Squaramides as novel class I and IIB histone deacetylase inhibitors for topical treatment of cutaneous t-cell lymphoma.

A series of squaramide-based hydroxamic acids were designed, synthesized and evaluated against human HDAC enzyme. Squaramides were found to be potent in the Hut78 cell line, but initially suffered from low solubility. Leads with improved solubility and metabolic profiles were shown to be class I, IIB and IV selective.

Mass spectrometry and DigiWest technology emphasize protein acetylation profile from Quisinostat-treated HuT78 CTCL cell line.

Histone deacetylases (HDACs) are key enzymes involved in epigenetic modulation and were targeted by HDAC inhibitors (HDACis) for cancer treatment. The action of HDACis is not restricted to histones and also prevents deacetylation of other proteins, supporting their wide biological actions. The HuT78 cell line is recognized as a key tool to support and understand cutaneous T-cell lymphoma (CTCL) biology and was used as a predictive model since HDACi such as Vorinostat and Panobinostat have both demonstrated apoptotic activities in HuT78 cells and in primary blood CTCL cells. In this study, Quisinostat (JNJ-26481585) a novel second-generation HDACi with highest potency for HDAC1, was tested on HuT78 cell line. Quantitative mass spectrometry (MS)-based proteomics after acetylated-lysine peptide enrichment and a targeted antibody-based immunoassay (DigiWest) were used as complementary technologies to assess the modifications of the acetylated proteome. As expected, several acetylated lysines of histones were increased by the HDACi. Additional acetylated non-histone proteins were modulated after treatment with Quisinostat including the nucleolin (a major nucleolar protein), the replication protein A 70 kDa DNA-binding subunit, the phosphoglycerate kinase 1, the stress-70 protein, the proto-oncogene Myc and the serine hydroxymethyltransferase. A better knowledge of histone and non-histone acetylated protein profile after Quisinostat treatment can strongly support the understanding of non-clinical and clinical results of this HDACi. These technological tools can also help in designing new HDACis in a pharmaceutical drug discovery program. SIGNIFICANCE: A better knowledge of histone and non-histone acetylated protein profile after HDAC inhibitors (HDACis) treatment can strongly support the understanding of non-clinical and clinical investigations in a pharmaceutical drug discovery program. Relative quantification using mass spectrometry -based proteomics after acetylated-lysine peptide enrichment and a targeted antibody-based immunoassay (DigiWest) are proposed as complementary technologies to assess the modifications of the acetylated proteome. Quisinostat (JNJ-26481585) a novel second-generation HDACi with highest potency for HDAC1 was better characterized in vitro in HuT78 cells to support and understand cutaneous T-cell lymphoma (CTCL) therapeutic research program.

Monitoring UV-induced signalling pathways in an ex vivo skin organ culture model using phospho-antibody array.

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis.

Platelet-rich plasma respectively reduces and promotes adipogenic and myofibroblastic differentiation of human adipose-derived stromal cells via the TGFß signalling pathway.

Autologous fat grafting is a gold standard therapy for soft tissue defects, but is hampered by unpredictable postoperative outcomes. Fat graft enrichment with adipose-derived stromal cell (ASCs) was recently reported to enhance graft survival. Platelet-rich plasma (PRP) has also emerged as a biologic scaffold that promotes fat graft viability. Combined ASC/PRP fat grafting enrichment is thus a promising new regenerative medicine approach. The effects of PRP on ASC proliferation are well documented, but the impact of PRP on ASC differentiation has yet to be investigated in depth to further elucidate the PRP clinical effects. Here we analyzed the human ASC fate upon PRP treatment. PRP was found to sharply reduce the potential of ASCs to undergo differentiation into adipocytes. Interestingly, the PRP anti-adipogenic effect was accompanied by the generation of myofibroblast-like cells. Among the various factors released from PRP, TGFß pathway activators played a critical role in both the anti-adipogenic and pro-myofibroblastic PRP effects. Overall, these data suggest that PRP participates in maintaining a pool of ASCs and in the repair process by promoting ASC differentiation into myofibroblast-like cells. TGFß may provide an important target pathway to improve PRP clinical outcomes.

Discovery of phenoxyindazoles and phenylthioindazoles as RORγ inverse agonists.

