The 6-AH family [d-Nle-X-Ile-NH-(CH2)5-CONH2; where X = various amino acids] of angiotensin IV (Ang IV) analogs binds directly to hepatocyte growth factor (HGF) and inhibit HGF's ability to form functional dimers. The metabolically stabilized 6-AH family member, d-Nle-Tyr-Ile-NH-(CH2)5-CONH2, had a t1/2 in blood of 80 min compared with the parent compound norleual [Nle-Tyr-Leu-Ψ-(CH2-NH2)3-4-His-Pro-Phe], which had a t1/2 in blood of <5 min. 6-AH family members were found to act as mimics of the dimerization domain of HGF (hinge region) and inhibited the interaction of an HGF molecule with a 3H-hinge region peptide resulting in an attenuated capacity of HGF to activate its receptor Met. This interference translated into inhibition of HGF-dependent signaling, proliferation, and scattering in multiple cell types at concentrations down into the low picomolar range. We also noted a significant correlation between the ability of the 6-AH family members to block HGF dimerization and inhibition of the cellular activity. Furthermore, a member of the 6-AH family with cysteine at position 2, was a particularly effective antagonist of HGF-dependent cellular activities. This compound suppressed pulmonary colonization by B16-F10 murine melanoma cells, which are characterized by an overactive HGF/Met system. Together, these data indicate that the 6-AH family of Ang IV analogs exerts its biological activity by modifying the activity of the HGF/Met system and offers the potential as therapeutic agents in disorders that are dependent on or possess an overactivation of the HGF/Met system.
The multifunctional growth factor hepatocyte growth factor (HGF) and its receptor Met are important mediators for mitogenesis, motogenesis, and morphogenesis in a wide range of cell types (Birchmeier et al., 2003), including epithelial (Kakazu et al., 2004), endothelial (Kanda et al., 2006), and hematopoietic cells (Ratajczak et al., 1997), neurons (Thompson et al., 2004), melanocytes (Halaban et al., 1992), and hepatocytes (Borowiak et al., 2004). Furthermore, dysregulation of the HGF/Met system often leads to neoplastic changes and to cancer (in both human and animal) where it contributes to tumor formation, tumor metastasis, and tumor angiogenesis (Christensen et al., 2005; Liu et al., 2008). Overactivation of this signaling system is routinely linked to poor patient prognosis (Liu et al., 2010). Therefore, molecules that inhibit the HGF/Met system can be expected to exhibit anticancer activity and attenuate malignant and metastatic transformations.
HGF is a vertebrate heteromeric polypeptide growth factor with a domain structure that closely resembles the proteinases of the plasminogen family (Donate et al., 1994). HGF consists of seven domains: an amino-terminal domain, a dimerization-linker domain, four kringle domains (K1–K4), and a serine proteinase homology domain (Lokker et al., 1992; Chirgadze et al., 1999). The single chain propolypeptide is proteolytically processed by convertases to yield a mature α- (heavy chain 55 kDa) and β-heterodimer (light chain 34 kDa), which are bound together by a disulfide link (Stella and Comoglio, 1999; Birchmeier et al., 2003; Gherardi et al., 2006). In addition to proteolytic processing, HGF requires dimerization to be fully activated (Lokker et al., 1992; Chirgadze et al., 1999; Youles et al., 2008). Several reports have shown that HGF forms dimers and/or multimers, which are arranged in a head-to-tail orientation before its interaction with Met (Gherardi et al., 2006). The dimer interface, which encompasses the interdomain linker amino acids (Lys122, Asp123, Tyr124, Ile125, Arg126, and Asn127), is referred to as the hinge region (Gherardi et al., 2006; Youles et al., 2008). Although both prepro-HGF and the active disulfide-linked heterodimer bind Met with high affinity, it is only the heterodimer that is capable of activating Met (Lokker et al., 1992; Sheth et al., 2008).
Recent studies from our laboratory (Yamamoto et al., 2010) have shown that picomolar concentrations of the Ang IV analog norleual [(Nle-Tyr-Leu-ψ-(CH2-NH2)3-4-His-Pro-Phe)] are capable of potently inhibiting the HGF/Met system and binding directly to the hinge region of HGF blocking its dimerization (Kawas et al., 2011). Moreover, a hexapeptide representing the actual hinge region possessed biochemical and pharmacological properties identical to norleuals (Kawas et al., 2011). The major implication of those studies was that molecules, which target the dimerization domain of HGF, could represent novel and viable anticancer therapeutics. Furthermore, these data support the development of such molecules using norleual and/or the hinge peptide as synthetic templates.
Despite its marked anticancer profile, norleual is highly unstable, making its transition to clinical use problematic. Thus, a family of metabolically stabile Ang IV-related analogs has been developed in our laboratory, which is referred to here as the 6-AH family because of 6-amnio hexanoic amide substituted at the carboxyl-terminal position. This substitution along with d-norleucine at the amino terminus enhances the metabolic resistance of family members.
