NVS-PAK1-1 A Chemical Probe For PAK1

NVS-PAK1-1 is available from Sigma and Tocris.

Its negative control (NVS-PAK1-C) is available for purchase from Tocris.

For any inquiries please contact proberequests@thesgc.org.

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Overview

Biology of the PAK1 kinase

PAK kinases belong belong to the family of STE kinases, named after the yeast STE7, STE11 and STE20 genes, which form the MAPK cascade, transducing signals from the surface of the cell to the nucleus. The PAK family itself consist of 6 members and can be subdivided into 2 groups Group I PAKs (PAK1-3) and Group II PAKs (PAK 4-6). Group I PAKs (1-3) contain a N-terminal PBD-AIN domain and a kinase domain as well as two SH3-domains and one non-classical SH3-domain.

PAK1 is involved in many intracellular signaling pathways downstream of integrins and receptor-type kinases and plays an important role in in cell adhesion, cytoskeleton dynamics, migration, proliferation, apoptosis, mitosis, and in vesicle-mediated transport processes. It also plays a role in the regulation of insulin secretion in response to elevated glucose levels. PAKs have been shown to activate components of ERK, AKT and WNT signaling pathway and functions as GTPase effector that links the Rho-related GTPases CDC42 and RAC1 to the JNK MAP kinase pathway. PAK1 phosphorylates and activates MAP2K1, and thereby mediates activation of downstream MAP kinases.

PAK1 has been found to be overexpressed and/or activated in many tumor types and its expression levels have been correlated with patient prognosis. In addition Group I PAKs have been implicated in neurodegenerative disorders like Alzheimer and Huntington diseases as well as FXS (fragile X syndrome).

We recommend using NVS1-PAK1-1 at 2.5 uM for PAK1/2 inhibition or 0.25 uM for PAK1 inhibition.

The inactive control at similar concentration has no effect on PAK1/2

NVS-PAK1-1: A Chemical Probe for PAK1

NVS1-PAK-1, a specific allosteric PAK1 inhibitor, has been developed by Novartis [1]

Chemical structure of NVS-PAK1-1 and its inactive control compound NVS-PAK1-C

Co-Crystal Structure

Details of the co-crystal structure of the hit compound and a related compound with the PAK1 kinase domain, click on the 'Co-Crystal structures' tab above for more details

Potency Against Target Family

NVS-PAK1-1 is a potent allosteric inhibitor of PAK1 with an IC 50 of 5 nM for dephosphorylated PAK1 and 6 nM for phosphorylated PAK1 as assessed in a Caliper assay. This data is in good agreement with KD of 7 nM assessed in DiscoverX kinome scan. Inhibition is ATP-competitive, most likely due to indirect competition due to incompatibility of ATP binding with the DFG-out binding conformation of the allosteric compound. The control compound NVS-PAK1-C has more than 100 fold less activity in the respective Caliper assays.

IC Activity against the closest PAK member, PAK2 is over 54 fold. NVS-PAK1-1 has an IC50 of 270 nM against dephosphorylated PAK2 and 720 nM against phosphorylated PAK2 respectively in Caliper assay.

Selectivity

Selectivity of NVS-PAK1-1 was assessed against a panel of 442 kinases (DiscoverX kinome scan) at 10 µM and the compound was found to be exquisitely selective.

Selectivity Beyond Target Family

NVS-PAK1-1 was found to be inactive vs 27 receptors (Novartis panel) at 10µM, with the most potent activity measured of 13 uM against H1 and M1. No activity at 10µM was found in a panel of 48 BRDs.

Cellular Activity

NVS-PAK1-1 potently inhibits autophosphorylation of PAK1 (S144) at 0.25 µM in the Su86.86 cell line and MEK S289 phosphorylation with an IC50 = 0.21 in Su86.86 cells in which PAK2 is downregulated.

Properties

NVS-PAK1-1

(S)-3-((2-chloro-5-(2,2-difluoroethyl)-8-fluoro-5H-dibenzo[b,e][1,4]diazepin-11-yl)amino)-N-isopropylpyrrolidine-1-carboxamide

For SDF click here

Potency against Target

Potency against Target

Caliper Assay

NVS-PAK1-1 is a potent allosteric inhibitor of PAK1 with an IC₅₀ of 0.005 µM for dephoshorylated PAK1 and 0.006 µM for phosphorylated PAK1 as shown in a Caliper in vitro dephosphorylation assay.

KINOME scan PAK1 Binding Assay.

NVS-PAK1-1 binds to PAK1 with a K_D of 7 nM.

Materials and Methods

Caliper assay

Inhibition of PAK1 kinase activity was measured using the Caliper (Caliper LC3000, PerkinElmer) assay. The assay was performed using 384-well microtiter plates. Compounds were tested as 8-point dose responses using PAK1 expressed in E.coli. IC50 values were derived from percent inhibition values at different compound concentrations by non-linear regression analysis.

Binding Assay

Binding assays were performed at DiscoverX coporation in the KINOMEscan assay. Inhibitor binding constants (Kd values) are calculated from duplicate 11-point dose-response curves using Hill equation. Curves were fitted using a non-linear least square fit with the Levenberg-Marquardt algorithm.

Selectivity Profile

Selectivity Within Target family

KINOMEscan

NVS-PAK1-1 is highly selective against 442 kinases tested at 10 µM with a selectivity score (S10-score) of 0.003.

