NVS-PAK1-1 is a specific allosteric PAK1 inhibitor discovered in a fragment-based screen. The process of finding inhibitors that target new allosteric kinase sites is very difficult. Once identified, these compounds may provide exceptional selectivity throughout the kinome. In comparison to other PAK isoforms and the kinome as a whole, NVS-PAK1-1 exhibits high selectivity for PAK1 inhibition. The biochemical Kds for PAK1 and PAK2 in NVS-PAK1-1 are 7 nM and 400 nM, respectively. Through outstanding selectivity profile against other known kinases, NVS-PAK1-1 demonstrates outstanding activity in biochemical assays.

Physicochemical Properties

Molecular Formula	C23H25CLF3N5O
Molecular Weight	479.925714254379
Exact Mass	479.17
Elemental Analysis	C, 57.56; H, 5.25; Cl, 7.39; F, 11.88; N, 14.59; O, 3.33
CAS #	1783816-74-9
Related CAS #	1783816-74-9
PubChem CID	137125241
Appearance	White to off-white solid powder
LogP	4.5
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	33
Complexity	726
Defined Atom Stereocenter Count	1
SMILES	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
InChi Key	OINGHOPGNMYCAB-INIZCTEOSA-N
InChi Code	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
Chemical Name	(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
Synonyms	NVS-PAK1-1; NVSPAK11; NVS PAK1 1
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	PAK1 (IC50 = 5 nM)
ln Vitro	NVS-PAK1-1 exhibits strong selectivity for PAK1 inhibition over the kinome as a whole and other PAK isoforms. Biochemically, NVS-PAK1-1 has a 400 nM PAK2 Kd and a 7 nM PAK1 Kd. In biochemical assays, NVS-PAK1-1 exhibits remarkable activity and a remarkable selectivity profile when compared to other known kinases. Merely 6–20 μM of NVS-PAK1-1 prevents the downstream substrate MEK1 Ser289 from being phosphorylated. In agreement with the findings, NVS-PAK1-1 only stops the Su86.86 cell line from proliferating at concentrations greater than 2 μM. On the other hand, a considerably lower 0.21 μM concentration of NVS-PAK1-1 and PAK2shRNA is used to inhibit downstream signaling and cell proliferation[1].
ln Vivo	NVS-PAK1-1 indicates a comparatively low stability in rat liver microsomes (RLM), which would restrict its use for in vivo research (t1/2 in RLM = 3.5 min)[1].
Cell Assay	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].
Animal Protocol
References	[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.

Solubility Data

Solubility (In Vitro)

DMSO: ≥125 mg/mL Water: <1 mg/mL Ethanol:

Solubility (In Vivo)

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

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0836 mL	10.4182 mL	20.8364 mL
	5 mM	0.4167 mL	2.0836 mL	4.1673 mL
	10 mM	0.2084 mL	1.0418 mL	2.0836 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.