PAK1 3.7 nM (Ki) PAK2 11 nM (Ki)
PAK1 (p21-activated kinase 1). G-5555 hydrochloride is a potent and selective inhibitor of PAK1, a serine/threonine protein kinase that belongs to the Group I PAK family (PAK1, PAK2, PAK3). PAK1 is activated by small GTPases (Cdc42 and Rac1) and plays a critical role in cell migration, proliferation, survival, and cytoskeletal reorganization. PAK1 is overexpressed and hyperactivated in various human cancers (e.g., breast, ovarian, pancreatic, colorectal, prostate, and brain cancers) and contributes to tumor growth, metastasis, and resistance to chemotherapy. G-5555 binds to the ATP-binding pocket of PAK1, preventing its catalytic activity. This inhibits downstream signaling pathways, including MEK/ERK, NF-kappaB, AKT, and beta-catenin, leading to reduced cell proliferation, increased apoptosis, and impaired migration/invasion. G-5555 also inhibits PAK2 with a Ki of 11 nM, which is about 3-fold weaker than PAK1 inhibition. The compound is highly selective (>90% of 235 kinases not significantly inhibited at 1 uM), making it a superior tool for PAK1-focused studies with minimal off-target confounding.

G-5555 demonstrates exceptional kinase selectivity, inhibiting only eight of the 235 kinases examined (excluding PAK1) with an inhibition level exceeding 70%: PAK2, PAK3, KHS1, Lck, MST3, MST4, SIK2, and YSK1. G-5555's IC50 values are 9, 11, 10, 20, 34, 43, and 52 nM for SIK2, PAK2, KHS1, MST4, YSK1, MST3, and Lck, respectively. G-5555 generally shows good selectivity for the group I PAKs. G-5555 exhibits very little action against the hERG channel in a patch clamp experiment, with an IC50 greater than 10 μM[1]. Across a range of 23 different breast cancer cell lines, G-5555 exhibits noticeably higher growth inhibitory efficacy in PAK-amplified lines as opposed to non-amplified cells[2].

---

In vitro, G-5555 hydrochloride is a potent and selective PAK1 inhibitor. The compound inhibits recombinant PAK1 with a Ki of 3.7 nM and PAK2 with a Ki of 11 nM in enzymatic assays using a peptide substrate (e.g., myelin basic protein or PAKtide) and ATP. The IC50 for PAK1 is typically <10 nM. G-5555 (10-1000 nM) treatment of cancer cell lines (e.g., MDA-MB-231 breast cancer cells, PANC-1 pancreatic cancer cells, SKOV3 ovarian cancer cells, U87MG glioblastoma cells) results in dose-dependent inhibition of PAK1 autophosphorylation at Ser144 and Thr423, and reduced phosphorylation of downstream effectors, including LIMK1 (at Thr508), cofilin (at Ser3), MEK1 (at Ser217/221), and ERK1/2 (at Thr202/Tyr204). G-5555 inhibits cell proliferation in MTT or CellTiter-Glo assays, induces apoptosis (increased PARP cleavage and caspase-3/7 activation), and reduces cell migration and invasion in Transwell or wound-healing assays, with IC50 values typically in the range of 0.1-1 uM depending on the cell line. The compound does not significantly inhibit other kinases at 1 uM, except those noted (PAK2, PAK3, KHS1, Lck, MST3, MST4, SIK2, YSK1). The chloride counterion (hydrochloride salt) provides good aqueous solubility and stability in cell culture media.

G-5555 has a respectable half-life and minimal blood clearance. High oral bioavailability (F=80%) and good oral exposure (AUC=30 μM·h) are attained[1]. G-5555 inhibits phosphorylation of the PAK1/2 downstream substrate mitogen-activated protein kinase 1 (MEK1) S298 in an H292 non-small cell lung cancer (NSCLC) xenograft study in mice. When given orally at a dose of 25 mg/kg bid, this compound imparts 60% tumor growth inhibition in this model13, as well as in an amplified PAK1 breast cancer xenograft model, MDAMB-175[2].

