PPARγ Ligand-binding Domain Research Articles

In-silico Docking and Dynamics Simulation Analysis of Peroxisome Proliferator-Activated Receptor-Gamma and β-Carotene

Background: Peroxisome proliferator-activated receptor-gamma (PPAR-γ) plays a crucial role in regulating lipid and glucose metabolism, cancer, and inflammation, making it an attractive target for drug development. Meanwhile, β-Carotene, known for its antioxidant, anticancer and antiinflammatory properties, holds promise for modulating PPAR-γ activity. Understanding their interaction is crucial. Objective: This study aims to explore the therapeutic potential of β-carotene in modulating PPAR-γ activity by investigating their binding interactions. Methods: Screening of bioactive compounds from PubChem was conducted using GlideXP to identify potential PPAR-γ (PDB: 2PRG) ligands. During this screening, both protein and bioactive compounds were prepared following established protocols. Subsequently, the compounds were docked into the ligand binding domain (LBD) of the protein using XP docking. Rosiglitazone was used as an internal control. β-Carotene emerged as a lead based on Lipinski’s rule, docking score, free energy, and LBD interactions. Molinspiration analysis assessed its drug likeness. Molecular dynamics (MD) simulations utilizing Desmond with OPLS 2005 force field were employed to examine the dynamics and stability of the PPAR-γ/β-carotene complex. Results: β-carotene had strong hydrophobic interactions with specific residues within the ligandbinding domain of PPAR-γ. The calculated binding affinity (-9.07 kcal/mol) indicated a strong interaction between β-carotene and PPAR-γ, suggesting that β-carotene may modulate the activity of PPAR-γ. On a time scale of 100 ns, the MD simulations provided insights into the conformational changes, flexibility, and intermolecular interactions within the complex. Conclusion: In silico docking and dynamics simulation analyses show that PPAR-γ and β-carotene can form a stable complex, suggesting potential implications for metabolic modulation.

Exploring the molecular pathways of the activation process in PPARγ recurrent bladder cancer mutants.

The intricate involvement of Peroxisome Proliferator-Activated Receptor Gamma (PPARγ) in glucose homeostasis and adipogenesis is well-established. However, its role in cancer, particularly luminal bladder cancer, remains debated. The overexpression and activation of PPARγ are implicated in tumorigenesis. Specific gain-of-function mutations (M280I, I290M, and T475M) within the ligand-binding domain of PPARγ are associated with bladder cancer and receptor activation. The underlying molecular pathways prompted by these mutations remain unclear. We employed a dual-basin structure-based model (db-SBM) to explore the conformational dynamics between the inactive and active states of PPARγ and examined the effects of the M280I, I290M, and T475M mutations. Our findings, consistent with the existing literature, reveal heightened ligand-independent transcriptional activity in the I290M and T475M mutants. Both mutants showed enhanced stabilization of the active state compared to the wild-type receptor, with the I290M mutation promoting a specific transition route, making it a prime candidate for further study. Electrostatic analysis identified residues K303 and E488 as pivotal in the I290M activation cascade. Biophysical assays confirmed that disrupting the K303-E488 interaction reduced the thermal stabilization characteristic of the I290M mutation. Our study demonstrates the predictive capabilities of combining simulation and cheminformatics methods, validated by biochemical experiments, to gain insights into molecular activation mechanisms and identify target residues for protein modulation.

Reversal of insulin resistance to combat type 2 diabetes mellitus by newer thiazolidinedione's in fructose induced insulin resistant rats

