PPARγ LBD (Ki = 1.2 μM); PPARγ LBD (EC50 = 680 nM (PPARγ LBD)

Pioglitazone (Pio) is an FDA-approved drug for type 2 diabetes that binds and activates the nuclear receptor PPARγ. yet it remains unclear how in vivo Pio metabolites affect PPARγ structure and function. In this study, researchers present a structure-function comparison of Pio and its most abundant in vivo metabolite, Leriglitazone (1-hydroxypioglitazone; PioOH). PioOH displayed a lower binding affinity and reduced potency in coregulator recruitment assays. X-ray crystallography and molecular docking analysis of PioOH-bound PPARγ ligand-binding domain (LBD) revealed an altered hydrogen bonding network, including formation of water-mediated bonds that could underlie its altered biochemical phenotype. NMR spectroscopy and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis coupled to activity assays revealed that PioOH better stabilizes the PPARγ activation function-2 (AF-2) coactivator binding surface and better enhances coactivator binding, affording slightly better transcriptional efficacy. These results indicating Pio hydroxylation affects its potency and efficacy as a PPARγ agonist contribute to our understanding of PPARγ-drug metabolite interactions[1].

In this study, researchers found that leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model[https://pubmed.ncbi.nlm.nih.gov/33171227/].

Circular dichroism (CD) spectroscopy[1]
CD wavelength scans and thermal denaturation experiments monitored at 222 nm were performed in CD buffer (10 mM potassium phosphate (pH 7.4) and 50 mM potassium fluoride) to determine the folding and stability of PPARγ LBD (10 µM) in the presence of one molar equivalent of Pio or Leriglitazone (1-hydroxypioglitazone; PioOH) on a Jasco J-815 spectropolarimeter. Thermal denaturation was monitored at 1°C intervals along a temperature gradient from 20°C to 80°C. Raw ellipticity was plotted using GraphPad Prism and fit to a set of equations based on the Gibbs-Helmholtz equation, as previously reported.

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TR-FRET competitive ligand displacement and coregulator interaction assays[1]
Time-resolved fluorescence resonance energy transfer (TR-FRET) assays were performed in black low-volume 384-well plate using a buffer containing 20 mM potassium phosphate (pH 7.4), 50 mM potassium chloride, 0.5 mM EDTA, and 5 mM TCEP, and 0.01% Tween-20. For the ligand displacement assay, each well (22.5 μL per well) contained 1 nM 6xHis-PPARγ LBD protein, 1 nM LanthaScreen Elite Tb-anti-His Antibody, and 5 nM Fluormone Pan-PPAR Green tracer ligand in TR-FRET buffer. For the TR-FRET coregulator interaction assay, each well contained 400 nM FITC-labeled TRAP220 or NCoR1 peptides, 4 nM 6xHis-PPARγ LBD protein, 1 nM LanthaScreen Elite Tb-anti-His Antibody, and 400 nM peptide in TR-FRET buffer in 22.5 μL total volume per well. Ligand stocks were prepared via serial dilution in DMSO, added to wells in triplicate to a final DMSO concentration of 1%, and the plates were incubated at 25 °C for ~1 h and read using BioTek Synergy Neo multimode plate reader. The Tb donor was excited at 340 nm; its fluorescence emission was monitored at 495 nm, and the acceptor FITC emission was measured at 520 nm. The TR-FRET ratio was calculated as the signal at 520 nm divided by the signal at 495 nm. Data were plotted using GraphPad Prism and fit to the appropriate equation: for the ligand displacement assay, data were fit to a competitive one site fit Ki equation using the known binding affinity of Fluormone™ Pan-PPAR Green tracer ligand (2.8 nM); and for the coregulator interaction assay data were fit to a sigmoidal dose response equation. For the competitive binding assay, significance was determined by F-test analysis of the Pio vs. Leriglitazone (1-hydroxypioglitazone; PioOH) fit Ki value. For the TR-FRET coregulator recruitment assays, significance was determined by unpaired t-test of EC/IC50 values from n=2 individual experiments.

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Fluorescence polarization coregulator interaction assays[1]
Fluorescence polarization assays were performed in black low-volume 384-well plates (Greiner) using a buffer containing 20 mM potassium phosphate (pH 7.4), 50 mM potassium chloride, 0.5 mM EDTA, 5 mM TCEP, and 0.01% Tween-20. Each well contained 100 nM FITC-labeled TRAP220 coactivator peptide, a serial dilution of PPARγ LBD (1.5 nM–90 µM), with a fixed concentration of vehicle control (1% DMSO) or ligand equal to the highest protein concentration (90 µM Pio or Leriglitazone (1-hydroxypioglitazone; PioOH)) in triplicate. Plates were incubated 2 hrs at 4°C and read using BioTek Synergy Neo multimode plate reader. Data were plotted in GraphPad Prism and fit to a sigmoidal dose response equation. Peptide affinity and polarization window from n=2 individual experiments was analyzed by unpaired t-test.

