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Methionine aminopeptidase-2 (MetAP2) processes N-terminal methionine from nascent cellular proteins. Inhibition of MetAP2 has been shown to block angiogenesis and suppress tumor growth in preclinical tumor models. However, the biological role of MetAP2 in cancer is not well understood. We examined the effect of three distinct chemical classes of MetAP2 inhibitors on the growth of a panel of human cancer cells in vitro. All MetAP2 inhibitors caused inhibition of tumor cell growth in both anchorage-dependent and, particularly, in anchorage-independent manner. These data prompted us to examine the possible roles of MetAP2 in cancers. Ectopic expression of MetAP2 in NIH-3T3 cells caused transformation, evidenced by the formation of foci in monolayer culture and growth of large colonies in soft agar. Overexpression of MetAP2 in an immortalized bronchial epithelial cell line NL20 accelerated growth. These phenotypes induced by the overexpression of MetAP2 were reversed by the treatment with MetAP2 inhibitors, indicating that the catalytic function of MetAP2 was essential. Accordingly, overexpression of a catalytically inactive MetAP2 resulted in growth retardation of HT1080 tumor cells, suggesting a dominant-negative role of the inactive MetAP2 mutant. Finally, we analysed the expression of MetAP2 in patient cancer samples by immunohistochemistry. Moderate-to-high staining was identified in the majority of breast, colon, lung, ovarian and prostate carcinomas examined. These data suggest that MetAP2 plays an important role in tumor cell growth and may contribute to tumorigenesis.

With the use of an NMR-based method, potent (IC50 < 25 nM) nonpeptide inhibitors of the matrix metalloproteinase stromelysin (MMP-3) were discovered. The method, called SAR by NMR (for structure−activity relationships by nuclear magnetic resonance), involves the identification, optimization, and linking of compounds that bind to proximal sites on a protein. Using this technique, two ligands that bind weakly to stromelysin (acetohydroxamic acid, KD = 17 mM; 3-(cyanomethyl)-4‘-hydroxybiphenyl, KD = 0.02 mM) were identified. On the basis of NMR-derived structural information, the two fragments were connected to produce a 15 nM inhibitor of this enzyme. This compound was rapidly discovered (less than 6 months) and required only a minimal amount of chemical synthesis. These studies indicate that the SAR by NMR method can be effectively applied to enzymes to yield potent lead inhibitorsan important part of the drug discovery process.

ABT-770 [(S)-N-[1-[[4'-trifluoromethoxy-[1,1'-biphenyl]-4-yl]oxy]methyl-2-(4,4-dimethyl-2,5-dioxo-1-imidazolidinyl)ethyl]-N-hydroxyformamide], a matrix metalloproteinase inhibitor (MMPI), produced generalized phospholipidosis in rats. Phospholipid accumulation was accompanied by retention of drug-related material and was associated with increased mortality. Generation of a successful drug candidate depended upon understanding the cause of the phospholipidosis and redesigning the chemical structure accordingly. ABT-770 and other MMPIs, plus several metabolites of each, were assayed for their ability to induce phospholipidosis in primary cultured rat and human hepatocytes. Phospholipid accumulation was detected by following the incorporation of a fluorescent phospholipid analogue into intracytoplasmic inclusion bodies characteristic of phospholipid storage disorders. At 24 and 48 hr, none of the parent compounds induced phospholipidosis in vitro in rat or human hepatocytes. Phospholipidosis was associated primarily with an amine metabolite of ABT-770. The amine metabolite of another MMPI, ABT-518 ([S-(R*,R*)]-N-[1-(2,2-dimethyl-1,3-dioxol-4-yl)-2-[[4-[4-(trifluoromethoxy)-phenoxy]phenyl]sulfonyl]ethyl]-N-hydroxyformamide), produced little phospholipidosis in rat and human hepatocytes even at concentrations up to 100 microM. The presence or absence of phospholipidosis in the in vitro assay correlated well with ultrastructural findings and drug accumulation in rat tissues. ABT-770, which produced phospholipidosis associated with its amine metabolite in vitro and in vivo, also generated a higher tissue to plasma distribution of metabolites particularly in tissues where phospholipidosis was observed. ABT-518 and its amine metabolite, however, produced low tissue to plasma ratios and induced little to no phospholipidosis in vitro or in vivo. These results demonstrate that the phospholipidosis observed for ABT-770 could be attributed to a cationic metabolite, and that altering the properties of such a metabolite, by modification of the parent compound, alleviated the disorder.

