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Regular Articles |
Cancer Research,
Advanced Technology,
Preclinical Safety, Global Pharmaceutical R & D, Abbott Laboratories, Abbott Park, Illinois,
Departments of Surgery and
Pediatric Oncology, The University of Pennsylvania School of Medicine and The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MetAP2 is one of the two MetAPs known in eukaryotes (25) . MetAPs are intracellular metalloenzymes responsible for removing the NH2-terminal initiator methionine residue from nascent proteins. They are required for protein cotranslational and/or posttranslational modifications, such as NH2-terminal myristoylation, and for protein stability (26 , 27) . Inhibition of MetAP activity could therefore affect protein biological activity, proper subcellular localization, and degradation and result in interference of normal cell signal transduction and cell cycle progression. Both MetAP1 and MetAP2 are expressed in all mammalian tissues and cells examined (7 , 28) , but only MetAP2 is up-regulated during cell proliferation (7) . MetAP2 is also found at higher concentrations in tumors as compared with normal cells (29 , 30) . Antisense of MetAP2 induces apoptosis in human mesothelioma cells (31) and in rat hepatoma cells (32) . These studies suggest that MetAP2 may play a critical role in cell proliferation and tumor growth. MetAP2 is a protein with two functions: processing initiator methionine from nascent proteins and stabilizing eukaryotic initiation factor 2 (32) . These dual functions of MetAP2 are mediated by different domains of the protein (32) . The TNP-470-inactivated MetAP2 retains its capacity to stabilize eukaryotic initiation factor 2 (2) , consistent with the hypothesis that MetAP2 catalytic activity is required for cell growth.
TNP-470 inhibits MetAP2 by forming a covalent adduct through epoxide alkylation of the His231 residue in the enzyme active site (3 , 4) . The short in vivo half-life (2–6 min in human; Ref. 24 ), the presence of multiple metabolites, and concerns that TNP-470 may alkylate additional proteins complicate the linking of MetAP2 enzyme inhibition to the observed in vivo therapeutic and toxic effects by this agent. To examine the effects of targeted MetAP2 inhibition in vitro and in vivo, we have synthesized highly specific and reversible MetAP2 inhibitors using structure-based design. Here we show that a reversible MetAP2 enzyme inhibitor induces cytostasis in tumor cells and ECs in vitro and inhibits tumor growth in murine models without the toxicities observed with TNP-470. The data show that reversible MetAP2 inhibitors are promising novel cancer therapeutics.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MetAP2 Inhibitor A-357300
Detailed chemistry of preparation of bestatin-type MetAP2 inhibitors has been described previously (34)
. A scheme used to synthesize A-357300 is shown below (Scheme 1
).
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Overexpression of MetAP2 in HT-1080 Cells
MetAP2 cDNA was cloned by reverse transcription-PCR using HMVEC total RNA as the template, as described previously (33)
. The primers used were 5'-att-aat-gct-agc-ccacc-atg-gcg-ggc-gtg-gag-gag-gta-gcg-gcct-3' and 5'-att-aat-ctc-gag-tct-aga-cggtccg-tta-ata-gtc-atc-tcc-tct-gct-gac-aact-3'. The amplified MetAP2 cDNA was cut with NheI and XbaI and cloned into pcDNA3.1 (Invitrogen). The DNA sequence of the final construct, pcDNA-MetAP2, was confirmed by standard sequencing techniques. Transient expression was carried out in HMVEC primary culture. The cells were grown in T-75 flasks to reach 
50% confluence. Four µg of DNA were incubated with 30 µl of LipofectAMINE (Invitrogen) and transfected into the HMVECs in a T-75 flask using the conditions recommended by the manufacturer. The cells were then incubated with EGM2 medium for 24 h, trypsinized, and used in the proliferation assay. Stable transfection was carried out in HT-1080 cells by electroporation. Briefly, 5 x 106 cells were mixed with 15 µg of DNA, and the mixture was subjected to 250 V and 950 µF electroporation. Transfected cells were selected by 300 µg/ml G418 (Invitrogen), and single-cell clones were obtained by limited dilution of the transfected cells.
Cellular MetAP2 Activity Assay
Inhibition of cellular MetAP2 activity was determined using the recently published method (33)
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EC Tube Formation in Fibrin Matrix
This protocol was adapted from a previously published method (37)
as described in detail (38)
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In Vivo Studies
Drug Formulation.
