Covalent Modifier Discovery Using Hydrogen/Deuterium Exchange-Mass Spectrometry.

Covalent ligands are generally filtered out of chemical libraries used for high-throughput screening, because electrophilic functional groups are considered to be pan-assay interference compounds (PAINS). Therefore, screening strategies that can distinguish true covalent ligands from PAINS are required. Hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for evaluating protein stability. Here, we report a covalent modifier screening approach using HDX-MS. In this study, HDX-MS was used to classify peroxisome proliferator-activated receptor γ (PPARγ) and vitamin D receptor ligands. HDX-MS could discriminate the strength of ligand-protein interactions. Our HDX-MS screening method identified LT175 and nTZDpa, which can bind concurrently to the PPARγ ligand-binding domain (PPARγ-LBD) with synergistic activation. Furthermore, iodoacetic acid was identified as a novel covalent modifier that stabilizes the PPARγ-LBD.

Liquid Crystal Monomer: A Potential PPARγ Antagonist.

Liquid crystal monomers (LCMs) are a large family of artificial ingredients that have been widely used in global liquid crystal display (LCD) industries. As a major constituent in LCDs as well as the end products of e-waste dismantling, LCMs are of growing research interest with regard to their environmental occurrences and biochemical consequences. Many studies have analyzed LCMs in multiple environmental matrices, yet limited research has investigated the toxic effects upon exposure to them. In this study, we combined in silico simulation and in vitro assay validation along with omics integration analysis to achieve a comprehensive toxicity elucidation as well as a systematic mechanism interpretation of LCMs for the first time. Briefly, the high-throughput virtual screen and reporter gene assay revealed that peroxisome proliferator-activated receptor gamma (PPARγ) was significantly antagonized by certain LCMs. Besides, LCMs induced global metabolome and transcriptome dysregulation in HK2 cells. Notably, fatty acid ß-oxidation was conspicuously dysregulated, which might be mediated through multiple pathways (IL-17, TNF, and NF-kB), whereas the activation of AMPK and ligand-dependent PPARγ antagonism may play particularly important parts. This study illustrated LCMs as a potential PPARγ antagonist and explored their toxicological mode of action on the trans-omics level, which provided an insightful overview in future chemical risk assessment.

Scavenging of Superoxide in Aprotic Solvents of Four Isoflavones That Mimic Superoxide Dismutase.

Isoflavones are plant-derived natural products commonly found in legumes that show a large spectrum of biomedical activities. A common antidiabetic remedy in traditional Chinese medicine, Astragalus trimestris L. contains the isoflavone formononetin (FMNT). Literature reports show that FMNT can increase insulin sensitivity and potentially target the peroxisome proliferator-activated receptor gamma, PPARγ, as a partial agonist. PPARγ is highly relevant for diabetes control and plays a major role in Type 2 diabetes mellitus development. In this study, we evaluate the biological role of FMNT, and three related isoflavones, genistein, daidzein and biochanin A, using several computational and experimental procedures. Our results reveal the FMNT X-ray crystal structure has strong intermolecular hydrogen bonding and stacking interactions which are useful for antioxidant action. Cyclovoltammetry rotating ring disk electrode (RRDE) measurements show that all four isoflavones behave in a similar manner when scavenging the superoxide radical. DFT calculations conclude that antioxidant activity is based on the familiar superoxide σ-scavenging mode involving hydrogen capture of ring-A H7(hydroxyl) as well as the π-π (polyphenol-superoxide) scavenging activity. These results suggest the possibility of their mimicking superoxide dismutase (SOD) action and help explain the ability of natural polyphenols to assist in lowering superoxide concentrations. The SOD metalloenzymes all dismutate O2•- to H2O2 plus O2 through metal ion redox chemistry whereas these polyphenolic compounds do so through suitable hydrogen bonding and stacking intermolecular interactions. Additionally, docking calculations suggest FMNT can be a partial agonist of the PPARγ domain. Overall, our work confirms the efficacy in combining multidisciplinary approaches to provide insight into the mechanism of action of small molecule polyphenol antioxidants. Our findings promote the further exploration of other natural products, including those known to be effective in traditional Chinese medicine for potential drug design in diabetes research.

Structural Biology Inspired Development of a Series of Human Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) Ligands: From Agonist to Antagonist.

