Small Molecule Inhibitors Targeting Tec Kinase Block Unconventional Secretion of Fibroblast Growth Factor 2^*

Biochemical Characterization of FGF2 Binding to Tec Kinase

A first set of experiments was based on biochemical pull-down experiments to probe for a direct interaction between FGF2 and Tec kinase as well as to define the domain in Tec kinase that binds to FGF2. FGF2 was expressed in Escherichia coli, purified to homogeneity (23, 24), and covalently coupled to epoxy beads (34). Various constructs of Tec kinase were expressed and purified from insect cells (Fig. 1A). These experiments revealed that the kinase domain (SH1) of Tec is sufficient for binding to FGF2 (Fig. 1B). Consistently, a construct containing the SH1, SH2, and SH3 domains of Tec (GST-NΔ173 Tec) did bind to FGF2 as well (Fig. 1B). By contrast, constructs restricted to either the PH and Tec homology (TH) domains (GST-PH-TH), the SH3/SH2 domains (GST-SH3-SH2), or GST alone did not bind to FGF2 (Fig. 1B). The Coomassie-stained gel shown in Fig. 1B was quantified using the LI-COR imaging system (Fig. 1C).

To determine affinity between FGF2 and Tec kinase, steady-state fluorescence polarization experiments were conducted. FGF2 was fluorescently labeled as explained under “Experimental Procedures,” and binding experiments with various Tec constructs were performed in solution (Fig. 2A). Similar to the results shown in Fig. 1, a direct interaction of FGF2 with both the SH1 kinase domain of Tec and GST-NΔ173 Tec was observed. The significance of these findings was confirmed by competition experiments using unlabeled FGF2. In the presence of 50 μm unlabeled FGF2, the interaction of labeled FGF2 with both the kinase domain (SH1) of Tec (Fig. 2B) and GST-NΔ173 Tec (Fig. 2C) was blocked. By contrast, FGF2 did not bind to GST-PH-TH, GST-SH3-SH2, or GST alone (Fig. 2A). To determine the affinity between FGF2 and Tec kinase, fluorescently labeled FGF2 (50 nm) was incubated with increasing concentrations (0.078–20 μm) of the various Tec constructs indicated (Fig. 2A). Data points were fitted, and dissociation constants were calculated as explained under “Experimental Procedures.” Dissociation constants of 1.434 μm ± 0.55 (S.E.) and 1.032 μm ± 0.29 (S.E.) were obtained for GST-NΔ173 Tec and the kinase domain (SH1) of Tec, respectively.

Large Scale Small Molecule Screening for Inhibitors That Block Binding of FGF2 to Tec Kinase

To identify small molecule inhibitors that prevent the interaction between FGF2 and Tec kinase, a screening assay was established based upon Alpha® technology (47). His-tagged FGF2 and GST-tagged NΔ173 Tec were used with glutathione donor and Ni-NTA acceptor beads, respectively. In a cross-titration experiment, suitable protein concentrations of FGF2 and NΔ173 Tec (see “Experimental Procedures”) were identified, providing a satisfying signal/noise ratio. Using these conditions, affinity between FGF2 and NΔ173 Tec was analyzed in a competition experiment. Based upon a titration curve with an untagged variant form of FGF2, NΔ25FGF2, a dissociation constant of 0.63 ± 0.03 μm (S.E.) was determined (Fig. 3). When analyzing an unrelated pair of interacting proteins, GST-Titin and His-tagged MBP-CARP, NΔ25FGF2 did not affect the Alpha® signal (Fig. 3). These findings establish a specific and direct interaction between FGF2 and NΔ173 Tec with a dissociation constant comparable with the results obtained in steady-state fluorescence polarization experiments (Fig. 2).

