Interaction of CCT020312 with paclitaxel.

<p><b>A) Paclitaxel-associated EIF2A phosphorylation in U-2OS and HCT116 cells.</b> U-2OS and HCT116 were exposed to 10 µM CCT020312 (CCT), DMSO or paclitaxel (taxol) for 4 hours. Cell lysates were analyzed by immunoblotting for P-S51-EIF2A and tubulin. <b>B) Proliferation inhibition by paclitaxel in the presence of CCT020312.</b> Cells were treated with increasing amounts (1–25 nM) of paclitaxel in the absence and presence of a fixed dose (2.5 µM) of CCT020312. Arrows denote proliferation inhibition in cells treated with 2.5 µM CCT020312 only. Cell lines were as indicated. <b>C) Multiple drugs effect analysis:</b> U-2OS and HCT116 cells were treated with escalating doses of paclitaxel, CCT020312 or their combination. Combination indices (CIs) for each dose are shown. The calculated mean CI and standard error is indicated. “CI excl” assumes agents act by a competing, mutually exclusive mechanism of action, “CI no-exl. assumes agents act through distinct, exclusive mechanisms of action <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028568#pone.0028568-Chou2" target="_blank">[57]</a>.</p

Mechanism of action predictions using cDNA microarray-based cluster analysis.

<p><b>A) Cluster analysis.</b> Data from CCT020312 and 22 other molecularly targeted agents, 296 samples in total, established using either the CRUK Human Whole Genome-wide Array v1.0.0 or the CRUKDMF_22K_v1.0.0 array were used. Genes that significantly varied with treatment (ANOVA p<0.05 with Bonferoni-correction) were used for hierarchical clustering. Clustering was performed using the Euclidian distance as a similarity measure. <b>B) Cluster deconvolution.</b> Conditions that cluster with (Cluster I) or adjacent to (Cluster II) CCT020312 are shown.</p

Effects of CCT020312 exposure on wild type and PERK KO MEFs.

<p><b>A) EIF2A phosphorylation.</b> SV40-immortalised wild type (WT) and PERK KO MEFs (PERK−/−) were treated with 10 µM CCT020312 (CCT), 2 µM thapsigargin (Tg) for the time indicated. Cell lysates were analysed using P-S51 EIF2A and pan EIF2A selective antibody. The position of P-S51 EIF2A is indicated with an arrow beneath an unrelated, high molecular weight, non-specific band (*). B) <b>Signal quantification for data shown in A).</b> Charts depict the background corrected signal for P-S51 EIF2A relative to that of pan EIF2A in the same samples. Signals were quantified using Image J. <b>C) Cell cycle response.</b> MEFs were treated with 10 µM CCT020312 or vehicle (DMSO) for 30 hrs and analysed for cell cycle distribution. Where indicated, Nocodazole (Noc) was added to the culture medium at a concentration of 1 µg/ml for the final 16 hours. Raw propidium iodide profiles are shown. Cells were treated in parallel to those analysed for A. <b>D) Quantitative analysis of cell cycle distribution.</b> Experiments were as described for C). Charts depict the percentage of cells with DNA content as indicated, nocodazole was added where indicated. Results for two independently run experiments are shown.</p

Cellular responses to CCT020312 treatment.

<p><b>A) Concentration-dependence of P-S608-pRB loss in CCT020312-exposed cells.</b> HT29 cells seeded in 96-well plates were exposed to CCT020312 for 24 hours. Ser608 pRB phosphorylation was quantified using the cell-based immunoassay for the detection of pRB-P-Ser608 as employed for the primary screen (Barrie et al. 2003). Signals normalized to protein content (BCA assay) are shown. Error bars represent the standard error of the mean (n−3). The range for linear response is indicated. <b>B) C) Effects of CCT020312 on cell cycle progression and DNA synthesis.</b> HT29 cells were treated for 16 and 24 hours with 10 µM CCT020312. Cells were stained with propidium iodide and analysed by flow-cytometry (B). Cells were treated with CCT020312 for 16 or 24 hours. BrdU was added to the medium for the final two hours. Cells were stained with anti-BrdU antibody and analysed by flow-cytometry (C). <b>D) Accumulation of Ser608 unphosphorylated pRB in CCT020312 exposed cells.</b> HT29 cells were incubated in the presence of the vehicle (MOCK) or CCT020312 for 24 hours and analysed by immunoblotting. NP-pRB denotes use of the antibody for detection of the non-phosphorylated Ser608 pRB site. <b>E) Marker expression 24 h post CCT020312 exposure.</b> HT29 cells were exposed to 10 µM CCT020312 or vehicle (MOCK) for 24 hours. Lysates were analysed by immunoblot for marker proteins as indicated. Membrane staining with amido black documents loading. <b>F) CCT020312 induces a rapid loss of D cyclin expression.</b> HT29 cells were treated with 10 µM CCT020312 for the times indicated and lysates analyzed as in E. Tubulin probing documents loading.</p

EIF2A phosphorylation in CCT020312-treated cells.

<p><b>A) Detection of EIF2A phosphorylation following CCT020312 treatment.</b> HT29 human colon cancer and MCF-7 human breast cancer cells were treated with 10 µM CCT020312 (+) or vehicle () for the times indicated. Lysates were prepared and immunoblot-analysis performed using antibodies to detect EIF2A and Ser51-phosphorylated EIF2A (P-S51EIF2A) as indicated. <b>B) Structure-activity relationships.</b> Representatives from different chemical series were tested using HT29 cells for their respective ability to activate EIF2A phosphorylation. <b>C) Analogue structures and potency of analogues for reducing P-S608-pRB and growth.</b></p

CCT020312 does not induce full ER stress signalling.