Members of the matrix metalloprotease (MMP) family are implicated in the progression of several malignancies including prostate cancer due to their ability to break down extracellular matrix (ECM) components. In this study, we have evaluated the ability of a synthetic MMP inhibitor (A-177430) to block tumor growth and metastases in a syngeneic model of rat prostate cancer. In an in vitro substrate assay, A-177430 exhibited nanomolar potency (IC(50) 2-6 nM) against the enzymatic activity of several MMPs. For in vivo studies, male Copenhagen rats were injected s.c. with Mat Ly Lu rat prostate cancer cells (1 x 10(6) cells ) into the right flank and animals were administered i.p.with different doses (10-100 mg/kg per day) of A-177430 for 16 days. Administration of A-177430 resulted in a dose-dependent decrease in tumor volume as compared to a control group of animals receiving vehicle alone. The maximum dose (100 mg/kg per day) of A-177430 exhibited complete arrest in tumor growth and prevented the development of macroscopic tumor metastases to lungs without exhibiting any noticeable side effects. Histologic examination of primary tumors from experimental animals showed extensive tumor necrosis and decreased tumor angiogenesis as determined by factor VIII staining of primary tumors following A-177430 treatment. These primary tumors from experimental animals also exhibited a significant increase in tumor cell DNA fragmentation as determined by TUNEL assay. Collectively, these results demonstrate the ability of MMP inhibitors to block tumor growth and metastases by blocking ECM degradation and by inhibiting tumor angiogenesis and promotion of prostate cancer cell apoptosis in vivo.

Studies conducted with the goal of discovering a second-generation platelet-activating factor (PAF) antagonist have identified a novel class of potent and orally active antagonists which have high aqueous solubility and long duration of action in animal models. The compounds arose from the combination of the lipophilic indole portion of Abbott's first-generation PAF antagonist ABT-299 (2) with the methylimidazopyridine heterocycle moiety of British Biotechnology's BB-882 (1) and possess the positive attributes of both of these clinical candidates. Structure-activity relationship (SAR) studies indicated that modification of the indole and benzoyl spacer of lead compound 7b gave analogues that were more potent, longer-lived, and bioavailable and resulted in the identification of 1-(N, N-dimethylcarbamoyl)-4-ethynyl-3-[3-fluoro-4-[(1H-2-methylimidazo[4,5-c] pyrid-1-yl)methyl]benzoyl]indole hydrochloride (ABT-491, 22 m.HCl) which has been evaluated extensively and is currently in clinical development.

ABT-299 is an aqueous soluble prodrug that is converted rapidly in vivo to A-85783, a novel, highly potent, specific platelet activating factor (PAF) antagonist. The K, for inhibiting PAF binding to rabbit platelet membranes is 3.9 and 0.3 nM for human platelets. Inhibition is selective and reversible and is correlated with functional antagonism of PAF-mediated cellular responses (calcium mobilization, priming of superoxide generation, aggregation and degranulation). The in vivo generation of A-85783 from ABT-299 leads to potent inhibition of PAF-induced inflammatory responses (increased vascular permeability, hypotension and edema) and PAF-induced lethality. When administered i.v., the potency (ED50) of ABT-299 for inhibiting PAF responses was between 6 to 10 micrograms/kg in the rat and mouse and 100 micrograms/kg in the guinea pig. A dose of 100 micrograms/kg in the rat provided greater than 60% protection for 8 to 16 hr against cutaneous and systemic PAF challenge. This duration was also evidenced by ex vivo inhibition of platelet aggregation in guinea pig and sheep. In addition to being active parenterally, ABT-299 exhibited p.o. activity in the rat and mouse (ED50 = 100 micrograms/kg in both species). Pharmacokinetic studies in the rat revealed that ABT-299 was converted rapidly to A-85783 and, in turn, metabolized to the corresponding pyridine-N-oxide and sulfoxide metabolites. These metabolites exhibited significant potency in vitro and in vivo and thus may contribute to the activity observed after administration of ABT-299.

3175 Background: ABT-869, a multi-targeted receptor tyrosine kinase inhibitor, has been shown to inhibit of all members of the VEGF and PDGF receptor families (e.g., KDR IC50 value of 4 nM), and ha...