In vitro transcriptional points of departure from human whole transcriptome and surrogate transcriptome (S1500+) targeted RNA-Seq assays are highly comparable

Poster presented to SOT Conference 2024: A New Approach Method (NAM) to Screen for the Impact of Endogenous Stress on Chemical Toxicity Search for CCTE records in EPA’s Science Inventory by typing in the title at this link.https://cfpub.epa.gov/si/si_public_search_results.cfm?advSearch=true&showCriteria=2&keyword=CCTE&TIMSType=&TIMSSubTypeID=&epaNumber=&ombCat=Any&dateBeginPublishedPresented=07/01/2017&dateEndPublishedPresented=&dateBeginUpdated=&dateEndUpdated=&DEID=&personName=&personID=&role=Any&journalName=&journalID=&publisherName=&publisherID=&sortBy=pubDate&count=25</p

New Pyrazolopyrimidine Inhibitors of Protein Kinase D as Potent Anticancer Agents for Prostate Cancer Cells

<div><p>The emergence of protein kinase D (PKD) as a potential therapeutic target for several diseases including cancer has triggered the search for potent, selective, and cell-permeable small molecule inhibitors. In this study, we describe the identification, <i>in vitro</i> characterization, structure-activity analysis, and biological evaluation of a novel PKD inhibitory scaffold exemplified by 1-naphthyl PP1 (1-NA-PP1). 1-NA-PP1 and IKK-16 were identified as pan-PKD inhibitors in a small-scale targeted kinase inhibitor library assay. Both screening hits inhibited PKD isoforms at about 100 nM and were ATP-competitive inhibitors. Analysis of several related kinases indicated that 1-NA-PP1 was highly selective for PKD as compared to IKK-16. SAR analysis showed that 1-NA-PP1 was considerably more potent and showed distinct substituent effects at the pyrazolopyrimidine core. 1-NA-PP1 was cell-active, and potently blocked prostate cancer cell proliferation by inducing G2/M arrest. It also potently blocked the migration and invasion of prostate cancer cells, demonstrating promising anticancer activities on multiple fronts. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and the inhibition of tumor cell invasion caused by 1-NA-PP1, indicating that its anti-proliferative and anti-invasive activities were mediated through the inhibition of PKD. Interestingly, a 12-fold increase in sensitivity to 1-NA-PP1 could be achieved by engineering a gatekeeper mutation in the active site of PKD1, suggesting that 1-NA-PP1 could be paired with the analog-sensitive PKD1^M659G for dissecting PKD-specific functions and signaling pathways in various biological systems.</p></div

Structures of 1-NA-PP1 analogs.

<p>Structures of 1-NA-PP1 analogs.</p

IKK-16 and 1-NA-PP1 were ATP-competitive inhibitors of PKD.

<p>PKD1 kinase activity was measured as a function of increasing concentrations of ATP in the presence of varying concentrations of 1-NA-PP1 (<b>A</b>) and IKK-16 (<b>B</b>). Lineweaver-Burke plots of the data are shown. Data presented were representative of three independent experiments.</p

1-NA-PP1 blocked prostate cancer cell migration and invasion.

<p><b>A.</b>1-NA- PP1 blocked prostate cancer cell migration. PC3 cells were grown to confluence in 6-well plates. Monolayer was wounded and imaged immediately (0 h). Cells were then treated in growth media containing a vehicle (DMSO) or 30 µM of 1-NA-PP1 for 22 h and wound closure was measured. Percentage wound healing was calculated as the percent of healed wound area as compared to the original wound. <b>B.</b> 1-NA-PP1 inhibited prostate cancer cell invasion. DU145 cells were incubated with 30 µM 1-NA-PP1 in Matrigel inserts. After 20 h, noninvasive cells were removed and invasive cells were fixed in 100% methanol, stained in 0.4% hematoxylin solution, and photographed. The number of cells that invaded the Matrigel matrix was determined by cell counts in 6 fields relative to the number of cells that migrated through the control insert. Percentage invasion was calculated as the percent of the cells invaded through Matrigel inserts vs. the total cells migrated through the control inserts. <b>C.</b> Overexpressed PKD1 and PKD3 reversed the inhibitory effects of 1-NA-PP1 on tumor cell invasion. DU145 cells were infected with null, PKD1, and PKD3 adenoviruses (Adv-null, Adv-PKD1, and Adv-PKD3) at 100 MOI. After 24 h, cells were replated in control and Matrigel inserts, and a Matrigel invasion assay was conducted as described above. The overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis (images below the graphs). All the above experiments were repeated at least three times and data from a representative experiment are shown.</p

Inhibition of PMA-induced activation of endogenous PKD1 by 1-NA-PP1 in cells.