442 Kinases tested at 10 uM

S(35), 3 hits, Sscore = 0.008

S(10), 1 hit, Sscore = 0.003

Selectivity Beyond Target Family

NVS-PAK1-1 shows no cross-reactivity in a panel of 53 proteases (tested at 10 µM), 22 receptors (IC₅₀> 13 µM against closest off-target Phosphodiesterase 4D) and a panel of 28 bromodomains.

Materials and Methods

Differential Scanning Fluorimetry (DSF)

Thermal melting experiments were carried out using an Mx3005p Real Time PCR machine (Stratagene). Proteins were buffered in 10 mM HEPES pH 7.5, 500 mM NaCl and assayed in a 96-well plate at a final concentration of 2 µM in 20 µl volume. Compounds were added at a final concentration of 10 µM. SYPRO Orange (Molecular Probes) was added as a fluorescence probe at a dilution of 1:1000. Excitation and emission filters for the SYPRO-Orange dye were set to 465 nm and 590 nm, respectively. The temperature was raised with a step of 3 °C per minute from 25 °C to 96 °C and fluorescence readings were taken at each interval. Data was analysed as previously reported [3]

KINOMEscan

NVS-PAK1-1 was profiled by KINOMEscan™, a platform that measures the interactions between test compounds and a panel of kinase assays (http://www.discoverx.com), against 442 kinases.

Cell-based Assay Data

The cellular efficacy of NVS-PAK-1-1 on PAK1 was investigated assessing autophosphorylation in the pancreatic duct carcinoma cell line Su86.86 expressing high levels of PAK1 and PAK2. PAK1 and PAK2 are activated by autophosphorylation on PAK1 (S144) and PAK2 (S141), respectively due to blockage of PDB-domain autoinhibition.

NVS-PAK-1-1 blocks autophosphorylkation at both sites in dose-dependent manner.

Substrate phosphorylation

NVS-PAK-1-1 also inhibits phosphorylation of the downstream substrate MEK1 at Ser289, but only at concentrations that inhibit both PAK1 and PAK2 (6-20 µM).

Antiproliferative effect

Pancreatic duct carcinoma Su86.86 cells treated with NVS-PAK1-1 for 5 days showed inhibition of proliferation with an IC₅₀of 2 μM due to inhibition of both PAK1 and partial inhibition of PAK2. NVS-PAK1-1 shows growth inhibition with an IC50 of 0.21 μM in SU86.86 cells treated with shPAK2.

We recommend using NVS1-PAK1-1 at 2.5 uM for PAK1/2 inhibition or 0.25 uM for PAK1 inhibition.

Materials and Methods

PAK1/2 SER141/144 TARGET MODULATION ASSAY

Su86.86 pancreatic cancer cell line was grown and seeded in 6-well plate at 500,000 cell density. 10 mM compound diluted in DMSO was further diluted to appropriate concentration (3 fold dilution for 8 concentrations) using growth media and added to each well with cells grown 1 day before. Cells were harvested after 30 minutes of compound treatment and lysed with RIPA buffer (Sigma cat# R0278). Protein concentration of lysates was normalized before loading on SDS-PAGE. Western blot was done as described. pPAK1/2 Ser144/141 antibodies were generated as described. PAK1 and PAK2 antibodies were purchased from Cell Signaling Technology (PAK1 Cat#2602; PAK2 Cat#2608) and were mixed in equal amounts for generation of PAK1/2 signal in western blot.

Co-crystal structures

The co-crystal structure of a related compound with PAK1 has been solved with a resolution of 1.99Å. The key features are

Binding in an allosteric binding site in DFG out conformation
NVS-PAK1-1 strengthens interaction with crucial β4 carbonyl
Shift of the aC helix opens pocket for tert-butyl group; non-conserved Asn affected by the move moving out into the solvent provides rationale for selectivity over PAK2, which has a Leu in the corresponding position

References

[1] Karpov AS, Amiri P, Bellamacina C, Bellance MH, Breitenstein W, Daniel D, Denay R, Fabbro D, Fernandez C, Galuba I, Guerro-Lagasse S, Gutmann S, Hinh L, Jahnke W, Klopp J, Lai A, Lindvall MK, Ma S, Mobitz H, Pecchi S, Rummel G, Shoemaker K, Trappe J, Voliva C, Cowan-Jacob SW, Marzinzik AL. Optimization of a Dibenzodiazepine Hit to a Potent and Selective Allosteric PAK1 Inhibitor. ACS medicinal chemistry letters. 2015;6:776-81.

[2] Radu M, Semenova G, Kosoff R, Chernoff J. PAK signalling during the development and progression of cancer.Nature reviews Cancer. 2014;14:13-25.

[3] Wang G, Zhang Q, Song Y, Wang X, Guo Q, Zhang J, Li J, Han Y, Miao Z, Li F. PAK1 regulates RUFY3-mediated gastric cancer cell migration and invasion. Cell death & disease. 2015;6:e1682.

[4] Fang F, Pan J, Li YP, Li G, Xu LX, Su GH, Li ZH, Feng X, Wang J. p21-activated kinase 1 (PAK1) expression correlates with prognosis in solid tumors: A systematic review and meta-analysis. Oncotarget. 2016.

[5] Pinto VI, Mohammadi H, Lee WS, Cheung AH, McCulloch CA. PAK1 is involved in sensing the orientation of collagen stiffness gradients in mouse fibroblasts. Biochimica et biophysica acta. 2015;1853:2526-38.