Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor

No therapeutic drugs are currently available for nonalcoholic steatohepatitis (NASH) that progresses from nonalcoholic fatty liver via oxidative stress-involved pathways. Three cognate peroxisome proliferator-activated receptor (PPAR) subtypes (PPARα/δ/γ) are considered as attractive targets. Although lanifibranor (PPARα/δ/γ pan agonist) and saroglitazar (PPARα/γ dual agonist) are currently under investigation in clinical trials for NASH, the development of seladelpar (PPARδ-selective agonist), elafibranor (PPARα/δ dual agonist), and many other dual/pan agonists has been discontinued due to serious side effects or little/no efficacies. This study aimed to obtain functional and structural insights into the potency, efficacy, and selectivity against PPARα/δ/γ of three current and past anti-NASH investigational drugs: lanifibranor, seladelpar, and elafibranor. Ligand activities were evaluated by three assays to detect different facets of the PPAR activation: transactivation assay, coactivator recruitment assay, and thermal stability assay. Seven high-resolution cocrystal structures (namely, those of the PPARα/δ/γ-ligand-binding domain (LBD)–lanifibranor, PPARα/δ/γ-LBD–seladelpar, and PPARα-LBD–elafibranor) were obtained through X-ray diffraction analyses, six of which represent the first deposit in the Protein Data Bank. Lanifibranor and seladelpar were found to bind to different regions of the PPARα/δ/γ-ligand-binding pockets and activated all PPAR subtypes with different potencies and efficacies in the three assays. In contrast, elafibranor induced transactivation and coactivator recruitment (not thermal stability) of all PPAR subtypes, but the PPARδ/γ-LBD–elafibranor cocrystals were not obtained. These results illustrate the highly variable PPARα/δ/γ activation profiles and binding modes of these PPAR ligands that define their pharmacological actions.


Introduction
There is serious concern regarding the medical/economic burden of the treatment of the globally expanding number of patients with nonalcoholic fatty liver disease (NAFLD)/ nonalcoholic steatohepatitis (NASH).NAFLD is defined by the evidence of hepatic steatosis (either through imaging or histology) and the absence of secondary causes of significant alcohol consumption, long-term use of a steatogenic medication, or monogenic hereditary disorders [1].The overall prevalence of nonalcoholic fatty liver (NAFL) worldwide is estimated to be 32.4% [2], while an estimated 10-25% of NAFL patients progress to the development of NASH (a condition characterized by ≥5% hepatic steatosis and inflammation with hepatic injury (e.g., ballooning) in the presence or absence of fibrosis [1]) in which oxidative stress plays a pivotal role by stimulating Kupffer cells, hepatic stellate cells, and hepatocytes [3].NASH can further progress to cirrhosis, end-stage liver disease, or hepatocellular carcinoma [4], and NAFLD is the leading cause of liver-related morbidity and mortality.NASH is the major cause of liver transplantation and, currently, there are no approved non-symptomatic therapies for NASH by the Food and Drug Administration (FDA) or the European Medicines Agency [5].NASH is a multifaceted condition with variable coexisting metabolic complications such as obesity and type 2 diabetes, thereby further complicating its treatment [4].Its therapeutic targets can be divided into four major categories: (i) metabolic targets, (ii) targets related to inflammation or cell injury, (iii) liver-gut axis targets, and (iv) targets related to fibrosis [5,6].In this respect, the peroxisome proliferator-activated receptors (PPARs) are attractive therapeutic targets that could simultaneously improve steatosis, ballooning, inflammation, and fibrosis [6,7].
PPARs belong to the nuclear receptor superfamily and the ligand-activated transcription factors; they exist in three subtypes in mammals (namely, PPARα, PPARβ/δ, and PPARγ) with considerable amino acid identity (54-71% in humans).The synthetic PPARα agonists known as "fibrates" have been widely used for the treatment of hypertriglyceridemia, while the synthetic PPARγ agonists known as "thiazolidinediones (glitazones)" are anti-diabetic drugs.In the guidelines of the American Association for the Study of Liver Diseases, pioglitazone has been proposed for the treatment of biopsy-proven NASH.The PPAR pan agonist lanifibranor (IVA-337) and the PPARα/γ dual agonist saroglitazar are currently in clinical trials for NASH (phase 3 in NCT04849728 and 2b in NCT05011305, respectively).However, the development of most glitazars (PPARα/γ dual agonists), including muraglitazar, tesaglitazar, and aleglitazar, has been abandoned due to serious safety concerns [8].The use of the PPARδ-selective agonist seladelpar (MBX-8025) for the treatment of NASH has been once discontinued at phase 2b [9], while that of the PPARα/δ dual agonist elafibranor against NASH has been discontinued due to its non-significant benefits [10].Therefore, the risks and the benefits of each PPAR-targeting drug should be carefully discussed based on detailed analyses at molecular levels.
This study was designed so as to provide functional and structural insights into the potency, efficacy, and selectivity against PPARα/δ/γ of lanifibranor, seladelpar, and elafibranor.We have found that all three agents can activate all PPAR subtypes with highly different preferences in the three different PPAR activation assays undertaken herein.Furthermore, we have characterized the high-resolution structures of the PPARα/δ/γ-ligandbinding domain (LBD)-lanifibranor, the PPARα/δ/γ-LBD-seladelpar, and the PPARα-LBD-elafibranor cocrystals through X-ray diffraction analyses.

