MT-SP1 was expressed and purified as described previously and stored at −20°C in 50mM Tris pH 8.0, 50mM NaCl, 10% glycerol (Takeuchi et al., 1999
). Mutants for crystallography and kinetic studies were expressed, purified, and stored in the same manner. Thrombin was purchased from Sigma and stored at −20°C in 50mM Tris pH 8.0, 50mM NaCl, 0.1 mg/mL BSA. The MT-SP1 substrates spectrazyme-tPA and spectrafluor-tPA were purchased from American Diagnostica and stored at −20 at 10 mM in H2
The materials for peptide synthesis including PyBOP, EDAC, HOBt, Fmoc-PEG20atom-OH, and 2-chlorotrityl chloride resin were purchased from NovaBiochem. All other chemicals were purchased from Sigma unless otherwise noted. Solvents including anhydrous ethanol (EtOH), choloroform (CHCl3), 1,4-dioxane, diethyl ether (Et2O), dichloromethane (DCM), trifluroethanol (TFE), trifluoroacetic acid (TFA) and N,N-dimethylformamide (DMF) were used as received. The peptides were synthesized following standard Fmoc-SPPS procedure on 2-chlorotrityl chloride resin.
Reactions were analyzed by LC-MS performed on a Waters Alliance liquid chromatography system with a Waters Micromass ZQ single-quadrupole mass spectrometer. HPLC purifications were carried out using an Agilent 1200 series system with C18 reversed-phase columns (Waters). Mobile phase consisted of 99.9:0.1% water/trifluoroacetic acid (solvent A) and 95:4.9:0.1% acetonitrile/water/trifluoroacetic acid (solvent B). All final compounds were characterized by Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry using an ABI 4700 MALDI-TOF-TOF mass spectrometer.
Compound numbers in bold refer to the structures shown in . Diphenyl [N-(benzyloxycarbonyl)amino](4-cyanophenyl)methanephosphonate (9) and NHS-biotin were synthesized according to literature procedure.(ref) No attempts were made to resolve the D,L-(4-AmPhGly)P(OPh)2 diastereomers.
Diphenyl-[N-(benzyloxycarbonyl)amino](4-ethylesterphenyl)methane phosphonate hydrochloride (10)
A batch of 9 (3.0g, 6.0mmol) was dissolved in 100 mL CHCl3 and placed in a 0° C bath. Next Ar was passed over the solution and under the flow of Ar, 3 mL of anhydrous EtOH (60mmol) and 100 mL of 4 M HCl in dioxane were added. The reaction was stirred under argon at 4°C for 5 days, after which 10 formed as a fine white precipitate formed. The precipitate was filtered, washed with Et2O, and dried under reduced pressure (2.32g, 72%). Mass calcd for C30H29N2O6P: 544.18; found 545.45 (M+H)+.
Diphenyl amino(4-amidinophenyl)methanephosphonate, trifluoroacetic acid salt (12)
A batch of 10 (1.0g, 1.8mmol) was dissolved in 25 mL 1,4-dioxane and 25 mL anhydrous EtOH. Next the reaction was purged with Ar and 10mL of 0.5M NH3 in dioxane (5 mmol) was added dropwise. The reaction was stirred under argon at 23° C for two days, and the solvent was removed completely under vacuum to afford the gummy white crude intermediate, diphenyl [N-(benzyloxycarbonyl)amino](4-amidinophenyl) methanephosphonate 11. This intermediate was dissolved in anhydrous EtOH (80mL) and concentrated HCl (305μL, 3.7mmol) and hydrogenated over 10% palladium on activated carbon for 5 h at room temperature. The catalyst was separated by filtration, and the solvent was removed under reduced pressure. The white powder thus isolated was dissolved in HPLC solvent (water with 0.1% TFA) with the aid of DMF and purified by reverse-phase HPLC. Lyophilization of fractions containing product afforded 0.49 g (27%) of 12 as a white powder. Mass calcd for C20H20N3O3P: 381.12, m/z found: 382.01 (M+H)+.