Here, we demonstrate that 6-AH family members have superior metabolic stability compared with norleual, bind to HGF with high affinity, and act as hinge region mimics, thus preventing HGF dimerization and activation. This interference translates into inhibition of HGF-dependent signaling, proliferation, and scattering in multiple cell types at concentrations in the picomolar range. A positive correlation was evident between the ability to block dimerization and the inhibition of the cellular outcomes of HGF activation. Finally, d-Nle-Cys-Ile-NH-(CH2)5-CONH2, a member of the 6-AH family, suppressed pulmonary colonization by B16-F10 murine melanoma cells, which are characterized by an overactive HGF/Met system.
These studies highlight the ability of Ang IV-like molecules to bind to HGF, block HGF dimerization, and inhibit the HGF/Met system. Moreover, these data encourage the development of Ang IV-related pharmaceuticals as therapeutic agents in disorders where inhibition of the HGF/Met system would be clinically advantageous.
Materials and Methods
C57BL/6 mice from Taconic Farms (Germantown, NY) were used in the lung colonization studies. Male Sprague-Dawley rats (>250 g) were obtained from Harlan (Indianapolis, IN) for use in pharmacokinetic studies. Animals were housed and cared for in accordance with the NIH guidelines as described in the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996).
d-Nle-X-Ile-NH-(CH2)5-COOH (where X = various amino acids) and norleual [Nle-Tyr-Leu-ψ-(CH2-NH2)3-4-His-Pro-Phe] were synthesized using Fmoc-based solid-phase methods in the Harding laboratory and purified by reverse-phase HPLC. Purity and structure were verified by LC-MS. HGF was purchased from R&D Systems (Minneapolis, MN).
Anti-Met was purchased from Cell Signaling Technology (Beverly, MA), and the phospho-Met antibody was purchased from Abcam Inc. (Cambridge, MA).
Human embryonic kidney cells 293 (HEK293) and Madin-Darby canine kidney cells (MDCK) were grown in DMEM and 10% fetal bovine serum (FBS). Cells were grown to 100% confluence before use. Human embryonic kidney and MDCK cells were serum-starved for 2 to 24 h before the initiation of drug treatment.
Blood Stability Studies
To compare the blood stability of norleual and d-Nle-Tyr-Ile-NH-(CH2)5-CONH2, a representative member of the 6-AH family, 20 μl of compound-containing vehicle [water (norleual) or 30% ethanol (d-Nle-Tyr-Ile-NH-(CH2)5-CONH2)] was added to 180 μl of heparinized blood and incubated at 37°C for various times. For norleual, incubations at 37°C were stopped at 0, 20, 40, and 60 min, and for d-Nle-Tyr-Ile-NH-(CH2)5-CONH2, incubations were stopped at 0, 1, 3, and 5 h.
At the end of each incubation, 20 μl of Nle1-Ang IV (100 μg/ml) was added to each sample as an internal standard. d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 samples were centrifuged at 4°C for 5 min at 2300g to pellet erythrocytes, and the plasma was transferred to clean tubes. The norleual and d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 samples were precipitated by adding 3 volumes of ice-cold acetonitrile, and the samples were vortexed vigorously. All samples were centrifuged at 4°C, 2300g for 5 min, and the supernatants were transferred to clean tubes. Samples were then evaporated to dryness in a Savant SpeedVac concentrator (Thermo Fisher Scientific, Waltham, MA); the residue was reconstituted in 225 μl of 35% methanol, vortexed briefly, and transferred to HPLC autosampler vials, and 100 μl was injected into the HPLC system.
Samples were then separated by HPLC on an Econosphere C18 column (100 × 2.1 mm) from Grace Davison Discovery Science (Deerfield, IL). Peaks were detected and analyzed by mass spectrographic methods using a LC-MS-2010EV mass spectrometer (Shimadzu, Kyoto, Japan). The mobile phase consisted of HPLC water (Sigma-Aldrich, St. Louis, MO) with 0.1% trifluoroacetic or 0.1% heptafluorobutyric acid (Sigma-Aldrich) and varying concentrations of acetonitrile or methanol. Separation was carried out using a gradient method at ambient temperature and a flow rate of 0.3 ml/min (see Chromatographic System and Conditions). Stability half-lives were determined assuming a normal single-phase exponential decay by use of Prism 5 graphical/statistical program (GraphPad Software, Inc., San Diego, CA).
Male Sprague-Dawley rats (>250 g) were allowed food (rodent diet; Harlan Teklad, Madison, WI) and water ad libitum in our Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-certified animal facility. Rats were housed in temperature-controlled rooms with a 12-h light/dark cycle. The right jugular veins of the rats were catheterized with sterile polyurethane Hydrocoat catheters (Access Technologies, Skokie, IL) under ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and isoflurane (Vet One; MWI, Meridian, ID) anesthesia. The catheters were exteriorized through the dorsal skin. The catheters were flushed with heparinized saline before and after blood sample collection and filled with heparin-glycerol locking solution [6 ml of glycerol, 3 ml of saline, 0.5 ml of gentamycin (100 mg/ml), and 0.5 ml of heparin (10,000 μ/ml)] when not used for more than 8 h. The animals were allowed to recover from surgery for several days before use in any experiment and were fasted overnight before the pharmacokinetic experiment.
