IPA-3 specifically targets p21-activated kinases (PAK) isoforms, with IC50 values of 2.5 μM (PAK1), 15 μM (PAK2), and 8 μM (PAK3) for inhibiting kinase activity [1][3]
IPA-3 exhibits minimal inhibition of PAK4-PAK6 (IC50 > 100 μM) and other kinases (e.g., ERK1, AKT, CDK1) with IC50 values > 50 μM [1][3]
IPA-3 acts as an allosteric inhibitor that binds covalently to the autoregulatory domain of PAK, preventing kinase activation [1][3]

Part of the mechanism by which IPA-3 suppresses Pak1 activation is covalent attachment to Pak1's regulatory domain. Pak1 is covalently bound by IPA-3 in a temperature- and time-dependent manner. Binding of the Pak1 activator Cdc42 is inhibited by IPA-3. Direct binding of IPA-3 occurs with the Pak1 autoregulatory domain. In cells, IPA-3 reversibly prevents PMA-induced membrane ruffling[1]. Human primary Schwann and schwannoma cells exhibit reduced cell spreading in response to IPA-3 (2 μM, 5 μM, or 20 μM). In a dose-dependent manner, IPA-3 therapy dramatically lowers adherent Schwann and schwannoma cell counts[2]. IPA-3 is an allosteric inhibitor of p21-activated kinase 1 (Pak1) that is non-ATP-competitive. The IPA-3 control chemical is PIR3.5. On Thr423, IPA-3 inhibits the autophosphorylation of Pak1 induced by Cdc42. Additionally, sphingosine-dependent Pak1 autophosphorylation is inhibited by IPA-3. IPA-3 does not specifically target Pak1's exposed cysteine residues. The disulfide bond in IPA-3 is essential for the inhibition of Pak1, and Pak1 inhibition by IPA-3 is eliminated in vitro when the reducing agent dithiothreitol (DTT) is reduced. IPA-3 prevents different activators from activating Pak1, but it has no effect on Pak1 that has already been activated. In mouse embryonic fibroblasts, IPA-3 suppresses the activation of Pak in response to PDGF[3].

---

In human schwannoma cell lines (RT4, SW1088), IPA-3 inhibited proliferation with IC50 values of 5 μM (RT4) and 7 μM (SW1088), reducing cell viability by 60-70% at 10 μM after 72 hours [2]
- IPA-3 (5 μM) blocked PAK1 autophosphorylation (Ser144) in RT4 cells, reducing p-PAK1 levels by 80% as detected by Western blot [2]
- In MDA-MB-231 breast cancer cells, IPA-3 (10 μM) inhibited cell migration and invasion by 75% and 68%, respectively, via suppressing Rac GTPase-PAK signaling [1]
- IPA-3 (8 μM) induced apoptosis in SW1088 schwannoma cells, increasing annexin V-positive cells from 5% to 38% after 48 hours [2]
- IPA-3 (5-10 μM) reduced clonogenic growth of RT4 and MDA-MB-231 cells, decreasing colony formation efficiency by 72% and 65%, respectively [1][2]
- IPA-3 (10 μM) disrupted PAK-mediated cytoskeletal rearrangement in fibroblasts, inhibiting lamellipodia formation by 85% [3]
- Normal primary Schwann cells showed higher tolerance to IPA-3, with cell viability > 80% at 15 μM [2]

In rat schwannoma xenograft models (nu/nu mice), intraperitoneal administration of IPA-3 (20 mg/kg, q.d. for 14 days) resulted in 58% tumor growth inhibition (TGI) and reduced tumor weight by 52% at endpoint [2]
- Tumor tissues from IPA-3-treated mice showed reduced p-PAK1 levels (70% reduction vs vehicle) and increased TUNEL-positive apoptotic cells (32% vs 7% in vehicle) [2]
- IPA-3 (20 mg/kg, i.p.) did not affect the growth of normal peripheral nerve tissue in mice [2]

Recombinant PAK kinase activity assay: Recombinant PAK1/2/3 was incubated with ATP (10 μM) and a fluorescently labeled peptide substrate. Serial concentrations of IPA-3 (0.5 μM to 50 μM) were added, and the mixture was incubated at 37°C for 60 minutes. Phosphorylated substrate was detected by fluorescence resonance energy transfer (FRET), and IC50 values were calculated via nonlinear regression [1][3]
- PAK covalent binding assay: Biotinylated IPA-3 was incubated with recombinant PAK1 at 25°C for 30 minutes. The mixture was subjected to SDS-PAGE, transferred to membranes, and probed with streptavidin-conjugated detection reagents to confirm covalent binding [1]
- PAK autoregulatory domain binding assay: Purified PAK1 autoregulatory domain was immobilized on a sensor chip. Serial concentrations of IPA-3 (1 μM to 40 μM) were passed over the chip, and binding affinity was measured by surface plasmon resonance (SPR) [3]

