Selective Inhibition of MAPKK Wis1 in the Stress-activated MAPK Cascade of Schizosaccharomyces pombe by Novel Berberine Derivatives

Abstract

Berberine alkaloids supposedly have diverse biological effects including anti-tumor activity in mice, anti-Candida activity, cytotoxic activities against human cancer cell lines, and inhibition of dopamine biosynthesis (1-4); however, their cytotoxic targets and biological mechanisms have yet to be identified. Since berberine has been shown to inhibit the growth of Candida albicans, Candida glabrata, and Candida tropicalis, 515 synthetic and semisynthetic berberine derivatives were produced and screened for more effective anti-fungal activity in C. albicans (5). C. albicans is the most frequently isolated fungal pathogen in humans. Its ability to change from a yeast form to a filamentous hypal or pseudohypal form during growth is thought to contribute to its virulence (6). HWY 289 and HWY 336 were selected because of their highly effective anti-Candida properties in the screen. Since C. albicans is a diploid fungus with no known sexual cycle and target validation by genetic approaches is limited, direct targets of HWY 289 and HWY 336 were identified in the fission yeast Schizosaccharomyces pombe, which is amenable for genetic studies. Studies using various mutations of the budding yeast S. cerevisiae have shown that yeast provides a good system for identifying targets of known compounds such as rapamycin and FK506 (7,8). Recently, the usage of budding yeast Saccharomyces cerevisiae has been expanded to include screenings for valuable chemical compounds or peptides for anticancer drugs and protein kinase inhibitors (9 -12). In this paper, we report that the novel berberine derivatives having anti-Candida properties, HWY 289 and HWY 336, selectively inhibit the in vivo and in vitro activities of a specific kinase in the stress-activated mitogen-activated protein kinase (MAPK) 1 cascade in S. pombe. MAPK (sometimes called extracellular signal-regulated kinase (ERK)) cascades are highly conserved signaling cassettes found in all eukaryotes. Eukaryotic cells respond to diverse extracellular stimuli such as growth factors, hormones, high temperature, osmotic shock, and hydroxyl radicals by activating distinct MAP kinase cascades, which lead to diverse regulatory events including proliferation, differentiation, and cell death. Therefore, kinases in distinct MAPK cascades serve as effective cytotoxic targets for a number of anti-fungal, antitumor, and anti-inflammatory drugs. Each MAPK cascade is composed of a series of three or more protein kinases, each phosphorylating, thereby activating, the next kinase in the pathway. MAPKs are activated by phosphorylation at conserved threonine and tyrosine residues in the catalytic domain by MAPK kinases (MAPKKs, sometimes called MEKs). These are in turn activated by phosphorylation of specific conserved serine/threonine residues by MAPK kinase kinases (MAPKKKs, sometimes called MEKKs). Several MAPK cascades exist in a cell. Each of them functions in parallel by mediating

A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

“…Consistent with the inhibitory effect of HWY336 on mammalian MKK4 and MKK7, HWY336 also inhibited the stress-activated MAPKK Wis1 in Schizosaccharomyces pombe [20]. Wis1 is a homologue of mammalian MKK4 and MKK7 in S. pombe , suggesting a similarity in the three-dimensional structures of these proteins, even though they only share 41% and 38% sequence homology, respectively.…”

Weak C—H...X(X= O, N) hydrogen bonds in the crystal structure of dihydroberberine

“…It has also been found to be effective in site-specific sustained release of drugs to the brain by under-going in vivo transformation into berberine (Bodor et al , 1981; Brewster & Bodor, 1983; Bodor & Brewster, 1983). Further derivatization of dihydroberberine by the introduction of hydroxy and cyano groups onto the reduced ring leads to active antimalarials (Vennerstrom & Klayman, 1988), avian myeblastosis virus inhibitors (AMV–RT) (Kusumoto et al , 1991), antitumour agents (Kim et al , 1997), antifungals (Dostál et al , 1999), mitogen-activated protein kinase inhibitors (MAPKK) (Jang et al , 2002), promoters of glucose metabolism and insulin secretion (Xu et al , 2011), and CD36 antagonists (Li et al , 2010). Dihydroberberine has found use as an intermediate in the synthesis of substituted berberine analogues which were found to act as anticancer agents (Zhang et al , 2012), pancreatic lipase inhibitory agents (Mohammad et al , 2013), antifungal agents (Li et al , 2013), Toxoplasma gondii inhibitors (Krivogorsky et al , 2012), and mitochondria-targeted antioxidants (Lyamzaev et al , 2011), and which have shown activity as antibabesial (Subeki et al , 2005) and antileishmania agents (Marquis et al , 2003) and as HIV-1 RT inhibitors (Vennerstrom et al , 1990).…”

Enhanced activity of strobilurin and fludioxonil by using berberine and phenolic compounds to target fungal antioxidative stress response

“…2000). The inhibition of the cellular stress response system by berberine was previously shown in the fission yeast, Schizosaccharomyces pombe (Jang et al. 2002), where berberine treatment disrupted MAPK kinase (Wis1p) of the stress‐activated protein kinase (SAPK) pathway.…”