To identify small molecule inhibitors of the interaction between FGF2 and NΔ173 Tec, a small molecule compound library was screened using the AlphaScreen® protein-protein interaction assay described in the legend to Fig. 3. This scaffold-based collection comprises 79,000 compounds. The screening assay was performed at a final compound concentration of 40 μm and revealed 661 compounds that inhibit the interaction between FGF2 and NΔ173 Tec by >40%. The Titin/CARP assay described in the legend to Fig. 3 was used to deselect unspecific compounds. In addition, compounds that were found active toward other targets in previous screening campaigns based on Alpha® technology were deselected. This procedure resulted in a preliminary hit list of 141 compounds inhibiting the interaction between FGF2 and NΔ173 Tec. These compounds were reordered and tested in dose-response experiments to determine IC₅₀ values, both for the FGF2/Tec validation assay and the Titin/CARP deselection assay (Fig. 3). A set of 28 compounds was found to be characterized by IC50 values of <100 μm. Based upon (i) inhibitory strength in the primary screening assay, (ii) lack of activity in the Titin/CARP deselection assay, and (iii) inhibitory potency in in vitro phosphorylation experiments (see Figs. 6 and 7), a final set of three highly active compounds (compounds 6, 14, and 21) was identified (Fig. 4) (58). In addition, two structurally related but inactive compounds (compounds 18 and 19) were selected as controls for all subsequent experiments. With regard to chemical identities, compounds 6, 14, and 19 are based on a 4H-pyrido[1,2-a]pyrimidin-4-one scaffold. Compounds 6 and 14 have a 2-hydroxymethyl substituent that is condensed to a substituted pyrrole carboxylic acid moiety via an ester function. This ester linkage is potentially metabolically labile in vivo due to cleavage by esterases. In addition, compound 6 contains a methyl ester on the pyrrole moiety that is likely to be cleaved in vivo. Compound 19 is a more stable analogue of compound 14 that replaces the ester linkage with a much more metabolically stable amide moiety. However, this appears to abolish all activity, although the absence of the bromo substituent in compound 19 makes a definitive conclusion regarding the importance of the linker difficult. It is clear, however, that the 1-(2,4-dimethyl-1H-pyrrol-3-yl)ethanone moiety alone is not sufficient for activity because it is found both in active compound 14 and inactive compound 19. Compound 21 has an alternative 3H-imidazo[4,5-b]pyridine scaffold and contains the 1-(2,4-dimethyl-1H-pyrrol-3-yl)ethanone moiety also found in compounds 14 and 19. The imidazo-pyridine scaffold of compound 21 is partially isosteric to the pyrido-pyrimidone scaffold of compound 14, and indeed this compound retains activity. In addition, the ester linkage is replaced by a sulfanyl-acetyl type link. This indicates that the labile ester linkage found in compounds 6 and 14 can be replaced with more metabolically stable alternatives. Compound 18 has a [1,2,4]triazolo[1,5-a]pyrimidine scaffold, which is also partially isosteric to the pyrido-pyrimidone scaffold of compound 14. Instead of the pyrrole substituent present in all other compounds, it contains a coumarin moiety. The simultaneous modification of both termini of the molecule plus a different amide linker makes this compound rather different from the other active compounds, and it can be considered as a negative control compound.

Determination of IC₅₀ Values of Active Compounds

In addition to dose-response experiments that were conducted under screening conditions (see above), we carefully analyzed the inhibitory potential of the identified compounds along with their inactive derivatives (Fig. 5). Using the Alpha® protein-protein interaction assay, IC₅₀ values of 8.9 ± 1.1 μm (S.E.) for compound 6 (Fig. 5A), 7.0 ± 1.1 μm (S.E.) for compound 14 (Fig. 5B), and 11.7 ± 1.0 μm (S.E.) for compound 21 (Fig. 5C) were determined. By contrast, the inactive compounds 18 (Fig. 5D) and 19 (Fig. 5E) did not exert significant inhibition of the interaction between His-tagged FGF2 and GST-NΔ173 Tec at concentrations of up to 200 μm. Importantly, neither the active nor the inactive compounds affected the deselection assay probing for a potential inhibitory activity toward the unrelated interaction between Titin and CARP (Fig. 5, A–E). Also, none of the compounds were active in a technical control assay in which a single fusion protein (GST-His-Biotin) was used to directly link Alpha® donor and acceptor beads (Fig. 5, A–E). Our combined findings from Fig. 5 establish compounds 6, 14, and 21 (Fig. 4) (58) as selective and potent inhibitors of the interaction between FGF2 and NΔ173 Tec that are characterized by IC50 values in the low micromolar range.