Catheterized rats were placed in metabolic cages before the start of the study, and time 0 blood samples were collected. Animals were then dosed intravenously via the jugular vein catheters, with d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 (24 mg/kg) in 30% ethanol. After dosing, blood samples were collected (times and blood volumes collected are listed in chronological order in Table 1). After each blood sample was taken, the catheter was flushed with saline solution, and a volume of saline equal to the volume of blood taken was injected (to maintain total blood volume).
Blood Sample Preparation.
Upon collection into polypropylene microcentrifuge tubes without heparin, blood samples were immediately centrifuged at 4°C, 2300g for 5 min to remove any cells and clots, and the serum was transferred into clean microcentrifuge tubes. A volume of internal standard (Nle1-Ang IV, 100 μg/ml) equal to 0.1 times the sample serum volume was added. A volume of ice-cold acetonitrile equal to four times the sample serum volume was then added, and the sample vortexed vigorously for 30 s. The supernatants were transferred to clean tubes, held on ice until the end of the experiment, and stored at 4°C afterward until further processing.
Serial dilutions of d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 in 30% ethanol were prepared from the stock used to dose the animals for standard curves. Twenty microliters of each serial dilution was added to 180 μl of blood on ice for final concentrations of 0.01, 0.1, 1, and 10 μg/ml. The samples were centrifuged at 4°C, 2300g for 5 min, and the serum was transferred into polypropylene microcentrifuge tubes. A volume of internal standard (Nle1-Ang IV, 100 μg/ml) equal to 0.1 times the sample serum volume was added. A volume of ice-cold acetonitrile equal to four times the sample serum volume was then added, and the sample vortexed vigorously for 30 s. The supernatants were transferred to clean tubes, and samples were stored at 4°C and processed alongside the pharmacokinetic study samples. All samples were evaporated to dryness in a Savant SpeedVac concentrator. The residue was reconstituted in 225 μl of 35% methanol and vortexed briefly. The samples were then transferred to HPLC autosampler vials, and 100 μl was injected into the HPLC system a total of two times (two HPLC/MS analyses) for each sample.
Chromatographic System and Conditions.
The HPLC/MS system used was from Shimadzu, consisting of a CBM-20A communications bus module, LC-20AD pumps, SIL-20AC autosampler, SPD-M20A diode array detector, and a LCMS-2010EV mass spectrometer. Data collection and integration were achieved using Shimadzu LC-MS solution software. The analytical column used was an Econosphere C18 column (100 × 2.1 mm) from Grace Davison Discovery Science. The mobile phase consisted of HPLC grade methanol and water with 0.1% trifluoroacetic acid. Separation was carried out using a nonisocratic method (40-50% methanol over 10 min) at ambient temperature and a flow rate of 0.3 ml/min. For MS analysis, a positive ion mode (scan) was used to monitor the m/z of d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 at 542 and the m/z of Nle1-Ang IV (used for internal standard) at 395. Good separation of d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 and the internal standard in blood was successfully achieved. No interfering peaks coeluted with the analyte or internal standard. Peak purity analysis revealed a peak purity index for d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 of 0.95 and the internal standard of 0.94. d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 eluted at 5.06 min and the internal standard at 4.31 min. Data were normalized based on the recovery of the internal standard.
Pharmacokinetic analysis was performed using data from individual rats. The means ± S.D. were calculated for the group. Noncompartmental pharmacokinetic parameters were calculated from serum drug concentration-time profiles by use of WinNonlin software (Pharsight, Mountain View, CA). The following relevant parameters were determined where possible: area under the concentration-time curve from time 0 to the last time point (AUC0-last) or extrapolated to infinity (AUC0-∞), Cmax concentration in plasma extrapolated to time 0 (C0), terminal elimination half-life (t1/2), volume of distribution (Vd), and clearance.
