Molecular Modeling Study of PPARγ Agonists: Dehydro-Di-Isoeugenol, Macelignan, Pioglitazone, Netoglitazone, and Rosiglitazone as Antidiabetic Drugs

The peroxisome proliferator-activated receptors (PPARs) are ligand-activated trasncription factors belonging to the nuclear receptor family. The objective of this study is to analyze the molecular aspects of PPARγ agonists which used to design of new antidiabetic drugs. The analysis method was comparing the interactions of ligands in the ligand binding domain of the PPARγ. This analysis showed that most known agonists of PPARγ interacted via hydrogen bond with Tyr473. Pioglitazone showed three hydrogen bonds with His323 and Tyr473. Netoglitazone showed four hydrogen bonds with Ser289, His323, His449, and Tyr473. Rosiglitazone showed five hydrogen bonds with Ser289, His323, His449, and Tyr473. AZ72, an agonist of PPARα and γ showed five hydrogen bonds with Ser289, His323, His449, and Tyr473. Molecular modeling was performed by redocking pioglitazone and rosiglitazone using AutoDock Vina. Docking showed that both pioglitazone (Ki 0.22 μM) and rosiglitazone (Ki 0.70 μM) occupied their origin sites and interacted with Tyr473. Docking simulation was also performed between dehydro-di-isoeugenol and macelignan to visualize the interaction with PPARγ. These two compounds are found in nutmeg’s seed (Myristica fragrans Hout) that have been proven had antidiabetic activity in vitro. It can be concluded that agonists of PPARγ should have hydrogen bond donor and acceptor groups for interacting with Tyr473. Tyr473 might be a critical site of interaction between the PPARγ ligand binding domain and its agonists.


Introduction
Diabetes mellitus (DM) is a group of clinical and genetic disorder that characterized by increasing levels of glucose in the blood.Among the various types of DM, more than 95% of people with diabetes is type 2 diabetes mellitus (T2DM) (American Diabets Association, 2009).T2DM is a combination of insulin resistance and pancreatic β cell insufficiency.One of the receptors target for the treatment of T2DM is peroxisome proliferator-activated receptor γ (PPARγ).
The peroxisome proliferator-activated receptors (PPARs) γ, β, and α compose a nuclear receptor subfamily that modulates the transcription of a large compendium of genes encoding proteins that regulate lipid metabolism, cell differentiation, and signal transduction in a ligand-dependent manner.PPARs bind as heterodimers with a retinoid X receptor and, upon binding agonist, interact with cofactors so the rate of transcription initiation is increased.The PPARs play a critical physiological role as lipid sensors and regulators of lipid metabolism (Berger & Moller, 2002).PPARγ has been shown to be a master regulator of adipogenesis and nutrient metabolism in adipocytes where it is highly expressed.PPARs are activated by fatty acids and eicosanoids, which have been identified as natural ligands for the PPARs, hence these receptors are targets for antidyslipidemic drugs and of antidiabetic agents (Cronet et al., 2001;Berger & Moller, 2002).More potent synthetic PPAR ligands, including the fibrates and thiazolidinediones (TZDs), have proven effective in the treatment of dyslipidemia and diabetes (Berger & Moller, 2002).TZDs or glitazones, a class of antidiabetic agents, have been reported as high affinity agonists of PPARγ (Willson et al., 2000).
TZDs were acted as insulin sensitizers.These compounds improve insulin resistance by increasing cell sensitivity to insulin.PPARγ agonists increase peripheral insulin sensitivity by increasing the transcription of genes, which, in turn, increase glucose uptake, also improving insulin-stimulated glucose disposal in muscle.PPARγ agonists also increase insulin signaling, reduce circulating levels of free fatty acids and stimulate adipocyte differentiation, thus favoring the formation of smaller, more insulin-sensitive adipocytes.TZDs have been proven effective in different experimental models to evaluate their effectiveness as an antidiabetic agent.There were excellent correlation between the hypoglycemic action of TZDs and their affinity for PPARγ (Vazquez et al., 2002).TZDs produce a conformational change in PPAR analogous to those produced by agonists of other nuclear hormone receptors.Antidiabetic actions of TZDs are directly mediated through binding to PPARγ and the resulting active conformation of the receptor.Therefore, binding and transactivation assays using PPARγ should serve to identify other novel therapeutic agents with potential antidiabetic activities (Berger et al., 1996).
Macelignan that had been isolated from nutmeg's seed (Myristica fragrans Hout) can activate the PPARγ (Han et al., 2008).Other studies indicate that macelignan not clearly detected in extracts of nutmeg because its concentration is very small.Based on screening of chemical compounds in nutmeg extracts and in vitro test with GAL-4/PPAR chimera assay and reported gene method, dehydro-di-isoeugenolhas proved can activate PPARγ (Lestari, 2010).This study is aimed to study the molecular aspects of PPARγ agonists' binding mode by comparing the interactions of ligands that were co-crystalized in the ligand binding domain of PPARγ for antidiabetic drug design.

Macromolecule Preparation and Molecular Modeling
3D structures of PPARγ crystallized using X-ray diffraction (code: 2XKW, 1FM6, 3B0Q, 1I7I, 3B0R, 3AN3) were downloaded from online Protein Data Bank (http://www.pdb.org) and viewed by using Jmol Viewer version 12.2.15.Ligand interactions were analyzed by using RSCB PDB Ligand Explorer Viewer 3.9 (powered by the MBT).The chain A of the protein and its co-crystallized inhibitors were separated by using SwissPDBViewer version 4.01 (http://www.expasy.org).The volume and position of PPARγ's binding site was calculated and located by using Q-SiteFinder.Q-SiteFinder was also used to predict amino acid residues in the ligand binding domain of PPARγ.Molecular docking was performed by using AutoDock Vina (Molecular Graphics Laboratory, The Scripps Research Institute, http://vina.scripps.edu/download.html).
Ligand preparation for dehydro-di-isoeugenol and macelignan begins with the design of 2D structure, then converted into a 3D structure by using ChemDraw Ultra v8.0.3 and Chem3D Ultra v8.0.3 in Chemoffice 2004 program.3D structure then optimized geometrically to obtain the most stable conformation.Conformational changes before and after optimization shown in Figure 2. The next step is the analysis of the ligand properties using Portable HyperChem Release 8.0.7.The results of the analysis are displayed in Table 1.

Conclusion
• Agonists of PPARγ should have hydrogen bond donor and acceptor for interacting with Tyr473 residue in the ligand binding domain of the receptor.Nitrogen and oxygen atoms of TZD's thiazolidine ring plays important role in this interaction.Tyr473 might be a critical site of interaction between PPARγ ligand binding domain and TZD derivates.
• Agonists of PPARγ with benzophenylpropanoic acid system interacted with Tyr327 instead of Tyr473.This phenomena confirmed a reversal of stereochemistry-transactivation activity of these class of compounds.
• Dehydro-di-isoeugenol and macelignan potentially can be developed into an oral antidiabetic drug because the value of energy interaction and inhibition constant are still relatively close to pioglitazone.

Figure 4 .
Figure 4. Molecular docking of (a) pioglitazone and (b) rosiglitazone with PPARγ.Hydrogen bond interactions of the ligands with Tyr473 were showed by black ovals