IN SILICO SCREENING OF POTENT PPARGAMMA AGONISTS AMONG NATURAL ANTICANCER COMPOUNDS OF INDIAN ORIGIN

ABSTRACTObjective: Naturally occurring anticancer compounds of Indian origin are well-known for potential therapeutic values. A better understanding ofthe intermolecular interactions of these compounds with peroxisome proliferator-activated receptor gamma (PPARγ) is essential, as its activity isreported in many of the cancers involving colon, breast, gastric, and lung. By this study, it is attempted to perform an in silico screening of naturalanticancer compounds of Indian origin with PPARγ ligand binding domain (LBD). The potential anticancer leads ranked in this study will also exertan additional advantage of PPARγ activity modulation. As PPARγ is also an important nuclear hormone receptor that modulates transcriptionalregulation of lipid and glucose homeostasis and also a key target for many of the anti-diabetic medications, the compounds ranked by this study willalso be utilized for other related therapeutic effects.Methods: This study features in silico screening of compounds from Indian Plant Anticancer compounds database against PPARγ LBD main performedSchrodinger glide virtual screening and docking module to delineate potential PPARγ agonists. Finally, the most potential lead was also subjected tomolecular dynamics simulation to infer the stability of complex formation.Results: The results reveal that majority of the top ranking compounds that interact with LBD was found to be flavonoids, and all these compoundswere found to interact with key residues involved in PPARγ agonist interactions.Conclusion: The leads from this study would be helpful in better understanding of the potential of naturally occurring anticancer compounds ofIndian origin toward targeting PPARγ.Keywords: Peroxisome proliferator-activated receptor-gamma, Agonists, Docking, Natural compounds, Anticancer.Â

Computational Studies and Design of PPARγ and GLUT1 Inhibitors

The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor of the nuclear receptor superfamily that controls the expression of a variety of genes involved in fatty acid metabolism, adipogenesis, and insulin sensitivity. PPARγ is a target for insulin-sensitizing drugs, and it plays a significant function in prostate cancer. PPARγ antagonists have anti-proliferative effects in a broad range of hematopoietic and epithelial cell lines. The ligand binding domain (LBD) of PPARγ is large and has orthosteric and allosteric binding sites. Several PPARγ-ligand co-crystal structures show two bound molecules, one to the orthosteric pocket and a second to the allosteric site. We ran docking studies against the orthosteric and allosteric binding sites to determine the most favorable binding site for PPARγ antagonists. We found that Glide docking performed well in predicting PPARγ antagonist binding affinities, and that the allosteric site of PPARγ was the most favorable binding site for antagonists. We also investigated PPARγ ligand-protein interactions to better define a structural basis for the binding selectivity of PPARγ antagonists. We found that Phe282, Arg288, and Lys367 interact with antagonists more than with agonists and partial agonists. We then identified several potential PPARγ antagonists by virtual screening of the PPARγ allosteric pocket. The glucose transporter 1 (GLUT1) is a uniporter protein that facilitates the transport of glucose across the plasma membranes of mammalian cells. As GLUT1 is overexpressed in numerous tumors, this transporter is a potential target for cancer treatment. GLUT1 works through conformational switching from an outward-open (OOP) to an inward-open (IOP) conformation passing through an occluded conformation. We sought to determine which conformation is favored for ligand binding by molecular docking studies of known GLUT1 inhibitors with the different GLUT1 conformers. Our data revealed that the IOP is the preferred conformation and that residues Phe291, Phe379, Glu380, Trp388, and Trp412 may play important roles in ligand binding to GLUT1. To identify new chemotypes targeting GLUT1, we built a pharmacophore model and searched against an NCI compound database. Sixteen hit molecules with good docking scores were screened for GLUT1 inhibition and anti-proliferative activities. From these, we identified four compounds that inhibited cell viability in an HCT116 colon cancer cell line. We also determined that one of these, NSC295720, inhibited GLUT1 in a biochemical assay

Structural basis for PPAR partial or full activation revealed by a novel ligand binding mode

The peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in the regulation of the metabolic homeostasis and therefore represent valuable therapeutic targets for the treatment of metabolic diseases. The development of more balanced drugs interacting with PPARs, devoid of the side-effects showed by the currently marketed PPARλ 3 full agonists, is considered the major challenge for the pharmaceutical companies. Here we present a structure-based virtual screening approach that let us identify a novel PPAR pan-agonist with a very attractive activity profile and its crystal structure in the complex with PPARα and PPARλ 3, respectively. In PPARα this ligand occupies a new pocket whose filling is allowed by the ligand-induced switching of the F273 side chain from a closed to an open conformation. The comparison between this pocket and the corresponding cavity in PPARλ 3 provides a rationale for the different activation of the ligand towards PPARα and PPARλ 3, suggesting a novel basis for ligand design

The application of molecular modelling in the safety assessment of chemicals: A case study on ligand-dependent PPARγ dysregulation.