Male rat liver microsomes were obtained from Celsis (Baltimore, MD). The protocol from Celsis for assessing microsomal-dependent drug metabolism was followed with minor adaptations. An NADPH-regenerating system was prepared as follows: 1.7 mg/ml NADP, 7.8 mg/ml glucose 6-phosphate, and 6 units/ml glucose-6-phosphate dehydrogenase were added to 10 ml of 2% sodium bicarbonate and used immediately. Solutions (500 μM) of norleual, d-Nle-Tyr-Ile-NH-(CH2)5-CONH2, piroxicam, verapamil, and 7-ethoxycoumarin (low, moderate, and highly metabolized controls, respectively) were prepared in acetonitrile. Microsomes were suspended in 0.1 M Tris buffer, pH 7.38, at 0.5 mg/ml, and 100 μl of the microsomal suspension was added to prechilled microcentrifuge tubes on ice. To each sample, 640 μl of 0.1 M Tris buffer, 10 μl of 500 μM test compound, and 250 μl of NADPH-regenerating system were added. Samples were incubated in a rotisserie hybridization oven at 37°C for the appropriate incubation time (10, 20, 30, 40, or 60 min). Five hundred microliters from each sample was transferred to tubes containing 500 μl of ice-cold acetonitrile with internal standard per incubation sample. Standard curve samples were prepared in incubation buffer, and 500 μl was added to 500 μl of ice-cold acetonitrile with internal standard. All samples were then analyzed by high-performance liquid chromatography/mass spectrometry. Drug concentrations were determined, and loss of parent relative to negative control samples containing no microsomes was calculated. Clearance was determined by nonlinear regression analysis for ke and t1/2, and the equation Clint = ke Vd. For in vitro-in vivo correlation, Clint per kilogram body weight was calculated using the following measurements for Sprague-Dawley rats: 44.8 mg of protein per g of liver, 40 g of liver per kg of body weight.
The binding of 6-AH analogs to HGF was assessed by competition using a soluble binding assay. PBS (250 μl) containing human HGF (1.25 ng) was incubated with 3H-hinge, the central dimerization domain of HGF, in the presence of varying concentrations of 6-AH analogs between 10−13 M and 10−7 M (half-log dilutions) for 40 min at 37°C. The incubates were then spun through Bio-Gel P6 spin columns (400-μl packed volume) for 1 min to separate free and bound 3H-hinge, and the eluent was collected. Five milliliters of scintillation fluid was added to the eluent, which contained the HGF-bound 3H-hinge, and was then counted using scintillation counter. Total disintegrations per minute of bound 3H-hinge were calculated based on machine-counting efficiency. The Ki values for the binding of the peptides were determined using the Prism 5. Competition binding curves were performed in triplicate. Preliminary kinetic studies indicated that equilibrium binding was reached by 40 min of incubation at 37°C. 3H-Hinge has recently been shown to bind to HGF with high affinity (Kawas et al., 2011).
HGF dimerization was assessed using PAGE followed by silver staining (Kawas et al., 2011). Human HGF at a concentration of 0.08 ng/μl with or without 6-AH analogs was incubated with heparin at a final concentration of 5 μg/ml. Loading buffer was then added to each sample, and the mixture was separated by native PAGE using gradient Criterion XT precast gels (4-12% Bis-Tris; Bio-Rad Laboratories, Hercules, CA). The gel was silver stained next for the detection of the HGF monomers and dimers. Bands were quantitated from digital images using a PhosphorImager (UVP, Inc., Upland, CA).
HEK293 cells were seeded in six-well tissue culture plates and grown to 95% confluence in DMEM containing 10% FBS. The cells were serum-deprived for 24 h before the treatment to reduce the basal levels of phospho-Met. After serum starvation, cocktails composed of vehicle and HGF with or without 6-AH analogs were prepared and preincubated for 30 min at room temperature. The cocktail was then added to the cells for 10 min to stimulate the Met receptor and downstream proteins. Cells were harvested using radioimmunoprecipitation assay lysis buffer (Millipore, Billerica, MA) fortified with phosphatase inhibitor cocktails 1 and 2 (Sigma-Aldrich). The lysate was clarified by centrifugation at 15,000g for 15 min, protein concentrations were determined using the BCA total protein assay (Thermo Fisher Scientific), and then appropriate volumes of the lysates were diluted with 2× reducing Laemmli buffer and heated for 10 min at 95°C. Samples containing identical amounts of protein were resolved using SDS-PAGE (Criterion; Bio-Rad Laboratories), transferred to nitrocellulose, and blocked in Tris-buffered saline containing 5% milk for 1 h at room temperature. The phospho-Met antibody was added to the blocking buffer at a final concentration of 1:1000 and incubated at 4°C overnight with gentle agitation. The membranes were then washed several times with water and Tris-buffered saline (PBS, 0.05% Tween 20), a 1:5000 dilution of horseradish-peroxidase conjugated goat anti-rabbit antiserum was added, and the membranes were incubated further for 1 h at room temperature. Proteins were visualized using the SuperSignal West Pico chemiluminescent substrate system (Thermo Fisher Scientific), and molecular weights were determined by comparison to protein ladders [Benchmark (Invitrogen, Carlsbad, CA) and Kaleidoscope (Bio-Rad Laboratories)]. Film images were digitized and analyzed using a UVP PhosphorImager.
