(S)-(+)-Dimethindene maleate Based on the inhibitory potency

Based on the inhibitory potency of C646 for HDAC6, we monitored α-tubulin acetylation; an HDAC6 substrate. C646 treatment provided pronounced inhibition of α-tubulin acetylation after both 6 and 20h of incubation, which argues against HDAC6 inhibition by C646 in RAW264.7 cells. The observed effect of C646 on α-tubulin acetylation may originate from inhibition of the HAT p300, which could directly reduce α-tubulin acetylation. However, p300 has not been described to acetylate α-tubulin directly. Alternatively, the results can be explained by the following. p300 has been described to mediate acetylation of HDAC6, which renders this HDAC less active [38], [39]. Therefore, inhibition of p300 by C646 could decrease HDAC6 acetylation, thereby increasing HDAC6 activity, and thereby resulting in less α-tubulin acetylation.
Acknowledgements
Introduction Candida albicans is a commensal fungus in the skin and mucosal microbiome of humans. It is also one of the most important opportunistic human pathogens, which causes mucosal and systemic candidiasis in individuals immunocompromised due to aging, infection or cancer and HIV treatments (Odds, 1988, Pande et al., 2013, Yapar, 2014). A striking feature of C. albicans is that it can undergo reversible morphogenetic transitions. It grows either as an unicellular yeast or in filamentous pseudohyphal and hyphal cell forms (Whiteway and Bachewich, 2007). This unique ability to switch from yeast to hyphal cell contributes greatly to its virulence (Gow et al., 2012). Hyphal (S)-(+)-Dimethindene maleate in C. albicans form in response to many environmental cues that mimic the diverse microenvironments it encounters in its human host (Sudbery, 2011). The yeast-to-hyphal transition in C. albicans is tightly correlated with the transcription of hyphal specific genes which are controlled by a number of transcriptional factors, such as Cph1, Efg1, Flo8, Sfl1, Sfl2 and Brg1 (Cao et al., 2006, Liu et al., 1994, Lu et al., 2012, Nobile et al., 2012, Stoldt et al., 1997, Znaidi et al., 2013). In addition to transcription factors, dynamic changes in chromatin structure also modulate gene regulation during hyphal development. We previously reported that in C. albicans the histone acetyltransferase (HAT) complex NuA4, which primarily acetylates H4 and H2A, was responsible for nucleosomal H4 acetylation in the promoters of hypha-specific genes during hyphal induction in an Efg1-dependent manner (Lu et al., 2008). Further, the catalytic subunit of the NuA4 HAT complex, Esa1, is known to be essential for hyphal development (Wang et al., 2013). Gcn5 is the first histone acetyltransferase (HAT) identified in Saccharomyces cerevisiae and is found to be the catalytic subunit of the HAT complexes SAGA (Spt-Ada-Gcn5-Acetyltransferase), ADA (Ada2-Gcn5-Ada3) and SLIK/SALSA (SAGA-like), which are involved in both negative and positive transcriptional regulation (Brownell et al., 1996, Daniel and Grant, 2007, Grant et al., 1997, Pray-Grant et al., 2002, Rando and Winston, 2012, Sterner et al., 2002). Gcn5 mainly targets N-terminal lysine residues in histones H3 and H2B respectively (Suka et al., 2001, Zhang et al., 1998). Gcn5 acts on free histones when alone and acetylates nucleosomal histones in association with other proteins (Grant et al., 1997, Grant et al., 1999, RuizGarcia et al., 1997). Gcn5 can also function as a lysine acetyltransferase (KAT) required for acetylation of Rsc4, a subunit of the RSC chromatin-remodeling complex, and contributed to regulation of gene expression (VanDemark et al., 2007). Homologs of Gcn5 have been identified in many fungal species and higher eukaryotes (Spedale et al., 2012, Wang and Dent, 2014). UmGcn5, a Gcn5 homolog in Ustilago maydis, was reported to be involved in dimorphism and virulence. Deletion of UmGCN5 resulted in the growth of long mycelial cells and fuzz-like colony formation in U. maydis (Gonzalez-Prieto et al., 2014). GcnE, a Gcn5 homolog in Aspergillus nidulans, is required for normal conidiophore development (Canovas et al., 2014). Mammals have two Gcn5 homologs, the Gcn5-like protein GCN5 (KAT2a) and p300/CREB-binding protein-associated factor PCAF (KAT2b) (Jin et al., 2014b, Rando and Winston, 2012, Spedale et al., 2012). GCN5 and PCAF are subunits in at least two types of multi-protein complexes, which possess global histone acetylation activity and locus-specific co-activator functions together with acetyl transferases activity on non-histone substrates (Scott et al., 2014, Weake and Workman, 2012). Together, GCN5 and PCAF can bind to many sequence-specific factors involved in cell growth and/or differentiation (Moore and Anderson, 2014). Recruiting GCN5 by cytoplasmic Myc-nick induces alpha-tubulin acetylation and drives cytoplasmic reorganization and differentiation (Conacci-Sorrell et al., 2010). The acetyltransferase activity and cellular location of PCAF are regulated through the acetylation of PCAF itself (Blanco-Garcia et al., 2009, Santos-Rosa, 2003). These two acetyltransferases have distinct and redundant roles in transcriptional regulation, signaling, adipogenesis, tumorigenesis and embryogenesis (Chen et al., 2013, Jin et al., 2014a, Jin et al., 2011, Jin et al., 2014b, Love et al., 2012, Scott et al., 2014, Weake and Workman, 2012).