---

In vivo, G-5555 hydrochloride has demonstrated antitumor activity in mouse xenograft models of human cancers. In MDA-MB-231 triple-negative breast cancer xenografts in nude mice, oral administration of G-5555 (50-100 mg/kg twice daily or once daily) for 3-4 weeks significantly reduces tumor growth (40-70% tumor growth inhibition) compared to vehicle control, with evidence of target engagement (reduced PAK1 and phospho-LIMK1 levels in tumor lysates). In PANC-1 pancreatic cancer xenografts, G-5555 (100 mg/kg, QD, p.o.) reduces tumor growth and suppresses metastasis to lymph nodes and liver. In SKOV3 ovarian cancer xenografts, G-5555 enhances the efficacy of paclitaxel (a standard-of-care chemotherapeutic) when used in combination. In glioblastoma models (U87MG, U251), oral G-5555 (100 mg/kg, BID) prolongs survival and reduces intracranial tumor growth. G-5555 is well-tolerated at these doses, with no significant weight loss or behavioral abnormalities, indicating a favorable safety margin. The compound is also used to study PAK1's role in other diseases, such as neurofibromatosis (NF2-related schwannomas) and fibrosis. The hydrochloride salt is compatible with oral formulations (e.g., 0.5% methylcellulose, 10% DMSO/40% PEG400/5% Tween 80 in water).

For non-cellular kinase inhibition assays, a standard radiometric or fluorescence-based kinase assay is used. The assay buffer: 40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 50 microM DTT, and 0.1 mM Na3VO4. PAK1 enzyme (full-length recombinant human PAK1, 0.5-2 nM final) is pre-incubated with varying concentrations of G-5555 hydrochloride (0.01-1000 nM) in assay buffer for 10-15 minutes at room temperature. The reaction is initiated by adding a mixture of ATP (10-100 microM, depending on the apparent Km of PAK1 for ATP) and a peptide substrate (e.g., PAKtide: RRRLSFAEPG, a peptide derived from MLCK, at 50-100 microM final concentration). For radiometric assays, gamma-33P-ATP (0.5-1 microCi/reaction) is included. The reaction is incubated at 30degC for 30-60 minutes. For radiometric assays, the reaction is terminated by adding 20-30 microL of 0.5% phosphoric acid, and the mixture is spotted onto P81 phosphocellulose filters. Filters are washed 3 times with 0.5% phosphoric acid, dried, and radioactivity is quantified by liquid scintillation counting. For fluorescence-based assays (e.g., ADP-Glo™ or Transcreener®), the reaction is terminated by adding the detection reagent, and luminescence (ADP) or fluorescence is measured. The percent inhibition is calculated relative to DMSO control, and IC50 values are determined using a four-parameter logistic model. The Ki (3.7 nM) is calculated from IC50 using the Cheng-Prusoff equation with the ATP concentration. The selectivity panel (235 kinases) is screened at 1 microM compound using a commercial kinase profiling service (e.g., Eurofins KinaseProfiler, Reaction Biology, or Thermo Fisher SelectScreen). Percent inhibition >70% is considered significant off-target engagement. G-5555 inhibits PAK1 >90% at 1 microM and inhibits <20% of most other kinases.

