Glucose-6-phosphate dehydrogenase (G6PD)

PDD4091 is a novel and selective inhibitor of G6PD activity

To determine whether G6PD inhibition reduces PH, we first established the maximum tolerated dose of G6PD inhibitor (PDD4091 ). The maximum tolerated dose in Hx mice was 15 mg kg−1 day−1, beyond which PDD4091 depressed LV function. More importantly, treatment with the G6PD inhibitor PDD4091 to Hx mice decreased the elevated RVSP and RVEDP in a dose-dependent manner (Fig. 1, B and C, top panel). PDD4091 had a reasonably wide therapeutic window (0.01–15 mg kg−1 day−1) with an EC50 of 0.26 ± 0.10, and 0.58 ± 0.36 mg kg−1 day−1 reduced both RVSP and RVEDP. Moreover, PDD4091 (1.5 mg kg−1 day−1) treatment to both Hx and Hx + SU mice efficaciously reduced the elevated RVSP and RVEDP (Fig. 1, B and C, bottom panel). Fulton’s index was increased in Hx and Hx + SU groups as compared with Nx and Nx + SU groups. G6PD inhibitor reduced elevated Fulton’s index in Hx and Hx + SU groups (Table 1). In addition, PDD4091 (1.5 mg kg−1 day−1) treatment to mice (n = 6) with preexisting Hx + SU–induced PH reduced RVSP (from Hx + SU: 74 ± 5.3 to Hx + SU + PDD4091: 41 ± 3.3 mm Hg; P < 0.05), RVEDP (from Hx + SU: 13 ± 3 to Hx + SU + PDD4091: 7 ± 1 mm Hg; P < 0.05), and Fulton’s index (from Hx + SU: 0.4 ± 0.01 to Hx + SU + PDD4091: 0.3 ± 0.01; P < 0.05). PDD4091 (1.5 mg kg−1 day−1) reduced G6PD activity by 20%. [1]

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G6PD Inhibition Relaxed Precontracted PA, Decreased PASMC Growth, and Reduced PA Remodeling in Hx + SU Mice. [1]
PA remodeling is the hallmark of severe PH. Hyperplasic and apoptosis-resistant PA endothelial cells and PASMCs contribute to hypertensive remodeling (Morrell et al., 2009). Previously, we and others proposed that SMCs switch from a differentiated to a dedifferentiated phenotype in PA of hypertensive patients and animals and contribute to PA remodeling (Zhou et al., 2009; Chettimada et al., 2015; Sahoo et al., 2016). Dedifferentiated SMCs are hyperproliferative, migratory, and secretory (Frismantiene et al., 2018). Previous studies show that the Hx + SU mouse model of PH has more severe PA remodeling than Hx mice (Vitali et al., 2014). Therefore, we determined whether G6PD inhibition relaxes PA in ex vivo studies, stunts the growth of PASMCs exposed to Hx and SU in cell culture, and reduces remodeling of PA in Hx + SU mice. Our results demonstrated PDD4091 dose-dependently relaxed PA precontracted with KCl (Fig. 2A). Application of PDD4091 [1 μmol/l, an EC50 dose (Hamilton et al., 2012)] for 48 hours to PASMCs cultured in normoxia decreased cell numbers (Fig. 2B) and in addition attenuated the cell growth evoked by Hx and Hx + SU (Fig. 2C). Treatment of Hx + SU mice with PDD4091 (1.5 mg kg−1 day−1) for 3 weeks abrogated the occlusive pulmonary vascular remodeling (Fig. 2D).

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G6PD Inhibition Decreased Expression of Cyp1a1 and Sufu Genes in Lungs of Mice and in Human PASMCs Exposed to Hx + SU. [1]
Next, we determined whether inhibition of G6PD activity decreases expression of Cyp1a1 and Sufu, which are increased in lungs of hypertensive mice (Fig. 3C) and Hx and Hx + SU mice and in human PASMCs exposed to Hx + SU. Treatment of PDD4091 (1.5 mg kg−1 day−1) for 3 weeks to mice and application of PDD4091 (1 μM) to human PASMCs for 48 hours rescinded the Hx + SU–induced Cyp1a1 and Sufu expression in lungs (Fig. 4, A and B) and in human PASMCs (Fig. 4C).

Human pulmonary artery smooth muscle cells were maintained at 37°C under 5% CO2 in smooth muscle basal media supplemented with growth factors (SMGM-2 smooth muscle singlequots kit). Once cells reached approximately 70% confluence, they were subcultured using 0.05% trypsin-EDTA into six-well plates at about 3 × 105 cells per well.

Animal Models and Experimental Protocols. [1]
Male and female C57BL/6J mice (18–32 g) were purchased from the Jackson Laboratory and were randomly divided into four groups: normoxia (Nx), normoxia + Sugen5416 (Nx + SU), hypoxia (Hx), and hypoxia + Sugen5416 (Hx + SU) groups. Mice in the Nx group were placed in a normoxic (21% O2) environment, and mice in the Hx group were placed in a normobaric hypoxic chamber (10% O2) for 6 weeks (Joshi et al., 2020). Mice in the Nx + SU and Hx + SU groups received subcutaneous injection of SU5416 (20 mg/kg) once weekly during 3 weeks of Nx (21% O2) or Hx (10% O2) as previously described (Vitali et al., 2014). Mice in the drug treatment groups received daily subcutaneous injection of a novel G6PD inhibitor, N-[(3β,5α)-17-oxoandrostan-3-yl]sulfamide (PDD4091; 1.5 mg kg−1 day−1) (Hamilton et al., 2012), for 3 weeks. To determine whether PDD4091 reduces PH in a dose-dependent manner, mice were randomized to receive low-dose (0.15 mg kg−1 day−1), medium-dose (1.5 mg kg−1 day−1), or high-dose (15 mg kg−1 day−1) injections of PDD4091. Hx and Hx + SU mice are preclinical models of PH (Stenmark et al., 2009). At the end of the treatment period, hemodynamic measurements were performed, tissue (lungs and arteries) was harvested, and blood samples were collected. Data analysis was performed in a blinded fashion.

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Reduced Representation Bisulfite Sequencing. [1]
To determine DNA methylation status in lungs of Nx, Hx, Hx + SU, and Hx + PDD4091 mice, genomic DNA was isolated from lungs using the Qiagen All Prep DNA/RNA/miRNA Universal kit according to the manufacturer’s instructions. DNA was quantified using the NanoDrop and Qubit Fluorometer. Genomic DNA quality was assessed using the Agilent TapeStation. Reduced representation bisulfite sequencing library construction was performed with the Premium Reduced Representation Bisulfite Sequencing Kit following the manufacturer’s instructions. Libraries were sequenced on the HiSeq2500 with paired-end reads of 125 nt. Raw reads generated from the Illumina HiSeq2500 sequencer were de-multiplexed using bcl2fastq version 2.19.0. Quality filtering and adapter removal are performed using Trim Galore version 0.4.4_dev with the following parameters: “–paired–clip_R1 3–clip_R2 3–three_prime_clip_R1 2–three_prime_clip_R2 2” (http://www.bioinformatics.babraham.ac.uk/projects/ trim_galore/). Processed and cleaned reads were then mapped to the mouse reference genome (mg38) using Bismark version 0.19.0 with the following parameters: “–bowtie2–maxins 1000.” Differential methylation analysis was performed using methylKit version 1.4.0 within an R version 3.4.1 environment. Bismark alignments were processed via methylKit in the CpG context with a minimum quality threshold of 10. Coverage was normalized after filtering for loci with a coverage of at least five reads and no more than the 99.9th percentile of coverage values. The coverage was then normalized across samples, and the methylation counts were aggregated for 500-nt windows spanning the entire genome. A unified window set across samples was derived such that only windows with coverage by at least one sample per group were retained. Differential methylation analysis between conditional groups was performed using the χ2 test and applying a q-value (SLIM) threshold of 0.05 and a methylation difference threshold of 25%.

[1]. Inhibition of Glucose-6-Phosphate Dehydrogenase Activity Attenuates Right Ventricle Pressure and Hypertrophy Elicited by VEGFR Inhibitor + Hypoxia. J Pharmacol Exp Ther. May 1, 2021, 377 (2) 284-292.

Pulmonary hypertension (PH) is a proliferative disease of pulmonary angiocytes. The pentose phosphate pathway (PPP) is an important pathway of glucose metabolism and is crucial for cell growth. Given the poor efficacy of current treatments for PH, our goal was to determine whether inhibiting the rate-limiting enzyme of PPP—glucose-6-phosphate dehydrogenase (G6PD)—could prevent aberrant gene expression that promotes smooth muscle cell (SMC) proliferation, reduce pulmonary artery remodeling, and restore hemodynamics to normal in an experimental PH model. We induced PH in mice by exposing them to 10% oxygen (Hx) or by weekly injections of the vascular endothelial growth factor receptor blocker [Sugen5416 (SU); 20 mg kg−1] (Hx + SU) during hypoxia exposure. During hypoxia (Hx) exposure, a novel G6PD inhibitor (N-[(3β,5α)-17-oxoandrostan-3-yl]sulfonamide; 1.5 mg kg−1) was administered daily. We measured right ventricular (RV) pressure, left ventricular pressure-volume relationship, and lung tissue gene expression in mice treated with normoxic oxygenation, hypoxia (Hx), hypoxia combined with sulfonamide (Hx + SU), and G6PD inhibitors. Compared with normoxic control mice, hypoxic (Hx) and hypoxia combined with sulfonamide (Hx + SU) mice showed increased right ventricular systolic and end-diastolic pressures. Hypoxia (Hx) and hypoxia combined with sulfonamide (Hx + SU) reduced the expression of epigenetic modifiers (writing and erasing factors), increased DNA hypomethylation, and induced aberrant gene expression in lung tissue. Compared with untreated pulmonary hypertension (PH), G6PD inhibitors reduced aberrant gene expression and smooth muscle cell (SMC) proliferation, decreased pulmonary vascular remodeling, and reduced right ventricular pressure. By modulating the activity of epigenetic modifiers and DNA methylation, pharmacological inhibition of G6PD activity effectively reduced right ventricular pressure load in Hx and Hx+SU mice and preclinical models of pulmonary hypertension, and appears to be a safe pharmacological treatment strategy. [1]

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In addition to inhibiting the expression of undesirable genes in pulmonary artery wall vascular cells and reducing cell growth in occlusive pulmonary disease, PDD4091 (a novel selective G6PD activity inhibitor (Hamilton et al., 2012)) also dose-dependently dilates preconstricted pulmonary arteries. We have previously demonstrated that inhibition of G6PD activity with nonspecific inhibitors (e.g., 17-ketosteroids [dehydroepiandrosterone (DHEA) and its metabolite epidermo-2-one]) and G6PD gene knockdown mediated by siRNA can dilate preconstricted pulmonary arteries (Gupte et al., 2002) and reduce right ventricular pressure in hypertensive rats (Chettimada et al., 2012, 2015). Therefore, these studies, along with our current findings, collectively demonstrate that G6PD inhibition can reduce elevated right ventricular pressure and pulmonary hypertension in Hx and Hx+SU mice by dilating pulmonary arteries and reducing pulmonary artery remodeling. In summary, our findings collectively indicate that G6PD activity is an important factor in differential DNA methylation, abnormal gene expression, and remodeling in the pulmonary arteries of Hx and Hx+SU mice. Inhibiting G6PD activity through drug intervention can eliminate pulmonary vascular remodeling and improve hemodynamics in a mouse model of pulmonary hypertension. Therefore, the G6PD inhibitor N-[(3β,5α)-17-oxoandrostan-3-yl]sulfonamide may be used as a drug for treating different types of pulmonary hypertension in the future. [1]

C19H32N2O3S

368.533984184265

368.21

C, 61.92; H, 8.75; N, 7.60; O, 13.02; S, 8.70

1373651-41-2

1373651-41-2;

Solid powder

C[C@@]12[C@@]3([C@]([C@]4([C@](C)(CC3)C(=O)CC4)[H])(CC[C@]1(C[C@@H](NS(N)(=O)=O)CC2)[H])[H])[H]

QLIJDTTVUNSTPX-LUJOEAJASA-N

InChI=1S/C19H32N2O3S/c1-18-9-7-13(21-25(20,23)24)11-12(18)3-4-14-15-5-6-17(22)19(15,2)10-8-16(14)18/h12-16,21H,3-11H2,1-2H3,(H2,20,23,24)/t12-,13-,14-,15-,16-,18-,19-/m0/s1

N-[(3beta,5alpha)-17-Oxoandrostan-3-yl]sulfamide

PDD4091; PDD 4091; PDD-4091

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)