In our pursuit of discovering new antidiabetic agents to manage type 2 diabetes mellitus (T2DM), our approach aimed to identify the bioactive feature/pharmacophore responsible for PPAR-γ expression, as it is accountable for the glucose homeostasis and lipid metabolism. This was achieved by pharmacophore model generation, screening of rationally designed newer thiazolidinedione's library, identifying synthesizing and characterizing the top ten molecules (5a-5j) for their (Invitro &invivo) antidiabetic activity. Preliminary screening of all the ligands by Invitro glucose uptake assay in L6 myotubes (skeletal muscle cell line of rats) revealed compound 5b and 5f stimulated the glucose uptake with 79.29 ± 1.02 % and 74.58 ± 1.02 % respectively compared to pioglitazone with 82.36 ± 0.98 %. This was validated by PPAR-γ TF expression assay, which highlighted a dose dependent increase in transactivation of PPAR-γ. These compounds 5b and 5f were evaluated in fructose induced insulin resistance rat model. Where the treatment with 5b and 5f markedly increased the exogenous clearance of glucose and exogenous insulin via OGTT and ITT respectively, also improved the glucose utilization by significantly increasing content of glycogen and uptake of glucose in rat hemidiaphragm and reversed insulin resistance. Likewise a significant decreased in the VLDL and triglyceride levels was seen in 5b and 5f treated groups compared to insulin resistant (IR) group. It improved glycogenesis by catabolism of glucose and maintained glycaemic control. Similarly it had marked action on enzymatic oxidative biomarkers. Compound 5b displayed better, improved T1/2 (half-life) of 4.21 h and Kel (elimination constant) of 0.381 was noticed in comparison to compound 5f indicating the pharmacokinetic profile. Insilico studies like DFT calculations refined the geometry of 5b and 5f ligands, docking and molecular simulation provided the insights in binding affinity, dynamic behaviour and stability of ligands in PPAR-γ ligand binding domain. MM/GBSA provided the energetics of 5b and 5f in binding pocket. Finally network pharmacology identified ADIPOQ (adiponectin), NR1C3 (PPAR-γ), SLC2A4 (GLUT4), and LEP (leptin) proteins associate with compound 5b and 5f and enriched in Adipocytokine pathway, and PPAR-γ signaling pathway.

Bis-chalcones obtained via one-pot synthesis as the anti-neurodegenerative agents and their effect on the HT-22 cell line

In the area of research on neurodegenerative diseases, the current challenge is to search for appropriate research methods that would detect these diseases at the earliest possible stage, but also new active structures that would reduce the rate of the disease progression and minimize the intensity of their symptoms experienced by the patient. The chalcones are considered in the context of candidates for new drugs dedicated to the fight against neurodegenerative diseases. The synthesis of bis-chalcone derivatives (3a-3d), as aim molecules was performed. Their structures were established by applying 1H NMR, 13C NMR, MS, FT-IR and UV–Vis spectra. All bis-chalcones were synthesized from terephthalaldehyde and appropriate aromatic ketone as substrates in the Claisen-Schmidt condensation method and evaluated in the biological tests and in silico analysis. Compounds exerted antioxidant activity using the HORAC method (3a-3d) and decreased the activities of GPx, COX-2 (3b-3d), GR (3a-3c) and CAT (3a,3b). The high anti-neurodegenerative potential of all four bis-chalcones was observed by inhibition of acetyl- (AChE) and butyrylcholinesterase (BChE) and a positive effect on the mouse hippocampal neuronal HT-22 cell line (LDH release and PGC-1α, PPARγ and GAPDH protein expression). TD-DFT method (computing a number of descriptors associated with HOMO–LUMO electron transition: electronegativity, chemical hardness and potential, first ionization potential, electron affinity) was employed to study the spectroscopic properties. This method showed that the first excited state of compounds was consistent with their maximum absorption in the computed UV–Vis spectra, which showed good agreement with the experimental spectrum using PBE1PBE functional. Using in silico approach, interactions of bis-chalcones with selected targets (aryl hydrocarbon receptor (AhR) PAS-A Domain, ligand binding domain of human PPAR-γ, soman-aged human BChE-butyrylthiocholine complex, Torpedo californica AChE:N-piperidinopropyl-galanthamine complex and the COX-2-celecoxib complex) were characterized. Results obtained in in silico models were consistent with in vitro experiments.

Structural Studies on the Binding Mode of Bisphenols to PPARγ.

Bisphenol A (BPA) and bisphenol B (BPB) are widely used in the production of plastics, and their potential adverse health effects, particularly on endocrine disruption and metabolic health, have raised concern. Peroxisome proliferator-activated receptor gamma (PPARγ) plays a pivotal role in metabolic regulation and adipogenesis, making it a target of interest in understanding the development of obesity and associated health impacts. In this study, we employ X-ray crystallography and molecular dynamics (MD) simulations to study the interaction of PPARγ with BPA and BPB. Crystallographic structures reveal the binding of BPA and BPB to the ligand binding domain of PPARγ, next to C285, where binding of partial agonists as well as antagonists and inverse agonists of PPARγ signaling has been previously observed. However, no interaction of BPA and BPB with Y437 in the activation function 2 site is observed, showing that these ligands cannot stabilize the active conformation of helix 12 directly. Furthermore, free energy analyses of the MD simulations revealed that I341 has a large energetic contribution to the BPA and BPB binding modes characterized in this study.