Recent progress in the structural and molecular pharmacological understanding of the nuclear receptor, peroxisome proliferator-activated receptor gamma (hPPARγ)-a transcription factor with pleiotropic effects on biological responses-has enabled the investigation of various graded hPPARγ ligands (full agonist, partial agonist, and antagonist). Such ligands are useful tools to investigate the functions of hPPARγ in detail and are also candidate drugs for the treatment of hPPARγ-mediated diseases, such as metabolic syndrome and cancer. This review summarizes our medicinal chemistry research on the design, synthesis, and pharmacological evaluation of a covalent-binding and non-covalent-binding hPPARγ antagonist, both of which have been created based on our working hypothesis of the helix 12 (H12) holding induction/inhibition concept. X-ray crystallographic analyses of our representative antagonists complexed with an hPPARγ ligand binding domain (LBD) indicated the unique binding modes of hPPARγ LBD, which are quite different from the binding modes observed for hPPARγ agonists and partial agonists.

Comparative Computational Screening of Natural-based Partial Agonists for PPARγ Receptor.

INTRODUCTION: The nuclear transcription factor PPARγ, which can modulate cell growth via proliferation and apoptosis-related mechanisms, is a promising target in cancer therapy. This study aims to focus on PPARγ as the target and use virtual screening to find hits. METHODS: A set of 5,677 flavonoid compounds were filtered by subjecting them to descriptor-based drug-likeness and ADMET strategies to discover drug-like compounds. The candidates' modes of binding to PPARγ were then evaluated using docking and MD simulation. PharmMapper was used to identify the potential targets of selected hits. The pharmacological network was constructed based on the GO and KEGG pathway analysis. RESULTS: In primary screening, 3,057 compounds met various drug-likeness criteria and docked well as partial agonists in the PPARγ-LBD. Five compounds (euchrenone b1, kaempferol-7-Orhamnoside, vincetoxicoside B, morusin, and karanjin) were selected with the use of ADMET profiles for further MD simulation investigation. Based on the PharmMapper findings, 52 proteins were then submitted to GO and KEGG enrichment analysis. As expected by GO and KEGG pathway enrichment studies, core targets were enriched in the PI3K-Akt signaling pathway (p < 0.01), indicating that certain chemicals may be involved in cancer processes. CONCLUSION: Our results suggested that the selected compounds might have sufficient drug-likeness, pharmacokinetics, and in silico bioactivity by acting as PPARγ partial agonists. Although much work remains to illuminate extensive cancer therapeutic/ chemopreventive efficacy of flavonoids in vivo, in silico methodology of our cheminformatics research may be able to provide additional data regarding the efficacy and safety of potential candidates for therapeutic targets.

Computational investigation of phytochemicals from Abrus precatorius seeds as modulators of peroxisome proliferator-activated receptor gamma (PPARγ).

Type 2 diabetes mellitus remains global health challenge with involvement of both insulin resistance and dysfunctional insulin secretion from the pancreatic ß-cell. Currently, peroxisome proliferator-activated receptor gamma (PPARγ) has been established to play a significant role in glucose homeostasis and insulin sensitization contributing to the pathogenesis of type 2 diabetes mellitus. Hence, this study used in-silico analysis to predict PPARγ antagonists from the natural compounds. ADMET screening, structure-based virtual screening and MM/GBSA calculations of phytochemicals from HPLC analysis of A. precatorius seeds were performed against PPARγ using Maestro Schrodinger suite, followed by the MD simulation of top hit compounds and reference ligand using GROMACS. The quantum chemical calculations of the compounds were performed using Spartan 14 computational chemistry software. The five compounds showed varying degree of binding affinity against PPARγ, the post-docking analysis confirmed strong interaction against the amino acid residues of the binding site of the target. Chlorogenic acid showed the highest docking score (-10.719 kcal/mol) among the compounds comparable to the reference ligand (acarbose = -10.634 kcal/mol). Additionally, MM/GBSA binding free energy (ΔGbind) calculations support the modulatory potential for the docked compounds, which exclusively revealed the highest binding energy for the compounds than the reference ligand (acarbose). The MD simulations suggested the stability of Chlorogenic acid and Quercetin in complex with PPARγ at least in the time period of 90 ns after initial equilibration state with more H-bond observed between the target-hit compounds complex compared to the Acarbose-PPARγ complex. ADMET profile revealed that the five compounds were favorably druggable and promising drug candidates. The quantum chemical calculations showed that the compounds possess better bioactivity and chemical reactivity with favorable intra-molecular charge transfer as electron-donor and electron-acceptor. This study revealed that bioactive compounds especially chlorogenic acid and quercetin identified from A. precatorius seeds demonstrated good modulatory potential against PPARγ compared to acarbose. Therefore, these compounds require further experimental validation for the discovery of new antagonist of PPARγ for developing new anti-diabetes therapy.Communicated by Ramaswamy H. Sarma.

