Synthesis, in Vitro, and in Cell Studies of a New Series of [Indoline-3,2′-thiazolidine]-Based p53 Modulators

Analogues of the previously described spiro­[imidazo­[1,5-<i>c</i>]­thiazole-3,3′-indoline]-2′,5,7­(6<i>H</i>,7a<i>H</i>)-trione p53 modulators were prepared to explore new structural requirements at the thiazolidine domain for the antiproliferative activity and p53 modulation. In cell, antiproliferative activity was evaluated against two human tumor cell lines. Derivative 5-bromo-3′-(cyclohexane carbonyl)-1-methyl-2-oxospiro­[indoline-3,2′-thiazolidine] (<b>4n</b>) emerged as the most potent compound of this series, inhibiting in vitro 30% of p53–MDM2 interaction at 5 μM and the cell growth of different human tumor cells at nanomolar concentrations. Docking studies confirmed the interactions of <b>4n</b> with the well-known Trp23 and Phe19 clefts, explaining the reasons for its binding affinity for MDM2. <b>4n</b> at 50 nM is capable of inducing the accumulation of p53 protein, inducing significant apoptotic cell death without affecting the cell cycle progression. Comparative studies using nutlin in the same cellular system confirm the potential of <b>4n</b> as a tool for increasing understanding of the process involved in the nontranscriptional proapoptotic activities of p53

Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1

Arginine methylation is a common post-translational modification that is crucial in modulating gene expression at multiple critical levels. The arginine methyltransferases (PRMTs) are envisaged as promising druggable targets, but their role in physiological and pathological pathways is far from being clear due to the limited number of modulators reported to date. In this effort, enzyme activators can be invaluable tools useful as gain-of-function reagents to interrogate the biological roles in cells and in vivo of PRMTs. Yet the identification of such molecules is rarely pursued. Herein we describe a series of aryl ureido acetamido indole carboxylates (dubbed “uracandolates”), able to increase the methylation of histone (H3) or nonhistone (polyadenylate-binding protein 1, PABP1) substrates induced by coactivator-associated arginine methyltransferase 1 (CARM1), both in in vitro and cellular settings. To the best of our knowledge, this is the first report of compounds acting as CARM1 activators

The effect of CaMKII selective peptide inhibitors in vivo in SHRs.

<p><b>A:</b> SHRs were subjected to intramural cardiac injections at the age of 13 week and repeated after 7 and 14 days. Transthoracic echocardiography was performed at each drug administration and once more at the day of euthanasia in untreated WKY rats, AntCaNtide SHRs, tat-CN17β SHRs, and control SHRs (SHR) using a dedicated small-animal high-resolution-ultrasound system (Vevo 770, VisualSonics). ANTCaNtide-SHRs and tat-CN17β-SHRs caused reduction of interventricularseptal (IVS) thickness compared to SHR (*<i>P</i><0.05 vs WKY; #<i>P</i><0.05 vs SHR). <b>B, C:</b> At the end of treatment, rats were weighed and then euthanized. Hearts were immediately removed, rinsed 3 times in cold PBS and blotted dry, weighed and then rapidly frozen. The HW/BW ratio (<b>B</b>) and LVM/BW ratio (<b>C</b>) were measured in ANTCaNtide-SHRs and tat-CN17β-SHRs and compared to WKY and SHR hearts (*<i>P</i><0.05 vs WKY; #<i>P</i><0.05 vs SHR). <b>D:</b> Cardiac Performance at the end of the treatment was assessed by ultrasound. All measurements were averaged on 5 consecutive cardiac cycles and analysed by two experienced investigators blinded to treatment (GS, MC). No differences were observed in LVFS and LVEF in ANTCaNtide and tat-CN17β SHRs when compared with SHR (*P<0.05 vs WKY). <b>E:</b> To evaluate the involvement of BP values in CaMKII–dependent regulation of cardiac hypertrophy, SBP and DBP values were assessed in AntCaNtide- and tat-CN17β- treated and control. The pressure values ​​in the graph represent the measurements made at the end of treatment (*<i>P</i><0.05 vs WKY-SBP; #<i>P</i><0.05 vs WKY-DBP). <b>F:</b> At the end of the treatment the total RNA was isolated from myocardial sample using TRIzol reagent, and cDNA was synthesized by means of a Thermo-Script RT-PCR System, following the manufacturer’s instruction. ANP gene expression was evaluated by real-time PCR in AntCaNtide- and tat-CN17β-SHR rats compared with WKY and SHR (*<i>P</i><0.05 vs WKY). Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>].</p

CaMKII isoforms.

