Levels of adiponectin,a marker for PPAR-gamma activity,correlate with skin fibrosis in systemic sclerosis: potential utility as biomarker?

Introduction

Progressive fibrosis in systemic sclerosis (SSc) is linked to aberrant transforming growth factor beta (TGF-beta) signaling. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) blocks fibrogenic TGF-beta responses in vitro and in vivo. Reduced expression and function of PPAR-gamma in patients with SSc may contribute to progression of fibrosis. Here we evaluated the levels of adiponectin, a sensitive and specific index of PPAR-gamma activity, as a potential fibrogenic biomarker in SSc.

Methods

Adiponectin levels were determined in the sera of 129 patients with SSc and 86 healthy controls, and serial determinations were performed in 27 patients. Levels of adiponectin mRNA in skin biopsies from SSc patients were assessed in an expression profiling microarray dataset. Regulation of adiponectin gene expression in explanted human subcutaneous preadipocytes and fibroblasts was examined by real-time quantitative PCR.

Results

Patients with diffuse cutaneous SSc had reduced serum adiponectin levels. A significant inverse correlation between adiponectin levels and the modified Rodnan skin score was observed. In longitudinal studies changes in serum adiponectin levels were inversely correlated with changes in skin fibrosis. Skin biopsies from a subset of SSc patients showed reduced adiponectin mRNA expression which was inversely correlated with the skin score. An agonist ligand of PPAR-gamma potently induced adiponectin expression in explanted mesenchymal cells in vitro.

Conclusions

Levels of adiponectin, reflecting PPAR-gamma activity, are correlated with skin fibrosis and might have potential utility as a biomarker in SSc.     

Repetitive Arg-Gly-Asp peptide as a cell-stimulating agent on electrospun poly(ϵ-caprolactone) scaffold for tissue engineering

Electrospun scaffolds derived from poly(ϵ-caprolactone) (PCL), a well known biodegradable material, have an architecture that is suitable for hosting cells. However, their biomedical applications are restricted because these scaffolds lack the bioactivity necessary to stimulate cell responses. In this work, a repetitive Arg-Gly-Asp (rRGD) peptide was produced as a cell-stimulating agent to provide the PCL scaffold with bioactivity. DNA encoding rRGD was amplified by polymerase chain reaction using overlap primers without a DNA template, and cloned into a protein expression vector to produce a His-tag fusion peptide. In an in vitro cell adhesion assay, the purified rRGD peptide, comprising 30 RGD repeats, promoted a 1.5-fold greater cell adhesion than the commercial tripeptide RGD. The rRGD peptide was immobilized onto an electrospun PCL scaffold that had been pretreated with argon plasma and graft-polymerized with acrylic acid. Fourier transform infrared (FTIR) analysis indicated that covalently linked rRGD peptide was present on the scaffold. The PCL scaffold with immobilized rRGD showed significantly changed hydrophilic properties and an enhanced adhesion and proliferation of mouse fibroblast cells by 2.3- and 2.9-fold, respectively, compared to the PCL scaffold alone. Through its ability to promote cell adhesion and proliferation, the rRGD peptide has great potential as a stimulant for improving the suboptimal cell-matrix interaction of polymeric scaffolds for tissue engineering applications.     

Discovery of N-ethylpyridine-2-carboxamide derivatives as a novel scaffold for orally active γ-secretase modulators

Gamma-secretase modulators (GSMs) are promising disease-modifying drugs for Alzheimer’s disease because they can selectively decrease pathogenic amyloid-β42 (Aβ42) levels. Here we report the discovery of orally active N-ethylpyridine-2-carboxamide derivatives as GSMs. The isoindolinone moiety of 5-[8-(benzyloxy)-2-methylimidazo[1,2-a]pyridin-3-yl]-2-ethyl-2,3-dihydro-1H-isoindol-1-one hydrogen chloride (1a) was replaced with a picolinamide moiety. Optimization of the benzyl group significantly improved GSM activity and mouse microsomal stability. 5-{8-[([1,1′-Biphenyl]-4-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-N-ethylpyridine-2-carboxamide hydrogen chloride (1v) potently reduced Aβ42 levels with an IC₅₀ value of 0.091 µM in cultured cells without inhibiting CYP3A4. Moreover, 1v demonstrated a sustained pharmacokinetic profile and significantly reduced brain Aβ42 levels in mice.     