In silico and in vitro analysis of PPAR - α / γ dual agonists: Comparative evaluation of potential phytochemicals with anti-obesity drug orlistat.

Obesity is an abnormal fat accumulation disorder in the metabolic syndrome constellation, and a risk factor for diabetes, cardiovascular disorders, non-alcoholic fatty liver disease (NAFLD), and cancer. Nuclear receptors (Peroxisome proliferator-activated receptor, PPAR) are implicated in metabolic syndrome and NAFLD, and have potential for therapeutic targeting. Nuclear receptors are ligand-dependent transcription factors that have diverse roles in metabolism, including regulating genes involved in lipid and glucose metabolism, modulating inflammatory genes, and are crucial for maintaining metabolic flexibility. PPAR activates adipose triglyceride lipase, which then releases fatty acids as ligands for PPAR, indicating the interdependency of nuclear receptors and lipases. Here, molecular docking was performed with selected phytochemical ligands that can bind with PPAR-α/γ (PDB ID: 2ZNN and 2ATH, respectively) using Glide module of Schrodinger software followed by molecular dynamics simulation study using Desmond module, and ADMET analysis. Interestingly, orlistat which is a well-known lipase and fatty acid synthase inhibitor also demonstrated favorable binding affinity with both PPAR-α/γ (-10.96 kcal/mol against PPARα and -10.26 kcal/mol against PPARγ). The highest docking scores were however shown by the flavonoids - rutin (-14.88 kcal/mol against PPARα and -13.64 kcal/mol against PPARγ), and its aglycone, quercetin (-10.08 kcal/mol in PPARα and -9.89 kcal/mol in PPARγ). The other phytochemicals (genistein, esculin, daidzin, naringenin, daidzein, dihydroxy coumarin, hydroquinone) showed lower binding affinity as dual agonists. The anti-obesity effects were experimentally validated in cultured adipocytes, which revealed better lipid inhibition by rutin and quercetin than orlistat (quercetin > rutin > orlistat) pointing to their strong potential in anti-obesity treatment.

Immobilization of peroxisome proliferator-activated receptor gamma and the application in screening modulators of the receptor from herbal medicine.

Screening and identification of potential compounds from herbal medicine is a prevailing way to find a lead for the development of innovative drugs. This promotes the development of new methods that are feasible in complex matrices. Here, we described a one-step reversible methodology to immobilize nuclear peroxisome proliferator-activated receptor gamma (PPARγ) onto amino microsphere coated with a DNA strand specifically binding to the receptor. The specific interaction allowed us to achieve the immobilization of PPARγ by mixing the DNA modified microspheres with E. coli lysates expressing the receptor. Characterization of the immobilized receptor was carried out by morphology and binding specificity analysis. Feasibility of immobilized PPARγ in the drug-receptor interaction analysis was performed by an injection amount-dependent method. Besides, immobilized PPARγ was also applied in screening modulators of the receptor from Coptidis Rhizoma extract. The binding of the screened compounds to PPARγ was examined by time-resolved fluorescence resonance energy transfer assay. The results showed that immobilized PPARγ was stable for thirty days with a high-specificity of ligand recognition at the subtype receptor level. Berberine and palmatine were the bioactive compounds of Coptidis Rhizoma specifically binding to PPARγ. The two compounds exhibited half maximal inhibitory concentrations of 4.11 and 2.98 µM during their binding to the receptor. We concluded that the current method is possible to become a common strategy for the immobilization of nuclear receptors, and the immobilized receptor is a high throughput method for recognizing and separating the receptor modulators from complex matrices including herbal medicine.

Phenolic Lipids Derived from Cashew Nut Shell Liquid to Treat Metabolic Diseases.