MDCK cells (5000) were seeded into 96-well plates in 10% FBS DMEM. To induce cellular quiescence, the cells were serum-deprived for 24 h before initiating the treatments. After serum starvation, 10 ng/ml HGF alone and with various concentrations of 6-AH analogs or PBS vehicle were added to the media. The cells were allowed to grow under these conditions for 4 days with a daily addition of 6-AH analogs. On the 4th day, 1 mg/ml 1-(4,5-dimethylthiazol-2-yl)3,5-diphenylformazan reagent (MTT; Sigma-Aldrich) prepared in PBS was added to the cells and incubated for 4 h. Dimethyl sulfoxide diluted in a 0.01 M glycine buffer was added to solubilize the cell membranes, and the absorbance of reduced MTT in the buffer was quantitated at 590 nm using a plate reader (Biotek Synergy 2; BioTek Instruments, Winooski, VT). HGF-dependent proliferation was determined by subtracting the basal proliferation (in the absence of HGF) from total proliferation rates in groups containing HGF.
MDCK cells were grown to 100% confluence on the coverslips in six-well plates and washed twice with PBS. The confluent coverslips were then aseptically transferred to new six-well plates containing 900 μl of serum-free DMEM. Norleual, hinge peptide, and/or HGF (20 ng/ml) was added to the appropriate wells. Control wells received PBS vehicle. Plates were incubated at 37°C with 5% CO2 for 48 h. Medium was removed, and cells were fixed with methanol. Cells were stained with Diff-Quik Wright-Giemsa (Dade Behring, Deerfield, IL), and digital images were taken. Coverslips were removed with forceps, and more digital images were captured. Pixel quantification of images was achieved using ImageJ software, and statistics were performed using Prism 5 and InStat, version 3.05 (GraphPad Software, Inc.).
Lung Colony Formation
Six- to 8-month-old C57BL/6 mice were injected with 400,000 B16-F10 cells in 200 μl of PBS by tail vein injection and subsequently received daily intraperitoneal injections of either d-Nle-X-Cys-NH-(CH2)5-CONH2 (10 and 100 μg/kg) or a PBS vehicle control. Two weeks later, mice were anesthetized, and lungs were perfused with PBS and removed. Photos were taken, and lungs were solubilized in 1% Triton X-100, 20 mM Tris, 0.15 M NaCl, 2 mM EDTA, and 0.02% sodium azide. Samples were disrupted by sonication (Mixonix, Farmingdale, NY) and spun. The supernatant was transferred to a 96-well plate, and melanin absorbance at 410 nm was measured using a plate reader.
Independent one-way analysis of variance (ANOVA) (InStat, version 3.05 and Prism 5) was used to determine differences among groups. Tukey-Kramer's or Bonferroni's multiple comparison post hoc tests were performed where necessary. Statistical comparisons of two groups were determined using the two-tailed Student's t test (InStat, version 3.05 and Prism 5).
The Ang IV Analog d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 Is More Metabolically Stable than Norleual (Nle-Tyr-Leu-ψ-(CH2-NH2)3-4-His-Pro-Phe).
The Ang IV-related peptidomimetic norleual was previously shown to possess anti-HGF/Met, antiangiogenic, and anticancer activities (Yamamoto et al., 2010). The presence of unprotected peptide bonds at both the amino- and carboxyl-terminal linkages predicts that norleual should have poor metabolic stability and rapid clearance for the circulation, properties that may limit its clinical utility. In an attempt to overcome this limitation, a family of compounds, the 6-AH family, was designed and synthesized to offer defense against exopeptidases. Figure 1 demonstrates that, as expected, norleual is unstable in heparinized blood, whereas d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 exhibited improved stability.
The Ang IV Analog d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 Has a Much Longer Circulating Half-Life than Norleual (Nle-Tyr-Leu-ψ-(CH2-NH2)3-4-His-Pro-Phe).
As anticipated from the in vitro blood stability data, d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 exhibited an extended in vivo elimination half-life of 1012 min after intravenous injection in rats. Other relevant pharmacokinetic parameters of d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 after a single intravenous bolus dose are summarized in Table 2. Serum data were modeled using WinNonlin software to perform noncompartmental analysis. d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 seemed to be extensively distributed outside the central blood compartment and/or bound within the tissues as proven by its large Vd. d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 is not expected to be highly bound to plasma proteins according to quantitative structure-activity relationship modeling and since total recovery from serum was greater than 35%. These results, which suggest that d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 is probably relatively hydrophobic, are in agreement with the outcome of quantitative structure-activity relationship-modeling estimates generated by ADMET Predictor (Simulations Plus, Inc., Lancaster, CA) that calculated an octanol/water partition coefficient of 28.18 for d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 (Table 3).
It is not surprising that because of its stability, hydrophobic character, and small size, d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 was predicted to be orally bioavailable. The Peff value represents the predicted effective human jejunal permeability of the molecule. The predicted Peff value for d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 (1.53) is intermediate between the predicted Peff values for enalapril (1.25) and piroxicam (2.14), two orally bioavailable drugs. d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 was also predicted to be 42.68% unbound to plasma proteins in circulation, thus making it available for distribution into the tissues.
