Comparison of average side chain lengths, SCn, and branching frequencies, Br Freq, for several strains of <i>C. glabrata</i> isolated by 2 different methods.

<p>Comparison of average side chain lengths, SCn, and branching frequencies, Br Freq, for several strains of <i>C. glabrata</i> isolated by 2 different methods.</p

Molecular modeling suggests one possible conformation that (1→6)-β-linked glucan side chains may exhibit, that is, a hook-like, bent structure.

<p>Structure A (top) is a linear polymer containing ten (1→3)-β-linked repeat units in the polymer chain. The linear (1→3)-β-linked glucan backbone structure assumes an open helical conformation. Structure B (bottom) is the same linear structure except a side chain branches from the third repeat unit. The side chain contains five (1→6)-β-linked repeat units. The curvature and hook-like structure of the side chain is evident in this model where the structure has been rotated slightly to optimize visualization of the curved side chain. The structures are rendered using JMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027614#pone.0027614-anonymous1" target="_blank">[39]</a>.</p

2D NMR spectra of the glycosidic linkages and non-reducing termini of the (1→3,1→6)-β-D-glucan isolated from <i>C. glabrata ace2</i> strain.

<p>(a) The three different (1→6)-β-linked glycosidic bonds from the side chain are detailed in the NOESY 2D NMR spectrum for SC1, SC Internal, and SC NRT glucosyl groups associated with H1 SC1, SC H1 and SC NRT H1. A: H6Br,H6′Br/H1SC1; B: H6SCn,H6′SCn/H1SC(n+1); C: H6SC(n-1),H6′SC(n-1)/H1SCNRT. (b) Similarity of the structures of the glycosyl group associated with SC NRT H1 and NRT H1 is indicated in the TOCSY 2D NMR spectrum.</p

Assignment of ¹³C and ¹H chemical shifts for the branchpoint repeat unit of the (1→3,1→6)-β-D-glucan isolated from <i>C. glabrata ace2</i> strain.

<p>HSQC-TOCSY spectrum indicates assignments and correlations for protons and carbons in the branch point repeat unit based upon correlations initially identified for C3Br (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027614#pone-0027614-g002" target="_blank">Figure 2</a>).</p

Initial identification of ¹³C and ¹H chemical shifts for the branchpoint repeat unit of the (1→3,1→6)-β-D-glucan isolated from <i>C. glabrata ace2</i> strain.

<p>HSQC-TOCSY 2D NMR spectrum shows correlations between C3 and C3Br carbons and methylene protons in the same spin system as C3Br.</p

Proton (top) and ¹³C (bottom) NMR chemical shifts (in ppm) for structural features characterized in the glucan isolated from <i>C. glabrata ace2 (HLS122)</i> strain.

<p>Proton (top) and ¹³C (bottom) NMR chemical shifts (in ppm) for structural features characterized in the glucan isolated from <i>C. glabrata ace2 (HLS122)</i> strain.</p

Conceptual model of the role of (1→6)-β-linked glucosyl side chains in the fungal cell wall structure.

<p>Possible arrangements of (1→6)-β-D-glucan branches within the glucan matrix include (a) direct covalent cross-linking between two (1→3)-β-linked polymers, or (b) non-covalent interactions by “hooking” across (1→6)-β-linked side chains, or (c) across (1→3)-β-linked polymers, or (d) “catching” a covalent (1→6)-β-linked cross-linking branch. The triple helix arrangement (e) with associated (1→6)-β-linked side chains may serve as points for attachment to chitin, mannan, mannoprotein, GPI protein anchor or other possible molecules.</p

2D NMR spectra show linkage between branchpoint and the first side chain repeat unit of the (1→3,1→6)-β-D-glucan isolated from <i>C. glabrata ace2</i> strain.

<p>Overlay of the 2D HSQC-TOCSY (black) and HMBC (blue) NMR spectra expanded around the C4 spectral region shows the correlation (C6Br/H1SC1) across the glycosidic link between C6Br of the branchpoint repeat unit and the anomeric proton (H1SC1) of the first (1→6)-β-linked repeat unit in the side chain as well as correlations across other (1→6)-β-linked side chain glycosidic linkages.</p

A WEE1 Inhibitor Analog of AZD1775 Maintains Synergy with Cisplatin and Demonstrates Reduced Single-Agent Cytotoxicity in Medulloblastoma Cells

The current treatment for medulloblastoma includes surgical resection, radiation, and cytotoxic chemotherapy. Although this approach has improved survival rates, the high doses of chemotherapy required for clinical efficacy often result in lasting neurocognitive defects and other adverse events. Therefore, the development of chemosensitizing agents that allow dose reductions of cytotoxic agents, limiting their adverse effects but maintaining their clinical efficacy, would be an attractive approach to treat medulloblastoma. We previously identified WEE1 kinase as a new molecular target for medulloblastoma from an integrated genomic analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. In addition, we demonstrated that WEE1 prevents DNA damage-induced cell death by cisplatin and that the WEE1 inhibitor AZD1775 displays synergistic activity with cisplatin. AZD1775 was developed as a WEE1 inhibitor from an initial hit from a high-throughput screen. However, given the lack of structure–activity data for AZD1775, we developed a small series of analogs to determine the requirements for WEE1 inhibition and further examine the effects of WEE1 inhibition in medulloblastoma. Interestingly, the compounds that inhibited WEE1 in the same nanomolar range as AZD1775 had significantly reduced single-agent cytotoxicity compared with AZD1775 and displayed synergistic activity with cisplatin in medulloblastoma cells. The potent cytotoxicity of AZD1775, unrelated to WEE1 inhibition, may result in dose-limiting toxicities and exacerbate adverse effects; therefore, WEE1 inhibitors that demonstrate low cytotoxicity could be dosed at higher concentrations to chemosensitize the tumor and potentiate the effect of DNA-damaging agents such as cisplatin