<p><b>A:</b> To evaluate the expression of CaMKII isoforms in ventricular adult cardiomyocytes from WKY rats, the total cell lysates were immunoprecipitated using anti-CaMKII antibody (lane a) or anti-CaMKII α, β γ or δ specific antibodies (lane b) and together to total samples from WKY cardiomyocytes (lane d) and H9C2 cardiomyoblasts (lane e) were analyzed by western blot with anti-CaMKII α, β γ or δ antibodies as indicated. Total extracts from rat brain (lane f), mouse brain (lane g) and mouse heart (lane h) were used as controls. Total lysates from WKY cardiomyocytes with specific antibody without A/G agarose beads were used as negative control (lane c). <b>B:</b> Total RNA from H9C2 cell line and isolated ventricular cardiomyocytes was extracted with standard methods. RT-PCR for CaMKII α, β, γ, and δ was performed as indicated in methods. The representative graph indicates the relative amounts of transcripts for CaMKII isoforms in H9C2s and ventricular adult myocytes from WKY rats. Cycle threshold (Ct) values from 3 independent experiments were normalized to the internal β-actin control. The ratio of fold change was calculated using the Pfaffl method. * = p<0.05 vs CaMKIIα; # = p<0.05 vs CaMKIIβ.</p

CaMKII/ERK-dependent regulation of the hypertrophy marker ANF.

<p><b>A:</b> H9C2 cells at ≈ 70% confluence were incubated 1 h at 37°C with 5 mL DMEM containing purified adenovirus at a multiplicity of infection (moi) of 100:1, encoding either the kinase-dead (CaMKII-DN, rCaMKIIdelta, K42M), or the wild type (CaMKII-WT, rCaMKIIdelta) variant of CaMKII or the empty virus as a negative control (Ctr). 48 h after the infection, the cells were stimulated with PE 100 nM for 24 h. Total RNA was isolated from H9C2s using TRIzol reagent, and cDNA was synthesized by means of a Thermo-Script RT-PCR System, following the manufacturer’s instruction. Then ANP gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>]. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. <b>B:</b> The H9C2s infected with adenoviruses encoding wilde type CaMKII (CaMKII-WT) and kinase dead CaMKII (CaMKII-DN) were stimulated with PE 100 nM for 24 h. Total cell lisates were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were corrected by Actin densitometry. Data from the immunoblots were quantified by densitometric analysis.* = p<0.05 vs Ctrl. Each data point in all graphs represents the mean±SEM of 3 independent experiments. <b>C:</b> H9C2 cells were pretreated with CaMK inhibitor KN93 (5 μmol/L), the selective inhibitors AntCaNtide (10 μmol/L) and tat-CN17β (10 μmol/L) and ERK specific inhibitor pathway UO126 (10 μmol/L) for 30 min. and then stimulated with PE (100 nmol/L) for 24 h. cDNA was synthesized from RNA obtained from H9C2s as indicated above. The ANF gene expression was evaluated by real-time PCR. Results are expressed as mean±SEM from 3 independent experiments. The ratio of fold change was calculated using the Pfaffl method[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130477#pone.0130477.ref036" target="_blank">36</a>]. * = p<0.05 vs Ctrl; # = p<0.05 vs PE.</p

PKA inhibition does not modify PE-induced CaMKII/ERK activation in H9C2 cardiomyoblasts.

