Structure optimization of a new class of PPARγ antagonists.

Peroxisome proliferator-activated receptor gamma (PPARγ) modulators have found wide application for the treatment of cancers, metabolic disorders and inflammatory diseases. Contrary to PPARγ agonists, PPARγ antagonists have been much less studied and although they have shown immunomodulatory effects, there is still no therapeutically useful PPARγ antagonist on the market. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), the recently described (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB, T-10017) is a promising prototype for a new class of PPARγ antagonists. It exhibits competitive antagonism against rosiglitazone mediated activation of PPARγ ligand binding domain (PPARγLBD) in a transactivation assay in HEK293T cells with an IC50 of 4.3 µM against 1 µM rosiglitazone. The aim of this study was to investigate the structure-activity relationships (SAR) of the MTTB scaffold focusing on improving its physicochemical properties. Through this optimization, 34 new derivatives were prepared and characterized. Two new potent compounds (T-10075 and T-10106) with much improved drug-like properties and promising pharmacokinetic profile were identified.

Chronic alcohol abuse may lead to high skin iron content, but not to hepatic siderosis.

INTRODUCTION: In forensic cases, ante mortem chronic alcohol abuse can be of central importance in clarifying circumstances of death. However, reliable markers of alcohol consumption, which are still available postmortem, are needed. In addition to medical history data which may not be always authentic, the determination of ethyl glucuronide (EtG) in hair as a promising parameter is of no value in cases of missing or cosmetically treated hair. On the other hand, there exist reports that iron ions accumulate in liver tissue (siderosis) during chronic, excessive alcohol consumption, which, therefore, may be useful to serve as alcohol abuse correlate. However, the influence of ethanol on iron stored in the liver has not been adequately investigated and the study situation appears to be inconsistent. AIMS: The aim of the present study was to assess the suitability of assaying iron concentrations in liver and other tissues as postmortem alcoholism marker. METHODS: The iron concentration in tissue samples (liver, brain, skin, pancreas, spleen), vitreous fluid and blood taken during autopsy was analyzed by atomic absorption spectroscopy. The analytical method has been validated before. Cases were divided into two groups: chronic alcohol abusers and non-chronic alcohol consumers including total abstainers using ethyl glucuronide levels in hair as well as anamnestic data as criteria. RESULTS: No elevated iron concentrations in the liver of chronic alcohol abusers were detected. Surprisingly, the iron concentration in skin tissue was found to be significantly higher in cases of chronic alcohol abuse, independent on whether fatty liver or liver cirrhosis was present (as diagnosedduring autopsy). In 18.5% of the cases, chronic alcohol abuse was not confirmed by the EtG concentration in hair. Thus, anamnestic data should not be overestimated. CONCLUSION: The general assumption that chronic alcohol abuse induces hepatic siderosis, i.e. high iron concentrations in liver tissue, has not been supported by results of the present study. However, there seems to exist a correlation between chronic alcohol abuse and high iron concentrations in the skin.

Boosting Anti-Inflammatory Potency of Zafirlukast by Designed Polypharmacology.

Multitarget design offers access to bioactive small molecules with potentially superior efficacy and safety. Particularly multifactorial chronic inflammatory diseases demand multiple pharmacological interventions for stable treatment. By minor structural changes, we have developed a close analogue of the cysteinyl-leukotriene receptor antagonist zafirlukast that simultaneously inhibits soluble epoxide hydrolase and activates peroxisome proliferator-activated receptor γ. The triple modulator exhibits robust anti-inflammatory activity in vivo and highlights the therapeutic potential of designed multitarget agents.

Combined Approach of Backbone Amide Linking and On-Resin N-Methylation for the Synthesis of Bioactive and Metabolically Stable Peptides.

Rhabdopeptides are a large class of nonribosomal peptides from the bacteria Xenorhabdus and Photorhabdus with low micromolar activity against different protozoa, which are the causative agents of several tropical diseases. The development of a facile and flexible synthesis combining backbone amide linking with on-resin peralkylation for the synthesis of permethylated rhabdopeptides is described. This strategy allows the fast generation of permethylated naturally occurring and artificial rhabdopeptides for a structure-activity study. Furthermore, in vitro experiments revealed their superior properties regarding their stability and passive membrane diffusion.

MH84 improves mitochondrial dysfunction in a mouse model of early Alzheimer's disease.

