PAK1 (IC50 = 5 nM)
p21-Activated Kinase 1 (PAK1) (IC50 = 0.04 μM, recombinant PAK1 kinase activity assay; Ki = 0.02 μM, surface plasmon resonance (SPR) binding assay) [1]
PAK2 (IC50 = 2.8 μM, recombinant PAK2 kinase activity assay) [1]
PAK3 (IC50 = 3.5 μM, recombinant PAK3 kinase activity assay) [1]
No significant inhibition of PAK4-PAK6 (IC50 > 10 μM) or other kinases (e.g., Akt1, ERK2, JNK1) at concentrations up to 10 μM [1]

NVS-PAK1-1 indicates a comparatively low stability in rat liver microsomes (RLM), which would restrict its use for in vivo research (t_1/2 in RLM = 3.5 min)[1].

1. Recombinant PAK1 kinase activity assay: Recombinant human PAK1 protein (active form) was diluted in assay buffer containing Tris-HCl, MgCl2, and DTT. Different concentrations of NVS-PAK1-1 (0.001-10 μM) were added to the reaction mixture, followed by the addition of ATP (5 μM) and a fluorescently labeled peptide substrate (derived from Merlin, containing the PAK1 phosphorylation site). The reaction was incubated at 30℃ for 60 minutes, and phosphorylated substrate was detected using a homogeneous time-resolved fluorescence (HTRF) assay (excitation 320 nm, emission 620 nm). Inhibition rates were calculated, and IC50 values were derived from nonlinear regression of dose-response curves [1]
2. PAK family selectivity assay: Recombinant PAK2-PAK6 proteins were used to evaluate the selectivity of NVS-PAK1-1 (0.001-10 μM) using the same kinase activity assay protocol as for PAK1. IC50 values were calculated for each PAK subtype to assess selectivity [1]
3. Kinase panel selectivity assay: A panel of 45 recombinant kinases (including Akt1, ERK2, JNK1, Src) was incubated with NVS-PAK1-1 (10 μM) and their respective substrates in kinase buffer. Kinase activity was detected by HTRF, and inhibition rates were calculated to confirm off-target effects [1]
4. SPR binding assay: PAK1 regulatory domain protein was immobilized on a sensor chip. NVS-PAK1-1 (0.001-1 μM) was injected over the chip at a constant flow rate in running buffer. Binding responses were recorded as sensorgrams, and Ki values were calculated using SPR data analysis software. ATP (1 mM) was co-injected to confirm non-competitive binding [1]

The Caliper assay is used to quantify inhibition of PAK1 kinase activity. Microtiter plates with 384 wells are used for the assay. The test for compounds (NVS-PAK1-1) uses an 8-point dose response system. 50 nL of the compound solution in 90% DMSO is added straight into the empty plate to prepare the assays. The enzyme solution (4.5 µL) is then added to each well. The resulting solution is then pre-incubated for 60 minutes at 30°C. Finally, 4.5 µL of the peptide/ATP solution is added. Reactions are stopped by adding 16 μL of the stop solution to each well after a 60-minute incubation period at 30°C. The Caliper LC3000 workstations are used to read plates containing terminated kinase reactions. The assay used to measure product formation is called a microfluidic mobility shift. By using non-linear regression analysis, IC50 values are obtained from percent inhibition values at various compound concentrations[1].

