Pan-PHLPP (PHLPP1 and PHLPP2) targeting the PP2C phosphatase domain [1].
Off-target: protein phosphatase 1 (PP1) (approx. 50% inhibition at 100 μM) [1].
Off-target: protein phosphatase 2Cα (PP2Cα) (approx. 50% inhibition at 100 μM) [1].
Off-target: Gβγ subunit (IC50 = 56 μM) [1].
NSC117079 targets the PHLPP1 and PHLPP2 phosphatases. PHLPP1 (Pleckstrin homology domain and leucine-rich repeat protein phosphatase 1) inhibits protein kinase B (AKT) survival signaling in neurons. By acting as a selective inhibitor of PHLPP1 and PHLPP2, NSC117079 prevents the dephosphorylation and inactivation of AKT, thereby promoting cell survival and proliferation. This mechanism makes it a valuable tool for studying neuroprotection and cancer.

Neutrophil adherence to plated fibrinogen was stimulated by NSC-117079 at 30 μM, increasing it from 9.0±2.4% to 27.0±8.0%. Similarly, 50 ng/mL GM-CSF induced neutrophil cell adhesion to 22.9± 6.0%, increasing it to 47.6±10.9%. Transendothelial migration of neutrophils occurs after neutrophil labeling. According to the findings, PHLPP NSC-117079 by itself can successfully stop Akt from becoming dephosphorylated in neutrophils, but PHLPP's Akt phosphatase can interfere with the migration of neutrophil markers [2].

---

NSC117079 (35 min treatment) increased basal neuronal AKT phosphorylation (pAKT473) in a dose-dependent manner in primary rat cortical neurons under supplement-starved conditions; did not significantly alter pERK1/2 levels [1].
NSC117079 (50 and 100 μM) blocked IGF-1-induced AKT activation in neurons and altered total AKT levels (Supplemental Fig. 1) [1].
In primary rat astrocytes, NSC117079 (50 μM, 35 min) decreased both basal pAKT473 and pERK1/2 levels [1].
NSC117079 dose-dependently reduced basal viability of primary rat astrocytes after 24 h incubation as measured by CellTiterBlue metabolism; induced a dose-dependent loss of astrocytes and altered cell morphology from large diffuse cell bodies to an elongated shape (Supplemental Fig. 3) [1].
NSC117079 (24 h co-treatment with staurosporine) protected primary rat cortical neurons from staurosporine-induced cell death at lower STS injury levels (50 nM, 100 nM, 200 nM STS); however, a 35-min pretreatment with NSC117079 failed to augment survival compared to injured control [1].
In vitro, NSC117079 is a potent inhibitor of PHLPP1 and PHLPP2. By inhibiting PHLPP activity, it activates neuronal AKT signaling. The compound has been shown to reduce mechanical allodynia and slow articular cartilage degeneration in a mouse model of joint injury. Animals treated with the PHLPP inhibitor maintained normal activity levels, while control animals showed reduced activity after joint injury. These results demonstrate the neuroprotective and chondroprotective effects of NSC117079.

In meniscally unstable joints, a single intra-articular injection of Phlpp twin NSC117079 reduces mechanical allodynia and interferes with joint damage. Seven weeks following injury, animals treated with Phlpp twins continued their regular activity levels, whereas three months following joint damage, animals with heart disease moved and walked less. More than 90% of human articular cartilage explants from osteoarthritis patients produce more cartilage extracellular matrix components (glucosamine antioxidants and aggregated proteins) when NSC117079 is added, and in human articular chondrocytes, Phlpp1 substrate increases the phosphorylation of substances (AKT2, ERK1/2, and PKC) [1].
In vivo, NSC117079 has demonstrated efficacy in a mouse model of joint injury. Single intra-articular injection of the PHLPP inhibitor NSC117079 reduced mechanical allodynia and slowed articular cartilage degeneration in mice with meniscal instability. Injured animals treated with the PHLPP inhibitor maintained normal activity levels, whereas control animals walked shorter distances and were less active three months after joint injury. These results demonstrate the therapeutic potential of PHLPP inhibition for joint injuries and osteoarthritis.

In vitro PHLPP phosphatase assays for NSC117079 involve measuring the dephosphorylation of a phosphopeptide substrate by purified PHLPP1 or PHLPP2 enzymes. The enzyme is incubated with the substrate in the presence of varying concentrations of NSC117079, and the amount of phosphate released is measured using a colorimetric assay (e.g., Malachite Green). IC₅₀ values are calculated from dose-response curves. Selectivity against other phosphatases is assessed to confirm specificity.