A-357300 was dissolved in 0.2% HPMC to yield a final working concentration of 5–10 mg/ml. This solution was sonicated for 30 min at 37°C and adjusted to pH 8.3. A-357300, dissolved in water, was used for mini-osmotic pump delivery (Alzet; Durect Corp., Cupertino, CA).
Mouse Cornea Angiogenesis.
On study day 0, twenty-four 30-g CF1 mice (Charles River Laboratories, Wilmington, MA) were anesthetized with ketamine and Rompum before surgery. After anesthesia, eyes were checked to insure that there was no evidence of active or recent ophthalmic infection/inflammation and that there were no residual capillary vessels within the corneal stroma. All whiskers and fur around the eyes were clipped, and the area was flushed with sterile saline. A corneal pocket was made by surgical incision approximately 0.7 mm from the limbus. Then one hydron-sucralfate pellet, containing either 30 ng of bFGF (right cornea) or 150 ng of VEGF (left cornea) was inserted into the top of the pocket, and Neosporin ointment was applied to each eye to prevent infection. Drug treatments were started on day 0. A-357300 was given by twice daily s.c. injections at 25, 75, and 150 mg/kg/day. On day 5 (right cornea with bFGF) and again on day 7 (left cornea with VEGF pellet), the eyes were vasodilated by injecting 0.1 ml of 100 nM Nitropress i.p. 60 min before imaging. The mice were anesthetized immediately before imaging, and their eyes were moistened with saline to reduce reflections and prevent drying. A magnified corneal image was obtained by using a digital camera attached to a slit lamp biomicroscope. The neovascular measurement field was the area between the pellet and the limbus where neovascularization had occurred. Data acquisition and storage were achieved with Leica imaging software, and the data computation was done with Microsoft Excel. The statistical significance was evaluated with a two-tailed t test.
CHP-134 Neuroblastoma Xenograft.
The CHP-134 cell line was derived from the diagnostic primary tumor biopsy from of a neuroblastoma patient whose tumor showed MYCN amplification, deletion of the distal short arm of chromosome 1, and unbalanced gain of chromosome 17q material, and its xenografting characteristics in athymic mice were as described previously (15)
. Tumor growth was observed within 14 days after inoculation in 90% of animals. Fourteen mice were randomized to receive either A-357300 or HPMC vehicle when tumor volumes reached 200 mm3 (3 weeks after inoculation). A dose of 50 mg/kg A-357300 was administered s.c. twice daily, and an equivalent volume of HPMC was given to control mice. Tumor measurements were made by Vernier calipers twice weekly, and tumor volumes were calculated using the ellipsoid formula: length x width x height x 0.52 (39)
. Treatment was continued for 30 days or until tumor volume exceeded 3.0 cm. Animals were sacrificed at this time and necropsied. These studies were approved by the Animal Care and Use Committee of the Children’s Hospital of Philadelphia.
HT-1080 Fibrosarcoma Xenograft.
Eight-week-old SCID-beige mice (Charles River Laboratories) were inoculated with HT-1080 human fibrosarcoma cells. The cells were reconstituted at 2 x 106 cells/ml in 20% saline and 80% phenol red-free Matrigel (Becton Dickinson Labware, Palo Alto, CA). Cells (0.25 ml; 0.5 million cells) were injected s.c. in the left upper abdominal quadrant. Dosing and tumor measurements were started on day 8 (after 7 days), when the tumor was established. Tumor measurements were made by Vernier calipers every other day.
MDA-435 Breast Carcinoma Xenograft.
