PKC (IC50 = 470 nM), PKA (IC50 = 140 nM), Ca2+/calmodulin-dependent kinase type II (IC50 = 270 nM), phosphorylase kinase (IC50 = 1.7 nM)

K-252a (3-100 nM, 8d) suppresses neurite outgrowth that is stimulated by NGF [5].
This study shows that K-252a, a potent protein kinase inhibitor, blocks NGF-induced neurite outgrowth and the changes in protein phosphorylation elicited by NGF. In the experiment with intact cells phosphorylated with 32P-orthophosphoric acid, an exposure of PC12h cells to NGF (50 ng/ml) caused an increase in the phosphorylation of tyrosine hydroxylase and a 35,000-D protein and a decrease in a 36,500-D protein. Pretreatment of PC12h cells with K-252a (100 nM) inhibited the effects of NGF on the phosphorylation of these three proteins. In the phosphorylation of cell-free extracts with [gamma-32P] ATP, treatment of PC12h cells with NGF (50 ng/ml) caused a decrease in the phosphorylation of Nsp100. Pretreatment of the cells with K-252a (30 nM) almost completely blocked the NGF effect on the phosphorylation of Nsp100 elicited by subsequent treatment of the cells with NGF. Treatment of PC12h cells with NGF promoted outgrowth of neurites. The addition of K-252a (100 nM) into the culture almost completely blocked the generation of neurites elicited by NGF. Earlier studies demonstrated that NGF-induced neurite outgrowth in PC12 cells involves at least two components: the first of these is transcription-dependent and the second is transcription-independent. To determine the component on which K-252a acts, experiments were carried out on NGF-induced priming or regeneration of neurites. When K-252a was present in the priming step, NGF induced only actinomycin D-sensitive neurites, showing that K-252a interferes with the transcription-dependent actions of NGF. When already primed cells were treated with NGF, actinomycin D-resistant neurites were formed and these were blocked by K-252a, showing that the inhibitor interferes with the transcription-independent actions of NGF as well. Although the exact mechanism of inhibition of NGF-promoted neurite formation by K-252a is unknown, the most probable explanation is that both transcription-dependent and -independent components are involved in at least one step of the activation of some specific protein kinase(s) that can be suppressed by K-252a[5].

TH-induced neuroprotection is lessened when K252a (20 mg/kg/day) inhibits the TrkB pathway [6].

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To further confirm the TrkB pathway in TH mediated effects, we further treated I/R mice with K252a to pharmacologically inhibit TrkB pathway. Then we evaluated the neurological functions of mice. The results suggested that attenuating TrkB pathway aggravated the neurological deficits of the mice (P < .05 compared with MCAO+TH group) (Figure 5A-E). What's more, downregulating TrkB increased brain oedema as well as apoptotic cell rate (P < .05 vs MCAO+TH group, Figure 5F,G). Therefore, those results suggested TrkB pathway exerted a crucial role in TH induced neuroprotection.[6]

K252a, an efficient serine/threonine protein kinase inhibitor (IC50s of 10 to 30 nM), has been shown to block the neuronal differentiation of rat pheochromocytoma PC12 cells induced by nerve growth factor (NGF). In this report, we demonstrate that K252a is a potent inhibitor (IC50 of 3 nM) of the tyrosine protein kinase activity of the NGF receptor gp140trk, the product of the trk protooncogene. K252a also inhibits the kinase activity of its transforming alleles, the trk oncogenes, and of the related neurotrophin receptors gp145trkB and gp145trkC, the products of the other known members of the trk gene family, trkB and trkC. In contrast, K252a has no effect (even at micromolar concentrations) on other tyrosine protein kinases such as the receptors for EGF and PDGF and the products of the v-src and v-fms oncogenes. In addition, K252a rapidly reverts the transformed phenotype of NIH3T3 cells transformed by either autocrine stimulation of the trk family of receptors by their cognate ligands or by expression of trk oncogenes isolated from human tumors. The selectivity of K252a for the catalytic activity of the trk family of kinases should help to establish the structural basis for the rational design of highly specific tyrosine protein kinase inhibitors[3].

Western blot analysis[4]
Cell Types: LINC00641-overexpression cell line.
Tested Concentrations: 1.7 nM (NGF (50 ng/mL)).
Incubation Duration: 6 hrs (hours).
Experimental Results: diminished levels of p-Akt and p-TrkB.

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Cell viability assay [5].
Cell Types: PC12 subclone h cells.
Tested Concentrations: 3 to 100 nM.
Incubation Duration: 8 days.
Experimental Results: Inhibition of NGF-promoted neurite outgrowth.

Animal/Disease Models: Mouse[6].
Doses: 20 mg/kg/day.
Route of Administration: intraperitoneal (ip) injection, one time/day for 5 days.
Experimental Results: Attenuation of TH-induced neuroprotection.

[1]. The structures of the novel protein kinase C inhibitors K-252a, b, c AND d Journal of Antibiotics 39(8), 1072-1078 (1986).

[2]. Inhibitors of protein kinase C. 1.1 2,3-bisarylmaleimides Journal of Medicinal Chemistry 35, 177-184 (1992).

[3]. K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene. 1992 Feb;7(2):371-81.

[4]. LncRNA LINC00641 Sponges miR-497-5p to Ameliorate Neural Injury Induced by Anesthesia via Up-Regulating BDNF. Front Mol Neurosci. 2020 Jun 30;13:95.

[5]. K-252a, a potent protein kinase inhibitor, blocks nerve growth factor-induced neurite outgrowth and changes in the phosphorylation of proteins in PC12h cells. J Cell Biol. 1988 Oct;107(4):1531-9.

