K-252b

K-252b potentiates the neurotrophic effects of neurotrophin-3 (NT-3) in primary cultures of rat central cholinergic and peripheral sensory neurons and in a rat pheochromocytoma PC12 cell line. The ligand and receptor specificity, and role of the low-affinity neurotrophin receptor (p75NTR) in the potentiation response induced by K-252b, are unknown. To address the issues of ligand and receptor specificity of K-252b potentiation, we have examined neurotrophin-induced DNA synthesis ([3H]-thymidine incorporation) in NIH3T3 cells expressing trkA, trkB, or trkC. Neither NT-3 nor K-252b alone could stimulate mitogenic activity in the trkA-overexpressing clone. However, coaddition of K-252b (EC50 of approximately 2 nM) with 10-100 ng/ml NT-3 led to incorporation of [3H]thymidine in trkA expressing cells to a level induced by optimal concentrations of nerve growth factor (NGF). The K-252b- and NT-3-induced [3H]thymidine incorporation correlated with an increase in the tyrosine autophosphorylation of the trkA receptor as well as tyrosine phosphorylation of trk-associated phospholipase C-gamma 1 and SH2-containing proteins. K-252b did not potentiate submaximal doses of NGF, or maximal doses of brain-derived neurotrophic factor (BDNF) or neurotrophin-4/5 (NT-4/ 5) in trkA-expressing cells. Furthermore, K-252b did not potentiate DNA synthesis by submaximal doses of BDNF, NT-4/5, or NT-3 in trkB- or trkC-expressing NIH3T3 cells, suggesting that the potentiation profile for K-252b was specific for NT-3 in trkA-expressing cells. We found no expression of p75NTR in the trk-expressing NIH3T3 cells. This is the first demonstration that K-252b potentiates a trkA-mediated biological nonneuronal response by NT-3 that occurs independent of p75NTR and appears to be both ligand and receptor specific.

We developed a practical preparation procedure for K-252a by methylating K-252b on an industrial scale. The water-insoluble K-252a, which was present in the cell mass, was converted to the water-soluble K-252b Na salt in an alkaline solution. The obtained K-252b was methylated with dimethylsulfate in the presence of potassium carbonate in dimethylacetamide. We have already used this method to manufacture 90 kg of K-252b from the fermentation broth, and regenerated 65 kg of K-252a from K-252b.

K-252b is a kinase inhibitor structurally related to K-252a, which is known to abolish selectively the effects of nerve growth factor (NGF) on PC12 cells and PNS neurons. We tested whether K-252b, K-252a, and staurosporine, another related compound, are effective and selective inhibitors of NGF actions on CNS neurons. All three compounds, at appropriate concentrations, completely and selectively prevented the NGF-mediated activity increase of the cholinergic marker enzyme choline acetyltransferase in cultures of rat basal forebrain cells. The stimulatory effects of basic fibroblast growth factor and insulin on choline acetyltransferase in these cultures and on dopamine uptake in cultures of dissociated ventral mesencephalon were not affected. No signs of toxicity were observed in cultures treated with K-252b. In contrast, K-252a and staurosporine, at concentrations required to block the NGF actions on cholinergic cells, were cytotoxic and produced cell loss. In addition, K-252a, at higher concentrations and in the absence of growth factors, increased cell numbers. Our study suggests that K-252b is a selective and nontoxic inhibitor of NGF actions in the brain and may become a useful tool to study these actions in vivo.