A lack of phase I metabolism for d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 also contributed to its slow removal from the blood. d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 exhibited no detectable metabolism over 90 min in an in vitro metabolism assay using rat liver microsomes (data not shown). Together these data indicate that d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 is more metabolically stable than norleual, possesses an elongated half-life in the circulation, and penetrates tissue effectively. Overall, these favorable pharmacokinetic properties justify the mechanistic and therapeutic evaluation of d-Nle-Tyr-Ile-NH-(CH2)5-CONH2 and related molecules.
d-Nle-X-Ile-NH-(CH2)5-CONH2 Analogs Bind HGF and Compete with the 3H-Hinge Peptide for HGF Binding.
Several members of the d-Nle-X-Ile-NH-(CH2)5-CONH2, 6-AH family, were analyzed for the capacity to compete for 3H-hinge binding to HGF. As is evident below, members of the 6-AH family display a varied ability to block the biological action of HGF. As such, the HGF binding properties of a selection of analogs with varying biological activities were assessed to determine whether there was a relationship between inhibitory activity and affinity for HGF. The hypothesis was created to prove that analogs are binding directly to HGF and affecting the sequestration of HGF in an inactive form. To begin the evaluation of this idea, we used a 3H-hinge peptide as a probe to assess direct HGF binding of the peptides. The use of 3H-hinge to probe the interaction was based on the ability of 3H-hinge to bind specifically and with high affinity to HGF (Kawas et al., 2011). A competition study was initiated with several derivatives of the d-Nle-X-Ile-NH-(CH2)5-CONH2 family. This study demonstrated that different analogs have variable abilities to bind HGF and that the analogs showing antagonism to HGF are acting as a hinge mimics. d-Nle-X-Ile-NH-(CH2)5-CONH2 derivatives were found to compete with hinge for HGF binding and exhibited a range of affinities for HGF, with Ki values ranging from 1.37 × 10−7 to 1.33 × 10−10 M (Fig. 2). As expected, it seems to be a relationship between a compound's ability to bind HGF and its capacity to block dimerization and inhibit HGF-dependent activities (see Figs. 4, 5, and 6).
d-Nle-X-Ile-NH-(CH2)5-CONH2 Analogs Block HGF Dimerization.
Several reports have shown that HGF needs to form homodimers and/or multimers before its activation of Met (Chirgadze et al., 1999; Gherardi et al., 2006). This dimer is arranged in a head to tail orientation; the dimer interface comprises a central region, the hinge region that is important for the proper dimer formation and orientation. A homologous sequence-conservation screen against all possible transcripts that were independent of and not derived from angiotensinogen looking for similarities to Ang IV identified partial homology with the hinge region (Yamamoto et al., 2010) of the plasminogen family of proteins, which include plasminogen itself, its antiangiogenic degradation product, angiostatin, and the protein hormones, HGF and macrophage-stimulating protein. Moreover, the Ang IV analog norleual, which is a potent inhibitor of the HGF/Met system, was shown to bind to HGF and block its dimerization (Kawas et al., 2011). This knowledge coupled with the demonstration that some members of the 6-AH family bound with high affinity to the hinge region of HGF led to the expectation that other active Ang IV analogs, such as 6-AH family members, could be expected to inhibit HGF dimerization and that the ability of an individual analog to bind HGF and inhibit HGF-dependent processes should be reflected in its capacity to attenuate dimerization. The data in Fig. 3 confirm this expectation by demonstrating that d-Nle-Cys-Ile-NH-(CH2)5-CONH2 and d-Nle-Tyr-Ile-NH-(CH2)5-CONH2, which bind HGF with high affinity (Fig. 2) and effectively attenuate HGF-dependent processes (Figs. 4, 5, and 6), completely block HGF dimer formation. In contrast, d-Nle-Met-Ile-NH-(CH2)5-CONH2, which has low affinity for HGF (Fig. 2) and exhibits little anti-HGF/Met activity, is unable to block dimerization at the concentration tested. The d-Nle-Trp-Ile-NH-(CH2)5-CONH2 analog, which exhibits intermediate inhibition of dimerization, predictably has a moderate affinity for HGF and a moderate ability to inhibit HGF-dependent processes (Figs. 4, 5, and 6). Together these data confirm the expectation that active 6-AH analogs can block dimerization and further that an analog's ability to inhibit dimerization translates, at least qualitatively, to its capacity to block HGF-dependent processes.
d-Nle-X-Ile-NH-(CH2)5-CONH2 Analogs Attenuates HGF-Dependent Met Signaling.