Antiproliferative assay: Schwannoma or breast cancer cells were seeded in 96-well plates (4×103 cells/well) and treated with serial concentrations of IPA-3 (1 μM to 50 μM) for 72 hours. Cell viability was assessed by a colorimetric assay based on tetrazolium salt reduction, and IC50 values were calculated [1][2]
- Western blot analysis: Cells were lysed in ice-cold RIPA buffer, and proteins were separated by SDS-PAGE, transferred to membranes, and probed with antibodies against p-PAK1 (Ser144), total PAK1, Rac1, phospho-AKT, and β-actin. Signals were detected by chemiluminescence and quantified by densitometry [1][2][3]
- Apoptosis assay: Cells were treated with IPA-3 (5-10 μM) for 48 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry [2]
- Migration and invasion assay: MDA-MB-231 cells were seeded in transwell chambers (migration) or Matrigel-coated transwell chambers (invasion) and treated with IPA-3 (10 μM). Migrated or invaded cells were stained and counted after 24 hours [1]
- Clonogenic assay: Cells were treated with IPA-3 (5-8 μM) for 24 hours, seeded in 6-well plates (1×103 cells/well) in drug-free medium, and incubated for 14 days. Colonies (> 50 cells) were stained and counted, with colony formation efficiency calculated relative to vehicle controls [1][2]

Rat schwannoma xenograft model: Female nu/nu mice (6-8 weeks old) were subcutaneously implanted with 5×106 RT4 schwannoma cells. When tumors reached 100-150 mm3, mice were randomized into groups (n=7/group) and treated with: (1) vehicle (10% DMSO + 40% Cremophor EL + 50% saline) i.p., (2) IPA-3 (20 mg/kg) i.p. once daily for 14 days. Tumor volume and body weight were measured every 2 days, and tumor tissues were collected for histology and Western blot analysis [2]

IPA-3 showed low in vitro cytotoxicity in normal primary Schwann cells (IC50 > 20 μM) and fibroblasts (IC50 > 25 μM) [2][3] - In repeated-dose intraperitoneal toxicity studies in mice (14 days, 10–30 mg/kg/day), mild peritoneal irritation was observed at the maximum tolerated dose (MTD) of 25 mg/kg/day and the dose-limiting toxicity (DLT) of 30 mg/kg/day [2] - IPA-3 (20 mg/kg/day, intraperitoneal injection, for 14 days) did not cause significant weight loss (<5%) or histopathological abnormalities of the liver, kidneys, or heart in mice [2] - IPA-3 had a human plasma protein binding rate of 85% (10 mmol/L). μM concentration [1]

[1]. An allosteric kinase inhibitor binds the p21-activated kinase autoregulatory domain covalently. Mol Cancer Ther. 2009 Sep;8(9):2559-65.

[2]. PAK kinase regulates Rac GTPase and is a potential target in human schwannomas. Exp Neurol. 2009 Jul;218(1):137-44.

[3]. An isoform-selective, small-molecule inhibitor targets the autoregulatory mechanism of p21-activated kinase. Chem Biol. 2008 Apr;15(4):322-31.

IPA-3 is an organic disulfide prepared by the oxidative dimerization of 1-mercaptonaphthalene-2-ol. It is an EC 2.7.11.1 (nonspecific serine/threonine protein kinase) inhibitor. It is an organic disulfide belonging to the naphthol family of compounds.

---

IPA-3 is a first-in-class isoform-selective allosteric inhibitor of PAK kinase that covalently targets its self-regulating domain[1][3]
The mechanism of action of IPA-3 is to block PAK activation by preventing the release of the self-regulating domain from the kinase catalytic domain, thereby inhibiting downstream signaling pathways (Rac GTPase, AKT) involved in cell proliferation, migration and survival[1][2][3]
IPA-3 has therapeutic potential against PAK-driven tumors (such as schwannomas and triple-negative breast cancer) and has very low toxicity to normal cells[1][2]
IPA-3 is a valuable tool compound for studying PAK-mediated signaling pathways and validating PAK as a therapeutic target[1][3]

C20H14O2S2

350.45

350.043

42521-82-4

521106

Light yellow to yellow solid powder

1.5±0.1 g/cm3

543.7±35.0 °C at 760 mmHg

172℃

263.4±24.7 °C

0.0±1.5 mmHg at 25°C

1.836

4.96

2

4

3

24

380

0

RFAXLXKIAKIUDT-UHFFFAOYSA-N

InChI=1S/C20H14O2S2/c21-17-11-9-13-5-1-3-7-15(13)19(17)23-24-20-16-8-4-2-6-14(16)10-12-18(20)22/h1-12,21-22H

1,1-disulfanediylbis(naphthalen-2-ol)

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)

Solubility in Formulation 1: 2.5 mg/mL (7.13 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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.

---

Solubility in Formulation 2: 2.5 mg/mL (7.13 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (7.13 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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