Small Molecule Inhibition of Tec Kinase-catalyzed Tyrosine Phosphorylation of FGF2

To analyze the potential of the active compounds beyond protein-protein interaction assays (Fig. 5), we tested whether they affect tyrosine phosphorylation of FGF2 mediated by Tec kinase (Fig. 6). Purified FGF2 and NΔ173 Tec were incubated under the conditions indicated. Tyrosine phosphorylation of both GST-NΔ173 Tec (autophosphorylation) and FGF2 was monitored by quantitative Western analysis using antibodies directed against a phosphopeptide derived from FGF2. These antibodies also recognized the autophosphorylated form of GST-NΔ173 Tec (Fig. 6A). Otherwise, they were found to be characterized by a low background recognition of non-phosphorylated FGF2 and Tec kinase (Figs. 6 and 7) and proved highly specific for the phosphorylated form of tyrosine 81 (Fig. 6, D–F), the key phosphorylation site targeted by Tec kinase (12, 24, 31). Protein amounts were monitored for all conditions employing SDS-PAGE and Coomassie staining (Fig. 6B). These experiments revealed potent inhibition of FGF2 tyrosine phosphorylation by the three active compounds 6, 14, and 21 (Fig. 6A). The Western analysis shown in Fig. 6A was quantified, revealing about 75% inhibition by compound 6 and almost quantitative inhibition by compounds 14 and 21 (Fig. 6C). Interestingly, autophosphorylation of NΔ173 Tec was affected as well, albeit less efficiently compared with FGF2 phosphorylation (Fig. 6, A and C). By contrast, the control compounds 18 and 19 affected neither NΔ173 Tec autophosphorylation nor FGF2 tyrosine phosphorylation (Fig. 6, A and C). Therefore, beyond the analysis of protein-protein interactions (Fig. 5), these experiments establish inhibitory potency of the identified active compounds with regard to FGF2 tyrosine phosphorylation mediated by Tec kinase.

Selectivity of Active Compounds Regarding Different Substrates of Tec Kinase

To test whether the active compounds 6, 14, and 21 selectively block Tec-mediated tyrosine phosphorylation of FGF2, we tested another established substrate of Tec, STAP1 (48, 49). In this independent set of experiments, the inhibitory potency of compounds 6, 14, and 21 toward Tec-mediated FGF2 tyrosine phosphorylation was confirmed (Fig. 7, A and D). Interestingly, the observed inhibition of Tec autophosphorylation was not only observed in the presence of FGF2 but, albeit less efficiently in case of compounds 6 and 21, in its absence as well (Fig. 7, B and E). This effect was particularly evident for compound 14. Finally, we tested whether the active compounds affect Tec-mediated tyrosine phosphorylation of STAP1 (Fig. 7, C and F). Intriguingly, in the presence of STAP1, neither autophosphorylation of NΔ173 Tec nor tyrosine phosphorylation of STAP1 was affected by any of the compounds we found active toward FGF2 phosphorylation. These findings suggest that the active compounds 6, 14, and 21 bind to Tec kinase and prevent recruitment of FGF2, resulting in a loss of FGF2 tyrosine phosphorylation. In the absence of a bound substrate, autophosphorylation of NΔ173 Tec is inhibited as well. However, in the presence of an unrelated substrate, such as STAP1, neither autophosphorylation of NΔ173 Tec nor tyrosine phosphorylation of STAP1 is affected by compounds 6, 14, and 21. Therefore, our combined findings establish selectivity of these inhibitors toward FGF2 as a substrate of Tec, reflecting the design of the original small molecule screening procedure aiming at FGF2/Tec protein-protein interaction inhibitors.

Active Compounds Targeting the Interaction between Tec Kinase and FGF2 Inhibit Tyrosine Phosphorylation of FGF2 in Cells

A first set of experiments in cells was conducted to analyze whether the active compounds 6, 14, and 21 inhibit tyrosine phosphorylation of FGF2 in cells (Fig. 8). Briefly, cells were induced with doxycycline to express FGF2-GFP in the presence of active (compounds 6, 14, and 21) and inactive (compounds 18 and 19) compounds as well as under mock conditions (for details, see “Experimental Procedures”). FGF2-GFP fusion proteins were affinity-purified under the conditions indicated using GFP trap magnetic beads and analyzed by quantitative Western blotting (Fig. 8A). Total FGF2-GFP was quantified with an affinity-purified anti-FGF2 antibody (26, 50). Tyrosine phosphorylation of FGF2-GFP was monitored using the anti-Tyr(P) antibody 4G10 (Merck Millipore). The LI-COR dual channel imaging platform was used to quantify tyrosine-phosphorylated FGF2-GFP normalized by the amount of total FGF2-GFP (Fig. 8B). These experiments revealed that all three active compounds inhibit tyrosine phosphorylation of FGF2 in cells. By contrast, compared with mock conditions, the inactive control compounds 18 and 19 did not inhibit this process to a significant extent. Intriguingly, although the active compounds 14 and 21 showed partial inhibition, the most active inhibitor was compound 6 that blocked tyrosine phosphorylation of FGF2 down to background levels (Fig. 8B). These experiments demonstrate that, beyond the in vitro reconstitution experiments shown in Figs. 5–7, the identified Tec/FGF2 protein-protein interaction inhibitors are capable of inhibiting tyrosine phosphorylation of FGF2 in a cellular context.

