Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N′-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N′-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM.

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The Saccharomyces cerevisiae genome encodes five sirtuins (Sir2 and Hst1–4), which constitute a conserved family of NAD-dependent histone deacetylases. Cells lacking any individual sirtuin display mild growth and gene silencing defects. However, hst3Δ hst4Δ double mutants are exquisitely sensitive to genotoxins, and hst3Δ hst4Δ sir2Δ mutants are inviable. Our published data also indicate that pharmacological inhibition of sirtuins prevents growth of several fungal pathogens, although the biological basis is unclear. Here, we present genome-wide fitness assays conducted with nicotinamide (NAM), a pan-sirtuin inhibitor. Our data indicate that NAM treatment causes yeast to solicit specific DNA damage response pathways for survival, and that NAM-induced growth defects are mainly attributable to inhibition of Hst3 and Hst4 and consequent elevation of histone H3 lysine 56 acetylation (H3K56ac). Our results further reveal that in the presence of constitutive H3K56ac, the Slx4 scaffolding protein and PP4 phosphatase complex play essential roles in preventing hyperactivation of the DNA damage-response kinase Rad53 in response to spontaneous DNA damage caused by reactive oxygen species. Overall, our data support the concept that chromosome-wide histone deacetylation by sirtuins is critical to mitigate growth defects caused by endogenous genotoxins.

A number of APOBEC family DNA cytosine deaminases can induce mutations in tumor cells. APOBEC3H haplotype I is one of the deaminases that has been proposed to cause mutations in lung cancer. Here, we confirmed that APOBEC3H haplotype I can cause uracil-induced DNA damage in lung cancer cells that results in γH2AX foci. Interestingly, the database of cancer biomarkers in DNA repair genes (DNArCdb) identified a single-nucleotide polymorphism (rs139298) of APOBEC3H haplotype I that is involved in lung cancer. While we thought this may increase the activity of APOBEC3H haplotype I, instead we found through computational modeling and cell-based experiments that this single-nucleotide polymorphism causes the destabilization of APOBEC3H Haplotype I. Computational analysis suggests that the resulting K121E change affects the structure of APOBEC3H leading to active site disruption and destabilization of the RNA-mediated dimer interface. A K117E mutation in a K121E background stabilized the APOBEC3H haplotype I, thus enabling biochemical study. Subsequent analysis showed that K121E affected catalytic activity, single-stranded DNA binding and oligomerization on single-stranded DNA. The destabilization of a DNA mutator associated with lung cancer supports the model that too much APOBEC3-induced mutation could result in immune recognition or death of tumor cells.

Human APOBEC3H is a single-stranded (ss)DNA deoxycytidine deaminase that inhibits replication of retroelements and HIV-1 in CD4+ T cells. When aberrantly expressed in lung or breast tissue APOBEC3H can contribute to cancer mutagenesis. These different activities are carried out by different haplotypes of APOBEC3H. Here we studied APOBEC3H haplotype II, which is able to restrict HIV-1 replication and retroelements. We determined how the dimerization mechanism, which is mediated by a double-stranded RNA molecule, influenced interactions with and activity on ssDNA. The data demonstrate that the cellular RNA bound by APOBEC3H does not completely inhibit enzyme activity, in contrast to other APOBEC family members. Despite degradation of the cellular RNA, an approximately 12 nt RNA remains bound to the enzyme, even in the presence of ssDNA. The RNA-mediated dimer is disrupted by mutating W115 on loop 7 or R175 and R176 on helix 6, but this also disrupts protein stability. In contrast, mutation of Y112 and Y113 on loop 7 also destabilizes RNA-mediated dimerization but results in a stable enzyme. Mutants unable to bind cellular RNA are unable to bind RNA oligonucleotides, oligomerize, deaminate ssDNA in vitro, but ssDNA binding is retained. Comparison of A3H wild type and Y112A/Y113A by fluorescence polarization, single molecule optical tweezer, and atomic force microscopy experiments demonstrates that RNA mediated dimerization alters the interactions of A3H with ssDNA and other RNA molecules. Altogether, the biochemical analysis demonstrates that RNA binding is integral to APOBEC3H function.