PPAR Activation Assay 3: Thermal Stability Assay Using Circular Dichroism Spectroscopy
PPARα/δ/γ-LBD proteins (10 µM) were incubated with different concentrations of ligands in buffer C consisting of 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM Tris 2-carboxyethylphosphine (TCEP)-HCl, and 10% glycerol.The circular dichroism (CD) spectra were monitored within a 200-260 nm range at increasing temperatures from 30 • C to 70 • C (2 • C/min) using a J-1500 spectropolarimeter equipped with a PTC-510 thermal controller (JASCO, Tokyo, Japan).The spectra of all PPARs displayed local minima at 208 and 222 nm, which is a typical feature of α-helical proteins [18].The thermal stability of the PPARs was investigated by continuously monitoring the ellipticity changes at 222 nm during the thermal denaturation [14,15], and a single-site sigmoidal dose-response curve fitting program (Prism 9) was used in order to obtain the melting temperature (T m ) that corresponds to the midpoint of the denaturation process.The ligand-induced increases in the T m values were defined as ∆T m .
All obtained cocrystals were briefly soaked in a cryoprotection buffer (each reservoir solution plus 20% glycerol for PPARα/δ-LBD crystals and 30% glycerol for PPARγ-LBD crystals).Subsequently, these were flash-cooled in a stream of liquid nitrogen until the X-ray diffraction analysis was conducted.

X-ray Diffraction: Data Collection and Model Refinement
Datasets were collected by a BL-5A or a BL-17A beamline at the Photon Factory (Tsukuba, Ibaraki, Japan) using synchrotron radiation of 1.0 Å. Diffraction data were collected at a 0.1 • oscillation per frame, and a total of 1800 frames (180 • ) were recorded for a 1.0-Å X-ray crystallography [14,15,17].Data processing and scaling were carried out using the XDS X-ray detector software (version February 5, 2021) [19] and AIMLESS (version 0.5.21)[20], respectively.Resolution cutoff values (R merge < 0.5, R pim < 0.3, and completeness > 0.9) were set by the highest resolution shell [14,15,17].All structures were determined by using the molecular replacement in PHASER (version 2.7.16) [21] with Protein Data Bank (PDB) IDs: 3SP6 for PPARα-LBD, 2ZNQ or 7WGL for PPARδ-LBD, and 1WM0 or 7WGO for all PPARγ-LBDs as the search model.Refinement was performed using the iterative cycles of the model adjustment in two programs: COOT (version 0.8.2) [22] and PHENIX (version 1.11.1-2575-000)[23].The structures were constructed using the PyMOL program (version 2.5.0).All collection data and refinement statistics are summarized in Supplementary Materials Table S1.