Cell viability assay (CellTiterBlue): For neurons, cells seeded in 96-well plates at high density (1.5×10^5/well). At DIV10-12, maintenance media replaced with drug treatment media containing different concentrations of staurosporine with or without NSC117079 for 24 h. Then 20 μl CellTiterBlue reagent added, incubated, and color change detected using a plate reader. For astrocytes, cells used at third passage, seeded onto poly-D-lysine coated 96-well plates, grown to confluence, then treated with increasing doses of NSC117079 for 24 h in DMEM/F12/10% FBS, followed by CellTiterBlue reagent incubation for 30-60 min and analysis [1].
Calcein AM live cell staining: For mixed neuron/astrocyte cultures, cells seeded on glass chamber slides, treated with DMSO or 50 nM staurosporine ± 200 μM NSC117079 for 24 h, then washed with DPBS, incubated with 2 μM calcein AM for ~10 min, and live cell images captured under fluorescent microscope. For astrocytes, cells seeded on glass chamber slides, incubated with NSC117079 at indicated concentrations for 24 h, then washed and stained with calcein AM, images captured [1].
Western blot: For neurons, cells grown on 6-well plates, supplement starved 2 h, then treated with increasing doses of NSC117079 for 35 min. Protein extracts harvested in RIPA buffer with EDTA, protease inhibitors, and phosphatase inhibitors. Samples sonicated, centrifuged, protein concentration determined by BCA assay. 20 μg protein per sample loaded onto 7.5% or 4-15% gradient SDS-PAGE gels, run at 200V for 2 h, transferred to PVDF membranes, blocked in 7.5% milk, incubated overnight at 4°C with primary antibodies (pAKT473, AKT total, pERK1/2, ERK1/2 total, GAPDH), then secondary antibodies, developed with ECL-2 Plus detection reagent, films exposed, densitometry analyzed using UN-SCAN-IT software [1].
For astrocytes, cells grown to confluence on 6-well plates, treated with 50 μM NSC117079 for 35 min, then western blot performed similarly [1].
In vitro cell-based assays for NSC117079 are performed using neuronal cell lines or primary neurons. Cells are cultured in appropriate media and treated with NSC117079 at various concentrations. AKT phosphorylation is assessed by Western blot to confirm target engagement. Cell viability is measured by MTT assays. The effects on cell survival and neuroprotection can be assessed in models of oxidative stress or excitotoxicity. For chondroprotection studies, chondrocyte cell lines or primary chondrocytes are used.

In vivo animal studies for NSC117079 have been conducted in a mouse model of meniscal instability-induced osteoarthritis. Mice undergo surgery to induce meniscal instability, and NSC117079 is administered via single intra-articular injection. Mechanical allodynia is assessed using von Frey filaments. Articular cartilage degeneration is evaluated histologically. Animal activity levels are monitored over time to assess functional outcomes.

Specific pharmacokinetic properties of NSC117079, such as half-life and bioavailability, are not extensively detailed in the available literature. As a small molecule with a molecular weight of 473.48 g/mol, it is expected to be absorbed and distributed to tissues following administration. It is soluble in DMSO and is typically formulated for in vivo administration. The compound's pharmacokinetic profile in animal models requires further elucidation.

NSC117079 reduced basal viability of primary rat astrocytes after 24 h incubation, induced dose-dependent loss of astrocytes, and altered cell morphology (elongated shape) [1].
Comprehensive toxicological data for NSC117079 are not widely available in public literature. In animal studies, single intra-articular injection of NSC117079 was tolerated and resulted in therapeutic benefits without reported adverse effects. However, as with all research compounds, standard safety precautions should be followed. NSC117079 is not intended for human therapeutic use.

[1]. Pharmacological inhibition of pleckstrin homology domain leucine-rich repeat protein phosphatase is neuroprotective: differential effects on astrocytes. J Pharmacol Exp Ther. 2013 Nov;347(2):516-28.

[2]. Regulation Of Neutrophil Adhesion And Migration By Ph Domain And Leucine Rich Repeat Protein Phosphatase. A35 RECENT ADVANCES IN PHAGOCYTE BIOLOGY / Thematic Poster Session / Sunday, May 20/8:15 AM-4:30 PM / Area G (Hall D, North Building, Lower Level), Moscone Center.

Inhibits leucine-rich repeat protein phosphatases with a pH domain; structure can be found in the first article.

---

NSC117079 is a pan-PHLPP inhibitor that targets the PP2C phosphatase domain of PHLPP1 and PHLPP2, thereby activating AKT in neurons but unexpectedly inhibiting AKT/ERK in astrocytes. It has off-target activity against PP1, PP2Cα, and Gβγ. The compound was neuroprotective in cortical neurons when co-treated with staurosporine but not when given as a brief pretreatment. It did not potentiate IGF-1-induced AKT activation; rather, it inhibited it at higher doses. These findings suggest that non-selective PHLPP inhibition may have detrimental effects on astrocytes due to blockade of PHLPP2-mediated pro-survival signaling. The study concludes that selective PHLPP1 inhibitors should be developed for optimal neuroprotection in global brain ischemia [1].
NSC117079 is a novel and potent inhibitor of PHLPP1 and PHLPP2. By inhibiting these phosphatases, it activates AKT survival signaling. The compound has demonstrated neuroprotective and chondroprotective effects in a mouse model of joint injury, reducing mechanical allodynia and slowing cartilage degeneration. NSC117079 is a valuable research tool for studying PHLPP function, neuroprotection, and osteoarthritis. This product is for research use only.

C20H15N3O7S2

473.47900223732

473.035

500363-63-3

272572

Pale purple to purple solid powder

2.1

4

10

4

32

972

0

S(C1=CC(=C2C(C3C=CC=CC=3C(C2=C1N)=O)=O)NC1C=CC=C(C=1)S(N)(=O)=O)(=O)(=O)O

LHSBZAWDPSTOEY-UHFFFAOYSA-N

InChI=1S/C20H15N3O7S2/c21-18-15(32(28,29)30)9-14(23-10-4-3-5-11(8-10)31(22,26)27)16-17(18)20(25)13-7-2-1-6-12(13)19(16)24/h1-9,23H,21H2,(H2,22,26,27)(H,28,29,30)

1-amino-9,10-dioxo-4-(3-sulfamoylanilino)anthracene-2-sulfonic acid

NSC117079; NSC-117079; NSC 117079

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)

DMSO : ~50 mg/mL (~105.60 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.28 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 (5.28 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.)