Small Molecule Inhibitors Blocking FGF2 Tyrosine Phosphorylation Inhibit FGF2 Secretion from Cells

Based upon the biochemical reconstitution experiments shown in Figs. 5–7 and the cell-based experiments shown in Fig. 8, we investigated whether the active compounds 6, 14, and 21 influence FGF2 secretion from cells (Fig. 9). Again, the inactive compounds 18 and 19 were taken as negative controls. To quantify FGF2 secretion, a well established biotinylation assay was used to detect FGF2 bound to heparan sulfates on cell surfaces (23, 26, 51). Briefly, following doxycycline-induced expression of FGF2-GFP, proteins present on cell surfaces were modified using a membrane-impermeable biotinylation reagent. Following cell lysis, biotinylated proteins (cell surface) were separated from non-biotinylated proteins (intracellular) using streptavidin beads. Samples were subjected to SDS-PAGE followed by a Western analysis of FGF2-GFP and GAPDH, the latter being a control for a protein restricted to the intracellular environment. Cell surface biotinylation of FGF2-GFP was analyzed under various experimental conditions, comparing a mock control with a titration (10, 25, and 50 μm) of both active (compounds 6, 14, and 21) and inactive (compounds 18 and 19) compounds (Fig. 9). Under these conditions, cell surface signals for FGF2-GFP were quantified using the LI-COR imaging platform. This analysis revealed all active compounds identified in the experiments shown in Figs. 5–8 to significantly inhibit FGF2 secretion from cells (Figs. 8 (B and C) and 9A). Importantly, the most active inhibitor (compound 6) identified in the cell-based experiments shown in Fig. 8 was also the most efficient one with regard to inhibition of FGF2 secretion. For compounds 6 and 14, inhibition was observed in a dose-dependent manner. In the case of the active compound 21, inhibition was stronger at 10 μm compared with higher concentrations. This observation might be related to solubility issues because aggregates of compound 21 were increasingly observable at concentrations of 25 and 50 μm (data not shown). Importantly, the inactive compounds 18 and 19 did not affect FGF2 secretion from cells (Fig. 9, D and E). In addition, neither the active nor the inactive compounds exerted substantial pleiotropic effects on cell proliferation (Fig. 10). Using an IncuCyte Zoom live cell imaging platform, we quantified cell proliferation under mock conditions versus the presence of active and inactive compounds. Although a modest decrease of cell proliferation could be observed, cells were viable and continued to proliferate under all experimental conditions (Fig. 10). Our combined findings establish the active compounds 6, 14, and 21 as potent and selective inhibitors of Tec kinase-mediated tyrosine phosphorylation (Figs. 5–8) that are also functional in a cellular context inhibiting FGF2 secretion (Figs. 9 and 10).

Expression and Purification of Recombinant Proteins

Several recombinant forms of both FGF2 and Tec kinase were used in this study. These included authentic FGF2 (154 amino acids; 18-kDa form), His-tagged FGF2 (including the variant forms Y81F, Y111F, and Y123F), and NΔ25FGF2 (129 amino acids) as well as four forms of Tec kinase with the domain structures depicted in Fig. 1A (GST-NΔ173 Tec, GST-PH-TH, GST-SH3-SH2, and the SH1 kinase domain).

All forms of FGF2 were expressed and purified from E. coli according to standard procedures (23, 24). Untagged FGF2 was expressed as an N-terminal GST fusion protein. Following binding to glutathione affinity beads (glutathione-Sepharose^TM 4 Fast Flow, GE Healthcare), the GST tag was cleaved off by thrombin treatment. The product was collected and loaded onto a heparin affinity column (HiTrap Heparin HP, GE Healthcare), and, following elution under high salt conditions, a homogeneous fraction of untagged FGF2 was collected. His-tagged FGF2 was purified by Ni-NTA affinity (HisTrap FF, GE Healthcare) and heparin affinity chromatography. N-terminally truncated FGF2 (NΔ25FGF2; 129 amino acids) lacking the first 25 amino acids was expressed and purified to homogeneity using heparin affinity chromatography. Purified His-STAP1 was purchased from Hölzel Diagnostika (catalog no. GWB-P0486E-100).