<p><b>A:</b> H9C2 cardiomyoblasts were transfected with PKA-I and then stimulated with 100 nm phenylephrine (PE) for 15 min. Total cell extracts were analyzed by western blot for phosphothreonine 286 CaMKII (pCaMKII) and CaMKII with specific antibodies. pCaMKII levels were corrected by CaMKII densitometry. *, <i>P</i> < 0.05 <i>vs</i>. Ctrl. <b>B:</b> H9C2s were stimulated with PE after transfection with PKA-I. Total lysates were analysed by WB for pERK with specific antibody. pERK1/2 levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl. <b>C:</b> To evaluate that PKA inhibition does not modify CaMKII/ERK interaction, H9C2s were stimulated with 100 nmol/L PE for 15 minutes following 30 min. pretreatment with PKA-I (10 μmol/L). CaMKII was immunoprecipitated from cell lysates using a specific anti- CaMKII antibody, and ERK1/2 was visualized by WB to evaluate its association with CaMKII. ERK1/2 levels were corrected by CaMKII densitometry. * = p<0.05 vs Ctrl. <b>D:</b> To confirm the interaction between CaMKII and ERK in H9C2s, total cell lysates were immunoprecipitated using anti-ERK1/2 antibody, and subjected to western blot using anti-CAMKII antibody. CaMKII levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl. <b>E:</b> H9C2s were stimulated with PE after trasfection with PKA-I. Subsequently, nuclear extract were prepared from the cells as indicated in the methods section. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl. <b>F:</b> To confirm that PE induced ERK nuclear localization was independent from PKA inhibition, H9C2s were transfected with PKA-I and then stimulated with PE. Nuclear extracts were analyzed by WB for total ERK1/2 with specific antibody. ERK1/2 levels were normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl. Data from all immunoblots were quantified by densitometric analysis. Each data point in all graphs represents the mean±SEM of 3 independent experiments.</p

Inhibition of ERK pathway downregulates PE-induced CaMKII activation in H9C2 cardiomyoblasts.

<p><b>A:</b> H9C2 cardiomyoblasts were exposed to UO126 (10 μmol/L) for 30 min., and then stimulated with 100 nM phenylephrine (PE) for 15 min. Total cell extracts were analyzed by western blot for phosphothreonine 286 CaMKII (pCaMKII) and CaMKII with specific antibodies. pCaMKII levels were corrected by CaMKII densitometry. *, <i>P</i> < 0.05 <i>vs</i>. Ctrl; #, <i>P</i> < 0.05 <i>vs</i>. PE. <b>B:</b> H9C2s were stimulated with PE after pretreatment with UO126 (10 μmol/L for 30 min.). Total lisates were analyzed by WB for pERK with specific antibody. pERK1/2 levels were corrected by ERK1/2 densitometry. * = p<0.05 vs Ctrl; # = p<0.05 vs PE. <b>C:</b> H9C2s were stimulated with 100 nmol/L PE for 15 minutes following 30 min. pretreatment with UO 126 (10 μmol/L). CaMKII was immunoprecipitated from cell lysates using a specific anti- CaMKII antibody, and ERK (ERK1/2) was visualized by WB to evaluate its association with CaMKII. ERK1/2 levels were corrected by CaMKII densitometry. * = p<0.05 vs Ctrl;# = p<0.05 vs PE. <b>D:</b> To confirm the interaction between CaMKII and ERK in H9C2s, total cell lysates were immunoprecipitated using anti-ERK1/2 antibody, and subjected to western blot using anti-CAMKII antibody. CaMKII levels were corrected by ERK1/2 densitometry.* = p<0.05 vs Ctrl; # = p<0.05 vs PE. <b>E:</b> After pharmacological inhibition of ERK and stimulation with PE, nuclear extract from H9C2s were prepared as indicated in the methods section. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl; # = p<0.05 vs PE. <b>F:</b> To confirm the effects PE induced ERK nuclear localization after pretreatment with UO126, nuclear extracts were analyzed by WB for total ERK1/2 with specific antibody. ERK1/2 levels were normalized to histone 3 densitometry. *, <b><i>P</i></b> < 0.05 <b><i>vs</i>.</b> Ctrl; # = p<0.05 vs PE. Data from all immunoblots were quantified by densitometric analysis. Each data point in all graphs represents the mean±SEM of 3 independent experiments.</p

Effects of CaMKII inhibition on CaMKII/ERK pathway <i>in vivo</i> in SHR.

