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Ras farnesylation by farnesyl protein transferase (FPT) is an intracellular event that facilitates the membrane association of the ras protein and is involved in the signal transduction process. FPT inhibition could be a novel, noncytotoxic method of treating ras dependent tumor growth. We report here three structural classes of 8-chlorobenzocycloheptapyridines as novel, nonpeptidic, nonsulfhydryl FPT inhibitors having antitumor activity in mice when dosed orally. We discuss structural and conformational aspects of these compounds in relation to biological activities as well as a comparison to the conformation of a bound tetrapeptide FPT inhibitor.

A comprehensive structure-activity relationship (SAR) study of novel tricyclic amides has been undertaken. The discovery of compounds that are potent FPT inhibitors in the nanomolar range has been achieved. These compounds are nonpeptidic and do not contain sulfhydryl groups. They selectively inhibit farnesyl protein transferase (FPT) and not geranylgeranyl protein transferase-1 (GGPT-1). They also inhibit H-Ras processing in Cos monkey kidney cells.

Novel tricyclic FPT inhibitors with submicromolar FPT activity are described. Greatly enhanced FPT activity is realized with phthaloyl derivatized amino compound 2k, which showed FPT inhibitory activity of IC50 = 0.66 μM. Sulfonamides 5g and 50 were also found to be potent FPT inhibitor. SAR resulting from a variety of tricyclic amino acids and sulfonamide derivatives is discussed.

A previously described cembranolide diterpene from Lobophytum cristagalli was identified as a potent (IC50 0.15 μM) inhibitor of farnesyl protein transferase (FPT). The compound showed selectivity for FPT as compared to the closely related enzyme geranylgeranyl protein transferase-1 (IC50 5.3 μM). Kinetic evaluation suggests that this compound competes with the protein/peptide farnesyl acceptor substrate, and not with farnesyl pyrophosphate for inhibition of FPT.

The condensation of indolo[2,3-a]-carbazole (12) with 2,5-dimethoxytetrahydrofuran derivatives gave cyclofuranosylated compounds (e.g. 13), which were converted via dibromocompounds to the dinitriles (e.g. 25). Hydrolysis, hydrolysis-reduction and thiolysis afforded imides, lactams (e.g. 27) and their thio analogs. These compounds were potent inhibitors of the protein kinase C family.

Rat 6 fibroblasts that stably overexpress cDNA for the beta 1 isozyme of protein kinase C (PKC3 cells) were used to determine the effect of protein kinase C (PKC) overexpression on hormonal stimulation of phospholipid hydrolysis. In control Rat 6 cells, inositol trisphosphate levels (InsP3) were increased 9-fold in 15 s in response to 10 nM alpha-thrombin, compared with only a 2-fold increase in PKC3 cells. PKC overexpression also inhibited thrombin-stimulated production of 1,2-diacylglycerol, the other product of phosphatidylinositol 4,5-bisphosphate hydrolysis, by 73% at 15 s. In permeabilized cells, PKC overexpression greatly reduced guanosine thiotriphosphate-stimulated InsP3 accumulation, but did not affect InsP3 stimulation by increased free calcium concentration. These data suggest that desensitization of thrombin-stimulated phosphoinositide-phospholipase C is enhanced by PKC-beta 1 overexpression and may involve modulation of G-protein/phospholipase C coupling. In contrast, thrombin was 4.5-fold more effective in stimulation of phosphatidylcholine-phospholipase D activity in PKC3 cells than in control cells, as determined by phosphatidylethanol formation. In permeabilized cells, guanosine thiotriphosphate also stimulated phospholipase D activity more effectively in PKC3 cells than in control cells, suggesting that upregulation of phospholipase D activity by PKC overexpression occurs distal to the thrombin receptor. These results suggest that PKC may act as a switch to up-regulate phosphatidylcholine-phospholipase D and down-regulate phosphoinositide-phospholipase C stimulations.

We examined the tyrosine kinase inhibitor erbstatin and several derivatives for their ability to inhibit serine/threonine protein kinases in vitro . Erbstatin was found to inhibit protein kinase C (PKC) with an ic 50 of 19.8 ± 3.2 μM. A trihydroxy derivative of erbstatin inhibited PKC with similar potency, whereas the corresponding methoxy derivatives were inactive. Inhibition by erbstatin was competitive with ATP ( K i = 11.0 ± 2.3 μM ) and non-competitive with the phosphate acceptor, either histone or the synthetic peptide kemptide. Action of erbstatin at the catalytic stie of PKC was further indicated by the findings that it inhibited the catalytic fragment of PKC but did not inhibit the interaction of phorbol ester with the intact enzyme. Erbstatin had a similar potency against three PKC isozymes (α, β, and γ) examined. In addition, erbstatin was found to inhibit other serine/threonine kinases (assayed at their K m , for ATP). The greatest potency was observed versus the cyclic nucleotide-dependent kinases, while lower potency was seen versus myosin light chain kinase. These observations are discussed in terms of the structure and kinetic properties of PKC and the epidermal growth factor receptor tyrosine kinase.

The autophosphorylation sites of the beta 2 isozyme of protein kinase C (PKC) were recently identified as Ser-16/Thr-17 near the NH2 terminus, Thr-314/Thr-324 in the hinge region, and Thr-634/Thr-641 near the COOH terminus [Flint, A.J., Paladini, R.D. & Koshland, D.E. (1990) Science 294, 408-411]. To define the role of autophosphorylation we constructed three site-directed mutants of PKC beta 1 isozyme in which each pair of phosphorylatable residues is changed to alanine. Wild-type PKC beta 1 and the mutant proteins were transiently overexpressed in COS cells, resulting in at least a 20-fold increase in [3H]phorbol 12,13-dibutyrate binding compared with control transfectants. Enzyme assays of PKC partially purified from transfected cells indicated at least a 5-fold increase in PKC activity upon expression of the wild-type protein or the NH2-terminal and hinge mutants. In contrast, no increased activity was detected upon expression of the COOH-terminal mutant. Immunoblot analysis using a beta isoform-specific antibody showed that wild-type, NH2-terminal mutant, and hinge mutant proteins are similarly distributed between the Triton-soluble and insoluble fractions. In contrast, the COOH-terminal mutant protein is largely Triton-insoluble. Immunoblot analysis also indicated that this mutant is resistant to down-regulation upon chronic exposure of cells to phorbol ester. Moreover, RNA blot analysis showed that overexpression of wild-type PKC but not of the COOH-terminal mutant enhances phorbol ester induction of c-FOS and c-JUN mRNA. Our results indicate that (i) alteration in the NH2-terminal and hinge autophosphorylation sites has no effect on PKC function by the criteria examined and (ii) the COOH-terminal autophosphorylation sites are critical for PKC function and possibly subcellular localization in COS cells.