Blockade of protein geranylgeranylation inhibits Cdk2-dependent p27Kip1 phosphorylation on Thr187 and accumulates p27Kip1 in the nucleus: implications for breast cancer therapy.

We describe the design of a potent and selective peptidomimetic inhibitor of geranylgeranyltransferase I (GGTI), GGTI-2418, and its methyl ester GGTI-2417, which increases the levels of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) and induces breast tumor regression in vivo. Experiments with p27(Kip1) small interfering RNA in breast cancer cells and p27(Kip1) null murine embryonic fibroblasts demonstrate that the ability of GGTI-2417 to induce cell death requires p27(Kip1). GGTI-2417 inhibits the Cdk2-mediated phosphorylation of p27(Kip1) at Thr187 and accumulates p27(Kip1) in the nucleus. In nude mouse xenografts, GGTI-2418 suppresses the growth of human breast tumors. Furthermore, in ErbB2 transgenic mice, GGTI-2418 increases p27(Kip1) and induces significant regression of breast tumors. We conclude that GGTIs' antitumor activity is, at least in part, due to inhibiting Cdk2-dependent p27(Kip1) phosphorylation at Thr187 and accumulating nuclear p27(Kip1). Thus, GGTI treatment might improve the poor prognosis of breast cancer patients with low nuclear p27(Kip1) levels.

Geranylgeranyltransferase I inhibitors target RalB to inhibit anchorage-dependent growth and induce apoptosis and RalA to inhibit anchorage-independent growth.

Geranylgeranyltransferase I inhibitors (GGTIs) are presently undergoing advanced preclinical studies and have been shown to disrupt oncogenic and tumor survival pathways, to inhibit anchorage-dependent and -independent growth, and to induce apoptosis. However, the geranylgeranylated proteins that are targeted by GGTIs to induce these effects are not known. Here we provide evidence that the Ras-like small GTPases RalA and RalB are exclusively geranylgeranylated and that inhibition of their geranylgeranylation mediates, at least in part, the effects of GGTIs on anchorage-dependent and -independent growth and tumor apoptosis. To this end, we have created the corresponding carboxyl-terminal mutants that are exclusively farnesylated and verified that they retain the subcellular localization and signaling activities of the wild-type geranylgeranylated proteins and that Ral GTPases do not undergo alternative prenylation in response to GGTI treatment. By expressing farnesylated, GGTI-resistant RalA and RalB in Cos7 cells and human pancreatic MiaPaCa2 cancer cells followed by GGTI-2417 treatment, we demonstrated that farnesylated RalB, but not RalA, confers resistance to the proapoptotic and anti-anchorage-dependent growth effects of GGTI-2417. Conversely, farnesylated RalA but not RalB expression renders MiaPaCa2 cells less sensitive to inhibition of anchorage-independent growth. Furthermore, farnesylated RalB, but not RalA, inhibits the ability of GGTI-2417 to suppress survivin and induce p27(Kip1) protein levels. We conclude that RalA and RalB are important, functionally distinct targets for GGTI-mediated tumor apoptosis and growth inhibition.

Synthesis and evaluation of potent, highly-selective, 3-aryl-piperazinone inhibitors of protein geranylgeranyltransferase-I.

A series of compounds based on the carboxyl-terminal CAAL sequence of PGGTase-I substrates was designed and synthesized. Using piperazin-2-one as a semi-rigid scaffold, we have introduced critical pharmacophores in a well-defined arrangement to mimic the CAAL sequence. High potency and exceptional selectivity were obtained for inhibition of PGGTase-I with structures such as 45 and 70. Potency of this series of GGTIs was dependent on the presence of an L-leucine residue with a free carboxyl terminus, as well as an S configuration of the 3-aryl group. The selectivity was significantly enhanced by 5-methyl substitution on the imidazole ring and fluorine substitution on the 3-aryl group. Modification of the 6-position of the piperazinone scaffold was found to be unfavorable. Compounds 44 and 69, the corresponding methyl esters of 45 and 70, were found to selectively block processing of Rap1A by PGGTase-I in whole cells with IC(50) values of 0.4 microM and 0.7 microM respectively.