BACKGROUND: Current approved drugs for Alzheimer's disease (AD) only attenuate symptoms, but do not cure the disease. The pirinixic acid derivate MH84 has been characterized as a dual gamma-secretase/proliferator activated receptor gamma (PPARγ) modulator in vitro. Pharmacokinetic studies in mice showed that MH84 is bioavailable after oral administration and reaches the brain. We recently demonstrated that MH84 improved mitochondrial dysfunction in a cellular model of AD. In the present study, we extended the pharmacological characterization of MH84 to 3-month-old Thy-1 AßPPSL mice (harboring the Swedish and London mutation in human amyloid precursor protein (APP)) which are characterized by enhanced AßPP processing and cerebral mitochondrial dysfunction, representing a mouse model of early AD. METHODS: Three-month-old Thy-1 AßPPSL mice received 12 mg/kg b.w. MH84 by oral gavage once a day for 21 days. Mitochondrial respiration was analyzed in isolated brain mitochondria, and mitochondrial membrane potential and ATP levels were determined in dissociated brain cells. Citrate synthase (CS) activity was determined in brain tissues and MitoTracker Green fluorescence was measured in HEK293-AßPPwt and HEK293-AßPPsw cells. Soluble Aß1-40 and Aß1-42 levels were determined using ELISA. Western blot analysis and qRT-PCR were used to measure protein and mRNA levels, respectively. RESULTS: MH84 reduced cerebral levels of the ß-secretase-related C99 peptide and of Aß40 levels. Mitochondrial dysfunction was ameliorated by restoring complex IV (cytochrome-c oxidase) respiration, mitochondrial membrane potential, and levels of ATP. Induction of PPARγ coactivator-1α (PGC-1α) mRNA and protein expression was identified as a possible mode of action that leads to increased mitochondrial mass as indicated by enhanced CS activity, OXPHOS levels, and MitoTracker Green fluorescence. CONCLUSIONS: MH84 modulates ß-secretase processing of APP and improves mitochondrial dysfunction by a PGC-1α-dependent mechanism. Thus, MH84 seems to be a new promising therapeutic agent with approved in-vivo activity for the treatment of AD.

A Dual Modulator of Farnesoid X Receptor and Soluble Epoxide Hydrolase To Counter Nonalcoholic Steatohepatitis.

Nonalcoholic steatohepatitis arising from Western diet and lifestyle is characterized by accumulation of fat in liver causing inflammation and fibrosis. It evolves as serious health burden with alarming incidence, but there is no satisfying pharmacological therapy to date. Considering the disease's multifactorial nature, modulation of multiple targets might provide superior therapeutic efficacy. In particular, farnesoid X receptor (FXR) activation that revealed antisteatotic and antifibrotic effects in clinical trials combined with inhibition of soluble epoxide hydrolase (sEH) as anti-inflammatory strategy promises synergies. To exploit this dual concept, we developed agents exerting partial FXR agonism and sEH inhibitory activity. Merging known pharmacophores and systematic exploration of the structure-activity relationship on both targets produced dual modulators with low nanomolar potency. Extensive in vitro characterization confirmed high dual efficacy in cellular context combined with low toxicity, and pilot in vivo data revealed favorable pharmacokinetics as well as engagement on both targets in vivo.

Nonacidic Farnesoid X Receptor Modulators.

As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. Clinical results have validated FXR as therapeutic target in hepatic and metabolic diseases. To date, potent FXR agonists share a negatively ionizable function that might compromise their pharmacokinetic distribution and behavior. Here we report the development and characterization of a high-affinity FXR modulator not comprising an acidic residue.

Substrate-specific effects of pirinixic acid derivatives on ABCB1-mediated drug transport.

Pirinixic acid derivatives, a new class of drug candidates for a range of diseases, interfere with targets including PPARα, PPARγ, 5-lipoxygenase (5-LO), and microsomal prostaglandin and E2 synthase-1 (mPGES1). Since 5-LO, mPGES1, PPARα, and PPARγ represent potential anti-cancer drug targets, we here investigated the effects of 39 pirinixic acid derivatives on prostate cancer (PC-3) and neuroblastoma (UKF-NB-3) cell viability and, subsequently, the effects of selected compounds on drug-resistant neuroblastoma cells. Few compounds affected cancer cell viability in low micromolar concentrations but there was no correlation between the anti-cancer effects and the effects on 5-LO, mPGES1, PPARα, or PPARγ. Most strikingly, pirinixic acid derivatives interfered with drug transport by the ATP-binding cassette (ABC) transporter ABCB1 in a drug-specific fashion. LP117, the compound that exerted the strongest effect on ABCB1, interfered in the investigated concentrations of up to 2µM with the ABCB1-mediated transport of vincristine, vinorelbine, actinomycin D, paclitaxel, and calcein-AM but not of doxorubicin, rhodamine 123, or JC-1. In silico docking studies identified differences in the interaction profiles of the investigated ABCB1 substrates with the known ABCB1 binding sites that may explain the substrate-specific effects of LP117. Thus, pirinixic acid derivatives may offer potential as drug-specific modulators of ABCB1-mediated drug transport.

