G-5555 hydrochloride is a potent and selective p21-activated kinase 1 (PAK1) inhibitor with a K_i of 3.7 nM.

G-5555 shows excellent kinase selectivity and inhibits only eight out of the 235 kinases tested other than PAK1 with inhibition >70%: PAK2, PAK3, KHS1, Lck, MST3, MST4, SIK2, and YSK1. The IC₅₀s of G-5555 against SIK2, PAK2, KHS1, MST4, YSK1, MST3 and Lck are 9, 11, 10, 20, 34, 43, 52 nM, respectively. In general, G-5555 demonstrates high selectivity for the group I PAKs. There is negligible activity for G-5555 against the hERG channel with IC₅₀ more than 10 μM in a patch clamp assay^[1]. In an array of 23 breast cancer cell lines, G-5555 has significantly greater growth inhibitory activity in cell lines that are PAK-amplified compared to non-amplified lines^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

G-5555 exhibits low blood clearance and an acceptable half-life. Good oral exposure (AUC=30 μM•h) and high oral bioavailability (F=80%) are achieved^[1]. In an H292 non-small cell lunger cancer (NSCLC) xenograft study in mice, G-5555 inhibits phosphorylation of the PAK1/2 downstream substrate mitogen-activated protein kinase 1 (MEK1) S298 and, when administered at an oral dose of 25 mg/kg b.i.d., imparts 60% tumor growth inhibition in this model13 and a PAK1 amplified breast cancer xenograft model, MDAMB-175^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

529.42

C₂₅H₂₆Cl₂N₆O₃

Room temperature in continental US; may vary elsewhere.

4°C, sealed storage, away from moisture

*In solvent : -80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)

In Vitro: 

DMSO : 100 mg/mL (188.89 mM; Need ultrasonic)

H₂O : 16.67 mg/mL (31.49 mM; Need ultrasonic)

*

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month (sealed storage, away from moisture)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

In Vivo:

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (4.72 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.72 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.5 mg/mL (4.72 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.72 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明

• [1]. Ndubaku CO, et al. Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety. ACS Med Chem Lett. 2015 Oct 31;6(12):1241-6.

  [2]. Rudolph J, et al. Chemically Diverse Group I p21-Activated Kinase (PAK) Inhibitors Impart Acute Cardiovascular Toxicity with a Narrow Therapeutic Window. J Med Chem. 2016 Jun 9;59(11):5520-41.

The 10 µL assay mixtures contain 50 mM HEPES (pH 7.5), 0.01% Brij-35, 10 mM MgCl₂, 1 mM EGTA, 2 µM FRET peptide substrate, and PAK enzyme (20 pM PAK1; 50 pM PAK2; 90 pM PAK4). Incubations are carried out at 22°C in black polypropylene 384-well plates. Prior to the assay, enzyme, FRET peptide substrate and serially diluted test compounds (G-5555) are preincubated together in assay buffer (7.5 µL) for 10 minutes, and the assay is initiated by the addition of 2.5 µL assay buffer containing 4x ATP (160 µM PAK1; 480 µM PAK2; 16 µM PAK4). Following the 60-minute incubation, the assay mixtures are quenched by the addition of development reagent, and 1 hour later the emissions of Coumarin (445 nm) and Fluorescein (520 nm) are determined after excitation at 400 nm using an Envision plate reader^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice: 3 mice in each of the two groups are administered 25 mg/kg oral suspension dose twice, with the second dose given 6 hours after the first dose. The dose volumes are 5 mL/kg for the IV group and 10 mL/kg for the PO groups. Following administration of G-5555 (12), 15 µL of blood is collected at each time point are stored at -70 to -80°C until analysis^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Ndubaku CO, et al. Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pK a Polar Moiety. ACS Med Chem Lett. 2015 Oct 31;6(12):1241-6.

  [2]. Rudolph J, et al. Chemically Diverse Group I p21-Activated Kinase (PAK) Inhibitors Impart Acute Cardiovascular Toxicity with a Narrow Therapeutic Window. J Med Chem. 2016 Jun 9;59(11):5520-41.