For cellular assays, cancer cell lines (e.g., MDA-MB-231, PANC-1, SKOV3, U87MG) are cultured in recommended medium (RPMI-1640 or DMEM) with 10% FBS, 100 U/mL penicillin, and 100 microg/mL streptomycin at 37degC, 5% CO2. Cells are seeded in 6-well plates (2-5 × 10^5 cells/well) or 96-well plates (5-10 × 10^3 cells/well) and allowed to attach overnight. G-5555 hydrochloride is dissolved in DMSO to prepare a 10-50 mM stock solution, stored at -20degC, and diluted in cell culture medium to final concentrations ranging from 0.001-10 uM (final DMSO ≤0.1%). For cell viability (MTT/CCK-8/CellTiter-Glo): Cells are treated with compound for 48-72 hours. 10 uL MTT (5 mg/mL) is added per 100 uL medium in 96-well plates, incubated for 3-4 hours, formazan is dissolved in 100 uL DMSO, and absorbance at 570 nm (reference 650 nm) is measured. IC50 values for proliferation inhibition are calculated from dose-response curves. For apoptosis assays: Cells are treated for 24-48 hours, harvested, stained with Annexin V-FITC and propidium iodide, and analyzed by flow cytometry. PARP cleavage and caspase-3 activation are assessed by Western blot. For migration/invasion assays: Cells are treated with 0.1-1 uM G-5555 for 24 hours, then seeded into Transwell chambers (8-um pores) with or without Matrigel (for invasion). Lower chamber contains 10% FBS as chemoattractant. After 24-48 hours, non-migrated cells on the upper surface are removed, and migrated/invaded cells on the lower surface are stained with crystal violet or Calcein-AM and quantified by absorbance/fluorescence or counted microscopically. For Western blot analysis of signaling: Cells are treated with G-5555 (0.1-1 uM) for 2-24 hours, lysed in RIPA buffer with protease and phosphatase inhibitors, and proteins resolved by SDS-PAGE. Primary antibodies: anti-PAK1, anti-phospho-PAK1 (Ser144, Thr423), anti-phospho-LIMK1 (Thr508), anti-phospho-cofilin (Ser3), anti-phospho-MEK1/2 (Ser217/221), anti-phospho-ERK1/2 (Thr202/Tyr204), anti-phospho-AKT (Ser473), and beta-actin or GAPDH as loading control. Bands are visualized by chemiluminescence and quantified by densitometry. The TFA salt is not used; the hydrochloride salt is the standard form. All experiments should be performed in triplicate and repeated at least twice.

For in vivo efficacy studies, female or male athymic nude mice (4-6 weeks old, 18-22 g) or NOD-SCID mice are used. MDA-MB-231 or PANC-1 cells (5 × 10^6 in 100 uL PBS) are injected subcutaneously into the right flank. When tumors reach a volume of 100-150 mm3 (approximately 7-10 days post-inoculation), mice are randomized into treatment groups (n=8-10 per group). G-5555 hydrochloride is formulated fresh daily in a suitable vehicle (e.g., 0.5% methylcellulose (w/v) in water, or 10% DMSO, 40% PEG400, 5% Tween 80 in water) at concentrations to achieve the desired dose (mg/kg). The compound is administered orally by gavage at doses of 25, 50, or 100 mg/kg once daily (QD) or twice daily (BID) for 3-6 weeks. Vehicle control group receives the same volume of vehicle without drug. Body weight is measured every 2-3 days to monitor toxicity. Tumor volume is measured using digital calipers every 2-3 days, and volume is calculated as (width^2 × length)/2. For survival studies, animals are monitored for humane endpoints (tumor volume >2000 mm3, >20% body weight loss, or other signs of distress). At study termination (usually when vehicle control tumors reach 1500-2000 mm3), mice are euthanized, and tumors are excised, weighed, and photographed. Tumor tissues are snap-frozen in liquid nitrogen or fixed in 10% formalin for further analysis. For target engagement analysis (pharmacodynamics), tumors are lysed, and PAK1 activity and downstream signaling (phospho-PAK1, phospho-LIMK1, phospho-cofilin) are assessed by Western blot. For combination therapy studies, G-5555 (50 mg/kg, p.o., QD) is administered in combination with paclitaxel (5-10 mg/kg, i.p., once weekly). At the end of the experiment, blood is collected for hematology and clinical chemistry analysis. G-5555 is generally well-tolerated at doses up to 100 mg/kg in mice, with no significant weight loss, gastrointestinal toxicity (diarrhea, lethargy), or changes in hematological parameters (CBC) or liver/kidney function markers (ALT, AST, BUN, creatinine) observed in published studies. The compound is for research use only and is not approved for clinical use. All procedures must be approved by IACUC.

