Synthesis of the Antiproliferative Agent Hippuristanol and Its Analogues from Hydrocortisone via Hg(II)-Catalyzed Spiroketalization: Structure–Activity Relationship

An efficient synthesis of hippuristanol (<b>1</b>), a marine-derived highly potent antiproliferative steroidal natural product, and nine closely related analogues has been accomplished from the commercially available hydrocortisone utilizing Hg­(II)-catalyzed spiroketalization of 3-alkyne-1,7-diol motif as a key strategy. This practical synthetic sequence furnished <b>1</b> in 11% overall yield from hydrocortisone in 15 linear steps. Modifications to the parent molecule <b>1</b> encompassed changing the functional groups on rings A and E. Each analogue was screened for their effects on inhibition of cap-dependent translation, and the assay results were used to establish structure–activity relationships. These results suggest that the stereochemistry and all substituents of spiroketal portion (rings E and F) and C3-α and C11-β hydroxyl functional groups on rings A and C, respectively, are critical for the inhibitory activity of natural product <b>1</b>

Additional file 1: Figure S1. of Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

Toxicity of NU7441, KU-0060648 and Scr7 on the cell lines used in this study. A The 293/TLR line was exposed to increasing concentrations of NU7441, KU-0060648, and Scr7 for 5 days at which point the viability of the cells was determined. Viability is plotted relative to vehicle controls. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *P ≤0.05; **P ≤0.01; ns, not significant. B Viability of Arf −/− MEFs exposed to NU7441 or KU-0060648. Experiments were performed as for 293/TLR cells in (A). (PDF 371 kb

Synthesis of Rocaglamide Hydroxamates and Related Compounds as Eukaryotic Translation Inhibitors: Synthetic and Biological Studies

The rocaglates/rocaglamides are a class of natural products known to display potent anticancer activity. One such derivative, silvestrol, has shown activity comparable to taxol in certain settings. Here, we report the synthesis of various rocaglamide analogues and identification of a hydroxamate derivative (−)-<b>9</b> having activity similar to silvestrol in vitro and ex vivo for inhibition of protein synthesis. We also show that (−)-<b>9</b> synergizes with doxorubicin in vivo to reduce Eμ-Myc driven lymphomas

MyoD binds to the endogenous eIF4AII promoter following induction of C2C12 differentiation.

<p>(<b>A</b>) Schematic representation of the eIF4A and eIF4AII promoters showing the relative position and nucleotide sequence of putative MyoD binding sites. Positions are relative to the transcription start site (+1). Arrows denote the relative position of the primers used in the ChIP assay. (<b>B</b>) ChIP assays performed with C2C12 extracts prepared on the indicated days following induction of differentiation. Equivalent amounts of crosslinked chromatin were immunoprecipitated using either an anti-MyoD antibody or an IgG control. The presence of eIF4AI and eIF4AII promoter sequences in the immunoprecipitations was evaluated by qPCR. Primers to the myogenin promoter were used as control and values are normalized to input levels. The input sample represents 5% of the initial DNA material following sonication before immunoprecipitation. n = 4±SEM. (<b>C</b>) Products of qPCRs from ChIP assays performed in (B) (In-Input, Ig- IgG elution, MD- MyoD elution).</p

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

<div><p>The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the <i>in vivo</i> target specificity of Cas9 is poorly understood and most studies rely on <i>in silico</i> predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the <i>Trp53</i> locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to ^5′NGG^3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. <i>In vitro</i> analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.</p></div

Expression of eIF4AI and eIF4AII during C2C12 differentiation.