<p><b>A</b>: After three weeks of treatments, heart were harvested, weighted, and samples from WKY, SHR- AntCaNtide, SHR- tat-CN17β, and SHR-Control total lysates were prepared from the left ventricular samples. Whole lysates were subjected western blotting analysis with anti‐CAMKII antibody. CAMKII levels were corrected by Actin densitometry.* = p<0.05 vs WKY. <b>B</b>: To assess CAMKII phosphorylation levels in LV after CaMKII inhibitors pretreatment, total lysate samples from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB for anti-phosphothreonine 286 CaMKII antibody (pCaMKII). pCaMKII levels were corrected by Actin densitometry.* = p<0.01 vs WKY. <b>C:</b> Total cell extracts of LV from WKY, SHR-AntCaNtide, SHR- tat-CN17β, and SHR-Control were analyzed by WB with anti-pERK (pERK1/2) or anti- total ERK1/2. pERK1/2 levels were corrected by total ERK1/2 densitometry. * = p<0.01 vs WKY. <b>D:</b> To evaluate the effects of CaMKII inhibition on CaMKII subcellular compartmentalization, nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were prepared as indicated in methods. Nuclear extracts were analyzed by WB for total CaMKII with specific antibody. CaMKII levels were averaged and normalized to histone 3 densitometry.*, <i>P</i> < 0.05 <i>vs</i>. WKY. <b>E:</b> The nuclear extract from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β were analyzed by WB for total ERK with specific antibody to test the effects of CaMKII inhibitors on ERK subcellular compartmentalization. CaMKII levels were averaged and normalized to histone 3 densitometry.*, <i>P</i> < 0.05 <i>vs</i>. WKY. <b>F</b>: To examine the association between CaMKII and ERK in the left ventricle from SHR following intramyocardial injections, total cell lysate from WKY, SHR, SHR-AntCaNtide and SHR-tat-CN17β, was immunoprecipitated using anti-CaMKII antibody and subjected to WB with anti-ERK antibody. ERK levels were averaged and normalized to IgG densitometry. *<i>P</i> < 0.05 vs. WKY. Data from all immunoblots presented here were quantified by densitometric analysis. Each data point in all graphs represent the mean±SEM of 3 independent experiments.</p

CaMKII selective peptide inhibitors reduces myocardial fibrosis in SHRs.

<p><b>A:</b> Paraffin-blocked tissues from left ventricle were sectioned and stained with Masson's trichrome after three weeks of CaMKII selective inhibitors (×20). SHR treated with AntCaNtide and tat-CN17β showed a decrease of interstitial fibrosis and reduced cardiomyocytes size when compared to SHR untreated group. The section stained with Masson's trichrome of WKY was used as control. <b>B:</b> Cardiomyocytes size was measured by Image J software, and means of areas are showed in the histogram ((*P<0.05 vs WKY; # P<0.05 vs SHR). Images are representative of 3 independent experiments (magnification ×60; black bar = 100 μm). <b>C:</b> Quantification of fibrosis was done by Image J software, and percent of fibrotic areas compared to WKY are shown in the histogram (*P<0.05 vs WKY). Images are representative of 3 independent experiments. <b>D, E:</b> To confirm that intramyocardial injection with CAMKII inhibitors AntCaNtide and tat-CN17β blunted interstitial fibrosis we have evaluated mRNA levels of fibrosis biomarkers such as collagen type I (<b>D</b>) and collagen type III (<b>E</b>) using RT-PCR. *<i>P</i><0.05 vs WKY; # <i>P</i><0.05 vs SHR. Results are the mean of 3 independent experiments.</p

Identification of a Potent Tryptophan-Based TRPM8 Antagonist With in Vivo Analgesic Activity

TRPM8 has been implicated in nociception and pain and is currently regarded as an attractive target for the pharmacological treatment of neuropathic pain syndromes. A series of analogues of <i>N</i>,<i>N</i>′-dibenzyl tryptamine <b>1</b>, a potent TRPM8 antagonist, was prepared and screened using a fluorescence-based in vitro assay based on menthol-evoked calcium influx in TRPM8 stably transfected HEK293 cells. The tryptophan derivative <b>14</b> was identified as a potent (IC₅₀ 0.2 ± 0.2 nM) and selective TRPM8 antagonist. In vivo, <b>14</b> showed significant target coverage in both an icilin-induced WDS (at 1–30 mg/kg s.c.) and oxaliplatin-induced cold allodynia (at 0.1–1 μg s.c.) mice models. Molecular modeling studies identified the putative binding mode of these antagonists, suggesting that they could influence an interaction network between the S1–4 transmembrane segments and the TRP domains of the channel subunits. The tryptophan moiety provides a new pharmacophoric scaffold for the design of highly potent modulators of TRPM8-mediated pain