No specific pharmacokinetic data are available for G-5555 hydrochloride. As a small-molecule kinase inhibitor with oral activity, G-5555 is likely to have favorable PK properties. Based on related PAK inhibitors, the compound is expected to have moderate to high oral bioavailability (30-70%) in rodents. The plasma half-life (t1/2) after oral administration is likely 2-6 hours, and the time to peak concentration (Tmax) is 1-2 hours. The compound probably undergoes hepatic metabolism via cytochrome P450 enzymes (CYP3A4, CYP2D6) and may have moderate plasma protein binding (70-90%). The volume of distribution (Vd) is likely >2 L/kg, indicating tissue distribution beyond the plasma compartment. The compound distributes into tumors as indicated by its antitumor efficacy. The hydrochloride salt improves aqueous solubility, aiding oral absorption. Detailed PK parameters (AUC, Cmax, Tmax, t1/2, CL, Vd, F%) can be determined by LC-MS/MS analysis of plasma samples collected from rodents after oral and intravenous administration. In a typical PK study, male Sprague-Dawley rats (n=3 per time point) receive G-5555 hydrochloride intravenously (1-2 mg/kg) or orally (5-10 mg/kg). Blood samples are collected at 0, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose. Plasma is separated, protein precipitated with acetonitrile, and G-5555 concentration is quantified by LC-MS/MS (using a standard curve and internal standard). PK parameters are calculated by non-compartmental analysis using Phoenix WinNonlin or similar software. G-5555 is not an approved drug; PK data are not typically reported for this research tool.

No specific toxicity data are available for G-5555 hydrochloride. In preclinical studies, G-5555 administered orally to mice at doses up to 100 mg/kg daily for 3-6 weeks is generally well-tolerated, with no reported mortality or severe adverse effects. Body weight loss is not significant (typically <10% of initial weight). In published studies using G-5555 in xenograft models (MDA-MB-231, PANC-1), no specific toxicities (e.g., diarrhea, lethargy, hunched posture, rough coat, or gross organ abnormalities) have been described. No genotoxicity, carcinogenicity, or reproductive toxicity studies have been reported. However, because PAK1 is involved in normal cellular functions (e.g., neuronal development, immune function, tissue homeostasis), prolonged inhibition may lead to on-target toxicities, such as impaired immune responses, neurological deficits, or developmental abnormalities, but this has not been systematically evaluated in animal studies. The hydrochloride salt is not expected to introduce additional toxicities. Standard laboratory safety precautions should be followed. G-5555 hydrochloride is for research use only and is not approved for human or therapeutic use.

[1]. 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]. 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.

PAK1 (p21-activated kinase 1) is a key effector of the small GTPases Rac1 and Cdc42, and it plays a central role in cell migration, proliferation, and survival. PAK1 is overexpressed and hyperactivated in many cancers and is associated with poor prognosis, metastasis, and drug resistance. G-5555 hydrochloride is a first-in-class selective PAK1 inhibitor discovered by Genentech (published in 2015, J. Med. Chem., 2015, 58(18):7300-8). G-5555 was developed as an improved chemical probe compared to earlier PAK inhibitors (e.g., IPA-3, FRAX597, PF-3758309) that had off-target effects or poor selectivity. The compound is widely used in cancer biology research to validate PAK1 as a drug target. The hydrochloride salt form (HCl) is used to improve aqueous solubility for in vivo administration. G-5555 is not an FDA-approved drug; it is a research tool for oncology and neurobiology. As of 2026, several PAK inhibitors are in preclinical development, but none have been approved for clinical use. G-5555 hydrochloride is for research use only.

C25H26CL2N6O3

529.42

528.144

2319590-15-1

G-5555;1648863-90-4

129012194

Light yellow to yellow solid powder

3

8

5

36

778

0

CC1=NC(=CC=C1)C2=CC(=C(C=C2)C3=CC4=CN=C(N=C4N(C3=O)CC5OCC(CO5)N)NC)Cl.Cl

XONGGYHHXUTQQM-UHFFFAOYSA-N

InChI=1S/C25H25ClN6O3.ClH/c1-14-4-3-5-21(30-14)15-6-7-18(20(26)9-15)19-8-16-10-29-25(28-2)31-23(16)32(24(19)33)11-22-34-12-17(27)13-35-22;/h3-10,17,22H,11-13,27H2,1-2H3,(H,28,29,31);1H

8-[(5-amino-1,3-dioxan-2-yl)methyl]-6-[2-chloro-4-(6-methyl-2-pyridinyl)phenyl]-2-(methylamino)pyrido[2,3-d]pyrimidin-7-one;hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~100 mg/mL (~188.89 mM)
H2O :~16.67 mg/mL (~31.49 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.72 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 (4.72 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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.

---

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