Evaluation of Molecular Interactions between PPAR-LBD Amino Acids and Ligands
Based on those X-ray cocrystal structures, proximity distances between each amino acid in PPARα/δ/γ-LBD and the three ligands were measured using PyMOL.All PPARα/δ/γ-LBD amino acids that have 4.5 Å or less proximity distances from the ligands were listed in Table S2.All molecular interactions between those amino acids and the ligands were evaluated with the MolDock scores using Ligand Energy Inspector programs in Molegro Virtual Docker (MVD) software (version 6.0; CLC bio, Aarhus, Denmark) (Table S2).The scoring function of MolDock is based on an extended piecewise linear potential including new hydrogen bonding and electrostatic terms.
be almost identical to their transactivation profiles, although each PPAR ligand displayed different activities toward PGC1α and SRC1.

Structures of the PPARα/δ/γ-LBD-Lanifibranor Complexes
After some trial and error using various cocrystallization techniques that we applied for PPARα-LBD and its numerous ligands [15], including conventional cocrystallization with multiple buffer sets, cross-seeding, soaking, coactivator addition, and delipidation in
The carboxylic groups of the PPAR ligands are known to stabilize the AF-2 helix 12 through hydrogen bonds (red dotted lines) and electrostatic interactions (blue dotted lines) with the four surrounding consensus amino acids [14]: Ser280/Tyr314/His440/Tyr464 in PPARα-LBD (Figure 4C), Thr253/His287/His413/Tyr437 in PPARδ-LBD (Figure 4F), and Ser289/His323/His449/Tyr473 in PPARγ-LBD (Figure 4I).However, a very close (2.3-Å) proximity was observed in the case of Y473 of PPARγ and lanifibranor (Figure 4I).We have previously defined five regions in PPARα-LBP (Arms I-III/X and Center) [15] and four regions (Arms II/III/X and Center) in PPARδ/γ-LBP [14].Lanifibranor was found to locate in the same position of the Center region in PPARα/δ/γ-LBD, although its benzothiazole moiety in PPARδ was flipped sideways when compared to that in PPARα/γ (Figure 4J).A previous cocrystallization study [31] located lanifibranor in the "Center" (for its benzothiazole ring), the "Arm II" (for its 5-chloroindole moiety), and the "Arm III" (for its carboxylic moiety) regions (yellow in Figure 4K); however, in this study, no electron density was observed in the "Arm III" region (Figure 4K).Our structure seems more reasonable because the interaction of the carboxylic groups of lanifibranor with the four consensus amino acids contributes to the stabilization of the AF-2 helix 12 so as to facilitate the recruitment of coactivators in all PPAR subtypes.