After establishing that the 6-AH family members exhibit a range of HGF binding and dimerization inhibitory profiles, we next determined whether these properties would parallel a compound's ability to inhibit Met signaling. Characteristic of tyrosine kinase-linked growth factor receptors such as Met is a requisite tyrosine residue autophosphorylation step, which is essential for the eventual recruitment of various Src homology 2 domain signaling proteins. Thus, we evaluated the ability of several 6-AH analogs to induce Met tyrosine phosphorylation. As anticipated, the data in Fig. 4 demonstrate that both d-Nle-Cys-Ile-NH-(CH2)5-CONH2 and d-Nle-Tyr-Ile-NH-(CH2)5-CONH2, which bind HGF with high affinity (Fig. 2) and effectively block its dimerization (Fig. 3), were able to block Met autophosphorylation. The d-Nle-Trp-Ile-NH-(CH2)5-CONH2 analog had intermediate inhibitory activity, and the d-Nle-Met-Ile-NH-(CH2)5-CONH2 analog showed no ability to have an effect on Met activation. Together, these data indicate that the capacity of 6-AH analogs to inhibit HGF-dependent Met activation paralleled their HGF binding affinity and their capacity to block dimerization.
d-Nle-X-Ile-NH-(CH2)5-CONH2 Analogs Affect HGF/Met-Stimulated MDCK Cell Proliferation.
Met activation initiates multiple cellular responses, including increased proliferation and motility, enhanced survival, and differentiation (Zhang and Vande Woude, 2003). As an initial test of the ability of 6-AH family members to alter HGF-dependent cellular activity, we evaluated the capacity of several members of the family to modify the proliferative activity of MDCK cells, a standard cellular model for investigating the HGF/Met system (Stella and Comoglio, 1999). As seen in Fig. 5, there is a wide range of inhibitory activity against HGF-dependent cellular proliferation. Similar to the results from the binding and dimerization experiments, the Cys2 and Tyr2 analogs exhibited marked inhibitory activity. The Asp2 analog, which had not been evaluated in the earlier studies, also exhibited pronounced inhibitory activity. The Trp2, Phe2, and Ser2 analogs all showed inhibitory activity, albeit less than that observed with the most potent analogs. The decrease in HGF-dependent MDCK proliferation below control levels for some compounds is not surprising because the experiment was carried in 2% serum, which probably contained some level of HGF. The hinge peptide (KDYIRN), which represents the dimerization domain of HGF, was included as a positive control. A recent study has demonstrated that hinge binds to HGF with high affinity, blocking its dimerization and acting as a potent inhibitor of HGF-dependent cellular activities, including MDCK proliferation (Kawas et al., 2011).
d-Nle-X-Ile-NH-(CH2)5-CONH2 Analogs Modify HGF/Met-Mediated Cell Scattering in MDCK Cells.
Cell scattering is the hallmark effect of HGF/Met signaling, a process characterized by decreased cell adhesion, increased motility, and increased proliferation. The treatment of MDCK cells with HGF initiates a scattering response that occurs in two stages. First, the cells lose their cell-to-cell adhesion and become polarized. Second, they separate completely and migrate away from each other. It is expected that if the 6-AH family members are capable of inhibiting the HGF/Met system, they should be able to modify HGF-dependent MDCK cell scattering.
Figure 6, A and B, indicates that those analogs that were previously found to block HGF dimerization were effective inhibitor of HGF/Met-mediated cell scattering in MDCK cells, whereas those analogs with poor affinity for HGF were ineffective. Figure 7 shows a correlation between the blockade of HGF dimerization and HGF binding affinity and the ability to prevent MDCK cell scattering.
d-Nle-Cys-Ile-NH-(CH2)5-CONH2 Inhibits B16-F10 Murine Melanoma Cell Migration and Lung Colony Formation.
To evaluate the prospective utility of the 6-AH family members as potential therapeutics, we examined the capacity of [d-Nle-Cys-Ile-NH-(CH2)5-CONH2], an analog that exhibits a strong inhibitory profile against HGF-dependent Met activation, to suppress the migratory and lung colony-forming capacity of B16-F10 murine melanoma cells. B16 melanoma cells overexpress Met (Ferraro et al., 2006) and were chosen for these studies because Met signaling is critical for their migration, invasion, and metastasis. As a final test for the physiological significance of the 6-AH family blockade of Met-dependent cellular outcomes, we evaluated the ability of d-Nle-Cys-Ile-NH-(CH2)5-CONH2 to inhibit the formation of pulmonary colonies by B16-F10 cells after tail vein injection in mice. Figure 8A illustrates the inhibitory response that was observed with daily intraperitoneal injections at two doses (10 and 100 μg/kg per day) of [d-Nle-Cys-Ile-NH-(CH2)5-CONH2]. Figure 8B provides a quantitative assessment of pulmonary colonization by measuring melanin content, which reflects the level of melanoma colonization. Together these data demonstrate that treatment of melanoma cells with d-Nle-Cys-Ile-NH-(CH2)5-CONH2 radically prevented lung colonization and highlight the potential utility of the use of 6-AH analogs as potential anticancer agents.
