Acknowledgements
The research leading to these results has received funding from the European Community’s 7th Framework
Program (FP7/2007-2013) COSMOS Project under grant agreement n° 266835 and from Cosmetics Europe.
Methods
www.cosmostox.eu
Toward better understanding of liver steatosis MoA:
molecular modelling study of PPAR receptor Merilin Al Sharif 1, Petko Alov 1, Mark Cronin 2, Elena Fioravanzo 3, Ivanka Tsakovska 1, Vessela Vitcheva 1,4,
Andrew Worth 5, Chihae Yang 6, Ilza Pajeva 1
1 Institute of Biophysics and Biomedical Engineering, Sofia, Bulgaria; 2 Liverpool John Moores University, Liverpool, UK; 3 S-IN Soluzioni Informatiche Srl, Vicenza, Italy; 4 US FDA CFSAN, College Park, MD, USA; 5 European Commission, Joint Research Centre, Institute for Health and Consumer Protection, Ispra, Italy; 6 Altamira LLC, Columbus, OH, USA
1. Literature search – main steps to identify key studies (Fig. 3) – all studies analysed and
ranked according to an array of carefully defined criteria; the selected studies paved the
way to describe PPARγ-dependent prosteatotic MoAs.
2. Extraction of all available PPAR complexes (118) from the PDB (http://www.rcsb.org)
3. MOE software (MOE 2012.10, http://www.chemcomp.com) used to: (i) characterise binding
pockets of the complexes; (ii) identify key protein-ligand interactions; (iii) perform
pharmacophore modelling.
Key words
Peroxisome proliferator-
activated receptor gamma
AND ("liver steatosis" OR
"fatty liver“)
220 papers
analysed Criteria
MIE
Pathway
Target protein
Endpoint
Species
SEARCH RESULTS ANALYSIS and RANKING SELECTED
Fig. 3. Main steps in the literature search to identify the major AOPs from PPAR activation to liver steatosis.
Results (1) - AOPs Results (2) – PDB analysis Four AOPs were generalised that have been shown to cause fatty liver triggered by PPARγ activation – transport of fatty acids, de
novo synthesis of fatty acids; triglyceride synthesis and lipid storage (Fig. 4);
The potential of the most studied target proteins to be starting points in a MoA leading to steatosis was evaluated and CD36, FSP27 and
aP2 were selected as prosteatotic factors downstream PPARγ signalization;
A model for the toxicological MoA of PPARγ ligand-dependent activation in hepatocytes mediated by CD36, one of the cornerstones in
the metabolic disregulation leading to fatty liver, was proposed (Fig. 5).
Fig. 6. Distribution of the structures according to the type of bound ligands.
Fig. 7. Distribution of the structures according to the form of the
receptors.
118 human PPARγ structures were
extracted from PDB.
The complexes were analyzed according to
the bound ligands and to the form of the
receptors (Fig. 6 and Fig. 7).
18 papers for all
ranked target
proteins, with the
highest number of
points for CD36
Activity data (Kd, EC50, IC50) for
30 full agonists and 26 partial agonists
were found in PDB and ChEMBLdb.
Results (3) – Molecular modelling
Fig. 1. Overview of fatty acids transport, metabolism and fate (FAT/CD36 – fatty acid
translocase/CD36; FABPpm – plasma membrane fatty acid binding protein; SLC 27A2 – solute
carrier family 27 (fatty acid transporter), member 2; SLC 27A5 – solute carrier family 27 (fatty acid
transporter), member 5; FFA – free fatty acids; TG – triglycerides; VLDL - very low-density
lipoprotein; LPL – lipoprotein lipase; LD – lipid droplet).
Fig. 4. Flow diagram of the MoA from PPAR γ ligand-dependent activation to liver steatosis (fatty liver).
Dark yellow marks CD36, which PPAR γ-mediated overexpression has been confirmed to be prosteatotic by
most experimental evidence, followed by aP2 and FSP27 (light yellow ).
Fig. 5. Model of ligand-dependent PPARγ activation as a potential MIE for liver steatosis through excessive
CD36 mediated fatty acid uptake and consequent hepatic triglyceride accumulation
The PPARγ binding pocket of the full
agonists is large, has a complex form,
can accommodate more than one
ligand and allows different binding
poses of the ligands. Polar parts of the
ligands are directed to H12 – a helix
proved to play a key role in binding of
coactivators (in cyan) (Fig. 8).
The key protein interactions of the most active
agonists include hydrogen bonding to 4/5 amino
acids in the receptor pocket either directly or
through water (Fig. 9).
The pharmacophore model outlines
hydrogen bonding, hydrophobic and
aromatic structural elements as most
important for the PPARγ binding of the
full agonists (Fig. 10, rosiglitazone – in
magenta, compound 544 - in green,
farglitazar - in gray).
Fig. 9. Specific ligand protein interactions of agonists with the key amino acids residues in the PPARγ binding pocket: A) for rosiglitazone; B) for the most active agonist in the
investigated group – compound 544.
A) B)
Fig. 8. The binding poses of all full agonists within the
PPARγ binding pocket (template complex PDB ID 1FM6) .
Conclusions
Fig. 10. Pharmacophore model of PPARγ full agonists.
Agonists of hepatic PPARγ can function as a
steatogenic inducer molecules.
Four significant AoPs for liver steatosis were
summarised triggered by PPARγ ligand-
dependent activation.
Model was proposed for toxicological MoA of
PPARγ ligand-dependent activation in
hepatocytes mediated by CD36.
Pharmacophore model was derived outlining
the importance of hydrogen bonding and
hydrophobic features for agonistic activity.
Correlation between the number of
pharmacophoric points and the agonistic
effect of the ligands (with known
experimental activity) was observed.
The results can be useful in ligand- and
structure-based screening of compounds
which binding to PPARγ could serve as a
MIE for disregulation of the PPARγ activity.
Introduction and Aims
Within the mode of action/adverse outcome pathway (MoA/AOP) framework the description and characterisation of the toxicological
MoAs leading to liver toxicity are of specific interest. Liver plays a central role in free fatty acids and triglyceride metabolism (Fig. 1).
Moreover, because of its unique function in the organism, the liver, and the hepatocyte in particular, is a major target for toxicity. Non-
alcoholic fatty liver disease is one potential repeated dose toxicity adverse effect, known to encompass both steatohepatitis - the more
aggressive form of the disease, and non-alcoholic fatty liver - grouping isolated steatosis and steatosis with mild lobular inflammation
alone. There are growing evidences for the steatogenic role of hepatic peroxisome proliferator-activated receptor gamma (PPAR), a
ligand-inducible transcription factor from the nuclear receptor superfamily (Fig. 2).
In this study AoPs from PPAR
activation to liver steatosis are
identified based on a systematic
literature analysis. Further, molecular
modelling study is performed for
the molecular initiating event (MIE) –
interaction between full agonsits and the PPAR receptor. It includes: (i) analysis of the 3D structural complexes of
human PPAR published in Protein Data Bank (PDB, http://www.rcsb.org); (ii) characterisation of the binding pocket of full agonists;
(iii) identification of the ligand-receptor interactions; (iv) development of pharmacophore models of full agonists to be
used inestablishing filtering rules for effective virtual screening of compounds with potential agonistic activity towards PPAR.
Fig. 2. Schematic structure of the functional domains of the PPAR isoforms.
PPARγ complexes
PPARγ complexes
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