Structure-based design of imidazole-containing peptidomimetic inhibitors of protein farnesyltransferase.

A series of imidazole-containing peptidomimetic PFTase inhibitors and their co-crystal structures bound to PFTase and FPP are reported. The structures reveal that the peptidomimetics adopt a similar conformation to that of the extended CVIM tetrapeptide, with the imidazole group coordinating to the catalytic zinc ion. Both mono- and bis-imidazole-containing derivatives, 13 and 16, showed remarkably high enzyme inhibition activity against PFTase in vitro with IC50 values of 0.86 and 1.7 nM, respectively. The peptidomimetics were also highly selective for PFTase over PGGTase-I both in vitro and in intact cells. In addition, peptidomimetics and were found to suppress tumor growth in nude mouse xenograft models with no gross toxicity at a daily dose of 25 mg kg(-1).

Design, synthesis, and evaluation of potent and selective benzoyleneurea-based inhibitors of protein geranylgeranyltransferase-I.

A series of novel protein geranylgeranyltransferase-I (PGGTase-I) inhibitors based on a benzoyleneurea scaffold has been synthesized. Using a benzoyleneurea scaffold as a mimetic for the central dipeptide (AA), we have developed CAAX peptidomimetic inhibitors that selectively block the activity of PGGTase-I over the closely related enzyme protein farnesyltransferase. In this new class of PGGTase-I inhibitors, compound (6c) with X=L-phenylalanine, displayed the highest inhibition activity against PGGTase-I with an IC50 value of 170 nM. The inhibitors described in this study represent novel and promising leads for the development of potent and selective inhibitors of mammalian PGGTase-I for potential application as antitumor agents.

In vitro and in vivo antimalarial activity of peptidomimetic protein farnesyltransferase inhibitors with improved membrane permeability.

A series of protein farnesyltransferase inhibitor ester prodrugs of FTI-2148 (17) were synthesized in order to evaluate the effects of ester structure modification on antimalarial activity and for further development of a farnesyltransferase inhibitor with in vivo activity. Evaluation against P. falciparum in red blood cells showed that all the investigated esters exhibited significant antimalarial activity, with the benzyl ester 16 showing the best inhibition (ED50=150 nM). Additionally, compound 16 displayed in vivo activity and was found to suppress parasitemia by 46.1% at a dose of 50 mg kg(-1) day(-1) against Plasmodium berghei in mice. The enhanced inhibition potency of the esters is consistent with improved cell membrane permeability compared to that of the free acid. The results of this study suggest that protein farnesyltransferase is a valid antimalarial drug target and that the antimalarial activity of these compounds derives from a balance between the hydrophobic character and the size and conformation of the ester moiety.

Design and synthesis of peptidomimetic protein farnesyltransferase inhibitors as anti-Trypanosoma brucei agents.

On the basis of the structure of the CVIM tetrapeptide substrate of mammalian protein farnesyltransferase, a series of imidazole-containing peptidomimetics was designed and synthesized, and their inhibition activity against Trypanosoma brucei protein farnesyltransferase (TbPFT) was evaluated. Peptidomimetics where the 5-position of the imidazole ring was linked to the hydrophobic scaffold showed over 70% inhibition activity at 50 nM in the enzyme assay, whereas the corresponding C-4 regioisomers were less potent. The ester prodrug 23 was found to be a potent inhibitor against cultured Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense cells with ED(50) values of 0.025 and 0.0026 microM, respectively. Furthermore, introducing a second imidazole group into 23 led to 31, which showed the highest inhibition activity against the parasite with an ED(50) of 0.0015 microM. The potency of the TbPFT inhibitors and the cytotoxicity of the corresponding esters to T. brucei cells were shown to be highly correlated. These studies validate TbPFT as a target for the development of novel therapeutics against African sleeping sickness.