Coupling of the biotinylated peptide to 12. General procedure
To a DCM/TFE(5:1) solution containing the biotin-PEG-peptide (0.03 mmol), EDAC (0.03 mmol) and HOBt (0.03 mmol), was added H-Bz-DPP 12 (0.03 mmol). After 2 h, an additional eq of EDAC and HOBt was added and the mixture stirred for 8 hr. Next the solvent was removed, the isolated oil was dissolved in MeCN/water (1:3), and purified by reverse-phase HPLC. The fractions with product were lyophilized down to yield the desired product as a white powder. The final probes were dissolved in DMSO and stored at −20° C. The concentration of the solution was determined by HABA biotin quantification kit (Pierce).
Reaction of Biotin-PEG-Gln(Trt)-Arg(Pbf)-Val-OH (0.10 g, 0.11 mmol), with 12 yielded 39 mg (72% yield) of Biotin-PEG-Gln(Trt)-Arg(Pbf)-Val-DPP as a white powder. Mass calcd for C92H119N14O18PS2: 1802.80, m/z found: 1802.10 (M+H)+. Next the powder was resuspended in 95% TFA, 2.5% TIS, 2.5% water and agitated for 2 hours, to obtain the desired probe 1. The product was precipitated into cold ether, pelleted by centrifugation, and purified by reversed-phase HPLC. Lyophilization of fractions containing product afforded 22 mg (79%) afforded 1 as a white powder. Mass calcd for C60H89N14O15PS: 1308.61, m/z found: 656.34 (M+2H)2+.
The protected peptide Biotin-PEG-Leu-Thr(tBu)-Pro-OH was reacted with 12 and resulted in Biotin-PEG-Leu-Thr(tBu)-Pro-Bz-DPP as a white powder (22 mg, 56% yield). Mass calcd for C63H93N10O15PS: 1292.63, m/z found: 1293.29 (M+H)+. Next the powder was resuspended in 95% TFA, 2.5% TIS, 2.5% water and agitated for 2 hours, to obtain the desired probe 2. The product was precipitated into cold ether, pelleted by centrifugation, and purified by reversed-phase HPLC. Lyophilization of fractions containing product afforded 8 mg (36% yield). Mass calcd for C59H85N10O15PS: 1236.57, m/z found: 1237.12 (M+H)+.
Reaction of Biotin-PEG-Gly-Ser-Gly-OH with 12 resulted in 3 as a white powder (22 mg, 65%). Mass calcd for C51H71N10O15PS: 1126.46, m/z found: 1126.92(M+H)+.
Biotin-PEG-Glu(OtBu)-Pro-Ile-OH was reacted with 12 to afford 23 mg (57% yield) of Biotin-PEG-Glu(OtBu)-Pro-Ile-Bz-DPP. Mass calcd for C64H93N10O16PS: 1320.62, m/z found: 1321.43 (M+H)+. Then the powder was resuspended in 95% TFA, 2.5% TIS, 2.5% water and agitated for 2 hours to obtain the desired probe. The product was precipitated into cold ether, pelleted by centrifugation, and purified by reversed-phase HPLC. Lyophilization of fractions containing product afforded 20 mg (90% yield) of 4. Mass calcd for C60H85N10O16PS: 1264.56, m/z found: 1266.31 (M+H)+.
Reaction of Biotin-PEG-OH with 12 yielded 5, which was isolated as white powder. (7mg, 25%). Mass calcd for C44H60N7O11PS: 925.38, m/z found: 926.17 (M+H)+.
A batch of Biotin-PEG-Val-OH was reacted with 12 to afford 6 as a powder after HPLC purification (7mg, 20%). Mass calcd for C49H69N8O13PS: 1024.45, m/z found: 1025.82 (M+H)+.