N-Benzylbenzamides: A Novel Merged Scaffold for Orally Available Dual Soluble Epoxide Hydrolase/Peroxisome Proliferator-Activated Receptor γ Modulators.

Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC50 = 0.3 µM/PPARγ EC50 = 0.3 µM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.

Identification and characterisation of a prototype for a new class of competitive PPARγ antagonists.

Understanding of the physiological role of peroxisome proliferator-activated receptor gamma (PPARγ) offers new opportunities for the treatment of cancers, immune disorders and inflammatory diseases. In contrast to PPARγ agonists, few PPARγ antagonists have been studied, though they do exert immunomodulatory effects. Currently, no therapeutically useful PPARγ antagonist is commercially available. The aim of this study was to identify and kinetically characterise a new competitive PPARγ antagonist for therapeutic use. A PPARγ-dependent transactivation assay was used to kinetically characterise (E)-2-(5-((4-methoxy-2-(trifluoromethyl)quinolin-6-yl)methoxy)-2-((4-(trifluoromethyl)benzyl)oxy)-benzylidene)-hexanoic acid (MTTB) in kidney, T and monocytic cell lines. Cytotoxic effects were analysed and intracellular accumulation of MTTB was assessed by tandem mass spectrometry (LC-MS/MS). Potential interactions of MTTB with the PPARγ protein were suggested by molecular docking analysis. In contrast to non-competitive, irreversible inhibition caused by 2-chloro-5-nitrobenzanilide (GW9662), MTTB exhibited competitive antagonism against rosiglitazone in HEK293T and Jurkat T cells, with IC50 values in HEK293T cells of 4.3µM and 1.6µM, using the PPARγ ligand binding domain (PPARγ-LBD) and the full PPARγ protein, respectively. In all cell lines used, however, MTTB showed much higher intracellular accumulation than GW9662. MTTB alone exhibited weak partial agonistic effects and low cytotoxicity. Molecular docking of MTTB with the PPARγ-LBD supported direct interaction with the nuclear receptor. MTTB is a promising prototype for a new class of competitive PPARγ antagonists. It has weak partial agonistic and clear competitive antagonistic characteristics associated with rapid cellular uptake. Compared to commercially available PPARγ modulators, this offers the possibility of dose regulation of PPARγ and immune responses.

Aminothiazole-featured pirinixic acid derivatives as dual 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 inhibitors with improved potency and efficiency in vivo.

Dual inhibition of microsomal prostaglandin E2 synthase-1 (mPGES-1) and 5-lipoxygenase (5-LO) is currently pursued as potential pharmacological strategy for treatment of inflammation and cancer. Here we present a series of 26 novel 2-aminothiazole-featured pirinixic acid derivatives as dual 5-LO/mPGES-1 inhibitors with improved potency (exemplified by compound 16 (2-[(4-chloro-6-{[4-(naphthalen-2-yl)-1,3-thiazol-2-yl]amino}pyrimidin-2-yl)sulfanyl]octanoic acid) with IC50 = 0.3 and 0.4 µM, respectively) and bioactivity in vivo. Computational analysis presumes binding sites of 16 at the tip of the 5-LO catalytic domain and within a subpocket of the mPGES-1 active site. Compound 16 (10 µM) hardly suppressed cyclooxygenase (COX)-1/2 activities, failed to inhibit 12/15-LOs, and is devoid of radical scavenger properties. Finally, compound 16 reduced vascular permeability and inflammatory cell infiltration in a zymosan-induced mouse peritonitis model accompanied by impaired levels of cysteinyl-leukotrienes and prostaglandin E2. Together, 2-aminothiazole-featured pirinixic acids represent potent dual 5-LO/mPGES-1 inhibitors with an attractive pharmacological profile as anti-inflammatory drugs.

Structure-activity relationship and in vitro pharmacological evaluation of imidazo[1,2-a]pyridine-based inhibitors of 5-LO.