Metabolic diseases are increasing at staggering rates globally. The peroxisome proliferator-activated receptors (PPARα/γ/δ) are fatty acid sensors that help mitigate imbalances between energy uptake and utilization. Herein, we report compounds derived from phenolic lipids present in cashew nut shell liquid (CNSL), an abundant waste byproduct, in an effort to create effective, accessible, and sustainable drugs. Derivatives of anacardic acid and cardanol were tested for PPAR activity in HEK293 cell co-transfection assays, primary hepatocytes, and 3T3-L1 adipocytes. In vivo studies using PPAR-expressing zebrafish embryos identified CNSL derivatives with varying tissue-specific activities. LDT409 (23) is an analogue of cardanol with partial agonist activity for PPARα and PPARγ. Pharmacokinetic profiling showed that 23 is orally bioavailable with a half-life of 4 h in mice. CNSL derivatives represent a sustainable source of selective PPAR modulators with balanced intermediate affinities (EC50 ∼ 100 nM to 10 µM) that provide distinct and favorable gene activation profiles for the treatment of diabetes and obesity.

A Novel Partial PPARγ Agonist Has Weaker Lipogenic Effect in Adipocytes and Stimulates GLUT4 Translocation in Skeletal Muscle Cells via AMPK-Dependent Signaling.

INTRODUCTION: Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are highly effective in treating insulin resistance. However, associated side effects such as weight gain due to increase in adipogenesis and lipogenesis hinder their clinical use. The aim of the study was to design and synthesize novel partial PPARγ agonists with weaker lipogenic effect in adipocytes and enhanced glucose transporter 4 (GLUT4) translocation stimulatory effect in skeletal muscle cells. METHODS: Novel partial PPARγ agonists (GS1, GS2, and GS3) were designed and screened to predict their binding interactions with PPARγ by molecular docking. The stability of the docked ligand-PPARγ complex was studied by molecular dynamics (MD) simulation. The cytotoxicity of synthesized compounds was tested in 3T3-L1 adipocytes and L6 myoblasts by MTT assay. The lipogenic effect was investigated in 3T3-L1 adipocytes using oil red O staining and GLUT4 translocation stimulatory effect in L6-GLUT4myc myotubes by an antibody-coupled colorimetric assay. RESULTS: The molecular docking showed the binding interactions between designed agonists and PPARγ. MD simulation demonstrated good stability between the GS2-PPARγ complex. GS2 and GS3 did not show any significant effect on cell viability up to 80 or 100 µM concentration. Pioglitazone treatment significantly increased intracellular lipid accumulation in adipocytes compared to control. However, this effect was significantly less in GS2- and GS3-treated conditions compared to pioglitazone at 10 µM concentration, indicating weaker lipogenic effect. Furthermore, GS2 significantly stimulated GLUT4 translocation to the plasma membrane in a dose-dependent manner via the AMPK-dependent signaling pathway in skeletal muscle cells. CONCLUSION: GS2 may be a promising therapeutic agent for the treatment of insulin resistance and type 2 diabetes mellitus without adiposity.

Probing intermolecular interactions and binding stability of kaempferol, quercetin and resveratrol derivatives with PPAR-γ: docking, molecular dynamics and MM/GBSA approach to reveal potent PPAR- γ agonist against cancer.

Peroxisome Proliferator-Activated Receptors-γ (PPAR-γ), a ligand-activated transcription factor, suggested having anti-inflammatory effects by activating the target genes when bound to the ligand. Herein, we examined a conformational analysis of 8708 derivatives of Kaempferol, Quercetin, and Resveratrol, the prime activators of PPAR-γ molecular target by employing molecular docking and dynamic simulation pipeline to screen out potential agonists. The structure-based docking procedure performed by FlexX tool shortlisted high binding affinities of these derivatives of Kaempferol, Quercetin and Resveratrol with the protein receptor with a score of -38.94 kcal/mol (4'-Carboxy-5, 7-Dihydroxyflavone-CDHF), -41.63 kcal/mol (Demethyltorosaflavone D- DMTF) and -31.52 kcal/mol (Resveratrol-O-disulphate- RD) respectively, signifying the selected derivatives forms interactions like H-bond, Aromatic H-Bond, Pi-Pi stacking and salt bridges with PPAR-γ. The PPAR-γ-derivative complex was stabilized by intermolecular hydrogen bonds and stacking interactions. A greater interaction was significantly observed between the binding affinities of derivatives compared to the standards. Based on the root mean square deviation (RMSD) and root mean square fluctuation (RMSF) carried by the means of high-speed molecular dynamics (MD) and simulation of best-docked poses, the ligand, DMTF attained the most favored interaction with PPAR-γ. Thus, it appeared to have high chemical scaffold diversity and may confer high drug-likeness. The binding free energy (ΔG) led us to manifest Quercetin derivative to have a key role for PPAR-γ receptor. The result obtained clearly indicates the exploitation of the promising new drug leads that may further influence in synthesizing and analyzing the development as anti-cancer agonists.Communicated by Ramaswamy H. Sarma.