Targeting the IL17 pathway and more specifically the nuclear receptor RORγ is thought to be beneficial in multiple skin disorders. The Letter describes the discovery of phenoxyindazoles and thiophenoxy indazoles as potent RORγ inverse agonists. Optimization of the potency and efforts to mitigate the phototoxic liability of the series are presented. Finally, crystallization of the lead compound revealed that the series bound to an allosteric site of the nuclear receptor. Such compounds could be useful as tool compounds for understanding the impact of topical treatment on skin disease models.

Transcriptional Analysis of Vitiligo Skin Reveals the Alteration of WNT Pathway: A Promising Target for Repigmenting Vitiligo Patients.

Vitiligo affects 1% of the worldwide population. Halting disease progression and repigmenting the lesional skin represent the two faces of therapeutic challenge in vitiligo. We performed transcriptome analysis on lesional, perilesional, and non-depigmented skin from vitiligo patients and on matched skin from healthy subjects. We found a significant increase in CXCL10 in non-depigmented and perilesional vitiligo skin compared with levels in healthy control skin; however, neither CXCL10 nor other immune factors were deregulated in depigmented vitiligo skin. Interestingly, the WNT pathway, which is involved in melanocyte differentiation, was altered specifically in vitiligo skin. We demonstrated that oxidative stress decreases WNT expression/activation in keratinocytes and melanocytes. We developed an ex vivo skin model and confirmed the decrease activation of the WNT pathway in human skin subjected to oxidative stress. Finally, using pharmacological agents that activate the WNT pathway, we treated ex vivo depigmented skin from vitiligo patients and successfully induced differentiation of resident stem cells into pre-melanocytes. Our results shed light on the previously unrecognized role of decreased WNT activation in the prevention of melanocyte differentiation in depigmented vitiligo skin. Furthermore, these results support further clinical exploration of WNT agonists to repigment vitiligo lesions.

Characterization of human knee and chin adipose-derived stromal cells.

Animal study findings have revealed that individual fat depots are not functionally equivalent and have different embryonic origins depending on the anatomic location. Mouse bone regeneration studies have also shown that it is essential to match the Hox code of transplanted cells and host tissues to achieve correct repair. However, subcutaneous fat depots from any donor site are often used in autologous fat grafting. Our study was thus carried out to determine the embryonic origins of human facial (chin) and limb (knee) fat depots and whether they had similar features and molecular matching patterns. Paired chin and knee fat depots were harvested from 11 subjects and gene expression profiles were determined by DNA microarray analyses. Adipose-derived stromal cells (ASCs) from both sites were isolated and analyzed for their capacity to proliferate, form clones, and differentiate. Chin and knee fat depots expressed a different HOX code and could have different embryonic origins. ASCs displayed a different phenotype, with chin-ASCs having the potential to differentiate into brown-like adipocytes, whereas knee-ASCs differentiated into white adipocytes. These results highlighted different features for these two fat sites and indicated that donor site selection might be an important factor to be considered when applying adipose tissue in cell-based therapies.

PPAR activators and COX inhibitors selectively block cytokine-induced COX-2 expression and activity in human aortic smooth muscle cells.

Atherosclerotic complications are related to the unstable character of the plaque rather than its volume. Vulnerable plaques often contain a large lipid core, a reduced content of smooth muscle cells (SMCs), and an accumulation of inflammatory cells. Regulation of this inflammatory response is an essential element in chronic inflammatory diseases such as atherosclerosis. Nuclear receptors and particularly peroxisome proliferator-activated receptors (PPARs) have emerged as therapeutic targets with a widespread impact on the treatment of metabolic disorders because they can modulate gene expression involved in lipid and glucose homeostasis and can exert anti-inflammatory properties. However, little is known about nuclear receptor effects on SMC inflammation, which produces large amounts of IL-6 and prostanoids. The aim of this study was to evaluate anti-inflammatory properties of nuclear receptor activators in a human physiological SMC model. We show that PPAR activators, as well as liver X receptor alpha, farnesoid X receptor and retinoid X receptor alpha activators, inhibit IL-1beta-induced SMC 6-keto PGF1alpha synthesis, an index of cyclooxygenase (COX)-2 activity, with IC(50) between 1 and 69 microM. In contrast, PPARgamma activators, as exemplified by rosiglitazone and pioglitazone, were unable to inhibit cytokine-induced 6-keto PGF1alpha synthesis. We also demonstrate for the first time that the COX-2 inhibitor rofecoxib can reduce 6-keto PGF1alpha production by both enzymatic inhibition and transcriptional repression. These results show that some nuclear receptor activators have SMC anti-inflammatory properties due to COX-2 inhibition which could participate in their anti-atherosclerotic properties beyond lipid impacts.