MDA-435-LM is a metastatic isolate derived from MDA-MB-435 (American Type Culture Collection). Approximately 7-week-old female SCID-C.B17 mice (C.B-17/IcrCrl-scid-BR; Charles River Laboratories) were inoculated s.c. in the right flank with 0.2 ml of 1 x 106 MDA-435 LM cells (1:1 Matrigel) on study day 0. All mice were ear tagged. Treatments were started at day 4. The tumors were measured by a pair of Vernier calipers twice a week after tumors were palpable (day 10), and the tumor volumes were calculated according to the formula V = L x W2/2 (V, volume; L, length; W, width). Perfusion fixation under halothane anesthesia was performed at the end of the study. Histology was performed on the major tissues/organs (bone marrow, brain, liver, lymphoid tissues, kidney, lung, and heart) as well as the mass.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. We observed that large hydrophobic amides at the R2 site provided potent inhibitors of MetAP2, despite the preference of MetAP2 for protein or peptide substrates with small amino acids adjacent to the NH2-terminal methionine residue. Modification of the link to the aromatic group from amide to diacyl hydrazine provided potent MetAP2 inhibitors with improved selectivity, particularly when combined with larger R1 groups. A-357300 was identified as the optimized compound (Fig. 1A)
. It selectively inhibited MetAP2 catalytic activity with an IC50 of 0.12 µM (Table 1)
and did not inhibit MetAP1 or leucine aminopeptidases at concentrations below 10 µM. The X-ray crystal structure of the enzyme-inhibitor complex of A-357300 (Fig. 1B)
indicates the mode of binding. The 2-hydroxy-3-aminoamide grouping interacts with the two manganese metal ions in the active site (33)
, with the oxygen substituent bridging between them. The thioether-containing side chain largely fills the adjacent hydrophobic site, whereas the 3-chlorophenyl aromatic group lies face to face with a histidine imidazole (His339) and occupies the adjacent opening of the active site. Both the R1 and R2 hydrophobic groups interact with MetAP2 near the site of an insertion of 60 amino acids (Tyr444 shown as an example), which is absent in MetAP1 (25)
. A modeled structure of MetAP1 indicates that the active site of MetAP1, lacking the insertion, has less room in this region, providing a basis for the selectivity observed. The chemical structure of TNP-470 (Fig. 1C)
and a crystal structure overlay of TNP-470 with A-357300 in MetAP2 active site (Fig. 1D)
are shown as a comparison.
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. Continuous exposure of A-357300 was required for its maximal potency against proliferation, and cells exposed to 10 µM A-357300 for only 4 h were not inhibited. The inhibition was cytostatic rather than cytotoxic because cell numbers did not fall below the initial values, and no cytotoxicity was observed with concentrations of A-357300 below 100 µM as measured by cellular lactate dehydrogenase release (data not shown). Furthermore, no apoptosis was detected by caspase-3 measurements in drug-treated cells. A-357300 also inhibited the proliferation of CECs (Table 2)
isolated from human peripheral blood (35)
that may contribute to angiogenesis (41)
. A-357300 inhibited murine and bovine ECs with IC50 values similar to those for HMVECs and HUVECs. The potency of A-357300 across species was not unexpected because there is >95% amino acid identity between human and mouse MetAP2. This high degree of similarity provided an advantage for generalizing from results of MetAP2 inhibitors in mouse models. Most of the nonendothelial human primary cells we examined were not inhibited by A-357300 (Table 2)
. The human tumor cell lines we tested showed distinct sensitivity to growth inhibition by A-357300 (Table 2)
. HT-1080 fibrosarcoma and CHP-134 neuroblastoma were among the most sensitive cells and were inhibited by A-357300 with IC50 values of 0.1–0.2 µM and displayed sensitivity similar to that of ECs. Other tumor cell lines such as PC3 prostate carcinoma and MCF7 breast carcinoma were not significantly inhibited by A-357300. The sensitivity of certain tumor cells to MetAP2 inhibition may augment the efficacy of MetAP2 inhibitors as anticancer agents because of the combination of an antiangiogenic effect with direct tumor cytostasis. Similar ranking of cell sensitivities to A-357300 and fumagillin (Table 2)
implies their common mechanism of action, i.e., inhibition of the MetAP2 enzyme.
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. Whereas the growth of HT-1080 cells was not affected by transfection of pcDNA-MetAP2 plasmid or vector control, the cells overexpressing MetAP2 (5-fold of control determined by Western blot; data not shown) showed there was an approximately 4-fold decrease in the potency of A-357300 or fumagillin, but not that of cytotoxic agents such as paclitaxel and 5-flurouracil (Table 3)
, supporting the hypothesis that A-357300 inhibits cell growth by inhibiting the cellular MetAP2 enzyme.
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, with an IC50 of 0.15 µM. The similar IC50 of A-357300 for inhibiting both cell proliferation and cellular MetAP2 enzyme activity further supports the postulated mechanism of action of this compound.