A structural basis for the activation of peroxisome proliferator-activated receptor gamma (PPARγ) by perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is a widespread environmental pollutant of the perfluoroalkyl substance (PFAS) class that is extremely resistant to environmental and metabolic degradation, leading to bioaccumulation. PFOA exposure has been linked to many health effects including endocrine disruption and metabolic dysregulation, but our understanding of the molecular mechanisms resulting in these outcomes remains incomplete. One target affected by PFOA is the ligand regulated nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) which plays a critical role in controlling metabolic homeostasis through regulating processes such as adipogenesis, glucose homeostasis, inflammation and osteogenesis. It has been previously established that PFOA activates PPARγ through binding to the PPARγ ligand binding domain (PPARγ LBD) leading to increased expression of PPARγ controlled target genes. However, the mechanism by which PFOA achieves this has remained elusive. Here, we employed a combination of X-ray crystallography and fluorescence polarization assays to provide a structural basis for PFOA mediated activation of PPARγ via binding to the PPARγ LBD. Using X-ray crystallography, the cocrystal structure of the PPARγ LBD:PFOA complex was solved. This revealed that PFOA occupies three distinct sites, two within the PPARγ LBD and one within the activation function 2 (AF2) on the protein surface. Structural comparison of PFOA binding with previously reported PPARγ:ligand complexes supports that PFOA activates PPARγ by a partial agonist mechanism at micromolar concentrations. Fluorescence polarization assays also revealed that PFOA binding to the AF2 is unlikely to occur in a cellular context and confirmed that PFOA behaves as a partial agonist in vitro, weakly recruiting a coactivator peptide to the AF2 of the PPARγ LBD. This discovery provides an advancement in understanding PFOA mediated regulation of PPARγ, giving new insight regarding regulation of PPARγ by PFAS and PFAS substitutes in general and can be applied to the design and assessment of safer PFAS.

Insights into the chirality-dependent recognition of Danshensu Bingpian Zhi stereoisomers with PPARγ.

Peroxisome proliferator-activated receptor γ (PPARγ), a nuclear receptor involved in metabolic processes, inflammation, and energy balance, represents a promising therapeutic target for cardiovascular diseases. Danshensu Bingpian Zhi (DBZ), a chiral compound derived from traditional Chinese medicine, exhibits potential as a PPARγ agonist. Using an ensemble-based docking approach, molecular dynamics (MD) simulations, and the molecular mechanics generalized born surface area (MM/GBSA) methods, we explored the binding modes and energetics of DBZ stereoisomers with the PPARγ ligand-binding domain (LBD). The results indicated that the right-handed stereoisomer (DBZR) binds like a full agonist, while the left-handed stereoisomer (DBZS) binds as a partial agonist with stronger binding energies (ΔGbind), indicating a robust interaction with PPARγ. Both the stereoisomers stabilize the β-sheet region of PPARγ-LBD, potentially protecting Ser245 from phosphorylation by Cdk5, a process implicated in atherosclerosis. Principal component analysis (PCA) and dynamic cross-correlation matrices (DCCM) revealed the complex structural dynamics within the Ω loop, β-sheet, and AF-2 region of PPARγ-LBD upon ligand binding, which may contribute to the unique binding mode and efficacy of DBZS. These findings provide insights into the molecular recognition of PPARγ-LBD by DBZ stereoisomers and their impact on the conformational dynamics of PPARγ, highlighting the therapeutic potential of DBZ and the significance of chirality in drug design.