The aim of this paper was to provide a proof of concept demonstrating that molecular modelling methodologies can be employed as a part of an integrated strategy to support toxicity prediction consistent with the mode of action/adverse outcome pathway (MoA/AOP) framework. To illustrate the role of molecular modelling in predictive toxicology, a case study was undertaken in which molecular modelling methodologies were employed to predict the activation of the peroxisome proliferator-activated nuclear receptor γ (PPARγ) as a potential molecular initiating event (MIE) for liver steatosis. A stepwise procedure combining different in silico approaches (virtual screening based on docking and pharmacophore filtering, and molecular field analysis) was developed to screen for PPARγ full agonists and to predict their transactivation activity (EC50). The performance metrics of the classification model to predict PPARγ full agonists were balanced accuracy=81%, sensitivity=85% and specificity=76%. The 3D QSAR model developed to predict EC50 of PPARγ full agonists had the following statistical parameters: q(2)cv=0.610, Nopt=7, SEPcv=0.505, r(2)pr=0.552. To support the linkage of PPARγ agonism predictions to prosteatotic potential, molecular modelling was combined with independently performed mechanistic mining of available in vivo toxicity data followed by ToxPrint chemotypes analysis. The approaches investigated demonstrated a potential to predict the MIE, to facilitate the process of MoA/AOP elaboration, to increase the scientific confidence in AOP, and to become a basis for 3D chemotype development

VIRTUAL SCREENING STUDIES OF SEAWEED METABOLITES FOR PREDICTING POTENTIAL PPARγ AGONISTS

Objective: Peroxisome Proliferator-Activated Receptor-gamma (PPARγ) is a crucial nuclear hormone receptor, which modulates the transcriptional regulation of lipid and glucose homeostasis. It plays a crucial role in many of the metabolic and inflammatory systems. It is a key target for many of the anti-diabetic medications. Perturbation of PPARγ activity is also observed in many of the cancers involving colon, breast, gastric and lung. Thus, it is considered to be the hub molecule for targeting many of these cellular disorders. Seaweed metabolites have been well documented to be novel structural entities with a broad spectrum of pharmacological values. However, it is yet to be utilized for screening PPARγ agonists.Methods: In this study, virtual screening of PPARγ Ligand Binding Domain (LBD) was performed against the datasets from SeaWeed Metabolite Database (SWMD) using Schrodinger Glide High Throughput Virtual Screening module to identify potential PPARγ agonists. Further, the most potential lead was also subjected to molecular dynamics simulation to infer the stability of complex formation.Results: The results have revealed that bromophenolic compounds from the genus Avrainvillea to interact with documented key residues of LBD involved in agonist interactions. Many other metabolites from the genus Rhodomela, Leathesia, Bifurcaria, Osmundaria, Cymopolia also showed significant interactions with LBD of PPARγ.Conclusion: The insights from this study will pave the way for further exploration of lead compounds from seaweed metabolites targeting PPARγ. Â

Optimisation of Tyrosine-based lead molecules capable of Modulation of the Peroxisome Proliferator-Activated Receptor Gamma

The peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone has recently been withdrawn from the European market and its use has been restricted in the US due to its undesirable effects which were considered to outweigh its benefits. Literature indicates that there are two agonist bound conformations of the PPARγ as exemplified by its binding to rosiglitazone (PDB ID; 1FM6) and to farglitazar (PDB ID; 1FM9). This study aims to explore these two conformations, and to evaluate whether they should be targeted separately in the context of drug design studies. Furthermore, it was aimed to design a series of molecules with the potential to act as leads in a drug design process and the capability of agonist activity at the PPARγ with an acceptable side effect profile. In silico ligand binding affinities (pKd) of rosiglitazone and farglitazar within their cognate receptors were 6.62 and 9.70 respectively. The farglitazar conformer that bound optimally within the rosiglitazone bound PPARγ ligand binding pocket was identified and its binding affinity (pKd) re-determined. An analogous conformational analysis of rosiglitazone within the farglitazar bound PPARγ ligand binding pocket was carried out. The binding affinities (pKd) for these optimum conformations were 8.12 and 6.16 respectively. De novo novel structures were generated in silico based on the tyrosine-agonist farglitazar and its cognate ligand binding pocket. Moreover, analysis of the binding modality of farglitazar indicates that this molecule accesses the PPARγ ligand binding pocket more completely than does rosiglitazone. Binding affinity studies have shown that the PPARγ ligand binding pocket adopts diverse ligand driven conformations.peer-reviewe

Identifying and modeling the contribution of nuclear receptors to environmental obesogen-induced toxicity in bone

