhodotorula, Fusarium, Trichosporum, and others.¹⁵ Also, TSA has been proposed for the treatment of invasive aspergillosis.¹⁶ In A. fumigatus, the proposed mechanism of action of TSA refers to modulation of acetylation of Hsp90, which led to defects in growth and conidiation, as well as hypersensitivity to geldanamycin (an inhibitor of Hsp90).¹⁷ In addition, TSA appears to potentiate the activity of caspofungin in A. fumigatus,¹⁶ which would broaden the antifungal strategies for treatment of aspergillosis. More recently, the HDAC inhibitor MGCD290, in combination with echinocandins, was show to impair the growth of echinocandin-resistant Candida spp. isolates.¹⁸ In C. albicans, pharmacological inhibition by TSA also led to alterations in the development of azole resistance.¹² Pharmacological inhibition of HDACs was also employed to characterize alterations of virulence traits in fungal pathogens. Treatment of C. neoformans and C. albicans with SB led to reduced biofilm formation and increased azole sensitivity. In addition, reduced germ tube formation was observed in C. albicans cells treated with SB¹⁹ and reduced adhesion to pneumocytes were found in these fungal cells treated with different HDACs inhibitors.²⁰ Despite the classical use of HDACs inhibitors in the treatment of cancer,³ HDACs inhibition appear to benefit immunological control of yeasts during host-pathogen interaction. At least in vitro, C. neoformans and C. albicans were found to be more sensitive to a SB-treated macrophage cell line (J774.16), and this effect was due to a raise in the macrophage cells levels of reactive nitrogen species.¹⁹ Altogether, the results presented summarize the potential of HDACs inhibition as a new venue for antifungal drug development.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.