<p><b>A) Schematic of unfolded protein response signalling.</b> Response biomarkers are indicated. <b>B) UPR response marker expression in HT29 colon and MCF7 breast cancer cells.</b> Cells were treated with CCT020312 (CCT, 10 µM) or thapsigargin (Tg, 2 µM) as indicated and analysed by immunoblotting for CHOP/GADD153 and GRP78/BIP. <b>C) UPR response marker expression following CCT020312 dose escalation.</b> HT29 cells were treated for 16 h. Lysates were analyzed as in B.</p

Chemical structure and properties of screen identified hits.

<p><b>A) Compound chemical structures.</b> Compounds are arranged in order of decreasing potency. <b>B) Summary of cellular effects and rationale for compound triage.</b> EC₅₀ values for inhibition of pRB phosphorylation and GI₅₀ values for inhibition of cell growth represent the calculated mean (n = 3). The cell-based immunoassay for the detection of pRB-P-Ser608 (Barrie et al. 2003) was run in HT29 colon carcinoma cells and used to quantify the EC50 for inhibition of pRB phosphorylation at 24 hours, with sulphorhodamine B staining to quantify the GI₅₀ values at 96 hours post compound addition. Protein remaining in wells at 24 hours was determined using bicinchoninic acid assays.</p

Signalling leading to CCT020312-dependent EIF2A phophorylation.

<p><b>A) Schematic showing EIF2A kinases and their regulation.</b> Pathway agonists thapsigargin, poly (I∶C) and NaAS2O3 and their locus of action are indicated. <b>B) Effect of EIF2AK3/PERK ablation on EIF2A phosphorylation by CCT020312.</b> U-2OS human osteosarcoma cells were transfected with either of two different EIF2AK3/PERK siRNA oligonucleotides (PERK-1, PERK-2) or an irrelevant control (NT) for 72 hours. Cells were treated with 10 µM CCT020312 (CCT) or 2 µM thapsigargin (Tg) for the indicated times. Lysates were analysed by immunoblot as indicated. <b>C) Quantitation of PERK mRNA expression in siPERK transfected cells.</b> PERK mRNA was quantified in siRNA tranfected cells using SYBR Green based quantitative PCR. The comparative cycle threshold method was used to determine the fold change in PERK mRNA relative to cells tranfected with irrelevant oligonucleotide. GAPDH was quantified in parallel and used to normalise between samples. Bars represent the mean fold change in triplicate technical replicates. <b>D) Ablation of EIF2AK3/PERK prevents CCT020312-mediated cyclin D loss and accumulation of underphosphorylated pRB.</b> HCT116 human colon cancer cells were transfected with EIF2AK3/PERK siRNA oligo ‘2’ or non-targeting siRNA (NT). After 72 hours cells were treated with 10 µM CCT020312, 2 µM thapsigargin (Tg) or DMSO for times indicated. Cell lysates were analysed by immunoblotting as indicated.</p

3‑(3,4-Dihydroisoquinolin-2(1<i>H</i>)‑ylsulfonyl)benzoic Acids: Highly Potent and Selective Inhibitors of the Type 5 17-β-Hydroxysteroid Dehydrogenase AKR1C3

A high-throughput screen identified 3-(3,4-dihydroisoquinolin-2­(1H)-ylsulfonyl)­benzoic acid as a novel, highly potent (low nM), and isoform-selective (1500-fold) inhibitor of aldo-keto reductase AKR1C3: a target of interest in both breast and prostate cancer. Crystal structure studies showed that the carboxylate group occupies the oxyanion hole in the enzyme, while the sulfonamide provides the correct twist to allow the dihydroisoquinoline to bind in an adjacent hydrophobic pocket. SAR studies around this lead showed that the positioning of the carboxylate was critical, although it could be substituted by acid isosteres and amides. Small substituents on the dihydroisoquinoline gave improvements in potency. A set of “reverse sulfonamides” showed a 12-fold preference for the <i>R</i> stereoisomer. The compounds showed good cellular potency, as measured by inhibition of AKR1C3 metabolism of a known dinitrobenzamide substrate, with a broad rank order between enzymic and cellular activity, but amide analogues were more effective than predicted by the cellular assay

Small Molecule Neuropilin‑1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFβ) Production in Regulatory T‑Cells

We report the design, synthesis, and biological evaluation of some potent small-molecule neuropilin-1 (NRP1) antagonists. NRP1 is implicated in the immune response to tumors, particularly in Treg cell fragility, required for PD1 checkpoint blockade. The design of these compounds was based on a previously identified compound EG00229. The design of these molecules was informed and supported by X-ray crystal structures. Compound <b>1</b> (EG01377) was identified as having properties suitable for further investigation. Compound <b>1</b> was then tested in several in vitro assays and was shown to have antiangiogenic, antimigratory, and antitumor effects. Remarkably, <b>1</b> was shown to be selective for NRP1 over the closely related protein NRP2. In purified Nrp1⁺, FoxP3⁺, and CD25⁺ populations of Tregs from mice, <b>1</b> was able to block a glioma-conditioned medium-induced increase in TGFβ production. This comprehensive characterization of a small-molecule NRP1 antagonist provides the basis for future in vivo studies