All Tec kinase-derived constructs depicted in Fig. 1A (GST-NΔ173 Tec, GST-PH-TH, GST-SH3-SH2, and the SH1 kinase domain) were expressed in SF9 insect cells using the baculovirus expression system (53). GST-NΔ173 Tec, GST-PH-TH, and GST-SH3-SH2 were purified by glutathione affinity chromatography (GSTrap^TM FF, GE Healthcare). By contrast, the SH1 kinase domain was purified based on an N-terminal His tag employing Ni-NTA affinity chromatography. The His tag was removed proteolytically followed by a second purification step based on size exclusion chromatography (HiLoad 16/600 Superdex^TM 200 pg, GE Healthcare), yielding a homogeneous preparation of the SH1 kinase domain.

Pull-down Experiments Using FGF2-conjugated Epoxy Beads and Various Forms of Tec Kinase (Fig. 1)

FGF2 (2 mg; untagged 18-kDa form) was covalently conjugated to epoxy-activated Sepharose^TM beads 6B (600 mg; catalog no. 17-0480-01, GE Healthcare) based on the manufacturer's instructions. FGF2-conjugated beads (final concentration = 0.2 μm FGF2) was incubated on a rotating platform for 2 h at room temperature with each of the Tec variant forms depicted in Fig. 1A (final concentration = 1.5 μm in a total volume of 200 μl). The binding buffer contained 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1 mm benzamidine, 0.05% Tween 20, 1 mm DTT. Proteins bound to beads were collected by centrifugation (7000 × g, 4 °C, 5 min). The beads were washed extensively with binding buffer. Bound proteins were eluted with SDS sample buffer, and, following boiling at 95 °C, bound (100%) and unbound (1%) fractions were analyzed by SDS-PAGE (Novex NuPAGE 4–12% BisTris/MOPS precast gels). Protein gels were stained with Coomassie InstantBlue (Expedeon). Marker proteins of 140, 115, 80, 65, 50, 40, 30, 25, 15, and 10 kDa (PageRuler prestained protein ladder; catalog no. 26616, Thermo Scientific) were used to monitor migration behavior of proteins.

Determining the Dissociation Constant for the Interaction between Tec Kinase and FGF2 Employing Steady-state Fluorescence Polarization (Fig. 2)

To determine binding affinity between Tec kinase and FGF2, steady-state fluorescence polarization was used (54). His-tagged FGF2 was labeled with fluorescein-5-maleimide (F150, Thermo Fisher Scientific) covalently modifying a cysteine residue on the molecular surface of FGF2. Briefly, 100 nmol of His-tagged FGF2 (dissolved in 50 mm Tris-HCl, 150 mm NaCl, pH 7.4) were incubated with a 10-fold molar excess of tris(2-carboxyethyl)phosphine hydrochloride (Pierce) at room temperature. Fluorescein-5-maleimide (44 nmol) was added, resulting in a final volume of 0.8 ml. Following incubation for 20 min at room temperature, residual amounts of free dye were removed using PD-10 desalting columns (GE Healthcare). The labeled protein was concentrated using a Vivaspin 500 system with a cut-off of 10 kDa (Sartorius). Based on the manufacturer's instructions (F150, Thermo Fisher Scientific) using the extinction coefficient of fluoroscein 5-maleimide and the absolute protein concentration, the degree of labeling of His-tagged FGF2 was determined to be 30%. The final preparation of fluorescein-labeled FGF2 had a protein concentration of 1.7 mg/ml. All proteins used in these assays were adjusted to the same buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.4) using PD-10 desalting columns (GE Healthcare). Absolute protein concentrations were determined based upon the extinction coefficients and the molecular weights of each protein measuring absorption at 280 nm using a NanoDrop 1000 spectrophotometer.

To determine the dissociation constants for the interaction of FGF2 with various forms of Tec kinase, fluorescein-labeled FGF2 (50 nm) was incubated with increasing concentrations (0–20 μm, as indicated) of GST-NΔ173 Tec, GST-PH-TH, GST-SH3-SH2, and the SH1 kinase domain of Tec (Fig. 2A). In addition, GST alone was taken as a negative control. Following incubation for 3 h at room temperature in binding buffer (50 mm Tris-HCl, 150 mm NaCl, pH 7.4, 0.1% BSA, 0.05% Tween 20; final volume = 10 μl), steady-state fluorescence polarization experiments were conducted by excitation of labeled FGF2 with polarized light at a wave length of 492 nm. The interaction between fluorescein-labeled FGF2 and unlabeled forms of Tec kinase was measured at 515 nm as the progressive increase of fluorescence polarization (ΔPolarization) as a function of the protein concentration of the various forms of Tec kinase. Samples were measured in black low volume 384-well plates (catalog no. 6008260, ProxiPlate^TM, PerkinElmer Life Sciences), and data were collected with a SpectraMax M5 plate reader from Molecular Devices equipped with SoftMax Pro software (version 5.4.5 for Macintosh OS X). Data were expressed as change in polarization (ΔPolarization) normalizing each individual replicate by the corresponding polarization value of fluorescein-labeled FGF2 in the absence of Tec kinase. The corresponding binding curve was used to calculate the dissociation constant with data fitting using the quadratic solution of the law of mass action,

