Mouse aquaporin-7 (mAQP7) water permeability (IC50 ~0.2 μM); mouse aquaporin-3 (mAQP3) water permeability (IC50 ~0.7 μM); mouse aquaporin-9 (mAQP9) water permeability (IC50 ~1.1 μM). [1]

Z433927330 has an IC50 of roughly 0.6 μM and inhibits triglyceride permeability[1].

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Z433927330 inhibits mouse AQP7 water permeability with an IC50 of approximately 0.2 μM and shows moderate efficacy. For mouse AQP3, the IC50 is approximately 0.7 μM with partial efficacy. For mouse AQP9, the IC50 is approximately 1.1 μM. (Figure 3B) [1]
The compound inhibits glycerol permeability in human erythrocytes, with an apparent IC50 of ~0.6 μM for the glycerol-induced swelling phase measured by stopped-flow light scattering. (Figure 5B) [1]
In CHO cells expressing mAQP3, Z433927330 inhibits glycerol permeability with an IC50 of ~11.5 μM, while in CHO cells expressing mAQP7, it inhibits glycerol permeability with an IC50 of ~2.6 μM, showing moderate selectivity for AQP7 over AQP3. (Figure 5D) [1]
Z433927330 reduces hydrogen peroxide (H2O2) permeability in CHO cell lines expressing mAQP3, mAQP7, or mAQP9, as measured by a slower increase in the 490/420 nm fluorescence ratio of the HyPer-3 sensor upon addition of 150 μM H2O2. The effect is isoform-cognate, and no clear effect is observed in control cells without ectopic AQP expression. (Figure 4) [1]
Cell proliferation and viability assays in CHO cells show that after 48 hours of incubation with Z433927330 at 2.5 μM, calcein retention is not affected, indicating no apparent negative effects. At 25 μM, the compound results in increased fluorescence (p < 0.001), suggesting increased proliferation or dye retention, rather than toxicity. (Figure 3D) [1]

For water permeability measurements, Chinese hamster ovary (CHO) cells with tetracycline-inducible expression of mouse AQP3, AQP7, or AQP9 were seeded in polylysine-coated 96-well plates at 6000 cells per well. After 24 hours, AQP expression was induced with tetracycline (titrated to achieve a cell shrinking half-time of ~1 s for uninhibited cells). Cells were grown for an additional 48 hours, then loaded with 2.5 μM calcein-AM in medium containing 5 mM probenecid for 45 minutes. The plate was transferred to a plate reader, and fluorescence intensity (excitation 485 nm, emission 520 nm) was recorded for 30 seconds. Osmotic cell shrinkage was induced 3.6 seconds after the start by adding 1 volume of 500 mM sucrose in assay buffer (containing 0.8 mM MgSO4, 5.0 mM KCl, 1.8 mM CaCl2, 25 mM NaHEPES, 111.5 mM NaCl, 5.0 mM probenecid, pH 7.4, and 1% DMSO to maintain constant DMSO concentration). The half-time of fluorescence quenching (t1/2) was used as an index of water permeability. Z433927330 was dissolved in DMSO and added to the assay buffer at final concentrations ranging from 0.01 to 25 μM, with a final DMSO concentration of 1%. [1]
For glycerol permeability assays in CHO cells, cells were preincubated in 500 mM glycerol buffer, then 500 mM membrane-impermeable sucrose was added. Fluorescence quenching (calcein) indicates cellular glycerol efflux. The half-time of quenching was measured to calculate IC50 values for Z433927330 against mAQP3 and mAQP7. [1]
For hydrogen peroxide permeability assays, CHO cells stably expressing the H2O2 sensor HyPer-3 (with or without ectopic AQP expression) were seeded similarly. After 48 hours of tetracycline induction (5 ng/mL), cells were not loaded with calcein but instead the HyPer-3 fluorescence ratio (excitation 420 nm and 490 nm, emission 520 nm) was recorded for 79 seconds. At 3.6 seconds, 150 μM H2O2 was added. Z433927330 was applied as described above, and the rate of fluorescence ratio increase was measured. [1]
For cytotoxicity/proliferation assays, CHO cells were grown in the presence of Z433927330 at indicated concentrations (2.5 μM and 25 μM) for 48 hours, then loaded with calcein-AM, and fluorescence was recorded at a single time point. No reduction in calcein retention was observed; at 25 μM, a significant increase in fluorescence was detected (p < 0.001). [1]

In CHO cell proliferation and viability assays, incubation with 2.5 μM Z433927330 for 48 hours showed no negative effect on calcein retention, indicating no apparent cytotoxicity. At 25 μM, increased fluorescence was observed (p < 0.001), suggesting increased cell proliferation or dye retention, not toxicity. (Figure 3D) [1]

[1]. Identification and characterization of potent and selective aquaporin-3 and aquaporin-7 inhibitors. J Biol Chem. 2019 May 3;294(18):7377-7387.

Z433927330 contains a methylurea linker and a right-hand side phenylpyrazole group. It is structurally similar to compound 9016645, and the additional pyrazole group shifts isoform specificity from AQP3 to AQP7, resulting in reversed potency and efficacy. Molecular docking homology models suggest that Z433927330 binds at the cytoplasmic side of the aquaglyceroporin pore, with hydrogen bonds formed between the inhibitor urea linker and backbone carbonyls of loop B, and oxygen from the ester functional group acting as a hydrogen bond acceptor for asparagine hydrogens. The predicted binding site is similar to that of RF03176 and DFP00173. The compound (along with DFP00173 and RF03176) crosses an electrically tight mpkCCD cell monolayer from basal to apical side at a rate comparable to DMSO, suggesting it can diffuse across the lipid bilayer to reach the cytoplasmic binding site. Z433927330 is commercially available, and its identity and purity were confirmed by mass spectrometry. [1]

C20H20N4O3

364.3978

364.153

1005883-72-6

51116434

White to off-white solid powder

2.6

2

4

7

27

486

0

O(C([H])([H])C([H])([H])[H])C(C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(N([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N1C([H])=C([H])C([H])=N1)=O)=O

KQUKHINHCUELQL-UHFFFAOYSA-N

InChI=1S/C20H20N4O3/c1-2-27-19(25)16-6-8-17(9-7-16)23-20(26)21-14-15-4-10-18(11-5-15)24-13-3-12-22-24/h3-13H,2,14H2,1H3,(H2,21,23,26)

ethyl 4-(3-(4-(1H-pyrazol-1-yl)benzyl)ureido)benzoate

Z433927330 Z-433927330 Z 433927330

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 : ≥ 280 mg/mL (~768.39 mM)

Solubility in Formulation 1: ≥ 2.33 mg/mL (6.39 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 23.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.33 mg/mL (6.39 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 23.3 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.)