Multi-conformational frame from molecular dynamics as a structure-based pharmacophore model for mapping, screening and identifying ligands against PPAR-γ: a new protocol to develop promising candidates.

Despite intensive research on clinical and molecular factors, the development of antidiabetic drugs in the last few decades is decelerating and as a result, the number of drugs approved by the US FDA is reduced. Hence, there is a persistent need for the innovative development of novel anti-diabetic drugs. Recent studies have provided ample proof that the peroxisome proliferator-activated receptor gamma (PPARγ), a ligand-activated transcription factor and its co-activator PGC-1 alpha may serve as good candidates for the treatment of several metabolic disorders. Therefore, in this study, 50 ns molecular dynamics (MD) simulations of the ligand-receptor complex were carried out and the most populated cluster of rosiglitazone bound to crucial amino acids during dynamics studies were selected to generate multi-conformation frame and further dynamic pharmacophore models. Finally, three pharmacophore models were generated, and 10 hits were retrieved as final lead candidates by virtual screening of ZINC database and molecular docking. The study reveals that the amino acids Met364, Lys367, His449, Leu453, Leu469, and Tyr473 play a crucial role in the binding of the compounds at the active site of PPARγ and the selected compounds from the ZINC database showed promising binding as compared to rosiglitazone. Further, ADMET studies were carried out to define the pharmacokinetic properties of promising PPARγ ligand candidates.Communicated by Ramaswamy H. Sarma.

Multi-Targeted Molecular Docking, Pharmacokinetics, and Drug-Likeness Evaluation of Okra-Derived Ligand Abscisic Acid Targeting Signaling Proteins Involved in the Development of Diabetes.

Diabetes mellitus is a global threat affecting millions of people of different age groups. In recent years, the development of naturally derived anti-diabetic agents has gained popularity. Okra is a common vegetable containing important bioactive components such as abscisic acid (ABA). ABA, a phytohormone, has been shown to elicit potent anti-diabetic effects in mouse models. Keeping its anti-diabetic potential in mind, in silico study was performed to explore its role in inhibiting proteins relevant to diabetes mellitus- 11ß-hydroxysteroid dehydrogenase (11ß-HSD1), aldose reductase, glucokinase, glutamine-fructose-6-phosphate amidotransferase (GFAT), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and Sirtuin family of NAD(+)-dependent protein deacetylases 6 (SIRT6). A comparative study of the ABA-protein docked complex with already known inhibitors of these proteins relevant to diabetes was compared to explore the inhibitory potential. Calculation of molecular binding energy (ΔG), inhibition constant (pKi), and prediction of pharmacokinetics and pharmacodynamics properties were performed. The molecular docking investigation of ABA with 11-HSD1, GFAT, PPAR-gamma, and SIRT6 revealed considerably low binding energy (ΔG from -8.1 to -7.3 Kcal/mol) and predicted inhibition constant (pKi from 6.01 to 5.21 µM). The ADMET study revealed that ABA is a promising drug candidate without any hazardous effect following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge.

The Demethoxy Derivatives of Curcumin Exhibit Greater Differentiation Suppression in 3T3-L1 Adipocytes Than Curcumin: A Mechanistic Study of Adipogenesis and Molecular Docking.