Phorbal-12-mysristate-13-acetate-induced inflammation is restored by protectin DX through PPARγ in human promonocytic U937 cells

AimsProtectin DX (PDX), a specialized pro-resolving mediator, is an important pharmaceutical compound with potential antioxidant and inflammation-resolving effects. However, the fundamental mechanism by which PDX's ameliorate chronic inflammatory diseases has not yet been elucidated. This study aims to evaluate the anti-inflammatory properties and PPARγ-mediated mechanisms of PDX in phorbal-12-mysristate-13-acetate (PMA)-stimulated human promonocytic U937 cells. Main methodsWe confirmed the effects of PDX on expressions of pro-inflammatory cytokines, mediators, and CD14 using conventional PCR, RT-qPCR, ELISA, and flow cytometry. Using western blotting, immunofluorescence, and reactive oxygen species (ROS) determination, we observed that PDX regulated PMA-induced signaling cascades. Molecular docking analysis and a cellular thermal shift assay were conducted to verify the interaction between PDX and the proliferator-activated receptor-γ (PPARγ) ligand binding domain. Western blotting was then employed to explore the alterations in PPARγ expression levels and validate PDX as a PPARγ full agonist. Key findingsPDX attenuated protein and mRNA expression levels of interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 in PMA-treated U937 cells. PDX acts as a PPARγ agonist, exerting a modulating effect on the ROS/JNK/c-Fos signaling pathways. Furthermore, PDX reduced human monocyte differentiation antigen CD14 expression levels. SignificancePPARγ exhibits pro-resolving effects to regulate the excessive inflammation. These results suggest that PDX demonstrates the resolution of inflammation, indicating the potential for therapeutic targeting of chronic inflammatory diseases.

Binding of Per- and Polyfluoroalkyl Substances (PFAS) to the PPARγ/RXRα-DNA Complex.

Nuclear receptors are the fundamental building blocks of gene expression regulation and the focus of many drug targets. While binding to DNA, nuclear receptors act as transcription factors, governing a multitude of functions in the human body. Peroxisome proliferator-activator receptor γ (PPARγ) and the retinoid X receptor α (RXRα) form heterodimers with unique properties and have a primordial role in insulin sensitization. This PPARγ/RXRα heterodimer has been shown to be impacted by per- and polyfluoroalkyl substances (PFAS) and linked to a variety of significant health conditions in humans. Herein, a selection of the most common PFAS (legacy and emerging) was studied utilizing molecular dynamics simulations for PPARγ/RXRα. The local and global structural effects of PFAS binding on the known ligand binding pockets of PPARγ and RXRα as well as the DNA binding domain (DBD) of RXRα were inspected. The binding free energies were predicted computationally and were compared between the different binding pockets. In addition, two electronic structure approaches were utilized to model the interaction of PFAS within the DNA binding domain, density functional theory (DFT) and domain-based pair natural orbital coupled cluster with perturbative triples (DLPNO-CCSD(T)) approaches, with implicit solvation. Residue decomposition and hydrogen-bonding analysis were also performed, detailing the role of prominent residues in molecular recognition. The role of l-carnitine is explored as a potential in vivo remediation strategy for PFAS interaction with the PPARγ/RXRα heterodimer. In this work, it was found that PFAS can bind and act as agonists for all of the investigated pockets. For the first time in the literature, PFAS are postulated to bind to the DNA binding domain in a nonspecific manner. In addition, for the PPARγ ligand binding domain, l-carnitine shows promise in replacing smaller PFAS from the pocket.

Design, synthesis and anti-hyperglycemic assessments of novel 5-benzylidenethiazolidine-2,4-dione derivatives as PPAR-γ agonist

The treatment of diabetes mellitus still relies predominantly on peroxisome proliferator-activated receptor-γ (PPAR-γ) therapy, known for its effectiveness. In this present study, a novel series of benzylidene-linked thiazolidine-2,4-dione compounds was conceptualized, synthesized, and subjected to assessment for their potential antidiabetic properties. Notably, among the compounds examined, 5-((Z)-4-(((E)-Propylidene)amino)benzylidene)thiazolidine-2,4-dione, designated as 4c, emerged as the most promising candidate, exhibiting the highest PPAR-γ agonistic activity with an EC50 value of 0.35 ± 0.52 μM. Additionally, molecular docking investigations were conducted on twelve 2,4-thiazolidinedione compounds, with a pyrazole moiety incorporated, within the ligand binding domain of PPAR-γ. Compound 4c, in particular, demonstrated a Glide XP score of −9.104, surpassing the score of the crystal ligand (Glide XP score = −7.482). Notably, both exhibited similar interactions with key amino acids, including HIE323, SER289, and GLN286. Furthermore, three derivatives (4a-c) with promising PPAR-γ agonistic activity and favorable molecular docking scores underwent in vivo evaluation for their capacity to lower blood glucose levels using a streptozotocin-induced diabetic mice model. In comparison to the reference drug pioglitazone, Compound 4c displayed commendable in-vivo performance across multiple parameters, encompassing serum HDL, serum LDL, serum total cholesterol, and serum triglyceride levels. Molecular dynamics simulations provided insights into the impact of ligand 4c, indicating its role in enhancing protein stability and rigidity. These findings emphasize its potential as a potent protein inhibitor, thus opening novel avenues for the intelligent design of molecules with high efficacy in the management of hyperglycemia.

