Characterization of Isoenzyme-Selective Inhibitors of Human Sphingosine Kinases

<div><p>Sphingosine kinases (SKs) are promising new therapeutic targets for cancer because they regulate the balance between pro-apoptotic ceramides and mitogenic sphingosine-1-phosphate. The functions of the two SK isoenzymes, SK1 and SK2, are not redundant, with genetic ablation of SK2 having more pronounced anticancer effects than removal of SK1. Although several small molecule inhibitors of SKs have been described in the literature, detailed characterization of their molecular and cellular pharmacology, particularly their activities against human SK1 and SK2, have not been completed. Computational modeling of the putative active sites of SK1 and SK2 suggests structural differences that might allow isozyme-selective inhibitors. Therefore, we characterized several SK-inhibitory compounds which revealed differential inhibitory effects on SK1 and SK2 as follows: SKI-II and ABC294735 are SK1/2-dual inhibitors; CB5468139 is a SK1-selective inhibitor; and ABC294640 is a SK2-selective inhibitor. We examined the effects of the SK inhibitors on several biochemical and phenotypic processes in A498 kidney adenocarcinoma cells. The SK2-selective inhibitor ABC294640 demonstrated the most pronounced effects on SK1 and SK2 mRNA expression, decrease of S1P levels, elevation of ceramide levels, cell cycle arrest, and inhibition of proliferation, migration and invasion. ABC294640 also down-regulated the expression or activation of several signaling proteins, including STAT3, AKT, ERK, p21, p53 and FAK. These effects were equivalent or superior to responses to the SK1/2-dual inhibitors. Overall, these results suggest that inhibition of SK2 results in stronger anticancer effects than does inhibition of SK1 or both SK1 and SK2.</p> </div

Substrate competition by SK inhibitors.

<p>SK1 (Panels A left and C) or SK2 (Panels A right and B) were assayed in the presence of the indicated concentrations of sphingosine and 0 (•), 1.56 (▴), 3.13 (▾), 6.25 (⧫), 12.5 (○), 25 (▪) or 50 (<b>X</b>) µM ABC294735 (Panel A) or ABC294640 (Panel B); or 0 (•), 0.78 (), 1.56 (▴), 3.13 (▾), 6.25 (⧫), 12.5 (○) or 25 (▪) µM CB5468139 (Panel C). Data are representative of at least 2 independent experiments for each compound.</p

Structural comparison of SK1 and SK2.

<p>Homology models for the catalytic domain of human SK1 and SK2 were created as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044543#s2" target="_blank">Materials and Methods</a> section. <b><i>A</i></b><i>)</i> The ribbon model for SK1 is shown in blue and SK2 in yellow. The expanded section highlights the extended loop found in the lipid binding domain of SK2, and bars indicate the distance between residues of SK1 and SK2. <b><i>B</i></b><i>)</i> The nucleotide and lipid binding domains of SK1 (left) and SK2 (right) were compared. Top Row: residues in the surface images of the catalytic domain are colored brown for hydrophobic and blue for hydrophilic. ADP is indicated by the ball-and-stick model, and S1P by the space-filling model. Middle Row: interaction diagrams of ADP binding to SK. Bottom Row: interaction diagrams of S1P binding to SK.</p

Biochemical and phenotypic effects of SK inhibitors.

<p><b><i>A</i></b><i>)</i> Cytotoxicity - After treatment with varying concentrations of the SK inhibitors for the indicated time periods, cell numbers were quantified. The IC₅₀ represents the concentration of the test compound that reduces cell number by 50% compared with DMSO-treated control cultures. In subsequent experiments, cells were incubated with each SK inhibitor at its IC₅₀s (DMS - 5 µM, SKI-II - 20 µM, ABC294735 - 35 µM, CB5468139 - 10 µM and ABC294640 - 40 µM) for 48 hours. <b><i>B</i></b><i>)</i> SK expression - After SK inhibitor treatment, mRNAs for SK1 (open bars) and SK2 (filled bars) compared to the vehicle controls (DMSO-treated cells) were calculated. <b><i>C</i></b><i>)</i> Sphingolipid profiling – After SK inhibitor treatment, cells were harvested and sphingolipids were analyzed by mass spectrometric. The bars represent the ratio of the amount of the indicated lipids in drug treated cells compared with control cells. Abbreviations are: C₂₆-ceramide (C26-Cer), C₂₄-ceramide (C24-Cer), C₂₀-ceramide (C20-Cer), C₁₈-ceramide (C18-Cer), C₁₆-ceramide (C16-Cer), sphingosine (Sph) and dihydrosphingosine (dhSph). <b><i>D</i></b><i>)</i> Cell cycle distribution – After SK inhibitor treatment, cells were harvested and analyzed by flow cytometry. The bars indicate the percentage of cells in each of the indicated cell cycle phases. <b><i>E</i></b><i>)</i> Cell migration and invasion – After SK inhibitor treatment, cells were harvested and migration, through unmodified filters (open bars) and invasion through Matrigel-coated filters (filled bars), were analyzed. The bars indicate the percentage of cells that had migrated compared to control cells. Data are the mean ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001 versus control.</p

SK kinetics and inhibition.