Structures of the PPARα/δ/γ-LBD-Seladelpar Complexes
Due to the fact that seladelpar is a relatively low-affinity PPARα/γ ligand (Figures 1C, 2C,D and 3C), its cocrystals were obtained by cocrystallization with a delipidized PPARα-LBD [15] or with PPARγ-LBD and SRC1 (Figure S1D,F).In contrast, the PPARδ-LBD-seladelpar cocrystals were obtained by employing cocrystallization without coactivators (Figure S1E).X-ray diffraction has revealed the monomeric structures for PPARα (Figure 5A,B) and PPARγ (Figure 5G,H), as well as the dimeric structure for PPARδ (Figures 5D,E and S2E,F).The structures of seladelpar bound to PPARα/δ/γ-LBD were solved in a monoclinic space group P2 1 at a 2.01 Å resolution (PDB ID: 8HUN), a monoclinic space group P2 1 at a 2.67 Å resolution (PDB ID: 8HUO), and an orthorhombic space group P2 1 2 1 2 1 at a 2.36 Å resolution (PDB ID: 8HUP), respectively (Table S1).The electron density maps located a single seladelpar molecule per PPARα/δ/γ-LBD protein monomer (Figures 5B,E,H, and S2F).Seladelpar was located in the similar positions of the asymmetric units (Figures 5B and S2F).The obtained PPARα/δ/γ-LBD helical structures were identical to those of previously reported active conformations described for lanifibranor above.Seladelpar was found to be located in the similar position of the "Center" and the "Arm II" regions in PPARα/δ/γ-LBD; however, its orientations differed (Figure 5J).Likewise, the orientations of the carboxylic group of seladelpar toward the four consensus amino acids in PPARα/δ/γ-LBD (Figure 5C,F,I) were different from those of lanifibranor (Figure 4C,F,I).The loss of the interaction with His323/His449/Tyr473 in PPARγ (Figure 5I) might explain the weak activity of seladelpar against PPARγ.(A-C), PPARδ-LBD (D-F), or PPARγ-LBD (G-I) were analyzed using X-ray diffraction.(A,D,G) Overall structures of the complexes deposited in PDB with IDs 8HUN, 8HUO, and 8HUP, respectively.The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (α-helix in red) are indicated by arrows (only in (G)) and arrowheads, respectively.The highest resolutions are labeled.(B,E,H) Magnified views of seladelpar located in the "Center" and the "Arm II" regions of PPARα/δ/γ-LBD.The electron density is shown in the mesh via F o -F c omit maps contoured at +3.0 σ.Water molecules are presented as cyan spheres in (B).(C,F,I) Hydrogen bonds and electrostatic interactions between seladelpar and the four consensus amino acid residues (that recognize the carboxyl moiety of seladelpar) are indicated by red and blue dotted lines, respectively, along with their distances (in Å). (J) Superposed view of seladelpar in PPARα (magenta)/PPARδ (green)/PPARγ (cyan)-LBD cocrystal structures.

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Structures of the PPARα-LBD-Elafibranor Complex
The PPARα-LBD-elafibranor cocrystals were obtained by soaking the PPARα-LBDclofibric acid-SRC1 cocrystals [15] in a buffer containing elafibranor (Figure S1G).X-ray diffraction analyses revealed the monomeric structure with a single elafibranor and SRC1 (Figure 6A,B).The complex structure was solved in a monoclinic space group P21 at a 1.65 Å resolution (Table S1; PDB ID: 8HUQ).The obtained structures were identical to the previously reported active conformations described above, while hydrogen bond/electrostatic interactions were observed between the carboxylic group of elafibranor and Ser280/Tyr314/His440/Tyr464 in PPARα-LBD (Figure 6C).Regrettably, we failed to obtain PPARδ/γ-LBD-elafibranor cocrystals (Figures S1H,I), although elafibranor did activate PPARδ/γ to some extent (Figures 1D and 2E,F).A-C) was analyzed using X-ray diffraction.(A) The 2.36 Å resolution overall structure of the complex deposited in PDB ID: 8HUQ.The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (αhelix in red) are indicated by an arrow and an arrowhead, respectively.(B) Magnified view of elafibranor located in the "Center" and the "Arm II" regions of PPARα-LBD.The electron density is shown in the mesh via Fo-Fc omit maps contoured at +3.0σ.Water molecules are presented as cyan spheres.(C) Hydrogen bonds and electrostatic interactions between elafibranor and the four consensus amino acid residues (that recognize the carboxyl moiety of elafibranor) are indicated by red and blue dotted lines, respectively, along with their distances (in Å).