GRK2 inhibits Flt-1+ macrophage infiltration and its proangiogenic properties in rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease with a complex etiology. Monocyte-derived macrophages (MDMs) infiltration are associated with RA severity. We have reported the deletion of G-protein-coupled receptor kinase 2 (GRK2) reprograms macrophages toward an anti-inflammatory phenotype by recovering G-protein-coupled receptor signaling. However, as more GRK2-interacting proteins were discovered, the GRK2 interactome mechanisms in RA have been understudied. Thus, in the collagen-induced arthritis mouse model, we performed genetic GRK2 deletion using GRK2f/fLyz2-Cre+/− mice. Synovial inflammation and M1 polarization were improved in GRK2f/fLyz2-Cre+/− mice. Supporting experiments with RNA-seq and dual-luciferase reporter assays identified peroxisome proliferator-activated receptor γ (PPARγ) as a new GRK2-interacting protein. We further confirmed that fms-related tyrosine kinase 1 (Flt-1), which promoted macrophage migration to induce angiogenesis, was inhibited by GRK2-PPARγ signaling. Mechanistically, excess GRK2 membrane recruitment in CIA MDMs reduced the activation of PPARγ ligand-binding domain and enhanced Flt-1 transcription. Furthermore, the treatment of mice with GRK2 activity inhibitor resulted in significantly diminished CIA pathology, Flt-1+ macrophages induced-synovial inflammation, and angiogenesis. Altogether, we anticipate to facilitate the elucidation of previously unappreciated details of GRK2-specific intracellular signaling. Targeting GRK2 activity is a viable strategy to inhibit MDMs infiltration, affording a distinct way to control joint inflammation and angiogenesis of RA.

Mechanisms of Nelumbinis folium targeting PPARγ for weight management: A molecular docking and molecular dynamics simulations study

The lotus leaf, Nelumbinis folium (NF), has frequently appeared in obesity clinical trials as an intervention to promote weight loss and improve metabolic profiles. However, the molecular mechanisms by which it interacts with important obesity targets and pathways, such as the peroxisome proliferator-activated receptor gamma (PPARγ) within the PPAR signalling pathway, were not well understood. This study aims to screen for candidate compounds from NF with desirable pharmacokinetic properties and examine their binding feasibility at the PPARγ ligand-binding domain (LBD). Ligand- and structure-based screening of NF compounds were performed, and a consensus approach has been applied to identify druggable candidates. By examining the pharmacokinetic profiles, a large proportion of NF compounds exhibited favourable drug-likeness and oral bioavailability properties. Furthermore, the binding affinity scores and poses provided new insights on the distinctive binding behaviours of NF compounds at the LBD of PPARγ in its inactive form. Several NF compounds could bind strongly to PPARγ at sub-pockets where partial agonists and antagonists were found to bind and may induce conformational changes that influence co-repressor binding, trans-repression, and gene expression inhibition. Subsequent molecular dynamics simulations of a candidate compound (NF129 narcissin) bound to PPARγ revealed conformational stability, residue fluctuation, and binding behaviours comparable to that of the known inhibitor, SR1664. Therefore, it can be proposed that narcissin exhibits characteristics of a PPARγ antagonist. Further experimental validation to support the development of NF129 as a future anti-obesity agent is warranted.

Molecular docking analysis of PPARγ with phytochemicals from Moroccan medicinal plants.