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Isothermal titration calorimetry[1]
A peptide derived from the TRAP220 coactivator (residues 638–656; NTKNHPMLMNLLKDNPAQD) was synthesized by Lifetein and resuspended at 500 μM in buffer containing 20 mM potassium phosphate (pH 7.4), 50 mM potassium chloride, 0.5 mM EDTA, and 5 mM TCEP. PPARγ LBD was prepared at 50 μM in identical buffer. 100 μM Pio or Leriglitazone (1-hydroxypioglitazone; PioOH) were added to PPARγ LBD and TRAP220 and incubated on ice 30 minutes before each experiment. TRAP220 peptide (syringe) was titrated into PPARγ LBD (sample cell). 20 total injections were made per experiment (0.4 μL for the first injection, 2.0 μL for subsequent injections), using a mixing speed of 1200 rpm, a reference power of 5 μcal/second, and a cell temperature of 25ºC. Two runs were performed for each ligand-bound condition. Experiments were performed using a MicroCal iTC200 (Malvern). Data were processed in NITPIC 45 to determine binding stoichiometry and further analyzed by unbiased global fitting of both replicate runs per ligand-bound condition in SEDPHAT 46, followed by export to GUSSI for publication-quality figure preparation. The SEDPHAT fitting model used was A + B to AB heteroassociation and the fit parameters were enthalpy (ΔH) and affinity (Kd).

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NMR spectroscopy[1]
Two dimensional [1H,15N] -transverse relaxation optimized spectroscopy (TROSY)-heteronuclear single quantum correlation (HSQC) data were collected at 298K using a Bruker 700 Mhz NMR instrument equipped with a QCI cryoprobe. Samples contained 200 µM 15N-labeled PPARγ LBD in a buffer (NMR buffer) containing 20 mM potassium phosphate (pH 7.4), 50 mM potassium chloride, 0.5 mM EDTA, 5 mM TCEP, and 10% D2O in the absence or presence of two molar equivalents of Pio or Leriglitazone (1-hydroxypioglitazone; PioOH). Data were processed and analyzed using Topspin 3.0 (Bruker) and NMRViewJ. NMR analysis was performed using previously described rosiglitazone-bound NMR chemical shift assignments (BMRB entry 17975) for well resolved residues with consistent NMR peak positions via the minimum chemical shift procedure.

Cell-based transactivation assays[1]
HEK293T cells cultured in DMEM media supplemented with 10% fetal bovine serum (FBS) and 50 units mL–1 of penicillin, streptomycin, and glutamine were grown to 90% confluency in a T-75 flask before seeding 4 million cells per well in 10-cm dishes. Seeded cells were transfected using transfection reagent containing 27 µL X-treme Gene 9 in serum-free Opti-mem reduced serum media (Gibco) with either 4.5 µg pCMV6-XL4 plasmid containing full-length human PPARγ2 and 4.5 µg 3X multimerized PPRE-luciferase reporter or 4.5 µg Gal4-PPARγ LBD and 4.5 µg 5X Upstream Activation Sequence (UAS) luciferase reporter. After 18 hrs incubation at 37 °C in a 5% CO2 incubator, the transfected cells were plated in quadruplicate in white 384-well plates at a density of 10,000 cells per well (20 µL volume) and incubated 4 hrs then treated with 20 µL of vehicle control (1% DMSO in DMEM media) or 1:2 serial dilution of each compound from 56 pM–10 µM (1% final DMSO concentration). After 18 hrs, luciferase activity was measured by addition of 20 µL Britelite Plus and luminescence was read using a BioTek Synergy Neo multimode plate reader. Data were plotted in GraphPad Prism as fold change in luminescence of compound-treated cells over DMSO-treated control cells vs ligand concentration and fit to a sigmoidal dose response equation. EC50 values from n=4 individual experiments for full length PPARγ and n=2 individual experiments for PPARγ-Gal4 were analyzed by unpaired t-test; the response window from individual experiments was analyzed by paired t-test.