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, displaying a dose response similar to that for cellular MetAP2 inhibition and proliferation inhibition. A-357300 also caused a reduction of cyclin A but had no effect on cyclin D1 concentrations (Fig. 2B)
. Similar results were seen with fumagillin (data not shown). Both HMVECs and HT-1080 tumor cells treated with A-357300 showed arrest at the G1 phase of the cell cycle as shown by flow cytometry (Fig. 2C)
. No accumulation of sub-G1 cell population was detected, consistent with the cytostatic effect of A-357300 seen in the proliferation assay. TNP-470 and fumagillin were reported to arrest cells at the G1 phase of cell cycle (5
, 6)
. Taken together, these observations with MetAP2 inhibitors support that MetAP2 inhibition leads to cytostasis by G1 arrest in susceptible cells.
A-357300 Inhibits Angiogenesis in Vitro and in Vivo.
We next evaluated A-357300 for its antiangiogenic activity both in vitro and in vivo. Sprout and tube formation in three-dimensional fibrin matrix is a feature of activated ECs, which provides a useful angiogenesis model in vitro (37)
. HMVECs attached to microcarrier beads embedded in fibrin gel were stimulated to grow, migrate, sprout, and form tubule structures in the presence of angiogenesis inducers VEGF and bFGF (Fig. 3A)
. A-357300 at 0.4 µM completely blocked this self-organization of HMVECs. In addition, A-357300 showed inhibition of mouse cornea angiogenesis in vivo. s.c. injections of A-357300 twice daily at 25, 75, and 150 mg/kg/day inhibited growth factor-induced cornea neovascularization in a dose-dependent manner against VEGF (Fig. 3, B and C)
, and against bFGF (Fig. 3D)
. A-357300 at 75 and 150 mg/kg/day inhibited VEGF-induced vessel area by 34% (P < 0.005) and 55% (P < 0.001), respectively, but showed no significant inhibition at the lowest dose of 25 mg/kg/day. It also inhibited bFGF-stimulated vessel area by 43%, 52%, and 60% (all P < 0.001) for the three doses, respectively. Plasma A-357300 concentrations measured at the 6 h time point after the terminal dose were 0.24, 0.38, and 2.2 µM for 25, 75, and 150 mg/kg/day groups, respectively, and correlated to efficacy in a dose-dependent manner. As a comparison, TNP-470 given near the maximal tolerated dose (30 mg/kg by s.c. injections every other day) resulted in a 54% inhibition of vessel area with bFGF induction.
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. A-357300-treated mice continued to gain weight, and no signs of adverse effects were observed.
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. Highest tumor growth inhibition (T/C of 0.34 at day 20; P < 0.001) was achieved with a 100 mg/kg/day dose, without overt signs of toxicity. When given via osmotic minipumps at 15 mg/kg/day, A-357300 also significantly inhibited HT-1080 tumor growth (Fig. 4C)
, giving a T/C value of 0.38 (P < 0.001) at day 23, although twice daily s.c. injections of double this daily dose had failed to produce a significant antitumor effect (Fig. 4B)
. These data suggest that in vivo activity of A-357300 was not driven by high Cmax values but was related to a continuous minimum drug exposure over a period of time. These observations were in agreement with our in vitro finding that continuous exposure to MetAP2 inhibitors was required for optimal inhibition of cell proliferation and consistent with the mode of action of MetAP2 inhibitors (namely, reversible cytostasis).