Reaction of Biotin-PEG-Arg(Pbf)-Val-OH with 12 afforded Biotin-PEG-Arg(Pbf)-Val-OH as a white powder (34 mg, 77%). Mass calcd for C68H99N12O17PS2,: 1450.64, m/z found: 1451.24 (M+H)+. Then the powder was dissolved in 95% TFA, 2.5% TIS, 2.5% water and agitated for 2 hours to obtain the desired probe. The product was precipitated into cold ether, pelleted by centrifugation, and purified by reversed-phase HPLC. Lyophilization of fractions containing product afforded 24 mg (90% yield) of 7. Mass calcd for C55H81N12O14PS: 1180.55, m/z found: 1181.64 (M+H)+.
Reaction of Biotin-PEG-Phe-Thr-Gly-Ser-Gly-OH with 12 afforded 13 as a white powder (45 mg, 40%). Mass calcd for C64H87N12O18PS: 1374.57, m/z found: 1376.27 (M+H)+.
All kinetic fluorescence measurements were taken in duplicate using a SpectraMax Gemini fluorescence spectrometer (Molecular Devices) with an excitation wavelength of 380nm, an emission wavelength of 460 nm, and a 435 nm cutoff filter. A solution of inhibitor was serially diluted over an appropriate concentration range and incubated with enzyme. Substrate was added at the end of 4 hours to initiate the reaction, and IC-50s were calculated. MT-SP1 was used at 0.2nM in a buffer containing 50mM Tris pH 8.0, 50mM NaCl, 0.01% Tween-20, with 200uM spectrafluor-tPA as the substrate. Thrombin was used at 0.5nM in a buffer containing 50mM Tris pH 8.0, 50mM NaCl, 0.01% Tween-20, 0.1 mg/mL BSA, with 200uM Boc-b-benzyl-Asp-Pro-Arg-AMC as the substrate. All reactions were run in duplicate.
Steady-state kinetics were used to determine the observed rate constants for the inhibition reaction. The inhibitors were serially diluted in a 96 well plate at an appropriate range of concentrations. Enzyme was added in hour intervals over 8-10 hours. The reaction was initiated by the addition of 200uM substrate, and the Vmax
recorded on a SpectraMax fluorescence spectrometer. Kobs
was determined at each inhibitor concentration by plotting Vmax
vs time, and KIapp
was determined by plotting Kobs
vs . Kinact
was determined using the equation
Crystals were grown at room temperature by vapour diffusion in hanging drops. A combination of micro and macro seeding was used to grow large single crystals in 4.0 M Na Formate at pH=7.0 and 25 mM FeCl3 as additive. Crystals belong to monoclinic space group C2 with one protease-inhibitor complex in the asymmetric unit corresponding to a solvent content of 50% and diffracted to better than 1.2Å resolution.
Structure resolution and refinement
Data were collected at beamline 8.3.1 at the Advanced Light Source in Berkeley on a single crystal cryoprotected in mother liquor supplemented with 20% glycerol. The data were indexed, scaled and reduced using Mosflm
(Holton and Alber, 2004
). The structure was solved by molecular replacement using Phaser
(McCoy et al., 2007
) with the previously solved protease structure (PDB 3BN9) as search probe (Farady et al., 2008
). Automatic building and refinement were performed in Phenix
(Adams et al., 2002
) using Phenix elBow
to generate the covalently bound ligand. Manual building was carried out in Coot
(Emsley and Cowtan, 2004
). The stererochemistry of the final model was validated using MolProbity
(Davis et al., 2007
Western blot labeling analysis
For recombinant protease labeling, enzyme was combined with varying concentrations of phosphonate inhibitor (3mM-30uM) at room temperature overnight. The reaction was stopped by the addition of SDS loading buffer and boiling for 10 minutes. Western blots were developed using the Vectastain ABC elite kit (Vector Labs).
