Structure-Activity Relationship for compound 8.

<p><b>(A)</b> Structure of compound <b>8</b>. The 2,9-diazaspiro[5.5]undecane spirocyclic core, the diphenylmethyl group and the 3,5-dimethylisoxazole moiety are highlighted in yellow, red and blue, respectively. <b>(B)</b> U87-MG cells treated with the indicated analogs were analyzed by immunoblot for GRP78 expression. GAPDH levels were used as housekeeping controls. Data are represented as GRP78/GAPDH ratio for each compound and normalized to the GRP78/GAPDH ratio for 20 μM thapsigargin. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s003" target="_blank">S3 Fig</a> for blot images. <b>(C)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated analog for 16 h. XBP-1 mRNA splicing was monitored by RT-PCR using XBP-1-specific primers, which amplify a spliced or unspliced fragment of 304 or 326 bp, respectively. <b>(D)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated compound for 24 h. CHOP and housekeeping GAPDH mRNA levels were monitored by qRT-PCR. Data is plotted relative to the DMSO treated sample set to 1. Error bars indicate the SD of three replicates. <b>(E)</b> U87-MG cells treated with the indicated compounds were tested for their ability to form colonies. Data are represented as the number of colonies, normalized to vehicle DMSO. Error bars indicate SE with n = 3. Note that <b>8e</b> is an inactive control. Analogs <b>8a</b> and <b>8d</b> were tested only at 20 μM. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s007" target="_blank">S7 Fig</a> for representative images.</p

Secondary assays identify <i>bona fide</i> ERSR inducers with cytotoxicity activity.

<p><b>(A)</b> U87-MG cells treated with the indicated compounds were analyzed by immunoblot for GRP78 expression. GAPDH levels were used as housekeeping controls. Data are represented as GRP78/GAPDH ratio for each compound and normalized to the GRP78/GAPDH ratio for 20 μM thapsigargin. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s003" target="_blank">S3 Fig</a> for blot images. <b>(B)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated compound for 16 h. XBP-1 mRNA splicing was monitored by RT-PCR using XBP-1-specific primers, which amplify a spliced or unspliced fragment of 304 or 326 bp, respectively. <b>(C)</b> U87-MG cells were treated with DMSO, 1 μM thapsigargin or 20 μM of indicated compound for 24 h. CHOP and housekeeping GAPDH mRNA levels were monitored by qRT-PCR. Data is plotted relative to the DMSO treated sample set to 1. Error bars indicate the SD of three replicates. <b>(D)</b> U87-MG cells treated with the indicated compounds were tested for their ability to form colonies. Data are presented as the number of colonies, normalized to vehicle DMSO. Error bars indicate SE with n = 3. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.s005" target="_blank">S5 Fig</a> for representative images.</p

Chemical structure and AC50 (μM) of hit compounds in qHTS assays.

<p>For Ca2+ mobilization, values obtained in + EGTA conditions are displayed. Active compounds are considered those with a high quality CRC and efficacy. Efficacy cutoffs are as follow: grp78-luciferase and UPRE-luciferase, >40%; Caspase 3/7 and Ca2+ mobilization, >30%; all cell viability assays, >50%.</p

Compound 8 and 6 synergize to reduce the viability of patient-derived glioma cell lines.

<p>Combination (6 x 6) response profiles for compound <b>8</b>, <b>6</b>, and <b>3</b> in JHH-136 (A) and JHH-520 (B) cells. Each response profile is displayed as heatmaps of cell viability (percent response normalized to thapsigargin control; left panels) and DBSumNeg analysis (right panels).</p

Compound 8 induces cell death in U87-MG spheroids.

<p>U87-MG spheroids were treated for 48 h with either compound <b>8</b> or inactive analog <b>8e</b> at the indicated concentrations. Spheroids were stained with Hoechst (blue) and propidium iodide (red) to mark nuclei and dead cells, respectively. For each spheroid, one single Z’ plane is shown.</p

Compound 8 analogs reduce the viability of cancer cell lines.

<p>CellTiter-Glo viability assay of glioma (U87-MG and LN-229), ovarian (IGROV-1 and HEY-A8) and colon (HT-29) cancer cell lines treated for 48 h with compound <b>8</b> analogs.</p

Chemical structure and AC50 (μM) of compound 8 analogs in qHTS assays.

<p>Efficacy cutoffs are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.g002" target="_blank">Fig 2</a>.</p

Schematic workflow for compound screening and triage.

<p>qHTS indicates assays performed in 1,536- and 384-well format. AC₅₀ obtained in qHTS assays are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.g002" target="_blank">Fig 2</a>. Results of remaining low-throughput assays are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161486#pone.0161486.g003" target="_blank">Fig 3</a>.</p

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH)

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as <b>63</b> exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug–target residence time was determined via SPR. Analysis of these data suggests that drug–target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target

Discovery and Optimization of Potent, Cell-Active Pyrazole-Based Inhibitors of Lactate Dehydrogenase (LDH)

We report the discovery and medicinal chemistry optimization of a novel series of pyrazole-based inhibitors of human lactate dehydrogenase (LDH). Utilization of a quantitative high-throughput screening paradigm facilitated hit identification, while structure-based design and multiparameter optimization enabled the development of compounds with potent enzymatic and cell-based inhibition of LDH enzymatic activity. Lead compounds such as <b>63</b> exhibit low nM inhibition of both LDHA and LDHB, submicromolar inhibition of lactate production, and inhibition of glycolysis in MiaPaCa2 pancreatic cancer and A673 sarcoma cells. Moreover, robust target engagement of LDHA by lead compounds was demonstrated using the cellular thermal shift assay (CETSA), and drug–target residence time was determined via SPR. Analysis of these data suggests that drug–target residence time (off-rate) may be an important attribute to consider for obtaining potent cell-based inhibition of this cancer metabolism target