Synthesis, computer modeling and biological evaluation of novel protein kinase C agonists based on a 7-membered lactam moiety.

4-Hydroxymethyl-5a-methyl-1,3,4,5,5a beta,6,7,8,9,9a alpha-decahydro-2H-benz[d]azepin-2-ones (4-12), which were designed to mimic the biologically active conformation of teleocidins and benzolactams, were synthesized and evaluated for the ability to compete with [3H]phorbol 12,13-dibutyrate in a PKC delta binding assay. Among the compounds, 10-12 showed potent binding affinity, with inhibition constants (Ki) of low nanomolar order. Computational docking simulation also indicates that the relative positions of the hydrogen-bonding sites and hydrophobic regions of the compounds are well matched to the PKC delta binding site.

Clarification of the binding mode of teleocidin and benzolactams to the Cys2 domain of protein kinase Cdelta by synthesis of hydrophobically modified, teleocidin-mimicking benzolactams and computational docking simulation.

Phorbol esters (12-O-tetradecanoylphorbol 13-acetate; TPA) and teleocidins are known to be potent tumor promoters and to activate protein kinase C (PKC) by binding competitively to the enzyme. The relationship between the chemical structures and the activities of these compounds has attracted much attention because of the marked structural dissimilarities. The benzolactam 5, with an eight-membered lactam ring and benzene ring instead of the nine-membered lactam ring and indole ring of teleocidins, reproduces the active ring conformation and biological activities of teleocidins. Herein we describe the synthesis of benzolactams with hydrophobic substituents at various positions. Structure-activity data indicate that the existence of a hydrophobic region between C-2 and C-9 and the steric factor at C-8 play critical roles in the appearance of biological activities. We also computationally simulated the docking of teleocidin and the modified benzolactam molecules to the Cys2 domain structure observed in the crystalline complex of PKCdelta with phorbol 13-acetate. Teleocidin and benzolactams fitted well into the same cavity as phorbol 13-acetate. Of the three functional groups hydrogen-bonding to the protein, two hydrogen-bonded with protein atoms in common with phorbol 13-acetate, but the third one hydrogen-bonded with a different protein atom from that in the case of phorbol 13-acetate. The model explains well the remarkable difference in activity between 5 and its analogue having a bulky substituent at C-8.

Mezerein and 12-deoxyphorbol 13-isobutyrate, protein kinase C ligands with differential biological activities, do not distinguish PKC-isotypes alpha, beta 1, beta 2, and gamma.

Previous in vitro experiments have shown that the phorbol-like diterpenes 12-deoxyphorbol 13-isobutyrate (dPB), and possibly mezerein, have multiple biological target sites which differ from one another in apparent affinity for dPB by 12.5-780 fold and for mezerein by 24-fold. These two compounds are thus very important ligands because of their potential PKC isotype-selectivity. In the present study they were found to have binding affinities differing by a maximum of only 1.6-fold among recombinant protein kinase C (PKC) isotypes alpha, beta 1, beta 2, and gamma (the "A-group") in a [3H]phorbol dibutyrate binding assay. The apparent Ki's were 92-140 nM for dPB and 68-92 nM for mezerein. Our results are consistent with short-term 12-deoxyphorbol ester-induced mouse skin inflammation being mediated at least in part by one or more A-group PKC isotypes. The data also indicate that the pharmacologically distinguishable target sites previously established for mezerein and dPB must include one or more binding sites not found in the A-group of PKC isotypes and that mezerein has a high-affinity, non-A-group target site in brain.

The structure and biological activities of the widely used protein kinase inhibitor, H7, differ depending on the commercial source.

The protein kinase inhibitor 1-(5'-isoquinolinesulfonyl)-2-methylpiperazine (H7) has been widely used because of its ability to inhibit cyclic AMP- and cyclic GMP-dependent protein kinases (PKA and PKG) and protein kinase C (PKC) at roughly equal concentrations; it is much less potent on other kinases. Previous studies in other laboratories have found that H7 samples from different commercial sources have different properties in cellular studies and protein kinase C inhibition assays. We now report the results of chemical and biological tests which show that H7 samples also differ in chemical structure, again depending on their commercial source. Chemical synthesis and NMR spectroscopy indicate that H7 from most suppliers has the structure originally proposed for H7, while "H7" from another supplier is in fact its 3-methylpiperazine positional isomer.

Different biological targets for resiniferatoxin and phorbol 12-myristate 13-acetate.

The phorbol-related diterpene ester resiniferatoxin is at least 100-fold more inflammatory for the mouse ear than is the potent tumor promoter phorbol 12-myristate 13-acetate but is nonpromoting. We report here that resiniferatoxin is 100- to 1000-fold less active than is phorbol 12-myristate 13-acetate in in vitro assays with both chicken and mouse fibroblasts. These results suggest that resiniferatoxin and phorbol 12-myristate 13-acetate have different primary target sites (receptors) and provide further evidence that the fibroblast target may be homologous to that involved in promotion.

Specific binding of phorbol ester tumor promoters.

[20-(3)H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a K(d) of 25 nM; this value is very close to the 50% effective dose (ED(50)), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20-(3)H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 x 10(4) molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate (K(i) = 2 nM), mezerein (K(i) = 180 nM), phorbol 12,13-dibenzoate (K(i) = 180 nM), phorbol 12,13-diacetate (K(i) = 1.7 muM), phorbol 12,13,20-triacetate (K(i) = 39 muM), and phorbol 13-acetate (K(i) = 120 muM). The measured K(i) values are all within a factor of 3.5 of the ED(50) values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 mug/ml) and 4alpha-phorbol 12,13-didecanoate (10 mug/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate 20-acetate (100 ng/ml). These data suggest that biological responses to the phorbol esters in chicken embryo fibroblasts are mediated by this binding activity and that the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 muM), phenol (1 mM), iodoacetic acid (1.7 muM), and cantharidin (75 muM), or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 muM), retinoic acid (10 muM), or prostaglandin E(2) (1 muM). These agents thus appear to act at a target distinct from that of the phorbol esters.