[Metabolic syndrome: which definition for what treatment(s)?]. / Syndrome métabolique: quelle définition pour quel(s) traitement(s)?

Metabolic syndrome is asymptomatic and results from the increasing prevalence of obesity. Although several definitions exist and complicate its diagnosis, metabolic syndrome is characterized by the clustering of moderate troubles of glucose, lipid metabolism, body weight, hypertension and vascular inflammation ; the synergy between 3 of them triggers type 2 diabetes, atherosclerosis and associated clinical events. Whatever the age but particularly in adolescence, the prevalence of metabolic syndrome is high ; beyond lifestyle interventions, the available treatments address essentially a single risk factor and an unmet medical need persists. The reduction of cardiovascular events in secondary prevention has been demonstrated for some antidiabetic, hypolipidemic and antihypertensive agents but any difference in efficacy between populations with and without metabolic syndrome has yet to be established. The approval of metabolic syndrome as a specific therapeutic target would need the characterization of its pathogenic mechanism, a clearer guidance for definition and diagnosis, and the clinical proof of concept of a novel molecule displaying multifactorial impacts. Some new mechanistic approaches are discussed and may represent a breakthrough particularly if positive results of Field and Proactive clinical studies related to PPARs are disclosed soon.

Human adipocyte fatty acid-binding protein (aP2) gene promoter-driven reporter assay discriminates nonlipogenic peroxisome proliferator-activated receptor gamma ligands.

Peroxisome proliferator-activated receptors (PPARs) regulate storage and catabolism of fats and carbohydrates. PPARgamma activity increases insulin sensitivity and adipocyte differentiation at the expense of adipogenesis and weight gain. The goal of this study was to 1) clone the promoter of the human adipocyte fatty acid binding protein (aP2) gene, namely fatty acid-binding protein-4, 2) characterize its pharmacological regulation, and 3) determine its putative predictability for adipogenesis. Among the selected PPAR agonists, rosiglitazone and pioglitazone displayed the highest maximal efficacy (E(max)) on reporter-gene assays in COS-7 cells cotransfected by either a galactosidase 4-response element-based or a human aP2 promoter-based Luc reporter vector, along with either chimeric or full-length human PPAR expression plasmids. The non-subtype-selective 2-(4-[2-(3-[2,4-difluorophenyl]-1-heptylureido)ethyl]phenoxy)-2-methyl-butyric acid (GW-2331) and the compounds [4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)-propoxyl]phenoxy]-acetic acid (L-165041), (4-((2S,5S)-5-(2-(bis(phenylmethyl)amino)-2-oxoethyl)-2-heptyl-4-oxo-3-thiazolidinyl)butyl)-benzoic acid (GW-0072), and indomethacin behaved as partial agonists relative to pioglitazone in full-length human aP2-PPARgamma2. Beyond their partial PPARgamma agonist properties, these compounds elicited a lower maximal up-regulation of mouse aP2 mRNA in 3T3-L1 adipocytes as compared with pioglitazone; these properties paralleled a time-dependent increase in neutral lipids. By contrast, the selective PPARalpha agonist 2,2-dichloro-12-(4-chlorophenyl)dodecanoic acid (BM-17.0744) neither stimulated the human aP2-PPARalpha promoter reporter-gene assay, thus demonstrating a specific interaction between PPARgamma and the aP2 promoter, nor affected lipogenesis in 3T3-L1 cells. Altogether, these data characterized a functional promoter of the human aP2 gene; its in vitro pharmacological regulation in PPARgamma-mediated reporter-gene assay may represent an interesting complement or an alternative to time-consuming procedures aiming at discriminating PPAR ligands with low lipogenic properties.

Cardiovascular drugs inhibit MMP-9 activity from human THP-1 macrophages.