LBP Regional Localization of the Five PPAR Ligands in PPARα/δ/γ-LBD
PPARα/δ/γ-LBP comprises five regions (Figure 7A) [14,15,17].In PPARα-LBP, seladelpar, elafibranor, and saroglitazar (PDB ID: 6LXB/6LXC using different crystallization methods) [15] were located in the "Center" and in the "Arm II/X" regions, whereas lanifibranor was only found in the "Center" (Figure 7B).In PPARδ-LBP, lanifibranor was located in the "Center," and seladelpar was located in the "Center" and the "Arm II" regions (Figure 7C).In PPARγ-LBP, lanifibranor was only found in the "Center" while seladelpar and saroglitazar (PDB ID: 7E0A) [17] were found in the "Center" and the "Arm II/X" regions.Mueller et al. deposited a PPARγ-LBD-pioglitazone structure in the PDB, in which two pioglitazone molecules were located in LBP (PDB ID: 2XKW; not published in a pa- 3.7.LBP Regional Localization of the Five PPAR Ligands in PPARα/δ/γ-LBD PPARα/δ/γ-LBP comprises five regions (Figure 7A) [14,15,17].In PPARα-LBP, seladelpar, elafibranor, and saroglitazar (PDB ID: 6LXB/6LXC using different crystallization methods) [15] were located in the "Center" and in the "Arm II/X" regions, whereas lanifibranor was only found in the "Center" (Figure 7B).In PPARδ-LBP, lanifibranor was located in the "Center," and seladelpar was located in the "Center" and the "Arm II" regions (Figure 7C).In PPARγ-LBP, lanifibranor was only found in the "Center" while seladelpar and saroglitazar (PDB ID: 7E0A) [17] were found in the "Center" and the "Arm II/X" regions.Mueller et al. deposited a PPARγ-LBD-pioglitazone structure in the PDB, in which two pioglitazone molecules were located in LBP (PDB ID: 2XKW; not published in a paper): one was in the "Center" and the "Arms II/X" regions and the other was in the "Arms II/III" (Figure 7D).These results indicate that each PPAR ligand has a flexible molecular frame allowing it to bind to different regions of the PPARα/δ/γ-LBPs.