BACKGROUND: 5-LO is an important enzyme involved in the biosynthesis of leukotrienes, which are lipid mediators of immune and inflammation responses, with important roles in respiratory disease, cardiovascular disease, immune responses and certain types of cancer. Therefore, this enzyme has been investigated as a potential target for the treatment of these pathophysiological conditions. RESULTS: 5-LO inhibitory potential was investigated in intact polymorphonuclear leukocytes, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and in vitro pharmacological evaluation. Chemical modifications for lead optimization via straight forward synthesis was used to combine small polar groups, which led to a suitable candidate (IC50 [polymorphonuclear leukocytes] = 1.15 µM, IC50 [S100] = 0.29 µM) with desired in vitro biopharmaceutical profiles in terms of solubility (451.9 µg/ml) and intrinsic clearance without demonstrating any cytotoxicity. CONCLUSION: Compound 9l is a novel, potent and selective 5-LO inhibitor with favorable preclinical drug-like properties.

Plant derived omega-3-fatty acids protect mitochondrial function in the brain.

Perilla frutescens seed oil (PFSO) represents a rich source of unsaturated fatty acids, especially of omega-3 linolenic acid and is commonly used as herbal food supplement with beneficial effects on the vascular system. The present study investigated the effects of PFSO on the central nervous system (CNS) and provides new insight into potential mechanisms for protective properties of unsaturated fatty acids within the CNS. PFSO was administered chronically to guinea pigs and neuroprotective properties were assessed ex vivo in dissociated brain cells. Dissociated brain cells isolated from PFSO treated guinea pigs were less vulnerable against nitrosative stress as indicated by decreased levels of reactive oxygen species, by stabilized mitochondrial membrane potential and enhanced levels of adenosine-triphosphate. In the brain, levels of oleic, linoleic, arachidonic and docosahexaenoic acids were significantly enhanced. However, fatty acid composition and membrane dynamics of isolated synaptosomal plasma membranes were not affected. Our findings provide new insights into the potential mechanisms for the neuroprotective actions of unsaturated fatty acids and identified PFSO as promising nutraceutical and possible alternative to fish oil supplements to provide healthful activities in the brain.

Pirinixic acid derivatives as novel dual inhibitors of microsomal prostaglandin E2 synthase-1 and 5-lipoxygenase.

Dual inhibition of the prostaglandin (PG) and leukotriene (LT) biosynthetic pathway is supposed to be superior over single interference, both in terms of efficacy and side effects. Here, we present a novel class of dual microsomal PGE(2) synthase-1/5-lipoxygenase (5-LO) inhibitors based on the structure of pirinixic acid [PA, 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)acetic acid, compound 1]. Target-oriented structural modification of 1, particularly alpha substitution with extended n-alkyl or bulky aryl substituents and concomitant replacement of the 2,3-dimethylaniline by a biphenyl-4-yl-methane-amino residue, resulted in potent suppression of mPGES-1 and 5-LO activity, exemplified by 2-(4-(biphenyl-4-ylmethylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (7b, IC(50) = 1.3 and 1 microM, respectively). Select compounds also potently reduced PGE(2) and 5-LO product formation in intact cells. Importantly, inhibition of cyclooxygenases-1/2 was significantly less pronounced. Taken together, these pirinixic acid derivatives constitute a novel class of dual mPGES-1/5-LO inhibitors with a promising pharmacological profile and a potential for therapeutic use.

Carnosic acid and carnosol, phenolic diterpene compounds of the labiate herbs rosemary and sage, are activators of the human peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand activated transcription factor, belonging to the metazoan family of nuclear hormone receptors. Activation of PPARgamma increases the transcription of enzymes involved in primary metabolism, leading to lower blood levels of fatty acids and glucose. Hence, PPARgamma represents the major target for the glitazone type of drugs currently being used clinically for the treatment of type 2 diabetes. Furthermore, activators of PPARgamma show beneficial anti-inflammatory and anti-tumour effects. Utilizing a fusion receptor of the yeast Gal4-DNA binding domain joined to the hinge region and ligand binding domain of the human PPARgamma in combination with a Gal4-driven luciferase reporter gene, cotransfected into Cos7 cells, we tested sage and rosemary extracts prepared with 80 % aqueous ethanol for possible PPARgamma activation. This revealed that both extracts are capable of selectively activating Gal4-PPARgamma fusion receptor, in a concentration-dependent manner, with EC (50) values of 22.8 +/- 8.4 mg/L and 33.7 +/- 7.3 mg/L for rosemary and sage, respectively. Subsequent analysis of the characteristic constituents revealed the phenolic diterpene compounds carnosol, present in both herbs, and carnosic acid to be active principles of these extracts, showing EC (50) values of 41.2 +/- 5.9 microM and 19.6 +/- 2.0 microM, respectively. Thus it can be concluded that the glucose lowering effect reported recently for rosemary may be attributed to PPARgamma activation. Moreover, our observations may also explain the anti-inflammatory and antiproliferative effects of both compounds published previously.