Bone is a dynamic tissue, where bone forming osteoblasts and bone resorbing osteoclasts maintain homeostasis. Research into bone toxicology has largely focused on pharmaceutical side effects adversely affecting bone development. However, many environmental toxicants can regulate bone homeostasis. Recently, the nuclear receptor peroxisome proliferator activated receptor gamma (PPARγ) has emerged as an important target of environmental toxicants. PPARγ dimerizes with the retinoid-X receptor alpha (RXRα), is a central transcription factor in adipogenesis, and in bone can transdifferentiate osteoblasts into adipocytes by suppressing osteogenic pathways. The central hypothesis of this dissertation is that environmental chemicals can adversely affect bone homeostasis by activating nuclear receptors in bone cells – particularly osteoblasts and osteoclasts – to perturb cellular differentiation and function. Three study aims were developed to test and refine this hypothesis. First, a set of structurally diverse environmental PPARγ agonists were individually applied to mouse primary bone marrow mesenchymal stromal cell cultures undergoing osteogenic differentiation. In vitro PPARγ ligand treatment suppressed osteogenesis and stimulated adipogenesis. Organotin compounds (tributyltin, triphenyltin) in particular more efficaciously suppressed osteogenesis. The second aim characterized the effects of in vivo tributyltin exposure on bone microarchitecture in female C57Bl/6 mice. Tributyltin exposure resulted in a thinner cortical bone, but significantly increased trabecular mineralization. Further analyses suggested that tributyltin did not suppress osteoclast numbers but rather changed osteoclast function, minimally attenuating the resorptive function and enhancing their ability to generate osteogenesis-stimulating factors. Furthermore, tributyltin activated not only PPARγ, but also RXR and liver X receptors. The third aim established the utility of Generalized Concentration Addition in modeling PPARγ activation by mixtures of full and partial PPARγ agonists. A complex mixture of multiple phthalate compounds activated an in vitro PPARγ reporter assay, and the individual dose-responses of each compound were used to construct modeled responses. The comparisons of empirical data and model predictions supported the use of Generalized Concentration Addition in modeling a complex mixture of environmental PPARγ agonists. Together, these studies support and establish important toxicological mechanisms related to PPARγ and RXRα activation in different aspects of bone biology and provide a basis for studying mixture effects of PPARγ agonists

Peroxisome Proliferator-Activated Receptor Alpha: Insight into the Structure, Function and Energy Homeostasis

Peroxisome proliferator-activated receptor alpha (PPAR alpha) belongs to the family of ligand-activated nuclear transcription factors and serves as a lipid sensor to regulate nutrient metabolism and energy homeostasis. The transcriptional activity of PPAR alpha is thought to be regulated by the binding of exogenous ligands (example, fenofibrate, TriCor), as well as endogenous ligands including fatty acids and their derivatives. Although long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) have been shown to activate PPAR alpha of several species, the true identity of high-affinity endogenous ligands for human PPAR alpha (hPPAR alpha) has been more elusive. This two part dissertation is a structural and functional evaluation of human and mouse PPAR alpha binding to LCFA and LCFA-CoA using biophysical and biochemical approaches of spectrofluorometry, circular dichroism spectroscopy, mutagenesis, molecular modelling and transactivation assays. The first goal of this dissertation was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for full-length hPPAR alpha. Data from spectrofluorometry suggests that LCFA and LCFA-CoA serve as physiologically relevant endogenous ligands of hPPAR alpha. These ligands bind hPPAR alpha and induce strong secondary structural changes in the circular dichroic spectra, consistent with the binding of ligand to nuclear receptors. Ligand binding is also associated with activation of hPPAR alpha, as observed in transactivation assays. The second goal of this dissertation was to determine whether there exist species differences for ligand specificity and affinity between hPPAR alpha and mouse PPAR alpha (mPPAR alpha). This is important because despite high amino acid sequence identity (\u3e90 precent), marked differences in PPAR alpha ligand binding, activation and gene regulation have been noted across species. Similar to previous observations with synthetic agonists, we reported differences in ligand affinities and extent of activation between hPPAR alpha and mPPAR alpha in response to saturated long chain fatty acids. In order to determine if structural alterations between the two proteins could account for these differences, we performed in silico molecular modeling and docking simulations. Modeling suggested that polymorphisms at amino acid position 272 and 279 are likely to be responsible for differences in saturated LCFA binding to hPPAR alpha and mPPAR alpha. To confirm these results experimentally, spectrofluorometry based-binding assays, circular dichroism, and transactivation studies were performed using a F272I mutant form of mPPAR alpha. Experimental data correlated with in silico docking simulations, further confirming the importance of amino acid 272 in LCFA binding. Although the driving force for evolution of species differences at this position are yet unidentified, this study enhances our understanding of ligand-induced regulation by PPAR alpha. Apart from demonstrating significant structure activity relationships explaining species differences in ligand binding, data in this dissertation identifies endogenous ligands for hPPAR alpha which will further help delineate the role of PPAR alpha as a nutrient sensor in regulating energy homeostasis