Discussion
This study investigated how lanifibranor, seladelpar, and elafibranor bind to and activate PPARα/δ/γ subtypes.Major antioxidant genes, including catalase, heme oxygenase-1, glutathione peroxidase 3/4, superoxide dismutase 2/3, thioredoxin, CD36, and uncoupling protein 2/3, contain PPAR responsive element (PPRE) in their promoter regions and are transcriptionally regulated by the PPARs [34][35][36].In addition, a putative PPRE is present in the promoter region of Nrf2 that is one of the most important regulators of cellular responses to oxidative stress and inflammation [37].Therefore, such PPAR agonists might potently modulate systemic redox homeostasis and inflammation as well as lipid/insulin signaling to counteract NASH.
Lanifibranor, the PPARα/δ/γ pan agonist developed by Inventiva (Daix, France) [31], was first described for its prevention of experimental skin [38] and lung fibrosis [39], and was then applied to liver fibrosis [40].In a phase 2b study involving 247 patients with active NASH (NCT03008070), lanifibranor significantly improved general NASH conditions [41].A large-scale (~1000 patients) phase 3 study, evaluating the long-term efficacy and safety of lanifibranor in adult NASH patients with Fibrosis 2/3 stage of liver fibrosis (NCT04849728) is currently underway, and its results have not been posted yet.The EC 50 values for PPARα/δ/γ were in small ranges (0.4-5 µM in Figure 1B and 3-14 µM in Figure 2A,B), and therefore, lanifibranor in clinical doses seems to be able to activate all PPAR subtypes at once.Due to the fact that its efficacies for PPARγ activation often match with those of pioglitazone (Figure 2A vs. Figure 2I, Figure 2B vs. Figure 2J, and Figure 3B vs. Figure 3F), PPARγ-related undesirable side effects (such as weight gain, edema, bone loss, and congestive heart failure) [42] should be closely monitored in the case of lanifibranor.In the cocrystal structures, indeglitazar, another PPARα/δ/γ pan agonist that resembles lanifibranor in structure, has also been reported to exist in the similar "Center" regions in PPARα/δ/γ (PDB ID: 3ET1, 3ET2, and 3ET3, respectively) [43] and some consensus amino acids in the Center region (such as Phe273/Ile354, Phe246/Ile327, and Phe282/Phe363 in PPARα/δ/γ-LBD, respectively) were highly stabilized by lanifibranor (Table S2); therefore, such molecular frames might be favorable for a PPAR pan activity.
Seladelpar is the novel PPARδ-selective agonist developed by CymaBay Therapeutics (Newark, NJ, USA) [44].Although its clinical trial against NAFLD/NASH was once discontinued at phase 2 due to abnormal findings on liver biopsy [9], the FDA thereafter lifted the injunction on July 2020 due to subsequent doubts about the relevance of the drug [45].The clinical trials have not resumed since then, and thus, its therapeutic potential against NASH remains unknown.In its clinical trial against hyperlipidemia (NCT00701883), seladelpar reduced the assessed blood lipid parameters (triglyceride, total cholesterol, LDLcholesterol, and free fatty acid levels), the alkaline phosphatase and γ-glutamyl transferase (GGT) activities, and the homeostatic model assessment-insulin resistance [46].Moreover, prompted by its positive results in its phase 2 clinical trial for primary biliary cholangitis, a phase 3 clinical trial is currently underway [44,47].In mice, seladelpar has been reported to reverse dyslipidemia and the hepatic storage of lipotoxic lipids, thereby improving the NASH pathology in atherogenic-diet-fed obese diabetic mice [48].Seladelpar could activate all PPAR subtypes; however, the EC 50 values for PPARδ-LBD were ~2 orders lower than those for PPARα/γ-LBD (Figures 1C and 2C,D), and this is why seladelpar can be used as a PPARδ-selective agonist in clinical use.In the cocrystal structures, seladelpar was bound to the "Center" and the "Arm II" regions of all PPARα/δ/γ-LBDs (Figure 5J), similar to the PPARδ-selective full agonist GW501516 in PPARδ-LBD (PDB ID: 5U46) [49].Both carboxylic and trifluoromethyl groups of seladelpar were located in positions similar to those of GW501516, thereby implying that amino acid residues in those regions were important for the full activation of PPARδ.Indeed, Val298, Leu303, Val312, and Ile328, which were reported to be important for the binding to GW501516 and other synthetic PPARδ-selective ligands [49], were all stabilized by seladelpar (Table S2).
Elafibranor, the PPARα/δ dual agonist developed by Genfit (Loos, France), has been abandoned at the phase 3 clinical trial against NAFLD/NASH due to its non-significant effect on the primary endpoint of the resolution of NASH without a worsening of fibrosis [10].In a phase 2 clinical trial (NCT01694849), its efficacy and safety at 80 and 120 mg/day doses for 52 weeks were confirmed among the 275 participating NASH patients [50].Elafibranor also reduced the fasting plasma triglyceride levels and GGT activities, increased HDL cholesterol, and decreased insulin resistance and fasting plasma glucose levels, in abdominally obese patients with either combined dyslipidemia or prediabetes [51].Further clinical trials have suggested that elafibranor can improve peripheral and hepatic insulin sensitivity [52].As its EC 50 values for PPARα/δ/γ-LBD were in small ranges (0.4-3 µM in Figure 1D and 5-16 µM in Figure 2E,F), elafibranor in clinical doses seems to be able to activate all PPAR subtypes at once.The efficacy of elafibranor for PPARγ activation was lower than that of pioglitazone in the cases of transactivation (Figure 1D,F), PGC1α (Figure 2E,I), and SRC1 recruitment (Figure 2F,J).Interestingly, the impact of elafibranor on the thermal stability of PPARα/δ/γ-LBD was much weaker than that of other ligands (Figure 3D).Regrettably, we failed to obtain PPARδ/γ-LBD-elafibranor cocrystals (Figure S1H,I).Therefore, it might be difficult for elafibranor to form stable complexes with any of the PPAR subtypes, especially with PPARδ/γ, and to induce enough clinical impact in some cases (e.g., NASH).
When compared to the LBPs of other nuclear receptors that have 600-1100 Å 3 cavities, the LBPs of PPARα/δ/γ have relatively large (1300-1440 Å 3 ) cavities that are able to accept 1-4 ligand molecules [15].The PPARα/δ/γ-LBD-various ligands' cocrystal structures registered in the PDB until 18 July 2023 (60, 52, and 288, respectively; among which 40, 8, and 25 registrations, respectively, derive from our laboratories) demonstrate their extremely diverse ligand binding modes.We have, herein, added seven novel structures (three, two, and two for PPARα/δ/γ, respectively) with altered ligand locations.However, unfortunately, we failed to provide a clear molecular basis (such as close interactions with specific PPAR-LBD amino acids; Table S2) for PPARα/δ/γ targeting preferences of these three drugs, perhaps in part for large PPAR-LBPs, which is the limitation of this study.Nevertheless, future drug discovery for NASH will undoubtedly benefit from functional and structural investigation of the PPARα/δ/γ-ligand molecular interactions such as in this study.Lanifibranor (rather than elafibranor) could be a prototype of PPAR pan agonists that bind to the similar positions in PPARα/δ/γ-LBD and thus have comparable preferences for PPARα/δ/γ, and seladelpar could be a lead compound of upcoming PPARδ-selective agonists with more preferences to PPARδ and less preferences to PPARα/γ.