<p><i>A)</i> The activities of SK1 (•) and SK2 (▴) were measured under initial velocity conditions at the indicated concentrations of sphingosine as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044543#s2" target="_blank">Materials and Methods</a> section. <i>B)</i> SK1 (left panel) and SK2 (right panel) activities were measured in the presence of the indicated concentrations of DMS (•), ABC294735 (▴), CB5468139 (⧫) or ABC294640 (▾) using the ADP Quest assay or SKI-II (▪) using the HPLC assay as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044543#s2" target="_blank">Materials and Methods</a> section. Data are mean ± SD of triplicates of a representative of three independent experiments.</p

Structures and potencies of SK inhibitors.

<p>Recombinant human SK1 and SK2 were assayed in the presence of varying concentrations of the indicated compounds, and K_is were calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044543#s2" target="_blank">Materials and Methods</a> section. Data are mean ± SEM of three independent experiments.</p

Effects of SK inhibitors on signaling proteins.

<p><b><i>A</i></b><i>)</i> Cells were incubated with the indicated SK inhibitors at their respective IC₅₀s for 48 hours and the levels of the indicated proteins were estimated by western blotting. Figures are representative blots of at least three independent experiments. <b><i>B</i></b><i>)</i> Immunoblotting was conducted with the indicated antibodies, and densitometric quantification was done using Image J. Relative expression is represented as the ratio to control after normalization to β-actin.</p

Development of Allosteric Hydrazide-Containing Class I Histone Deacetylase Inhibitors for Use in Acute Myeloid Leukemia

One of the biggest hurdles yet to be overcome for the continued improvement of histone deacetylase (HDAC) inhibitors is finding alternative motifs equipotent to the classic and ubiquitously used hydroxamic acid. The <i>N</i>-hydroxyl group of this motif is highly subject to sulfation/glucoronidation-based inactivation in humans; compounds containing this motif require much higher dosing in clinic to achieve therapeutic concentrations. With the goal of developing a second generation of HDAC inhibitors lacking this hydroxamate, we designed a series of potent and selective class I HDAC inhibitors using a hydrazide motif. These inhibitors are impervious to glucuronidation and demonstrate allosteric inhibition. In vitro and ex vivo characterization of our lead analogues’ efficacy, selectivity, and toxicity profiles demonstrate that they possess low nanomolar activity against models of acute myeloid leukemia (AML) and are at least 100-fold more selective for AML than solid immortalized cells such as HEK293 or human peripheral blood mononuclear cells

High-Throughput Screening for Novel Inhibitors of <em>Neisseria gonorrhoeae</em> Penicillin-Binding Protein 2

<div><p>The increasing prevalence of <em>N. gonorrhoeae</em> strains exhibiting decreased susceptibility to third-generation cephalosporins and the recent isolation of two distinct strains with high-level resistance to cefixime or ceftriaxone heralds the possible demise of β-lactam antibiotics as effective treatments for gonorrhea. To identify new compounds that inhibit penicillin-binding proteins (PBPs), which are proven targets for β-lactam antibiotics, we developed a high-throughput assay that uses fluorescence polarization (FP) to distinguish the fluorescent penicillin, Bocillin-FL, in free or PBP-bound form. This assay was used to screen a 50,000 compound library for potential inhibitors of <em>N. gonorrhoeae</em> PBP 2, and 32 compounds were identified that exhibited >50% inhibition of Bocillin-FL binding to PBP 2. These included a cephalosporin that provided validation of the assay. After elimination of compounds that failed to exhibit concentration-dependent inhibition, the antimicrobial activity of the remaining 24 was tested. Of these, 7 showed antimicrobial activity against susceptible and penicillin- or cephalosporin-resistant strains of <em>N. gonorrhoeae</em>. In molecular docking simulations using the crystal structure of PBP 2, two of these inhibitors docked into the active site of the enzyme and each mediate interactions with the active site serine nucleophile. This study demonstrates the validity of a FP-based assay to find novel inhibitors of PBPs and paves the way for more comprehensive high-throughput screening against highly resistant strains of <em>N. gonorrhoeae</em>. It also provides a set of lead compounds for optimization of anti-gonococcal agents.</p> </div

Structures of seven compounds that exhibited antimicrobial activity against <i>N. gonorrhoeae</i>.

<p>Compound numbers correspond with those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044918#pone-0044918-t002" target="_blank">Table 2</a>.</p