Synthesis and protein kinase C inhibitory activities of acyclic balanol analogs that are highly selective for protein kinase C over protein kinase A.

A series of balanol analogs in which the perhydroazepine ring and the p-hydroxybenzamide moiety were combined into an acyclic linked unit have been prepared and evaluated for their inhibitory properties against the serine/threonine kinase PKC. Several low-micromolar to low-nanomolar inhibitors of the alpha, beta I, beta II, gamma, delta, epsilon and eta PKC isozymes were prepared. In general, these acyclic balanol analogs were found to be highly selective for PKC over the serine/threonine kinase PKA. The type and number of atoms linking the benzophenone ester to the p-hydroxyphenyl group necessary for optimal PKC inhibition were investigated. The most potent compounds contained a three-carbon linker in which the carboxamide moiety of balanol had been replaced by a methylene group. The effect of placing substituents on the three-carbon chain was also investigated. The preferred compounds contained either a 2-benzenesulfonamido (6b) or a 1-methyl (21b) substituent. The preferred compounds 6b and 21b were tested against a panel of serine/threonine kinases and found to be highly selective for PKC. The more active enantiomer of 6b, (S)-12b, was 3-10-fold more active than the R-enantiomer against the PKC isozymes. The effect of making the analogs more rigid by making the three-carbon chain part of a five-membered ring, but with retention of the methylene replacement for the carboxamide moiety, led to potent PKC inhibitors including anti-substituted pyrrolidine analog 35b and the most potent PKC inhibitor in the series, anti-substituted cyclopentane analog 29b. The anti cyclopentane analog 29b, was a low-micromolar inhibitor of the PMA-induced superoxide burst in neutrophils, and its carboxylic ester was a high-nanomolar inhibitor of neutrophils. Finally esterification of 21b, (S)-12b, and 35b turned these potent PKC inhibitors into low-micromolar inhibitors of neutrophils.

Inhibition of leukotriene B4 (LTB4) in human neutrophils by L-threo-dihydrosphingosine.

The sphingosine analog L-threo-dihydrosphingosine has been shown to inhibit protein kinase C (PKC) isoenzymes in mixed micelle and vesicle assays. This compound also inhibited the reactive oxygen intermediates (ROI) released from isolated neutrophils (IC50 approximately 2 microM) and phorbol ester-induced edema and neutrophil influx in the mouse ear model (ED50 approximately 11 mg/kg). Based on the anti-inflammatory activity of this compound, studies were done to determine its effect on arachidonate metabolism by the lipoxygenase pathway. Neutrophils were preincubated with test agents or vehicle for one minute and then incubated with 1 microM calcium ionophore A23187 for two minutes. Supernatants were assayed for LTB4 using a radioimmunoassay. The reference lipoxygenase inhibitor nordihydroguaiaretic acid exhibited 98.3% inhibition at 1 microM (n = 2) and prevented ROI production (IC50 approximately 6 microM). In contrast, the potent PKC inhibitor staurosporine was inactive against LTB4 in these studies (< 23% inhibition at 10 microM, n = 2), but inhibited ROI formation (IC50 approximately 3nM). L-threo-dihydrosphingosine inhibited LTB4 production 96.9 +/- 1.3%, at 10 microM (IC50 = 6 microM, n = 2). These data suggest that L-threo-dihydrosphingosine blocks the release of LTB4 from human neutrophils via a mechanism independent of PKC.

A cell-based reporter assay for the identification of protein kinase C activators and inhibitors.

Transmission of extra cellular signals across biological membranes results in the generation of lipid metabolites which in turn influence specific cellular events such as cell growth or differentiation. Many of these lipid messengers can activate protein kinase C (PKC) isozymes of which one function is to perpetuate the extracellular signals to the nucleus by phosphorylating other targets proteins. We have engineered mammalian cell lines to identify and evaluate activators and inhibitors of PKC-dependent and independent signal transduction pathways. The A31 mouse fibroblast cell line, has been stably transfected with a construct containing a triplet repeat of the TPA response element (TRE) upstream of a thymidine kinase promoter fused to the human growth hormone (hGH) gene. A31 cells containing this reporter construct exhibit significant increases in hGH secretion following stimulation by phorbol esters or other mitogens. The levels of hGH secretion are modulated in this system using different pharmacological agents. We demonstrate that this assay can be used to identify specific and general inhibitors as well as activators of the signal transduction pathway mediated by PKC isozymes.

Naturally occurring protein kinase C inhibitors; II. Isolation of oligomeric stilbenes from Caragana sinica.

The oligomeric stilbenes (+)-alpha-viniferin (1), miyabenol C (2), and kobophenol A (3) have been isolated from Caragana sinica (Buchoz) Rehd (Leguminosae). (+)-alpha-Viniferin (1) and miyabenol C (2) exhibited protein kinase C inhibitory activity at low micromolar concentrations. (+)-alpha-Viniferin inhibited keratinocyte proliferation (0.4 microM) and free radical release in whole blood (47 microM), in vitro, and may be useful in treating hyperproliferative or inflammatory skin diseases.