Antioxidants 2023 , 18 Figure 4 .
Figure 4. PPARα/δ/γ-LBD-lanifibranor cocrystal structures.Cocrystals of lanifibranor and PPARα-LBD (A-C), PPARδ-LBD (D-F), or PPARγ-LBD (G-I) were analyzed using X-ray diffraction.(A,D,G) Overall structures of the complexes deposited in PDB with IDs: 8HUK, 8HUL, and 8HUM, respectively.The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (α-helix in red) are indicated by arrows (only in (G)) and arrowheads, respectively.The highest resolutions are labeled.(B,E,H) Magnified views of lanifibranor located in the "Center" region of PPARα/δ/γ-LBD.The electron density is shown in the mesh via Fo-Fc omit maps contoured at +3.0 σ.A water molecule is presented as a cyan sphere in (H).(C,F,I) Hydrogen bonds and electrostatic interactions between lanifibranor and the four consensus amino acid residues (that recognize the carboxyl moiety of lanifibranor) are indicated by red and blue dotted lines, respectively, along with their distances (in

Figure 6 .
Figure 6.PPARα-LBD-elafibranor cocrystal structures.A cocrystal of elafibranor and PPARα-LBD (A-C) was analyzed using X-ray diffraction.(A) The 2.36 Å resolution overall structure of the complex deposited in PDB ID: 8HUQ.The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (αhelix in red) are indicated by an arrow and an arrowhead, respectively.(B) Magnified view of elafibranor located in the "Center" and the "Arm II" regions of PPARα-LBD.The electron density is shown in the mesh via Fo-Fc omit maps contoured at +3.0σ.Water molecules are presented as cyan spheres.(C) Hydrogen bonds and electrostatic interactions between elafibranor and the four consensus amino acid residues (that recognize the carboxyl moiety of elafibranor) are indicated by red and blue dotted lines, respectively, along with their distances (in Å).

Figure 6 .
Figure 6.PPARα-LBD-elafibranor cocrystal structures.A cocrystal of elafibranor and PPARα-LBD (A-C) was analyzed using X-ray diffraction.(A) The 2.36 Å resolution overall structure of the complex deposited in PDB ID: 8HUQ.The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (α-helix in red) are indicated by an arrow and an arrowhead, respectively.(B) Magnified view of elafibranor located in the "Center" and the "Arm II" regions of PPARα-LBD.The electron density is shown in the mesh via F o -F c omit maps contoured at +3.0 σ.Water molecules are presented as cyan spheres.(C) Hydrogen bonds and electrostatic interactions between elafibranor and the four consensus amino acid residues (that recognize the carboxyl moiety of elafibranor) are indicated by red and blue dotted lines, respectively, along with their distances (in Å).