Exploration of 2-benzylbenzimidazole scaffold as novel inhibitor of NF-κB

For finding the novel inhibitor of nuclear factor κB activity, a series of benzimidazole derivatives were rationally designed, synthesized and systematically studied for their in vitro activities against LPS induced NF-κB inhibition in RAW 264.7 cells using the SEAP assay based on the flexible chalcone JSH ((E)-1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxy phenyl)prop-2-en-1-one) which was previously reported. Although most of the benzimidazole derivatives showed strong inhibitory activity in low micromolar potency, 2-(4-methoxybenzyl)-1H-benzo[d]imidazole (3m; IC₅₀ = 1.7 μM) and 2-(2-methoxybenzyl)-1H-benzo[d]imidazole (3n; IC₅₀ = 2.4 μM) showed the best inhibition. The structure activity relationship revealed that 2-benzylbenzimidazole scaffold with hydrogen bonding acceptor on phenyl ring appears as a pharmacophore.     

Optimization and biological evaluation of imidazopyridine derivatives as a novel scaffold for γ-secretase modulators with oral efficacy against cognitive deficits in Alzheimer’s disease model mice

Gamma-secretase modulators (GSMs) selectively lower amyloid-β42 (Aβ42) and are therefore potential disease-modifying drugs for Alzheimer’s disease (AD). Here, we report the discovery of imidazopyridine derivatives as GSMs with oral activity on not only Aβ42 levels but also cognitive function. Structural optimization of the biphenyl group and pyridine-2-amide moiety of compound 1a greatly improved GSM activity and rat microsomal stability, respectively. 5-{8-[(3,4′-Difluoro[1,1′-biphenyl]-4-yl)methoxy]-2-methylimidazo[1,2-a]pyridin-3-yl}-N-methylpyridine-2-carboxamide (1o) showed high in vitro potency and brain exposure, induced a robust reduction in brain Aβ42 levels, and exhibited undetectable inhibition of cytochrome p450 enzymes. Moreover, compound 1o showed excellent efficacy against cognitive deficits in AD model mice. These findings suggest that compound 1o is a promising candidate for AD therapeutics.     

Evidence for adenylate cyclase as a scaffold protein for Ras2–Ira interaction in Saccharomyces cerevisie

Data in literature suggest that budding yeast adenylate cyclase forms a membrane-associated complex with the upstream components of the cAMP/PKA pathway. Here we provide evidences that adenylate cyclase (Cyr1p) acts as a scaffold protein keeping Ras2 available for its regulatory factors. We show that in a strain with deletion of the CYR1 gene (cyr1Δ pde2Δ msn2Δ msn4Δ) the basal Ras2-GTP level is very high and this is independent on the lack of feedback inhibition that could result from the absence of adenylate cyclase activity. Moreover, strains effected either in the intrinsic adenylate cyclase activity (fil1 strain) or in the stimulation of adenylate cyclase activity by active G-proteins (lcr1 strain) had a normal basal and glucose-induced Ras2-GTP level, indicating that adenylate cyclase activity does not influence the Ras2 activation state and suggesting that Cyr1 protein is required for the proper interaction between Ras2 and the Ira proteins. We also provide evidence that the two Ras-binding sites mapped on Cyr1p are required for the signalling complex assembly. In fact, we show that the cyr1Δ strain expressing CYR1 alleles lacking either the LRR region or the C-terminal domain still have a high basal and glucose-induced Ras2-GTP level. In contrast, a mutant expressing a Cyr1 protein only missing the N-terminal domain showed a normal Ras2 activation pattern. Likewise, the Ras2-GTP levels are comparable in the wild type strain and the srv2Δ strain, supporting the hypothesis that Cap is not essential for the Ras-adenylate cyclase interaction.     