PPARγ agonists play a crucial role in regulating metabolic homeostasis for treating type-2 diabetes (T2D). Due to the adverse side effects associated with thiazolidinediones, a class of PPARγ agonists, there is a growing interest in identifying natural compounds from medicinal plants that have the potential to bind PPARγ. In this study, we extensively investigated Moroccan phytochemicals using computational structure-based screening with the crystal structure of the PPARγ ligand-binding domain (PDB ID: 7awc) to discover novel phytochemicals targeting PPARγ. The docking results of 540 Moroccan phytochemicals were integrated into online databases for further exploitation through in-depth studies. Drug-likeness analysis was performed to assess the phytochemicals drug-like properties. Two promising phytochemicals, 3,4-dicaffeoylquinic acid and Chlorogenic acid, were identified, both exhibiting high docking affinity and unique binding site interactions compared to the established PPARγ full agonist, rosiglitazone. Molecular dynamics simulations of 100 ns were conducted to examine the stability of the complexes formed by both compounds within the PPARγ active site, and their dynamic behavior was compared to the reference structure of PPARγ alone and with rosiglitazone. Binding free energy calculations demonstrated that 3,4-dicaffeoylquinic acid and Chlorogenic acid exhibited higher binding free energy than the reference agonist, suggesting their potential as candidates for experimental validation in future drug discovery efforts targeting PPARγ for the treatment of T2D and metabolic syndrome.

Deoxyelephantopin—a novel PPARγ agonist regresses pressure overload-induced cardiac fibrosis via IL-6/STAT-3 pathway in crosstalk with PKCδ

Pathological cardiac hypertrophy is associated with ventricular fibrosis leading to heart failure. The use of thiazolidinediones as Peroxisome Proliferator-Activated Receptor-gamma (PPARγ)-modulating anti-hypertrophic therapeutics has been restricted due to major side-effects. The present study aims to evaluate the anti-fibrotic potential of a novel PPARγ agonist, deoxyelephantopin (DEP) in cardiac hypertrophy. AngiotensinII treatment in vitro and renal artery ligation in vivo were performed to mimic pressure overload-induced cardiac hypertrophy. Myocardial fibrosis was evaluated by Masson's trichrome staining and hydroxyproline assay. Our results showed that DEP treatment significantly improves the echocardiographic parameters by ameliorating ventricular fibrosis without any bystander damage to other major organs. Following molecular docking, all-atomistic molecular dynamics simulation, reverse transcription-polymerase chain reaction and immunoblot analyses, we established DEP as a PPARγ agonist stably interacting with the ligand-binding domain of PPARγ. DEP specifically downregulated the Signal Transducer and Activator of Transcription (STAT)-3-mediated collagen gene expression in a PPARγ-dependent manner, as confirmed by PPARγ silencing and site-directed mutagenesis of DEP-interacting PPARγ residues. Although DEP impaired STAT-3 activation, it did not have any effect on the upstream Interleukin (IL)-6 level implying possible crosstalk of the IL-6/STAT-3 axis with other signaling mediators. Mechanistically, DEP increased the binding of PPARγ with Protein Kinase C-delta (PKCδ) which impeded the membrane translocation and activation of PKCδ, downregulating STAT-3 phosphorylation and resultant fibrosis. This study, therefore, for the first time demonstrates DEP as a novel cardioprotective PPARγ agonist. The therapeutic potential of DEP as an anti-fibrotic remedy can be exploited against hypertrophic heart failure in the future.

PPARγ Corepression Involves Alternate Ligand Conformation and Inflation of H12 Ensembles.

Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.

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.

Assessment of lipid metabolism-disrupting effects of non-phthalate plasticizer diisobutyl adipate through in silico and in vitro approaches.

Diisobutyl adipate (DIBA), as a novel non-phthalate plasticizer, is widely used in various products. However, little effort has been made to investigate whether DIBA might have adverse effects on human health. In this study, we integrated an in silico and in vitro strategy to assess the impact of DIBA on cellular homeostasis. Since numerous plasticizers could activate peroxisome proliferator-activated receptor γ (PPARγ) pathway to interrupt metabolism systems, we first utilized molecular docking to analyze interaction between DIBA and PPARγ. Results indicated that DIBA had strong affinity with the ligand-binding domain of PPARγ (PPARγ-LBD) at Histidine 499. Afterwards, we used cellular models to investigate in vitro effects of DIBA. Results demonstrated that DIBA exposure increased intracellular lipid content in murine and human hepatocytes, and altered transcriptional expression of genes related to PPARγ signaling and lipid metabolism pathways. At last, target genes regulated by DIBA were predicted and enriched for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Protein-protein interaction (PPI) network and transcriptional factors (TFs)-genes network were established accordingly. Target genes were enriched in Phospholipase D signaling pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and Epidermal growth factor receptor (EGFR) signaling pathway which were related to lipid metabolism. These findings suggested that DIBA exposure might disturb intracellular lipid metabolism homeostasis via targeting PPARγ. This study also demonstrated that this integrated in silico and in vitro methodology could be utilized as a high throughput, cost-saving and effective tool to assess the potential risk of various environmental chemicals on human health.

