The crystallographic structure of acetyl-Cys-Val-Ile-selenoMet-COOH and alpha-hydroxyfarnesylphosphonic acid (alphaHFP) complexed with rat farnesyl protein transferase (FPT) (space group P61, a = b = 174. 13 A, c = 69.71 A, alpha = beta = 90 degrees, gamma = 120 degrees, Rfactor = 21.8%, Rfree = 29.2%, 2.5 A resolution) is reported. In the ternary complex, the bound substrates are within van der Waals contact of each other and the FPT enzyme. alphaHFP binds in an extended conformation in the active-site cavity where positively charged side chains and solvent molecules interact with the phosphate moiety and aromatic side chains pack adjacent to the isoprenoid chain. The backbone of the bound CaaX peptide adopts an extended conformation, and the side chains interact with both FPT and alphaHFP. The cysteine sulfur of the bound peptide coordinates the active-site zinc. Overall, peptide binding and recognition appear to be dominated by side-chain interactions. Comparison of the structures of the ternary complex and unliganded FPT [Park, H., Boduluri, S., Moomaw, J., Casey, P., and Beese, L. (1997) Science 275, 1800-1804] shows that major rearrangements of several active site side chains occur upon substrate binding.

Ras proteins are small GTP-binding proteins which are critical for cell signaling and proliferation. Four Ras isoforms exist: Ha-Ras, N-Ras, Ki-Ras4A, and Ki-Ras4B. The carboxyl termini of all four isoforms are post-translationally modified by farnesyl protein transferase (FPT). Prenylation is required for oncogenic Ras to transform cells. Recently, it was reported that Ki-Ras4B is also an in vitro substrate for the related enzyme geranylgeranyl protein transferase-1 (GGPT-1) (James, G. L., Goldstein, J. L., and Brown, M. S. (1995) J. Biol. Chem. 270, 6221-6226). In the current studies, we compared the four isoforms of Ras as substrates for FPT and GGPT-1. The affinity of FPT for Ki-Ras4B (K m ‫؍‬ 30 nM) is 10 -20-fold higher than that for the other Ras isoforms. Consistent with this, when the different Ras isoforms are tested at equimolar concentrations, it requires 10 -20-fold higher levels of CAAX-competitive compounds to inhibit Ki-Ras4B farnesylation. Additionally, we found that, as reported for Ki-Ras4B, N-Ras and Ki-Ras4A are also in vitro substrates for GGPT-1. Of the Ras isoforms, N-Ras is the highest affinity substrate for GGPT-1 and is similar in affinity to a standard GGPT-1 substrate terminating in leucine. However, the catalytic efficiencies of these geranylgeranylation reactions are between 15-and 140-fold lower than the corresponding farnesylation reactions, largely reflecting differences in affinity. Carboxyl-terminal peptides account for many of the properties of the Ras proteins. One interesting exception is that, unlike the full-length N-Ras protein, a carboxylterminal N-Ras peptide is not a GGPT-1 substrate, raising the possibility that upstream sequences in this protein may play a role in its recognition by GGPT-1. Studies with various carboxyl-terminal peptides from Ki-Ras4B suggest that both the carboxyl-terminal methionine and the upstream polylysine region are important determinants for geranylgeranylation. Furthermore, it was found that full-length Ki-Ras4B, but not other Ras isoforms, can be geranylgeranylated in vitro by FPT. These findings suggest that the different distribution of Ras isoforms and the ability of cells to alternatively process these proteins may explain in part the resistance of some cell lines to FPT inhibitors.Ras proteins are small GTP-binding proteins that play critical roles in cell signaling, differentiation, and proliferation (1). Ras signaling is regulated by a GDP-GTP cycle. Binding of GTP to Ras is required for its productive interaction with Raf-1 and other downstream effector proteins (2). Ras proteins are activated by nucleotide exchange factors such as SOS-1 which stimulate the exchange of GDP for GTP. The lifetime of activated Ras is limited by its intrinsic GTPase activity, which hydrolyzes GTP to GDP. GTPase-activating proteins, such as p120 Ras-GAP and NF-1, stimulate this activity and thereby facilitate inactivation of Ras proteins (2). Transforming mutations of Ras which decrease the rate of GTP hydrolysis result in its constitutive activation. S...