<p><b>A</b>. LNCaP cells were pretreated with different doses of inhibitors for 45 min, followed by PMA stimulation at 10 nM for 20 min. Cell lysates were subjected to immunoblotting for p-S⁹¹⁶-PKD1 and p-S^744/748-PKD1. Tubulin was blotted as loading control. The experiment was repeated three times and the representative blots are shown. <b>B</b>. Determination of the IC₅₀. Western blots were quantified using densitometry analysis. The data were plotted and IC₅₀ values were derived the concentration-response curves using GraphPad. One of the three concentration-response curves was shown.</p

Mutating the gatekeeper amino acid sensitized PKD1 to the inhibition of 1-NA-PP1.

<p><b>A.</b>Alignment of the primary sequences containing the gatekeeper amino acid in PKD. Arrow indicates the consensus gatekeeper amino acid “Methionine” (M) in a shaded rectangle. <b>B.</b> Expression of wild-type and mutant PKDs. HEK293 cells were transfected with wild-type and two gatekeeper mutants of Flag-PKD1 (Flag-PKD1^M659G and Flag-PKD1^M659A). Two days after transfection, cells were lysed and subjected to Western blotting for PKD1 and tubulin (loading control). <b>C.</b> 1-NA-PP1 concentration-dependently inhibited PMA-induced activation of Flag-PKD1 and Flag-PKD1^M659G. HEK293 cells transfected with Flag-PKD1 and Flag-PKD1^M659G were serum-starved for 24 h and pre-treated with 1-NA-PP1 at increasing concentrations in serum-free medium for 45 min, followed by stimulation with PMA at 10 nM for 20 min. The cells were harvested and subjected to immunoblotting for p-S⁹¹⁶-PKD1, PKD1, and tubulin. The experiment was repeated three times and representative images from one experiment are shown.</p

Primary hits identified in a PKD1 inhibitor screen of a targeted library.

<p>A targeted protein kinase inhibitor library of 80 compounds was screened for PKD1 inhibitory activity at 1 µM using an <i>in vitro</i> radiometric PKD1 kinase assay. Sixteen compounds were selected as primary hits based on their ability to inhibit PKD1 at or above 50% at 1 µM. The % PKD1 inhibition referred to the percent inhibition of the total kinase activity measured in the absence of inhibitors (DMSO). Kb-NB142-70, a previously validated PKD inhibitor, was used as a positive control. Experiments were performed with triplicate determinations at 1 µM for each compound.</p

Synthesis and SAR analysis of 1-NA-PP1 analogs.

<p><b>A</b>. 4-Zone model for 1-NA-PP1 analog synthesis. <b>B</b>. Synthesis of 1<i>H</i>-pyrazolo[3,4-<i>d</i>]pyrimidines <b>1</b>.</p

1-NA-PP1 inhibited PC3 cell proliferation, survival, and arrested cells in G2/M.

<p><b>A.</b>1-NA-PP1 blocked PC3 cell proliferation. PC3 cells were plated in triplicates in 24-well plates. Cells were allowed to attach overnight. A cell count at day 1 was made, and then either a vehicle (DMSO) or 1-NA-PP1 at 10 µM was added. Cells were counted daily for a total of 5 days. Fresh media and inhibitor were added every 2 days. The means of triplicate determinations were plotted over time. The experiment was repeated twice and results from one representative experiment are shown. <b>B</b>. 1-NA-PP1 induced cell death in PC3 cells. PC3 cells were seeded into 96-well plates (3000 cells/well) and were then incubated in media containing 0.3–100 µM inhibitors for 72 h. MTT solution was added to each well and incubated for 4 h. Optical density was read at 570 nm to determine cell viability. The IC₅₀ was determined as the mean of two independent experiments for each compound. <b>C.</b> 1-NA-PP1 caused G2/M phase cell cycle arrest. PC3 cells were treated with either vehicle (DMSO), or 10 µM 1-NA-PP1 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. Statistical significance was determined by unpaired t-test and is indicated. **, <i>p</i><0.01; ***, <i>p</i><0.001.</p