Protein kinase C (PKC) is a family of closely related serine and threonine kinases. Overactivation of some PKC isozymes has been postulated to occur in several diseases states, including diabetic complications. Selective inhibition of overactivated PKC isozymes may offer a unique therapeutic approach to disease states such as diabetic retinopathy. A novel series of 14-membered macrocycles containing a N-N'-bridged bisindolylmaleimide moiety is described. A panel of eight cloned human PKC isozymes (alpha, beta I, beta II, gamma, delta, epsilon, sigma, eta) was used to identify the series and optimize the structure and associated activity relationship. The dimethylamine analogue LY333531 (1), (S)-13-[(dimethylamino)methyl]-10,11,14,15-tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene++ +-1,3(2H)-dione, inhibits the PKC beta I (IC50 = 4.7 nM) and PKC beta II (IC50 = 5.9 nM) isozymes and was 76- and 61-fold selective for inhibition of PKC beta I and PKC beta II in comparison to PKC alpha, respectively. The additional analogues described in the series are also selective inhibitors of PKC beta. LY333531 (1) exhibits ATP dependent competitive inhibition of PKC beta I and is selective for PKC in comparison to other ATP dependent kinases (protein kinase A, calcium calmodulin, caesin kinase, src tyrosine kinase). The cellular activity of the series was assessed using bovine retinal capillary endothelial cells. Retinal endothelial cell dysfunction has been implicated in the development of diabetic retinopathy. Plasminogen activator activity stimulated by a phorbol ester (4 beta-phorbol 12,13-dibutyrate) in endothelial cells was inhibited by the compounds in the series with ED50 values ranging from 7.5 to 0.21 microM. A comparison of the PKC isozyme and related ATP dependent kinase inhibition profiles is provided for the series and compared to the profile for staurosporine, a nonselective PKC inhibitor. The cellular activity of the series is compared with that of the kinase inhibitor staurosporine.

Bax is a major player in the apoptotic process, being at the core of the mitochondria permeabilization events. In spite of the major recent advances in the knowledge of Bax organization within the membrane, the precise behavior of the C-terminal helix α9 remains elusive, since it was absent from the resolved structure of active Bax. The Proline 168 (P168) residue, located in the short loop between α8 and α9, has been the target of site-directed mutagenesis experiments, with conflicting results. We have produced and purified a recombinant mutant Bax-P168A, and we have compared its behavior with that of wild-type Bax in a series of tests on Large Unilamellar Vesicles (LUVs) and isolated mitochondria. We conclude that Bax-P168A had a greater ability to oligomerize and bind to membranes. Bax-P168A was not more efficient than wild-type Bax to permeabilize liposomes to small molecules but was more prone to release cytochrome c from mitochondria.

Edited by Barry HalliwellBax-dependent mitochondrial permeabilization during apoptosis is controlled by multiple factors, including the phosphorylation by the protein kinase AKT. We used the heterologous co-expression of human Bax and AKT1 in yeast to investigate how the kinase modulates the different steps underlying Bax activation. We found that AKT activated Bax and increased its cellular content. Both effects were dependent on Ser184, but a phosphorylation of this residue did not fully explain the effects of AKT. Additional experiments with mutants substituted on Ser184 suggested that the regulation of Bax dynamic equilibrium between the cytosol and mitochondria might be more tightly regulated by Bcl-xL when Bax is phosphorylated.