Fluorescent substrate synthesis
Substrates corresponding to each inhibitor were synthesized by solid phase peptide synthesis. ACC-Rink-amide resin was obtained from Kimia Corp. The first amino acid was coupled using 5 equivalents each of amino acid, HATU, and collidine in dry DMF under argon for 16 hours with agitation. The full-length peptide was synthesized using a Symphony Quartet peptide synthesizer (Protein Technologies, Inc.), acetylated with 8 equivalents each of acetic anhydride and DIPEA, and cleaved with 95% TFA/2.5% water/ 2.5% triisopropyl silane. Cleaved peptides were precipitated into cold ether, collected by centrifugation, and purified by HPLC.
Cell culture and propagation
PC3 cells were obtained from the American Type Culture Collection (ATCC) and propagated in F12K Nutrient mixture with Kaighn’s Modification (1X) and L-Glutamine (GIBCO). Media was supplemented with 10% FBS and 1X Penicillin:Streptomycin. MCF7 cells were obtained from the ATCC and propagated in Dulbecco’s Modified Eagle Medium with high-glucose (D-ME H21) without phenol red (GIBCO). Media was supplemented with 10% FBS, 10ug/mL Insulin, and 1X Penicillin:Streptomycin. PDAC2.1 cells were isolated from p48-Cre/+, LSL-KrasG12D/+, Trp53F/+ transgenic mice according to (Nolan-Stevaux et al., 2009
). PDAC2.1 cells were propagated in D-ME H21 (GIBCO) supplemented with 10% FBS and 1X Penicillin:Streptomycin.
Cells were grown to confluency in 6-well cell culture-treated dishes using complete media appropriate for each specific cell line. Media was then aspirated and replaced with Opti-MEM serum-free medium (GIBCO). All experiments were done in triplicate. FTGSGBz-DPP (13) was added to the appropriate wells at a final concentration of 50mM. To test proteolysis, ABP was added in the presence of 1X Complete Protease Inhibitor Cocktail (Roche) dissolved in Opti-MEM. The cells were then incubated at 37°C for approximately 20hrs. After incubation, ABP-containing media was aspirated and cells were washed 3x with Opti-MEM. Cells were detached from the surface of the wells with Enzyme-free Cell Dissociation Buffer (GIBCO) for 15min. Detached cells were washed 2x with Opti-MEM, resuspended in 200uL, and incubated for 20min with Streptavidin:AlexaFluor 488 conjugate (Invitrogen) at 4°C. Cells were washed 3x with Opti-MEM and resuspended in 500uL Opti-MEM and assayed for fluorescence using a BD FACSCalibur (BD Biosciences). Data was analyzed and population mean fluorescence values were obtained using FlowJo Flow Cytometry Analysis Software (TreeStar, Inc.).
Cells were grown to confluency in 9.5cm2
glass bottom microwell dishes (MatTek) in complete media. Media was then aspirated and replaced with Opti-MEM. Phosphonate was added to each dish to a final concentration of 50mM. FTGSGBz-DPP (13
) was incubated for 20hrs at 37°C and QRVBz-DPP (1
) was incubated for 3hrs at 37°C, respectively. Cells were washed 3x with Opti-MEM and then incubated for 20min with Streptavidin:AlexaFluor 488 conjugate and tetramethylrhodamine-conjugated wheat germ agglutinin (Invitrogen), simultaneously, at 4°C. Cells were then washed 3x with Opti-MEM and imaged. Fluorescence microscopy was carried out in the wide field using a Nikon Diaphot with a Nikon 60x lens, numerical aperture 1.4, objective and standard interference filter sets (Omega Optical). Images were collected using a 12-bit cooled charge-coupled device camera (Princeton Instruments) interfaced to a computer running Micro-Manager 1.3 software (http://micro-manager.org
). Images were processed using Adobe Photoshop to assemble dual color image files. Brightness/Contrast was adjusted, where necessary, to improve image quality and clarity.
- Synthesis of peptide phosphonate ABPs has been optimized to increase yield.
- Peptide length is a key determinant of phosphonate inhibition.
- 3-D structure reveals an important steric contribution to phosphonate potency.
- ABPs were shown to label active S1A proteases on the surface of live cancer cells.