Design,synthesis, and biological evaluation of dihydroartemisinin–fluoroquinolone conjugates as a novel type of potential antitubercular agents

Tuberculosis remains a global public health problem in recent years. To develop novel type of potential antitubercular agents, twelve novel dihydroartemisinin–fluoroquinolone (DHA–FQ) conjugates (three types of molecules) were gradually designed and conveniently synthesized. All the newly synthesized conjugates were well characterized and evaluated against different Mycobacterium tuberculosis strains in vitro. The screening results showed that five DHA–FQ conjugates were active toward M. tuberculosis H37Rv, and compound 3a exhibited the strongest inhibitory activity (MIC = 0.0625 μg/mL), which was comparable to the positive control Moxifloxacin and even stronger than Ofloxacin. Conjugates 2a and 3a also displayed comparable activities against various clinically isolated sensitive and resistant M. tuberculosis strains (MIC = 0.125–16 μg/mL) to Moxifloxacin. All target compounds possessed selective anti-M. tuberculosis ability. Preliminary structure–activity relationship demonstrated that short linker between DHA and FQ was favorable for strong antitubercular activity. This study provides a new clue for the development of novel antitubercular lead molecules.     

β-CA-specific inhibitor dithiocarbamate Fc14–584B: a novel antimycobacterial agent with potential to treat drug-resistant tuberculosis

Inhibition of novel biological pathways in Mycobacterium tuberculosis (Mtb) creates the potential for alternative approaches for treating drug-resistant tuberculosis. In vitro studies have shown that dithiocarbamate-derived β-carbonic anhydrase (β-CA) inhibitors Fc14–594?A and Fc14–584B effectively inhibit the activity of Mtb β-CA enzymes. We screened the dithiocarbamates for toxicity, and studied the in vivo inhibitory effect of the least toxic inhibitor on M. marinum in a zebrafish model. In our toxicity screening, Fc14–584B emerged as the least toxic and showed minimal toxicity in 5-day-old larvae at 300?µM concentration. In vitro inhibition of M. marinum showed that both compounds inhibited growth at a concentration of 75?µM. In vivo inhibition studies using 300?µM Fc14–584B showed significant (p?>?.05) impairment of bacterial growth in zebrafish larvae at 6?days post infection. Our studies highlight the therapeutic potential of Fc14–584B as a β-CA inhibitor against Mtb, and that dithiocarbamate compounds may be developed into potent anti-tuberculosis drugs.     

TRPV1: a therapeutic target for novel analgesic drugs?

The vanilloid receptor TRPV1 is now recognized as a molecular integrator of painful stimuli ranging from noxious heat to endovanilloids in inflammation. Pharmacological blockade of TRPV1 represents a new strategy in pain relief. TRPV1 antagonists are expected to prevent pain by silencing receptors where pain is generated rather than stopping the propagation of pain, as most-traditional pain killers do. This hypothesis has already being tested in the clinic by administering small molecule TRPV1 antagonists (e.g. GlaxoSmithKline SB-705498) for migraine and dental pain. Paradoxically, in some murine models of chronic pain, TRPV1-deficient mice exhibit more pain-related behavior than their wild-type littermates, indicating that the understanding of TRPV1 in pain is still incomplete. Moreover, there is mounting evidence to suggest the existence of functional TRPV1 both in the brain and in various non-neuronal tissues. The biological role of these receptors remains elusive, but their tissue distribution clearly indicates that they are involved in many more functions than just pain perception. Here, we review the potential therapeutic indications and adverse effects of TRPV1 antagonists.     