Recently interest has grown in developing therapeutics targeting the HGF/Met system. At present, this interest has been primarily driven by the realization that overactivation of the HGF/c-Met system is a common characteristic of many human cancers (Comoglio et al., 2008; Eder et al., 2009). The potential utility of anti-HGF/Met drugs, however, goes well beyond their use as anticancer agents. For example, the recognized involvement of the HGF/c-Met system in the regulation of angiogenesis (see review in You and McDonald, 2008) supports the potential utility of HGF/Met antagonists for the treatment of disorders in which control of tissue vascularization would be clinically beneficial. These could include hypervascular diseases of the eye such as diabetic retinopathy and the wet type of macular degeneration. In both cases, antiangiogenic therapies are currently in use (Jeganathan, 2011; see reviews in Barkmeier and Carvounis, 2011). Antiangiogenics are also being examined as treatment options in a variety of other disorders ranging from obesity where adipose tissue vascularization is targeted (Daquinag et al., 2011) to chronic liver disease (Coulon et al., 2011) and psoriasis where topical application of antiangiogenic drugs is being considered (Canavese et al., 2010).
Currently, the pharmaceutical industry is using two general approaches to block Met-dependent cellular activities (Eder et al., 2009; Liu X et al., 2010). The first involves the development of single-arm humanized antibodies to HGF (Burgess et al., 2006; Stabile et al., 2008) or Met (Martens et al., 2006). The second approach uses “kinase inhibitors” that block the intracellular consequences of Met activation. These kinase inhibitors are small hydrophobic molecules that work intracellularly to compete for the binding of ATP to the kinase domain of Met, thus inhibiting receptor autophosphorylation (Morotti et al., 2002; Christensen et al., 2003; Sattler et al., 2003). Despite the promise of the biologic and kinase-inhibitor approaches, which are currently represented in clinical trials, both have limitations arising from toxicity or specificity considerations and/or cost (Hansel et al., 2010; Maya, 2010).
A third approach that our laboratory has been pursuing exploits a step in the activation process of the HGF-Met system; namely the need for HGF to predimerize before it is able to activate Met. Thus, we have targeted the dimerization process by developing molecules that mimic the dimerization domain, the hinge region, with idea that they can act as dominant negative replacements. Recent studies have validated this general approach, demonstrating that molecules designed around angiotensin IV (Yamamoto et al., 2010) or the hinge sequence itself (Kawas et al., 2011) can bind HGF, block its dimerization, and attenuate HGF-dependent cellular actions. The studies described herein represent a first step toward producing useful therapeutics targeted at HGF dimerization. The primary focus of this study was to improve the pharmacokinetic characteristics of a parent compound norleual (Yamamoto et al., 2010) while maintaining biological activity. To this end, we successfully synthesized and evaluated a family of new molecules, the 6-AH family [d-Nle-X-Ile-NH-(CH2)5-COOH]. A subset of these molecules not only had improved metabolic stability and circulating t1/2 but exhibited excellent in vitro and in vivo activity.
In addition to characterizing a new family of HGF/Met antagonists, the present investigation demonstrated a qualitative relationship between the ability of a compound to bind HGF and block HGF dimerization and its observed in vitro biological activity. Moreover, these studies provide initial structure-activity data and pave the way for more extensive evaluation. The chemical modifications that were made at the amino and carboxyl termini of the Ang IV molecule and the resultant improvement in metabolic stability highlight the critical role played by exopeptidases in the metabolism of Ang IV-derived molecules. The demonstrated importance of protecting the terminals to pharmacokinetic characteristics suggests numerous additional synthetic approaches that may be applicable, including the insertion of nonpeptide linkages (see Sardinia et al., 1994) between the first and second amino acids, the replacement of the amino-terminal amino acid with a non-α-amino acid and amino-terminal acylation.
In summary, these studies further validate the notion that targeting the dimerization domain of HGF is an effective means of inhibiting the HGF/Met system. Furthermore, they demonstrate that molecules with favorable pharmacokinetic characteristics can be produced, thus highlighting their potential clinical utility.
Participated in research design: Kawas, McCoy, Yamamoto, Wright, and Harding.
Conducted experiments: Kawas, McCoy, and Yamamoto.
Preformed data analysis: Kawas, McCoy, Yamamoto, and Harding.
Wrote or contributed to the writing of the manuscript: Kawas, McCoy, Wright, and Harding.
This work was supported by a grant from the Adler Foundation (to J.W.H.).
J.W.W. and J.W.H. are founders and shareholders in M3 Biotechnology, LLC, which is developing pharmaceuticals based on this technology.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- hepatocyte growth factor
- high-performance liquid chromatography
- human embryonic kidney 293
- liquid chromatography-mass spectrometry
- Madin-Darby canine kidney
- Dulbecco's modified Eagle's medium
- fetal bovine serum
- 2-[bis(2 hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
- reduced peptide bond
- volume of distribution
- phosphate-buffered saline
- polyacrylamide gel electrophoresis
- bissulfosuccinimidyl suberate
- Nle1-Ang IV
- 1-(4,5-dimethylthiazol-2-yl)3,5-diphenylformazan reagent
- analysis of variance.
- Received September 16, 2011.
- Accepted November 29, 2011.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics