PDE4; Long-form isoforms of cyclic AMP-specific phosphodiesterase 4 (PDE4), including PDE4A4, PDE4B1, PDE4C3, and PDE4D5 [1]

MR-L2 activates representative long-form PDE4 isoforms from all four PDE4 subfamilies (PDE4A4, PDE4B1, PDE4C3, PDE4D5) in a concentration-dependent manner. For PDE4D5, a ∼60% increase in activity over basal levels was observed at maximally effective concentrations. [1]
MR-L2 does not activate short PDE4 isoforms (PDE4A1, PDE4B2, PDE4D1/2) or an engineered construct comprising solely the core catalytic domain of PDE4D without UCR1, UCR2, and C-terminal tail. [1]
MR-L2 fails to enhance the activity of exemplars from any of the other 10 families within the PDE superfamily, demonstrating selectivity for the PDE4 family. [1]
Kinetic analysis of MR-L2 action on PDE4D5 revealed that it acts as a reversible, noncompetitive activator, increasing the apparent Vmax for cAMP hydrolysis without affecting the apparent Km for cAMP. [1]
The S126D phosphomimetic mutant of PDE4D5 (constitutively activated to mimic PKA phosphorylation) exhibited near-complete ablation of sensitivity to activation by MR-L2. [1]
The S133D phosphomimetic mutant of PDE4D5 (mimicking MK2 phosphorylation) was activated by MR-L2 to a similar extent as wild-type PDE4D5. The double mutant S126D:S133D-PDE4D5 showed partial restoration of sensitivity to activation by MR-L2 compared to the S126D single mutant. [1]
The S651D phosphomimetic mutant of PDE4D5 (mimicking Erk phosphorylation) was activated by MR-L2 to a comparable level as wild-type PDE4D5. [1]
The monomeric DD1-R499D-PDE4D5 mutant (dimerization-deficient) showed complete loss of sensitivity to activation by MR-L2. [1]
In MDCK cells, MR-L2 (3 μM, 1 h pretreatment) significantly suppressed forskolin (3 μM, 15 min)-stimulated cAMP accumulation (mean of n=7 independent experiments). [1]
In MDCK cells, the suppressive effect of MR-L2 (3 μM) on forskolin-stimulated cAMP accumulation was ablated by co-treatment with the PDE4 inhibitor roflumilast (100 nM). [1]
In MDCK cells, MR-L2 (3 μM, 1 h pretreatment) significantly reduced extracellular cAMP levels following forskolin (3 μM, 15 min) challenge, indicating that reduced intracellular cAMP is not due to enhanced excretion. [1]
In 2D culture, MR-L2 exhibited no cytotoxic effects on MDCK cell proliferation as assessed by xCELLigence impedance-based assay. [1]
In MDCK cyst assays, the PDE4 inhibitor rolipram exacerbated agonist-driven cyst formation in a concentration-dependent manner. [1]
In MDCK cyst assays, MR-L2 suppressed PGE2 (300 nM)-stimulated cyst formation in a concentration-dependent manner with an EC50 of 1 μM. [1]
In MDCK cyst assays, co-treatment with roflumilast (100 nM) ablated the suppressive effect of MR-L2 (3 μM) on PGE2-stimulated cyst growth. [1]
In MDCK cyst assays, MR-L2 (3 μM) reduced the DNA-normalized ATP level by 15.6% (±30% SD) in cystic cultures. [1]
In MDCK cells, MR-L2 (3 μM) suppressed forskolin-induced cyst formation across a range of forskolin concentrations. [1]
In MDCK cells, MR-L2 suppressed PGE2-stimulated CFTR-mediated membrane depolarization in a concentration-dependent manner, with the dose dependency closely matching that observed for cyst suppression. [1]
In OX161 cells (immortalized ADPKD patient-derived cell line), the PDE4 inhibitor rolipram exacerbated cyst expansion. [1]
In OX161 cells, MR-L2 suppressed PGE2-stimulated cyst formation in a concentration-dependent manner. [1]
In primary human ADPKD patient-derived kidney epithelial cells, MR-L2 suppressed spontaneous cyst formation and vasopressin (10 nM)-exacerbated cyst formation without adversely affecting cell viability (as assessed by ATP-based luminescence assay). [1]

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In MDCK cells, MR-L2 (0.3-10 μM; 1 h) effectively reduces the development of cysts and rise of cAMP[1].

cAMP phosphodiesterase assays were conducted using cell lysates. Cells were collected in KHEM buffer (50 mM KCl, 10 mM EGTA, 50 mM Hepes pH 7.2, 1.92 mM MgCl2) and lysed by mechanical disruption. Lysates were precleared by centrifugation at 2,000 × g for 10 min, followed by centrifugation at 100,000 × g for 30 min. Protein concentration of the supernatant was determined by BCA assay. Assays were conducted using protein concentrations (typically 200 ng to 1 μg per reaction) and incubation times (10 min) that yielded linear rates of reaction. The activity of PDE4 isoforms was assessed in the presence of varying concentrations of MR-L2. [1]
Kinetic analysis was performed using Eadie-Hofstee plots to determine Vmax and Km values for cAMP hydrolysis by PDE4D5 in the presence of increasing concentrations of MR-L2 (3, 10, 30, and 100 μM). [1]

cAMP ELISA: Assays were conducted using a commercial cAMP ELISA kit following the manufacturer’s instructions. MDCK cells were pretreated with MR-L2 for 1 hour, then challenged with forskolin (3 μM) for 15 minutes. [1]
MDCK cyst assay: Collagen type I from rat tail was used to form a 3D matrix (final concentration 1 mg/mL). MDCK cells were grown in 24- or 96-well plate format. Images were captured at 4× magnification, and cyst diameter was recorded. Cyst volume (V) was calculated using the formula V = 4/3πr³, where r is the radius. Typically, 100-300 cysts were measured per treatment condition. For ATP measurement, DNA was first labeled with 20 μM Hoechst for 1 hour at 37°C and quantified by fluorescence measurement at 361-486 nm. ATP levels were then assessed using a CellTiter-Glo 3D reagent. [1]
OX161 cyst assay: OX161 cells were cultured, and assays were conducted as previously described (42, 61). [1]
Primary human kidney cell cyst assay: Single-cyst-derived or tissue-derived primary cultures from ADPKD patients were grown in biogels containing proprietary media. Manual imaging and cyst counting were conducted in 96-well plates, and automated imaging of entire wells of 384-well plates was conducted. [1]
CFTR assay: MDCK cells were seeded in 96-well clear-bottom assay plates 4 days before the assay. Assays were conducted in low chloride buffer (140 mM Na gluconate, 5 mM K gluconate, 10 mM glucose, 10 mM Hepes free acid, 1 mM CaCl2, 1 mM MgCl2, pH 7.4 with NaOH) supplemented with FLIPR Membrane Potential Assay Kit Blue and amiloride (10 μM). Real-time fluorescence measurements were recorded at excitation 530 nm and emission 565 nm using a Flex Station 3. [1]
xCELLigence assay: MDCK cells were plated at a density of 5,000 cells per well and allowed to adhere for 24 hours before treatment with MR-L2. Cell proliferation was monitored using impedance-based measurement. [1]
Western blot: Cells were lysed for 20 minutes in whole-cell lysis buffer [1% Triton X-100, 25 mM Hepes, 2.5 mM EDTA, 150 mM NaCl, 50 mM NaF, 30 mM NaPPi] containing a protease inhibitor mixture. Insoluble material was removed by centrifugation at 14,000 × g. Protein concentration was measured by BCA assay before SDS-PAGE and immunoblotting using antisera raised against PDE4 isoforms and β-actin. [1]

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Cell Viability Assay[1]
Cell Types: Madin-Darby Canine Kidney (MDCK) cell line
Tested Concentrations: 0.3, 1 , 3 and 10 μM
Incubation Duration: 1 hour
Experimental Results: Suppressed cAMP elevation but not enhanced cAMP excretion in MDCK cells. Suppressed cysts formation in MDCK cells with an EC50 value of 1.2 µM. Suppressed the number of cysts formation in 3D culture for both unstimulated and vasopressin-treated culture condition while demonstrated no effect on cell viability.

In 2D culture, MR-L2 exhibited no cytotoxic effects on MDCK cell proliferation as assessed by xCELLigence impedance-based assay. [1]
In primary human ADPKD patient-derived kidney epithelial cells, MR-L2 treatment did not adversely affect cell viability as assessed by ATP-based luminescence assay. [1]

[1]. Small-molecule allosteric activators of PDE4 long form cyclic AMP phosphodiesterases. Proc Natl Acad Sci U S A. 2019 Jul 2;116(27):13320-13329.

MR-L2 is an N-substituted-2-(3-aryl-1H-1,2,4-triazol-1-yl)acetamide derivative that acts as a selective allosteric activator of long-form PDE4 isoforms, phenocopying the stimulatory effect exerted by PKA phosphorylation on dimeric PDE4 long isoforms. [1]
The mechanism of action of MR-L2 requires the dimeric state adopted by long (but not short) PDE4 isoforms and involves modulation of the autoinhibitor UCR2 cross-capping within the PDE4 long-form dimeric assembly. [1]
MR-L2 demonstrates the ability to lower intracellular cAMP levels and suppress cyst formation in multiple in vitro models of autosomal dominant polycystic kidney disease (ADPKD), including MDCK cells, immortalized ADPKD patient-derived OX161 cells, and primary human ADPKD patient-derived kidney epithelial cells. [1]
The compound suppresses PGE2-stimulated CFTR-mediated membrane depolarization in MDCK cells, consistent with reduced PKA activity due to lowered local cAMP levels. [1]
The potency of MR-L2 in cellular assays (cAMP suppression and cyst formation) was higher than in the biochemical enzyme activation assay, suggesting enhanced sensitivity due to cellular context-dependent conformational and/or post-translational modification status of the target PDE4 long isoforms. [1]

C19H16CL3FN4O

441.71394443512

440.037

2374703-19-0

138911347

White to light yellow solid powder

5.4

1

4

6

28

521

0

ClC1C=C(C=C(C=1)CNC(CN1C(CC)=NC(C2C=CC(=C(C=2)F)Cl)=N1)=O)Cl

JXACCOKEEPXHCF-UHFFFAOYSA-N

InChI=1S/C19H16Cl3FN4O/c1-2-17-25-19(12-3-4-15(22)16(23)7-12)26-27(17)10-18(28)24-9-11-5-13(20)8-14(21)6-11/h3-8H,2,9-10H2,1H3,(H,24,28)

2-[3-(4-chloro-3-fluorophenyl)-5-ethyl-1,2,4-triazol-1-yl]-N-[(3,5-dichlorophenyl)methyl]acetamide

MR-L2; MR-L-2; 2374703-19-0; MR L2; CHEMBL5407478; C19H16Cl3FN4O;

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)

DMSO: 83.33 mg/mL (188.65 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.71 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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