[6]. Tenacissoside H promotes neurological recovery of cerebral ischaemia/reperfusion injury in mice by modulating inflammation and oxidative stress via TrkB pathway. Clin Exp Pharmacol Physiol. 2021 May;48(5):757-769.

K-252a is an organic heterocyclic octane compound, a potent inhibitor of protein kinase C, isolated from Nocardiopsis sp. It possesses multiple functions, including as an EC 2.7.11.13 (protein kinase C) inhibitor, an antibacterial agent, a tropomyosin-associated kinase B receptor antagonist, and a bacterial metabolite. It is an organic heterocyclic octane compound belonging to the bridging compounds, γ-lactams, and methyl esters. The antibiotic K-252a has been reported to exist in Actinomadura, Nocardiopsis, and Streptomyces longisporoflavus, with relevant data available. K-252a is an indolecarbazole alkaloid and astrosporin analog, isolated from Nocardia and Actinoma sp., and possesses kinase inhibitory activity. K252a inhibits a variety of enzymes, including but not limited to protein kinases A (PKA), C (PKC), and G (PKG), calcium (Ca2+)/calmodulin-dependent kinase type II (CaMKII), phosphorylase kinase (PhK), tropomyosin receptor kinase (Trk; neurotrophic tyrosine receptor kinase; NTRK), myosin light chain kinase (MLCK; MYLK), mixed lineage protein kinase 3 (MLK3), receptor tyrosine protein kinase FLT3 (CD135; fms-like tyrosine kinase 3; fetal liver kinase-2; FLK2), and actin-regulated kinase PRK1 (PAK1). Inhibition of these kinases prevents the activation of signaling pathways in which they play crucial roles. Nerve growth factor (NGF) promotes neuronal differentiation in PC12 pheochromocytoma cells. One of the most prominent features of neuronal differentiation is the growth of neurites. The mechanism by which NGF induces neurite formation in cells remains unclear. This study demonstrates that the potent protein kinase inhibitor K-252a can block NGF-induced neurite growth and NGF-induced changes in protein phosphorylation. In whole-cell assays phosphorylated with 32P-orthophosphate, PC12h cells exposed to NGF (50 ng/ml) showed increased phosphorylation levels of tyrosine hydroxylase and a 35,000-D protein, while decreasing phosphorylation levels of a 36,500-D protein. Pretreatment of PC12h cells with K-252a (100 nM) inhibited the effect of NGF on the phosphorylation of these three proteins. In cell-free extracts phosphorylated with [γ-32P]ATP, treatment of PC12h cells with NGF (50 ng/ml) resulted in decreased Nsp100 phosphorylation levels. Pretreatment of cells with K-252a (30 nM) almost completely blocked the effect of NGF on subsequent Nsp100 phosphorylation. NGF treatment of PC12h cells promotes neurite growth. The addition of K-252a (100 nM) to the culture medium almost completely blocked NGF-induced neurite formation. Previous studies have shown that NGF-induced neurite growth in PC12 cells involves at least two components: the first is transcription-dependent, and the second is transcription-independent. To determine the mechanism of action of K-252a, we conducted NGF-induced neurite initiation or regeneration experiments. When K-252a was present in the initiation phase, NGF induced only actinomycin D-sensitive neurites, indicating that K-252a interferes with the transcription-dependent effect of NGF. When initiated cells were treated with NGF, actinomycin D-resistant neurites formed, and K-252a blocked the formation of these neurites, indicating that this inhibitor also interferes with the transcription-independent effect of NGF. Although the exact mechanism by which K-252a inhibits NGF-promoted neurite formation is unclear, the most likely explanation is that both transcription-dependent and non-transcription-dependent components are involved in at least one step of the activation of certain protein kinases, which can be inhibited by K-252a. [5]

C27H21N3O5

467.47274

467.148

C, 69.37; H, 4.53; N, 8.99; O, 17.11

99533-80-9

99533-80-9; 97161-97-2;

3035817

Typically exists as White to light yellow solids at room temperature

1.7±0.1 g/cm3

685.3±55.0 °C at 760 mmHg

368.2±31.5 °C

0.0±2.2 mmHg at 25°C

1.841

4.23

2

5

2

35

977

3

C[C@@]12[C@](C(OC)=O)(O)C[C@@H](O1)N3C4=CC=CC=C4C5=C3C6=C(C7=C5C(NC7)=O)C8=CC=CC=C8N62

KOZFSFOOLUUIGY-SOLYNIJKSA-N

InChI=1S/C27H21N3O5/c1-26-27(33,25(32)34-2)11-18(35-26)29-16-9-5-3-7-13(16)20-21-15(12-28-24(21)31)19-14-8-4-6-10-17(14)30(26)23(19)22(20)29/h3-10,18,33H,11-12H2,1-2H3,(H,28,31)/t18-,26+,27+/m1/s1

methyl (5S,6R,8R)-6-hydroxy-5-methyl-13-oxo-5,6,7,8,14,15-hexahydro-13H-16-oxa-4b,8a,14-triaza-5,8-methanodibenzo[b,h]cycloocta[jkl]cyclopenta[e]-as-indacene-6-carboxylate

K-252a; K 252a; K252a; SF-2370; SF 2370; k-252a; 99533-80-9; Antibiotic K 252a; K252a; Antibiotic SF 2370; (+)-Antibiotic K 252a; IV7H45AM5B; SF2370.

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 (~106.96 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.35 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.45 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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