<p>(<b>A</b>) Phase contrast images of C2C12 cells grown in the presence of DM for the indicated number of days (d). Scale bars represent 50 µm. (<b>B</b>) Western Blot analysis documenting expression levels of the indicated proteins during C2C12 cell differentiation. Long (l.e.) and short (s.e.) exposures of the eIF4AII Western blot are presented. (<b>C</b>) Quantification of changes in eIF4AI and eIF4AII protein levels relative to those obtained on day 0. n = 3±SEM. (<b>D</b>) ³⁵S-methionine/cysteine incorporation into TCA-insoluble protein. C2C12 cells were induced to differentiate and protein extracts were prepared at the indicated time points. Cells were labeled for 30 min and the amount of radiolabeled protein quantitated by TCA precipitation. Values are standardized against total protein content. n = 3±SEM. (<b>E</b>) eIF4AI/II are efficiently incorporated into the eIF4F complex during C2C12 differentiation. m⁷GTP affinity purification of the eIF4F complex from C2C12 cells at the indicated days following induction of differentiation. Western blots to the indicated proteins were performed on an aliquot of input extract (lanes 1–4), GDP eluents (lanes 5–8), and m⁷GTP eluents (lanes 9–12).</p

The proximal eIF4AII promoter is activated by MyoD.

<p>(<b>A</b>) Schematic representation of Renilla Luciferase reporters linked to eIF4AI and eIF4AII proximal promoter sequences. The relative position of putative MyoD binding sites is indicated. (<b>B</b>) Transactivation assays involving eIF4AI and eIF4AII reporter constructs and MyoD expression vectors. NIH-3T3 cells were transfected as indicated in the Materials and Methods and extracts prepared on the indicated days following differentiation. Relative light units (RLU) were standardized to protein levels. The ratio of MyoD dependent expression relative to empty expression vector is plotted. n = 4±SEM.</p

Transcriptional changes in eIF4AII mRNA levels during C2C12 differentiation.

<p>(<b>A</b>) Changes in eIF4AI and eIF4AII mRNA levels during C2C12 cell differentiation. mRNA levels were determined by RT-qPCR and are standardized to GAPDH levels. n = 3±SEM. (B) Transcriptional changes in eIF4AI and eIF4AII mRNA levels during primary myoblast differentiation. mRNA levels were determined by RT-qPCR and are standardized to GAPDH levels. n = 4±SEM. (<b>C</b>) Nuclear Run-On analysis of GAPDH, MyoD and eIF4AII transcription in C2C12 cells at days 0 and 3 after induction of differentiation. Probes targeting the 5′ and 3′ UTRs of eIF4AII were used to distinguish the transcript from that of eIF4AI. (<b>D</b>) Quantiation of nuclear run-on experiments. Changes in eIF4AII transcription was quantified using a Typhoon Scanner (GE Healthcare) (values are normalized to GAPDH mRNA levels which did not change over this time period).</p

Base Complementarity of the PAM distal target region and the 5′ crRNA end affects engagement of Cas9 nuclease activity.

<p><b>A.</b> Sequence comparison of oligonucleotides harboring the wt p53 [Exon 7] target motif (underlined) and mutants harboring mismatches at nucleotides 16–20 of the crRNA guide target. Flanking 5′ and 3′ regions indicated by dots were maintained constant and are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109213#pone-0109213-g002" target="_blank">Figure 2A</a>. <b>B</b>. <u>Left panel:</u> Assessment of Cas9 binding to oligonucleotides shown in Panel A by EMSA. <u>Right panel:</u> Cleavage reactions of oligonucleotides shown in Panel A. The “-RNA” lanes indicate the absence of crRNA and tracrRNA. Quantifications were performed on a Typhoon Trio Variable Mode Imager with a Fuji imaging screen. n = 3±SD.</p

Number and Percentage of Total, Aligned, Duplicate, and Processed Reads Obtained from Chromatin Immunoprecipitates or Whole Cell Extracts (WCE) of Arf^−/− MEFs infected with pQdmCiG, pQdmCiG/sgp53-1, or pQdmCiG/sgp53-3.

<p>*After mapping and removal of duplicates.</p><p>Number and Percentage of Total, Aligned, Duplicate, and Processed Reads Obtained from Chromatin Immunoprecipitates or Whole Cell Extracts (WCE) of Arf^−/− MEFs infected with pQdmCiG, pQdmCiG/sgp53-1, or pQdmCiG/sgp53-3.</p