Heat shock protein–peptide complexes as immunotherapy for human cancer

Heat shock proteins (Hsps), ubiquitous in nature, act as chaperones for peptides and other proteins. They have been implicated in loading immunogenic peptides onto major histocompatibility complex molecules for presentation to T cells. When isolated from tumor cells, Hsps are complexed with a wide array of peptides, some of which serve as tumor-specific antigens. Animal studies have demonstrated that heat shock protein–peptide complexes (HSPPCs) from tumor cells can act as vaccines to prevent or treat tumors. Potent and specific tumor antigens have long been the holy grail in cancer immunotherapy; HSPPCs from tumor cells could become a safe and reliable source of tumor-specific antigens for clinical application.     

Design,recombinant expression,and antibacterial activity of a novel hybrid magainin–thanatin antimicrobial peptide

Antimicrobial peptides are small molecule polypeptides with biological activity, which can avoid the drug resistance. Magainin and thanatin are antimicrobial peptides with a broad spectrum of inhibitory microbes, and the core sequence of magainin is linked to a core sequence of thanatin. Here, the hybrid magainin–thanatin (MT) antimicrobial peptide was designed through bioinformatics analysis. The recombinant MT antimicrobial peptide was successfully expressed and purified in Escherichia coli BL21 (DE3). The molecular weight of the hybrid MT antimicrobial peptide was about 3.35?kDa. Moreover, the target protein indeed has an inhibitory effect on Staphylococcus aureus, E. coli DH5α, and Bacillus subtilis, with the minimum inhibitory concentrations 16.5, 20, and 9?μM, respectively. The rational designed hybrid MT antimicrobial peptide will hopefully provide large-scale fermentable antimicrobial peptides in the industrial production in the future.     

NFκB signaling in carcinogenesis and as a potential molecular target for cancer therapy

It has become increasingly clear that deregulation of the NFκB signaling cascade is a common underlying feature of many human ailments including cancers. The past two decades of intensive research on NFκB has identified the basic mechanisms that govern the functioning of this pathway but uncovering the details of why this pathway works differently in different cellular contexts or how it interacts with other signaling pathways remains a challenge. A thorough understanding of these processes is needed to design better and more efficient therapeutic approaches to treat complex diseases like cancer. In this review, we summarize the literature documenting the involvement of NFκB in cancer, and then focus on the approaches that are being undertaken to develop NFκB inhibitors towards treatment of human cancers.     

α-Fucosidase as a novel convenient biomarker for cellular senescence

Due to its role in aging and antitumor defense, cellular senescence has recently attracted increasing interest. However, there is currently no single specific marker that can unequivocally detect senescent cells. Here, we identified α-L-fucosidase (α-Fuc) as a novel sensitive biomarker for cellular senescence. Regardless of the stress stimulus and cell type, α-Fuc activity was induced in all canonical types of cellular senescence, including replicative, DNA damage- and oncogene-induced senescence. Strikingly, in most models the degree of α-Fuc upregulation was higher than the induction of senescence-associated β-galactosidase (SA-β-Gal), the current gold standard for senescence detection. As α-Fuc is convenient and easy to measure, we suggest its utility as a valuable marker, in particular in cells with low SA-β-Gal activity.     

Old drugs as lead compounds for a new disease? Binding analysis of SARS coronavirus main proteinase with HIV, psychotic and parasite drugs

The SARS-associated coronavirus (SARS-CoV) main proteinase is a key enzyme in viral polyprotein processing. To allow structure-based design of drugs directed at SARS-CoV main proteinase, we predicted its binding pockets and affinities with existing HIV, psychotic and parasite drugs (lopinavir, ritonavir, niclosamide and promazine), which show signs of inhibiting the replication of SARS-CoV. Our results suggest that these drugs and another two HIV inhibitors (PNU and UC2) could be used as templates for designing SARS-CoV proteinase inhibitors.     

Novel tacrine-related drugs as potential candidates for the treatment of Alzheimer’s disease

A summary of the recently published efforts on tacrine derivatives as a renewed potential therapeutic approach for the treatment of Alzheimer’s disease is presented.