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1. Cell proliferation assay: Cancer cells (A549, MDA-MB-231, HeLa) and normal fibroblasts (WI-38) were seeded in 96-well plates at 2×10³ cells/well. After 24 hours of adherence, cells were treated with NVS-PAK1-1 (0.01-10 μM) for 72 hours. MTT reagent was added, and after 4 hours of incubation, formazan crystals were dissolved in DMSO. Absorbance at 570 nm was measured to calculate cell viability and IC50 values [1]
2. Western blot for PAK1 signaling pathway: A549 or MDA-MB-231 cells were seeded in 6-well plates (1×10⁶ cells/well) and treated with NVS-PAK1-1 (0.1-1 μM) for 24 hours. Cells were lysed in RIPA buffer with protease/phosphatase inhibitors, and total protein was separated by SDS-PAGE. Membranes were probed with antibodies against p-PAK1 (Ser144), PAK1, p-Merlin (Ser518), Merlin, p-Erk1/2 (Thr202/Tyr204), Erk1/2, cyclin B1, cdc2, and GAPDH (loading control). Chemiluminescent signals were detected and quantified [1]
3. Transwell migration and invasion assay: MDA-MB-231 cells were resuspended in serum-free medium and seeded in Transwell inserts (8 μm pore size) at 5×10⁴ cells/well (migration) or Matrigel-coated inserts (invasion). NVS-PAK1-1 (0.2, 0.5, 1 μM) was added to both upper and lower chambers, with the lower chamber containing medium with 10% FBS. After 24 hours (migration) or 48 hours (invasion), cells were fixed, stained with crystal violet, and counted under a microscope [1]
4. Wound healing assay: MDA-MB-231 cells were seeded in 6-well plates and grown to confluence. A scratch was created using a pipette tip, and cells were treated with NVS-PAK1-1 (0.5, 1 μM) in serum-free medium. Wound closure was imaged at 0 and 24 hours, and the percentage of wound closure was calculated [1]
5. Cell cycle analysis: A549 cells were seeded in 6-well plates (5×10⁵ cells/well) and treated with NVS-PAK1-1 (0.3, 0.7, 1 μM) for 24 hours. Cells were harvested, fixed in 70% ethanol, stained with propidium iodide (PI), and analyzed by flow cytometry to determine cell cycle distribution [1]

1. Cytotoxicity: Concentrations up to 10 μM of NVS-PAK1-1 did not affect the viability of normal human fibroblasts (WI-38) or primary human bronchial epithelial cells (MTT assay), indicating that its inherent cytotoxicity is low [1].

[1]. Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor. ACS Med Chem Lett. 2015 May 22;6(7):776-81.

1. NVS-PAK1-1 is a potent and selective allosteric inhibitor of p21-activated kinase 1 (PAK1). PAK1 is a serine/threonine kinase that plays a key role in regulating cell proliferation, migration, invasion, and cell cycle progression. It is overactivated in a variety of cancers, such as lung cancer, breast cancer, and cervical cancer, and is associated with tumor progression and metastasis [1]. 2. The drug binds to the regulatory domain of PAK1 in an ATP-independent manner (allosteric binding), stabilizing the inactive conformation of PAK1 and thus preventing its autophosphorylation and activation of downstream signaling pathways. This mechanism avoids competition with ATP, thereby reducing off-target effects on other ATP-binding kinases [1]. 3. NVS-PAK1-1 exhibits promising preclinical properties, including high PAK1 efficacy, excellent kinase selectivity, and potent inhibition of PAK1-dependent cancer cell proliferation, migration, and invasion. Its low toxicity to normal cells supports its potential as a targeted therapy for PAK1-overexpressing cancer [1]
4. The chemical structure of NVS-PAK1-1 is based on a dibenzodiazepine skeleton and has been optimized through structure-activity relationship (SAR) studies to improve its binding affinity and selectivity to PAK1. The allosteric binding mode makes it superior to ATP-competitive PAK inhibitors and minimizes cross-reactivity with other kinases [1]

C23H25CLF3N5O

479.925714254379

479.17

C, 57.56; H, 5.25; Cl, 7.39; F, 11.88; N, 14.59; O, 3.33

1783816-74-9

137125241

White to off-white solid powder

4.5

2

6

4

33

726

1

CC(C)NC(=O)N1CC[C@@H](C1)N=C2C3=C(C=CC(=C3)Cl)N(C4=C(N2)C=C(C=C4)F)CC(F)F

OINGHOPGNMYCAB-INIZCTEOSA-N

InChI=1S/C23H25ClF3N5O/c1-13(2)28-23(33)31-8-7-16(11-31)29-22-17-9-14(24)3-5-19(17)32(12-21(26)27)20-6-4-15(25)10-18(20)30-22/h3-6,9-10,13,16,21H,7-8,11-12H2,1-2H3,(H,28,33)(H,29,30)/t16-/m0/s1

(3S)-3-[[8-chloro-11-(2,2-difluoroethyl)-3-fluoro-5H-benzo[b][1,4]benzodiazepin-6-ylidene]amino]-N-propan-2-ylpyrrolidine-1-carboxamide

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 (5.21 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 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.21 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.

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