Purpose Melanoma, the most aggressive form of skin cancer, accounts for 75% of all skin cancer-related deaths and current therapeutic strategies are not effective in advanced disease. In the current study, we have investigated the efficacy of orally active small-molecule antagonist targeting CXCR2/CXCR1. Experimental Design Human A375SM melanoma cells were treated with SCH-479833 or SCH-527123, and their effect on proliferation, motility, and invasion was evaluated in vitro. We examined the downstream signaling events in the cells following treatment with antagonists. For in vivo studies, A375SM cells were implanted subcutaneously into athymic nude mice followed by administration of SCH-479833, SCH-527123, or hydroxypropyl-β-cyclodextrin (20%) orally for 21days and their effect on tumor growth and angiogenesis was evaluated. Results Our data show that SCH-479833 or SCH-527123 inhibited the melanoma cell proliferation, chemotaxis, and invasive potential in vitro.Treatment of melanoma cells with SCH-479833 or SCH-527123 also inhibited tumor growth. Histologic and histochemical analyses showed significant (P < 0.05) decreases in tumor cell proliferation and microvessel density in tumors. Moreover, we observed a significant increase in melanoma cell apoptosis in SCH-479833- or SCH-527123-treated animals compared with controls. Conclusion Together, these studies show that selectively targeting CXCR2/CXCR1 with orally active small-molecule inhibitors is a promising therapeutic approach for inhibiting melanoma growth and angiogenesis.

Sch527123 [2-hydroxy-N,N-dimethyl-3-[[2-[[1(R)-(5-methyl-2-furanyl)propyl]amino]-3,4-dioxo-1-cyclobuten-1-yl]amino]benzamide] is a potent, selective antagonist of the human CXCR1 and CXCR2 receptors (Gonsiorek et al., 2007). Here we describe its pharmacologic properties at rodent CXCR2 and at the CXCR1 and CXCR2 receptors in the cynomolgus monkey, as well as its in vivo activity in models demonstrating prominent pulmonary neutrophilia, goblet cell hyperplasia, and mucus production. Sch527123 bound with high affinity to the CXCR2 receptors of mouse (K d ϭ 0.20 nM), rat (K d ϭ 0.20 nM), and cynomolgus monkey (K d ϭ 0.08 nM) and was a potent antagonist of CXCR2-mediated chemotaxis (IC 50 ϳ3-6 nM). In contrast, Sch527123 bound to cynomolgus CXCR1 with lesser affinity (K d ϭ 41 nM) and weakly inhibited cynomolgus CXCR1-mediated chemotaxis (IC 50 ϳ1000 nM). Oral treatment with Sch527123 blocked pulmonary neutrophilia (ED 50 ϭ 1.2 mg/kg) and goblet cell hyperplasia (32-38% inhibition at 1-3 mg/kg) in mice following the intranasal lipopolysaccharide (LPS) administration. In rats, Sch527123 suppressed the pulmonary neutrophilia (ED 50 ϭ 1.8 mg/kg) and increase in bronchoalveolar lavage (BAL) mucin content (ED 50 ϭ Ͻ0.1 mg/kg) induced by intratracheal (i.t.) LPS. Sch527123 also suppressed the pulmonary neutrophilia (ED 50 ϭ 1.3 mg/kg), goblet cell hyperplasia (ED 50 ϭ 0.7 mg/kg), and increase in BAL mucin content (ED 50 ϭ Ͻ1 mg/kg) in rats after i.t. administration of vanadium pentoxide. In cynomolgus monkeys, Sch527123 reduced the pulmonary neutrophilia induced by repeat bronchoscopy and lavage (ED 50 ϭ 0.3 mg/kg). Therefore, Sch527123 may offer benefit for the treatment of inflammatory lung disorders in which pulmonary neutrophilia and mucus hypersecretion are important components of the underlying disease pathology.Chronic obstructive pulmonary disease (COPD) is the fourth major cause of death in the United States and is characterized by irreversible airflow limitation due to chronic bronchitis or emphysema (Barnes and Stockley, 2005). The pathological hallmarks of COPD include peripheral airway inflammation dominated by neutrophils, the destruction of the lung parenchyma, submucosal gland hypertrophy, and goblet cell hyperplasia, as well as an increase in proinflammatory cytokines and chemokines (Barnes and Stockley, 2005). Current therapies for COPD are similar to those for asthma and include the use of ␤-adrenoceptor agonists, theophylline, muscarinic antagonists, and corticosteroids (Barnes and Stockley, 2005). However, these treatments do not prevent the progressive decline in lung function and demonstrate clinical activity in only a subpopulation of COPD patients. There is a strong correlation between disease Article, publication date, and citation information can be found at