Curcumin is a known anti-adipogenic agent for alleviating obesity and related disorders. Comprehensive comparisons of the anti-adipogenic activity of curcumin with other curcuminoids is minimal. This study compared adipogenesis inhibition with curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC), and their underlying mechanisms. We differentiated 3T3-L1 cells in the presence of curcuminoids, to determine lipid accumulation and triglyceride (TG) production. The expression of adipogenic transcription factors and lipogenic proteins was analyzed by Western blot. A significant reduction in Oil red O (ORO) staining was observed in the cells treated with curcuminoids at 20 µM. Inhibition was increased in the order of curcumin < DMC < BDMC. A similar trend was observed in the detection of intracellular TG. Curcuminoids suppressed differentiation by downregulating the expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), leading to the downregulation of the lipogenic enzymes acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS). AMP-activated protein kinase α (AMPKα) phosphorylation was also activated by BDMC. Curcuminoids reduced the release of proinflammatory cytokines and leptin in 3T3-L1 cells in a dose-dependent manner, with BDMC showing the greatest potency. BDMC at 20 µM significantly decreased leptin by 72% compared with differentiated controls. Molecular docking computation indicated that curcuminoids, despite having structural similarity, had different interaction positions to PPARγ, C/EBPα, and ACC. The docking profiles suggested a possible interaction of curcuminoids with C/EBPα and ACC, to directly inhibit their expression.

The therapeutic potential of inhibiting PPARγ phosphorylation to treat type 2 diabetes.

A promising approach for treating type 2 diabetes mellitus (T2DM) is to target the Peroxisome Proliferator-Activated Receptor γ (PPARγ) transcription factor, which regulates the expression of proteins critical for T2DM. Mechanisms involved in PPARγ signaling are poorly understood, yet globally increasing T2DM prevalence demands improvements in drug design. Synthetic, nonactivating PPARγ ligands can abolish the phosphorylation of PPARγ at Ser273, a posttranslational modification correlated with obesity and insulin resistance. It is not understood how these ligands prevent phosphorylation, and the lack of experimental mechanistic information can be attributed to previous ambiguity in the field as well as to limitations in experimental approaches; in silico modeling currently provides the only insight into how ligands block Ser273 phosphorylation. The future availability of experimental evidence is critical for clarifying the mechanism by which ligands prevent phosphorylation and should be the priority of future T2DM-focused research. Following this, the properties of ligands that enable them to block phosphorylation can be improved upon to generate ligands tailored for blocking phosphorylation and therefore restoring insulin sensitivity. This would represent a significant step forward for treating T2DM. This review summarizes current knowledge of the roles of PPARγ in T2DM as well as the effects of synthetic ligands on the modulation of these roles. We hypothesize potential factors that contribute to the reduction in recent developments and summarize what has currently been done to shed light on this critical field of research.

Research progress of indole compounds with potential antidiabetic activity.

New types of antidiabetic agents are continually needed with diabetes becoming the epidemic in the world. Indole alkaloids play an important role in natural products owing to their variable structures and versatile biological activities like anticonvulsant, anti-inflammatory, antidiabetic, antimicrobial, and anticancer activities, which are a promising source of novel antidiabetic drugs discovery. The synthesized indole derivatives possess similar properties to natural indole alkaloids. In the last two decades, more and more indole derivatives have been designed and synthesized for searching their bioactivities. This present review describes comprehensive structures of indole compounds with the potential antidiabetic activity including natural indole alkaloids and the synthetic indole derivatives based on the structure classification, summarizes their approaches isolated from natural sources or by synthetic methods, and discusses the antidiabetic effects and the mechanisms of action. Furthermore, this review also provides briefly synthetic procedures of some important indole derivatives.

Acankoreagenin and acankoreosides, a family of lupane triterpenoids with anti-inflammatory properties: an overview.

Acankoreagenin (ACK, also known as acankoreanogenin and HLEDA) and impressic acid are two lupane-type triterpenes that can be isolated from various Acanthopanax and Schefflera species. They efficiently block activation of the NF-κB signaling pathway and the release of proinflammatory cytokines and/or the action of inflammation mediators (HMGB1, iNOS, and NO). These effects are the basis for the antiviral and anticancer activities reported with these pentacyclic compounds or their various glycoside derivatives. More than 15 acankoreosides (Ack-A to -O, and -R) and a few other mono- and bidesmosidic saponins (acantrifoside A and acangraciliside S) derive from the ACK aglycone. Compounds like Ack-A and -B are remarkable anti-inflammatory agents, inhibiting cytokine release from activated macrophages. Despite their effectiveness, ACK and impressic acid are far much less known and studied than the structurally related compounds betulinic acid and 23-hydroxybetulinic acid (anemosapogenin). The structural differences (notably the R/S stereoisomerism of the 3-hydroxyl group) and functional similarities of these compounds are discussed. The complete series of acankoreosides is presented for the first time. These natural products deserve further attention as anti-inflammatory agents, and ACK is recommended as a template for the design of new anticancer and antiviral drugs.