In this study, researchers assess whether MIN-102 (INN: Leriglitazone (1-hydroxypioglitazone; PioOH)), a novel brain penetrant and orally bioavailable PPARγ agonist with an improved profile for central nervous system (CNS) diseases, rescues phenotypic features in cellular and animal models of FRDA. In frataxin-deficient dorsal root ganglia (DRG) neurons, leriglitazone increased frataxin protein levels, reduced neurite degeneration and α-fodrin cleavage mediated by calpain and caspase 3, and increased survival. Leriglitazone also restored mitochondrial membrane potential and partially reversed decreased levels of mitochondrial Na+/Ca2+ exchanger (NCLX), resulting in an improvement of mitochondrial functions and calcium homeostasis. In frataxin-deficient primary neonatal cardiomyocytes, leriglitazone prevented lipid droplet accumulation without increases in frataxin levels. Furthermore, leriglitazone improved motor function deficit in YG8sR mice, a FRDA mouse model. In agreement with the role of PPARγ in mitochondrial biogenesis, leriglitazone significantly increased markers of mitochondrial biogenesis in FRDA patient cells. Overall, these results suggest that targeting the PPARγ pathway by leriglitazone may provide an efficacious therapy for FRDA increasing the mitochondrial function and biogenesis that could increase frataxin levels in compromised frataxin-deficient DRG neurons. Alternately, leriglitazone improved the energy metabolism by increasing the fatty acid β-oxidation in frataxin-deficient cardiomyocytes without elevation of frataxin levels. This could be linked to a lack of significant mitochondrial biogenesis and cardiac hypertrophy. The results reinforced the different tissue requirement in FRDA and the pleiotropic effects of leriglitazone that could be a promising therapy for FRDA.https://pubmed.ncbi.nlm.nih.gov/33171227/

[1]. Structural Basis of Altered Potency and Efficacy Displayed by a Major in Vivo Metabolite of the Antidiabetic PPARγ Drug Pioglitazone. J Med Chem. 2019 Feb 28;62(4):2008-2023.

Leriglitazone Hydrochloride is the hydrochloride salt form of leriglitazone, an orally bioavailable, blood-brain-barrier (BBB) penetrable, selective peroxisome proliferator-activated receptor (PPAR) subtype gamma agonist, with potential neuroprotective activity that could be used for certain central nervous system (CNS) diseases, such as adrenomyeloneuropathy, cerebral adrenoleukodystrophy (cALD), Friedreich's ataxia, and certain other CNS diseases. Upon oral administration, leriglitazone selectively targets, binds to and activates PPARgamma, thereby regulating the expression of genes involved in mitochondrial biogenesis. This modulates pathways leading to the restoration of mitochondrial function in which dysfunction is caused by the accumulation of very long-chain fatty acids (VLCFAs), and increases energy production, decreases oxidative stress, decreases nuclear factor kappa B (NF-kB) levels, inhibits neuroinflammation, protects the BBB integrity, prevents demyelination and axonal degeneration, increases neuronal survival, increases myelination and oligodendrocyte survival and improves motor function. Mutations in the ABCD1 gene, which encodes the peroxisomal membrane adrenoleukodystrophy protein, cause a defective function of the ABCD1 transporter leading to an accumulation of VLCFA. VLCFA accumulation contributes to membrane destabilization of the myelin sheath, mitochondrial dysfunction, oxidative stress, neuroinflammation and compromised BBB integrity.

C19H21CLN2O4S

408.899042844772

408.091

C, 55.81; H, 5.18; Cl, 8.67; N, 6.85; O, 15.65; S, 7.84

146062-46-6

Leriglitazone;146062-44-4; Leriglitazone hydrochloride;146062-46-6;Leriglitazone-d4;1188263-49-1; 146062-44-4

86748889

White to light yellow solid powder

3

6

7

27

496

0

SBHOQYCDAHAMDW-UHFFFAOYSA-N

InChI=1S/C19H20N2O4S.ClH/c1-12(22)14-4-5-15(20-11-14)8-9-25-16-6-2-13(3-7-16)10-17-18(23)21-19(24)26-17;/h2-7,11-12,17,22H,8-10H2,1H3,(H,21,23,24);1H

5-[[4-[2-[5-(1-hydroxyethyl)pyridin-2-yl]ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione;hydrochloride

1-Hydroxy Pioglitazone Hydrochloride; Leriglitazone hydrochloride; WN9K42SAM6; Leriglitazone (hydrochloride); Leriglitazone hydrochloride [USAN]; 5-[[4-[2-[5-(1-Hydroxyethyl)pyridin-2-yl]ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione;hydrochloride; HYDROXYPIOGLITAZONE HYDROCHLORIDE;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~122.28 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)