We further examined both the efficacy and toxicity of A-357300 in MDA-435-LM human breast carcinoma s.c. xenograft model in SCID mice, and we compared it with TNP-470. A-357300 or TNP-470 was administered starting on day 4 after tumor inoculation. A-357300 inhibited tumor growth in a dose-dependent manner, with T/C values on day 32 of 0.59 (P < 0.01) and 0.32 (P < 0.01), respectively, for 50 and 100 mg/kg/day doses by twice daily s.c. injections (Fig. 5A)
. The efficacy of A-357300 at 100 mg/kg/day (T/C value of 0.32 on day 32; P < 0.01) was superior to that of TNP-470 (T/C value of 0.5 on day 32; P < 0.01; Fig. 5B
), which was given at 16 or 20 mg/kg by s.c. injections every other day in the SCID mice. No overt signs of toxicity were observed during the 28 days of drug treatment with A-357300. In contrast, TNP-470-treated mice exhibited severe skin irritation at the injection site, dehydration, rough coat, and diminished weight gain. Histological evaluation of these chronically treated mice on termination revealed that TNP-470, at doses of 16 and 20 mg/kg given by s.c. injection every other day, caused pleural (Fig. 5C)
and epicardial fibrosis, as well as suppurative vasculitis with medial hypertrophy at nontumor sites. None of these lesions were seen in animals treated with A-357300 at any doses, suggesting that these toxic effects of TNP-470 are not related to MetAP2 inhibition but rather are effects related to the chemical nature of TNP-470.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The molecular mechanism of MetAP2 inhibition leading to the activation of p53 pathway (8
, 42)
and subsequent G1 cell cycle arrest has not been established. MetAP2 is one of the two known enzymes responsible for the removal of the NH2-terminal initiator methionine from nascent proteins. Whereas MetAP1 and MetAP2 share an enzyme activity and might compensate for each other, MetAP2 is >1000-fold more efficient at catalyzing methionine removal from peptide sequences of glyceraldehyde-3-phosphate dehydrogenase (9)
. Other MetAP2-specific cellular substrate proteins, such as cyclophilin A and 14-3-3
, have been identified (9
, 43)
. Therefore, MetAP2 inhibition may result in unprocessed initiator methionine in only a small subset of cellular proteins, which are very poor substrates for MetAP1. Altered NH2 termini of these proteins may interfere with subsequent modification(s) and affect their function, subcellular localization, or turnover. For example, removal of initiator methionine is required for protein N-myristoylation. Proteins destined to become myristoylated begin with the NH2-terminal sequence Met-Gly. The initiator Met is removed by MetAPs before myristate is linked to the Gly via an amide bond catalyzed by N-myristoyl transferase. Protein N-myristoylation is required for membrane binding of many important signal transduction proteins including Src family tyrosine kinases, Abl tyrosine kinases, Ser/Thr kinases such as cAMP-dependent protein kinase, phosphatases such as calcineurin B, guanine nucleotide-binding proteins, myristoylated alanine-rich C kinase substrate protein, and others (44)
. Protein turnover in a given cell follows the N-end rule that relates the half-life of a protein to the identity of its NH2-terminal residue (45)
. MetAP2 inhibitors could affect protein turnover due to the uncleaved NH2-terminal methionine. It has been speculated that the effect of MetAP2 inhibitors may stem from inhibition of the NH2-terminal Met-Cys cleavage in a normally short-lived regulator of angiogenesis that is targeted by the N-end rule pathway through its NH2-terminal Cys residue (46)
. Therefore, we postulate that removal of the initiator methionine in specific cellular proteins by MetAP2 cannot be compensated by MetAP1 and that this first step of protein posttranslational modification may be required for the activity, stability, or subcellular localization of these MetAP2-specific substrate proteins essential for the cell cycle progression in susceptible cells. Application of proteomics technology should help delineate the critical proteins in the MetAP2 pathway and may uncover novel cell cycle regulators or controlling mechanisms.
We have demonstrated that a reversible MetAP2-specific inhibitor showed potent single agent antitumor efficacy in xenograft models of carcinoma, sarcoma, and neuroblastoma. These data support the view that reversible MetAP2 inhibitors are promising therapeutics for cancer treatment. MetAP2 inhibitors as novel anticancer agents may have several potential advantages over the chemotherapeutics in the clinic and other agents in development. First, MetAP2 inhibition blocks multiple growth signals. MetAP2 inhibitors arrest EC growth in the presence of FBS and many other added growth factors. Tumor cells secrete multiple angiogenesis inducers, and it is important that effective angiogenesis inhibitors block ECs in response to multiple stimulators. Next, MetAP2 inhibition also results in an antiproliferative effect in many tumor cells (Table 2)
. The sensitivity of these tumor cells to MetAP2 inhibitors does not seem to correlate strictly with their p53 status. MCF7 cells have wild-type p53 but are not inhibited by A-357300 or fumagillin, whereas p53-null cells (HCT-15 and DLD1) are inhibited by these agents. p53 pathway has been shown to be required for inhibition of primary mouse ECs by TNP-470 (8
, 42)
. However, MetAP2 inhibitors may use alternative mechanisms to block proliferation in tumor cells without functional p53. This hypothesis is also supported by a previously published observation that the p53-null prostate cancer PC3 cells in monolayer culture were insensitive to TNP-470 but were inhibited when grown in soft agar (47)
. The combined antiangiogenic and direct antitumor properties of a MetAP2 inhibitor could prove to be a significant advantage. MDA-435-LM cells are less sensitive to A-357300 in vitro, whereas this agent significantly inhibited the growth of MDA-435-LM tumor in vivo, which could be attributed to the antiangiogenic effect of A-357300. Conventional cytotoxic drugs may also have this dual effect, but they are less attractive because they kill other normal cells in addition to ECs. MetAP2 activity seems less critical for the growth of most nonendothelial primary cells. Finally, MetAP2 inhibition is exclusively cytostatic. MetAP2 inhibitors induce cell arrest at the G1 phase of cell cycle without apoptosis. They do not cause cytotoxicity at concentrations up to 1000-fold of the IC50 for inhibition of cellular MetAP2 and proliferation. A-357300 could be dosed for 100 days continuously in the murine models without overt signs of toxicity in the treated mice (data not shown). Thus, MetAP2 inhibitors may be tolerated for chronic use in the clinic because of the selective cytostatic mechanism of action. These biological features of MetAP2 distinguish it from other known cell growth controlling targets. MetAP2 inhibitors therefore are a promising new generation of anticancer therapeutics.