SIRT6 transcriptionally regulates fatty acid transport by suppressing PPARγ.

Pathological lipid accumulation is often associated with enhanced uptake of free fatty acids via specific transporters in cardiomyocytes. Here, we identify SIRT6 as a critical transcriptional regulator of fatty acid transporters in cardiomyocytes. We find that SIRT6 deficiency enhances the expression of fatty acid transporters, leading to enhanced fatty acid uptake and lipid accumulation. Interestingly, the haploinsufficiency of SIRT6 is sufficient to induce the expression of fatty acid transporters and cause lipid accumulation in murine hearts. Mechanistically, SIRT6 depletion enhances the occupancy of the transcription factor PPARγ on the promoters of critical fatty acid transporters without modulating the acetylation of histone 3 at Lys 9 and Lys 56. Notably, the binding of SIRT6 to the DNA-binding domain of PPARγ is critical for regulating the expression of fatty acid transporters in cardiomyocytes. Our data suggest exploiting SIRT6 as a potential therapeutic target for protecting the heart from metabolic diseases.

Heterodimer formation with retinoic acid receptor RXRα modulates coactivator recruitment by peroxisome proliferator-activated receptor PPARγ.

Nuclear receptors (NRs) activate transcription of target genes in response to binding of ligands to their ligand-binding domains (LBDs). Typically, in vitro assays use either gene expression or the recruitment of coactivators to the isolated LBD of the NR of interest to measure NR activation. However, this approach ignores that NRs function as homo- as well as heterodimers and that the LBD harbors the main dimerization interface. Cofactor recruitment is thereby interconnected with oligomerization status as well as ligand occupation of the partnering LBD through allosteric cross talk. Here we present a modular set of homogeneous time-resolved FRET-based assays through which we investigated the activation of PPARγ in response to ligands and the formation of heterodimers with its obligatory partner RXRα. We introduced mutations into the RXRα LBD that prevent coactivator binding but do not interfere with LBD dimerization or ligand binding. This enabled us to specifically detect PPARγ coactivator recruitment to PPARγ:RXRα heterodimers. We found that the RXRα agonist SR11237 destabilized the RXRα homodimer but promoted formation of the PPARγ:RXRα heterodimer, while being inactive on PPARγ itself. Of interest, incorporation of PPARγ into the heterodimer resulted in a substantial gain in affinity for coactivator CBP-1, even in the absence of ligands. Consequently, SR11237 indirectly promoted coactivator binding to PPARγ by shifting the oligomerization preference of RXRα toward PPARγ:RXRα heterodimer formation. These results emphasize that investigation of ligand-dependent NR activation should take NR dimerization into account. We envision these assays as the necessary assay tool kit for investigating NRs that partner with RXRα.

Thiazolidinedione "Magic Bullets" Simultaneously Targeting PPARγ and HDACs: Design, Synthesis, and Investigations of their In Vitro and In Vivo Antitumor Effects.

Monotargeting anticancer agents suffer from resistance and target nonspecificity concerns, which can be tackled with a multitargeting approach. The combined treatment with HDAC inhibitors and PPARγ agonists has displayed potential antitumor effects. Based on these observations, this work involves design and synthesis of molecules that can simultaneously target PPARγ and HDAC. Several out of 25 compounds inhibited HDAC4, and six compounds acted as dual-targeting agents. Compound 7i was the most potent, with activity toward PPARγ EC50 = 0.245 µM and HDAC4 IC50 = 1.1 µM. Additionally, compounds 7c and 7i were cytotoxic to CCRF-CEM cells (CC50 = 2.8 and 9.6 µM, respectively), induced apoptosis, and caused DNA fragmentation. Furthermore, compound 7c modulated the expression of c-Myc, cleaved caspase-3, and caused in vivo tumor regression in CCRF-CEM tumor xenografts. Thus, this study provides a basis for the rational design of dual/multitargeting agents that could be developed further as anticancer therapeutics.