Targeting p75 neurotrophin receptors ameliorates spinal cord injury-induced detrusor sphincter dyssynergia in mice

Aims: To determine the role of p75 neurotrophin receptor (p75NTR) and the therapeutic effect of the selective small molecule p75NTR modulator, LM11A-31, in spinal cord injury (SCI) induced lower urinary tract dysfunction (LTUD) using a mouse model. Methods: Adult female T8-T9 transected mice were gavaged daily with LM11A-31 (100mg/kg) for up to 6 weeks, starting 1 day before, or 7 days following injury. Mice were evaluated in vivo using urine spot analysis, cystometrograms (CMGs), and external urethral sphincter (EUS) electromyograms (EMGs); and in vitro using histology, immunohistochemistry, and Western blot. Results: Our studies confirm highest expression of p75NTRs in the detrusor layer of the mouse bladder and lamina II region of the dorsal horn of the lumbar-sacral (L6-S1) spinal cord which significantly decreased following SCI. LM11A-31 prevented or ameliorated the detrusor sphincter dyssynergia (DSD) and detrusor overactivity (DO) in SCI mice, significantly improving bladder compliance. Furthermore, LM11A-31 treatment blocked the SCI-related urothelial damage and bladder wall remodeling. Conclusion: Drugs targeting p75NTRs can moderate DSD and DO in SCI mice, may identify pathophysiological mechanisms, and have therapeutic potential in SCI patients

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

SD-208 arrested cells in G2/M and regulated the levels and activities of cell cycle regulatory proteins at the G2/M phase of cell cycle in prostate cancer cells.

<p><b>A-B. SD-208 induced G2/M cell cycle arrest in prostate cancer cells</b>. DU145 cells <b>(A)</b> and PC3 cells <b>(B)</b> were treated with either vehicle (DMSO) or 30 μM SD 208 for 48 h. Cell cycle distribution was determined by flow cytometry after propidium iodide labeling of fixed cells. The cell cycle plots are representative of three independent experiments (<i>left</i>). Data in the bar graph are the mean ± SEM of three independent experiments (<i>right</i>). Statistical significance was determined using the unpaired t-test and is indicated. **, p<0.01; ***, p<0.001. <b>C-D. Effects of SD-208 on the expression and activities of G2/M cell cycle regulatory proteins</b>. DU145 (<b>C</b>) and PC3 (<b>D</b>) cells were treated with DMSO or 5–30 µM SD-208 for 24 and 48 h. At the end of each treatment, cells were harvested and subjected to immunoblotting for the cell cycle regulatory proteins as indicated. GAPDH was blotted as loading control. The densitometry data (mean ± SEM from four experiments) were plotted as ‘fold change’ over the control after normalization with GAPDH. The experiments were repeated four times and representative blots from one experiment are shown.</p

SD-208 inhibited prostate cancer cells proliferation, survival, and invasion and the anti-proliferative effect of SD-208 was mediated through the inhibition of PKD.

<p><b>A-B. SD-208 inhibited PC3 (A) and LNCaP (B) prostate cancer cell proliferation</b>. PC3 and LNCaP 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 SD-208 at 30 μM was added. Cells were counted daily for a total of 6 days. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. <b>C</b>. <b>SD-208 inhibited PC3 prostate cancer cell survival</b>. 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 three independent experiments for each compound. <b>D. SD-208 inhibited prostate cancer cell invasion</b>. DU145 cells were incubated with 30 μM SD 208 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. Data are the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ***, p<0.001. <b>E-F. Overexpression of PKD1 and PKD3 in prostate cancer cells rescued the anti-proliferative effects of SD-208</b>. PC3 (0.5 million) cells were seeded in a 60 mm dish and infected the next day with 50 and 100 MOI of PKD1 and PKD3 adenoviruses (Adv-PKD1 and Adv-PKD3). Empty adenovirus (Adv-null) was used as control. After 24 h, 3000 cells/well were plated in 96-well plates and treated with and without 10 and 30 μM SD-208 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 overexpression of PKD1 and PKD3 was confirmed by Western blotting analysis. This experiment was repeated three times and data are the mean ± S.E. of all three independent experiments. Statistical significance between DMSO and inhibitor treatment was determined using the unpaired t-test.*, p<0.05; **, p<0.01; ***, p<0.001. <b>G. PKD mediated Hsp27 activity in prostate cancer cells was inhibited by SD-208</b>. DU145 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-S⁹¹⁰-PKD1 and p-S^738/742-PKD1. GAPDH was blotted as loading control. The experiment was repeated two times and the representative blots are shown.</p

Overview of structures and yields of intermediates 3 and 4 (shown in “S1 Fig.”) and analogs 5 (as described in “<b><i>Supporting Information</i></b>”) with PKD1 inhibition data for analogs 5.

<p>^aPiperazine is directly connected to the pteridine core without a secondary amine.</p><p>^bVia the PyBOP route.</p><p>^cVia the chlorination/substitution route (2-step yield).</p><p>Overview of structures and yields of intermediates 3 and 4 (shown in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119346#pone.0119346.s001" target="_blank">S1 Fig.</a>”) and analogs 5 (as described in “<b><i>Supporting Information</i></b>”) with PKD1 inhibition data for analogs 5.</p

SD-208 was a cell-active, ATP-competitive PKD inhibitor and did not inhibit PKC and CAMK.

<p><b>A. Determination of PKD kinase activity <i>in vitro</i></b>. Inhibition of recombinant human PKD1, 2 and 3 was assayed in the presence of 10 different concentrations of SD-208 by an <i>in vitro</i> radiometric PKD kinase assay. The IC₅₀ values were calculated as the mean ± SEM of at least three independent experiments with triplicate determinations at each compound concentration in each experiment. The data were plotted as a function of inhibitor concentration and a representative graph is shown. <b>B</b>. <b>SD-208 inhibited PMA-induced PKD1 activation in prostate cancer cells</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-S⁹¹⁰-PKD1 and p-S^738/742-PKD1. Tubulin was blotted as loading control. The experiment was repeated three times and the representative blots are shown. <b>C. Determination of cellular IC</b>_<b>50</b><b> </b>. Western blots from ‘B” were quantified using densitometry analysis. The data were plotted and IC₅₀ values were derived from the concentration-response curves using GraphPad. One of the three concentration-response curves is shown. <b>D. SD-208 is an ATP-competitive kinase inhibitor</b>. PKD1 kinase activity was measured as a function of increasing concentrations of ATP in the presence of varying concentrations of SD-208. Lineweaver-Burke plots of the data are shown. Data presented were the mean ± S.E. of three independent experiments with triplicate determinations at each data point in each experiment. <b>E-F. Selectivity of SD-208 against related kinases</b>. Inhibition of PKCα (<b>B</b>) or PKCδ (<b>C</b>) was determined at 10 nM, 100 nM, 1μM, and 10 μM. As controls, the PKC inhibitor GF109203X potently inhibited PKCα and PKCδ activity. Data are the mean ± SEM of two independent experiments. <b>G</b>. Inhibition of CAMKIIα was measured by the radiometric CAMK kinase assay. Data are the mean ± S.E. of two independent experiments with triplicate determinations at each data point in each experiment. Statistical significance was determined using the unpaired t-test. ns, not significantly significant; *, p<0.05; **, p<0.01; ***, p<0.001.</p

Scope of first-generation SAR studies on SD-208.

<p>2-Zone model for SD-208 analog synthesis.</p