The potential utility of MetAP2 inhibitors in the treatment of cancer is underscored by clinical observations with TNP-470. Anticancer activity of TNP-470 was observed in four patients in a Phase I study of 18 patients with inoperable recurring metastatic squamous cell cancer of the cervix (20
, 21)
. Patients with lung metastasis appeared to benefit most frequently from TNP-470 treatment. Four of seven patients with lung metastasis demonstrated clinical benefit (three patients experienced disease stabilization, and one patient obtained complete resolution of all her pulmonary metastases). In another study, regression of metastatic breast cancer in multiple organs in a patient treated with TNP-470 was observed (22)
. In addition, three patients experienced disease stabilization in a Phase I trial of TNP-470 in patients who had solid tumors refractory to the best available treatment or with a high risk of recurrence (23
, 24) . In a Phase I study published in 2002, the combination of TNP-470 and paclitaxel in 16 NSCLC patients produced a favorable effect on patient survival when compared with published reports for paclitaxel alone (48)
. However, TNP-470 has significant liabilities that limit its usefulness in the clinic. The mean plasma half-lives of TNP-470 and its principal metabolite AGM-1883 were extremely short (harmonic mean t1/2 of 2 and 6 min, respectively). Although in vitro data suggest that TNP-470 is cytostatic at low concentrations, this drug resembles a cytotoxic agent in vivo. Neurotoxicity (weakness, nystagmus, diplopia, and ataxia) observed with TNP-470 was sudden and dose-limiting but appeared to be reversible. TNP-470 caused pleural and epicardial fibrosis, as well as suppurative vasculitis with medial hypertrophy at nontumor sites in mice (Fig. 5C)
. None of these lesions were seen in animals treated with A-357300, which produced superior antitumor efficacy (Fig. 5)
. We postulate that the chemical properties of TNP-470 cause it to be difficult to deliver, metabolized quickly, and exhibit toxicities, thus limiting its utility in the clinic. Rationally designed reversible MetAP2 inhibitors should overcome these liabilities and be amenable to chronic use in the clinic to suppress tumor growth and improve patient survival.
In summary, we have discovered a potent, selective, and reversible MetAP2 inhibitor that suppresses tumor growth in murine models. It does not cause the adverse pathological changes observed with TNP-470, supporting the hypothesis that the toxicity of TNP-470 is unrelated to MetAP2 inhibition. Because MetAP2 appears to act as a cellular rheostat for signaling of multiple growth factors, targeted inhibition of MetAP2 enzyme represents a novel anticancer strategy that has the potential to combine antiangiogenesis and direct tumor stasis. Therefore, further development of reversible and selective MetAP2 inhibitors may offer a promising new cancer therapy with a high therapeutic index.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Requests for reprints: Jieyi Wang, R48R, AP9, Abbott Laboratories, Abbott Park, Illinois 60064. Phone: (847) 938-0434; Fax: (847) 937-4150; E-mail: Jieyi.Wang{at}abbott.com
1 The abbreviations used are: EC, endothelial cell; MetAP, methionine aminopeptidase; HMVEC, human microvascular endothelial cell; CEC, circulating endothelial cell; T/C, the ratio of tumor volume of drug-treated versus vehicle control groups; VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; FBS, fetal bovine serum; HPMC, hydroxypropyl methylcellulose; SCID, severe combined immunodeficient; Rb, retinoblastoma; HUVEC, human umbilical vein endothelial cell. 
Received 5/15/03. Revised 7/11/03. Accepted 9/ 2/03.
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