It is now recognized that atherosclerosis complications are related to the unstable character of the plaque rather than its volume. Vulnerable plaques often contain a large lipid core, a reduced content of smooth muscle cells, and accumulation of inflammatory cells. Colocalization of macrophages and active matrix metalloproteinases (MMPs) is likely relevant for atherosclerotic lesion disruption. Nevertheless, MMP activity and regulation by cardiovascular drugs remains poorly defined. In this study, we evaluated the effects of avasimibe, fluvastatin, and peroxisome proliferator-activated receptor (PPAR) ligands on 92-kDa gelatinase B (MMP-9) secretion by human THP-1 macrophages. THP-1 macrophages were treated with compounds for 48 h, and secreted MMP-9 protein was quantified by immunoassay. Avasimibe, fluvastatin, and PPARalpha agonists (fenofibric acid and Wy-14643) significantly reduced, in a concentration-dependent manner, MMP-9 protein (up to 67 +/- 5% for fenofibric acid). In these assays, the PPARgamma selective agonist rosiglitazone displayed a lower efficacy than other compounds. Enzymatic activity of MMP-9 was also decreased by all cardiovascular drugs tested. MMP-9 protein/activity inhibition by cardiovascular drugs was due, at least in part, to a decrease in MMP-9 mRNA. These results show that THP-1 macrophages could be an useful cellular model to investigate effects of compounds on plaque vulnerability through MMP-9 activity.

PPARalpha and PPARdelta activators inhibit cytokine-induced nuclear translocation of NF-kappaB and expression of VCAM-1 in EAhy926 endothelial cells.

Endothelium injury is a primary event in atherogenesis, which is followed by monocyte infiltration, macrophage differentiation, and smooth muscle cell migration. Peroxisome proliferator-activated receptors (PPARs) are transcription factors now recognized as important mediators in the inflammatory response. The aim of this study was to develop a human endothelial model to evaluate anti-inflammatory properties of PPAR activators. PPAR proteins (alpha, delta and gamma) are expressed in EAhy926 endothelial cells (ECs). Pirinixic acid (Wy-14643), fenofibrate, fenofibric acid, the Merck ligand PPARdelta activator L-165041, 15-deoxy-Delta(12,14)-prostaglandin J2, but not rosiglitazone (BRL-49653) inhibited the induced expression of vascular cell adhesion molecule-1 (VCAM-1), as measured by enzyme linked immunosorbent assay (ELISA), and monocyte binding to activated-EAhy926 cells. The PPARdelta activator L-165041 had the greatest potency to reduce cytokine-induced monocyte chemotactic protein-1 (MCP-1) secretion. All PPAR activators tested which impaired VCAM-1 expression reduced significantly nuclear p65 amount. These results show that EAhy926 endothelial cells are an adequate tool to substantiate and characterize inflammatory impacts of PPAR activators.

Anti-atherosclerotic properties of the acyl-coenzyme A:cholesterol acyltransferase inhibitor F 12511 in casein-fed New Zealand rabbits.

SUMMARY: The anti-atherosclerotic properties of F 12511, a novel acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitor, were studied in rabbits that were fed a cholesterol-free casein-rich diet and developed endogenous hypercholesterolemia and fibrofatty preatheroma lesions. After 6 weeks of casein feeding, an endothelial abrasion was performed in the abdominal aorta; at week 8, a control group was maintained on this diet while F 12511 (8 mg/kg/d) was administered as a diet admixture for the subsequent 24 weeks. Total plasma cholesterol level rose to 250-300 mg/dl in both groups before starting the treatment; F 12511 time-dependently reduced total plasma cholesterol by 50%, and also decreased by 50% the incidence of lesions and macrophage accumulation in uninjured aorta (thoracic arch, celiac bifurcation). Residual lesions in the treated group were characterized by few macrophages, essentially under the endothelium, and by a larger content of smooth muscle cells. Quantitative image analysis of serial sections of mechanically injured abdominal aorta revealed a 20% surface covered by preatheroma lesions in the placebo group; F 12511 significantly reduced this surface. These data suggest that the combination of endogenous hypercholesterolemia with endothelial injury in the rabbit may offer a useful model to study atherosclerosis; lipid lowering by F 12511 reduces the incidence of vascular lesions and macrophage infiltration and may reinforce the fibrous skeleton of the atheroma.