Structural basis of the activation of PPARγ by the plasticizer metabolites MEHP and MINCH

Di-2-ethylhexyl phthalate (DEHP) and its substitute 1,2-cyclohexane dicarboxylic acid diisononyl ester (DINCH) are widely used as plasticizers but may have adverse health effects. Via hydrolysis of one of the two ester bonds in the human body, DEHP and DINCH form the monoesters MEHP and MINCH, respectively. Previous studies demonstrated binding of these metabolites to PPARγ and the induction of adipogenesis via this pathway. Detailed structural understanding of how these metabolites interact with PPARγ and thereby affect human health is lacking until now. We therefore characterized the binding modes of MINCH and MEHP to the ligand binding domain of PPARγ by X-ray crystallography and molecular dynamics (MD) simulations. Both compounds bind to the activating function-2 (AF-2) binding site via an interaction of the free carboxylates with the histidines 323 and 449, tyrosine 473 and serine 289. The alkyl chains form hydrophobic interactions with the tunnel next to cysteine 285. These binding modes are generally stable as demonstrated by the MD simulations and they resemble the complexation of fatty acids and their metabolites to the AF-2 site of PPARγ. Similar to the situation for these natural PPARγ agonists, the interaction of the free carboxylate groups of MEHP and MINCH with tyrosine 473 and surrounding residues stabilizes the AF-2 helix in the upward conformation. This state promotes binding of coactivator proteins and thus formation of the active complex for transcription of the specific target genes. Moreover, a comparison of the residues involved in binding of the plasticizer metabolites in vertebrate PPARγ orthologs shows that these compounds likely have similar effects in other species.

PPARγ lipodystrophy mutants reveal intermolecular interactions required for enhancer activation

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipocyte differentiation, and mutations that interfere with its function cause lipodystrophy. PPARγ is a highly modular protein, and structural studies indicate that PPARγ domains engage in several intra- and inter-molecular interactions. How these interactions modulate PPARγ’s ability to activate target genes in a cellular context is currently poorly understood. Here we take advantage of two previously uncharacterized lipodystrophy mutations, R212Q and E379K, that are predicted to interfere with the interaction of the hinge of PPARγ with DNA and with the interaction of PPARγ ligand binding domain (LBD) with the DNA-binding domain (DBD) of the retinoid X receptor, respectively. Using biochemical and genome-wide approaches we show that these mutations impair PPARγ function on an overlapping subset of target enhancers. The hinge region-DNA interaction appears mostly important for binding and remodelling of target enhancers in inaccessible chromatin, whereas the PPARγ-LBD:RXR-DBD interface stabilizes the PPARγ:RXR:DNA ternary complex. Our data demonstrate how in-depth analyses of lipodystrophy mutants can unravel molecular mechanisms of PPARγ function.

Molecular Modeling of Allosteric Site of Isoform-Specific Inhibition of the Peroxisome Proliferator-Activated Receptor PPARγ.

The peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor and controls a number of gene expressions. The ligand binding domain (LBD) of PPARγ is large and involves two binding sites: orthosteric and allosteric binding sites. Increased evidence has shown that PPARγ is an oncogene and thus the PPARγ antagonists have potential as anticancer agents. In this paper, we use Glide Dock approach to determine which binding site, orthosteric or allosteric, would be a preferred pocket for PPARγ antagonist binding, though antidiabetic drugs such as thiazolidinediones (TZDs) bind to the orthosteric site. The Glide Dock results show that the binding of PPARγ antagonists at the allosteric site yielded results that were much closer to the experimental data than at the orthosteric site. The PPARγ antagonists seem to selectively bind to residues Lys265, Ser342 and Arg288 at the allosteric binding site, whereas PPARγ agonists would selectively bind to residues Leu228, Phe363, and His449, though Phe282 and Lys367 may also play a role for agonist binding at the orthosteric binding pocket. This finding will provide new perspectives in the design and optimization of selective and potent PPARγ antagonists or agonists.