where P₀ and P_m represent minimum and maximum values for the polarization, x is the Tec concentration, L is the concentration of labeled FGF2, and K is the dissociation constant. Fitting was performed using GraphPad Prism (version 5.0 for Macintosh OS X).

To establish a specific interaction between FGF2 and GST-NΔ173 Tec as well as the SH1 kinase domain of Tec, competition experiments with unlabeled FGF2 were performed (Fig. 2, B and C). Experiments were conducted at 50 nm fluorescein-labeled His-FGF2 in the presence and absence of 1.25 μm GST-NΔ173 Tec or the SH1 kinase domain under the conditions described above. In addition, conditions were varied based upon the presence or absence of an unlabeled FGF2 competitor molecule at 50 μm.

A Protein-Protein Interaction Assay Designed to Screen Small Molecule Libraries for Compounds Inhibiting FGF2 Binding to Tec Kinase (AlphaScreen® Assay)

To quantify the interaction of FGF2 with Tec kinase under screening conditions, an assay was established based upon Alpha® technology (47). Briefly, this methodology is based on a pair of proteins of interest that bind to Alpha® donor and acceptor beads, respectively. Upon excitation of donor beads at 680 nm, singlet oxygen species are produced that can diffuse within a range of about 200 nm. If donor and acceptor beads are in close proximity due to the protein-protein interaction, singlet oxygen species will transfer energy to acceptor beads. This in turn causes emission of light with a wavelength of 520 nm that is detected as the Alpha® signal.

For all individual preparations and experiments of His-tagged FGF2 and GST-tagged NΔ173 Tec, optimal protein concentrations in AlphaScreen® assays were determined in cross-titration experiments. Typically, GST-tagged NΔ173 was used at a concentration of 30 nm, and His-tagged FGF2 was used in a range between 62.5 and 125 nm (data not shown). To quantify binding affinity between His-tagged FGF2 and GST-NΔ173 Tec and to assess the specificity of the Alpha® signal for their interaction, competition experiments were conducted with an untagged version of FGF2 (NΔ25-FGF2; Fig. 3). As specificity control for the competition by untagged FGF2, an Alpha® assay was conducted using an unrelated protein-protein interaction pair (GST-Titin/His-MBP-CARP) at a final concentration of 20 nm each. Briefly, after incubation of the proteins for 75 min at room temperature in PBS (supplemented with 0.1% BSA, 0.05% Tween 20), glutathione donor beads (catalog no. 6765300, PerkinElmer Life Sciences) and Ni-NTA acceptor beads (catalog no. 6760619, PerkinElmer Life Sciences) were added at a concentration of 7.5 μg/ml in a final volume of 15 μl. Following incubation for an additional 2 h, Alpha® protein interaction signals were recorded. All Alpha® assays were carried out in white low volume 384-well plates (catalog no. 6008280, ProxiPlate^TM) using an EnVision plate reader (PerkinElmer Life Sciences). The obtained data points were fitted with GraphPad Prism (version 5.0 for Macintosh OS X) using the cubic solution of the law of mass action for competitive binding of two ligands to one binding site (cubic equation (55)) to calculate the dissociation constant for the interaction between GST-NΔ173 Tec and His-tagged FGF2.

Screening of a Collection of Small Molecules for Inhibitors of the Interaction between FGF2 and Tec Kinase

AlphaScreen® experiments were performed as described above. GST-NΔ173 Tec (30 nm) and His-tagged FGF2 (125 nm) were combined in screening buffer (20 mm Tris-HCl, 150 mm NaCl, 1 mm DTT, 0.1% BSA, 0.05% Tween 20) and preincubated for 60 min in the presence of compounds or under mock control conditions (DMSO). Afterward, glutathione-coated donor and Ni-NTA-coated acceptor beads were added. Following 60 min of incubation, Alpha® signals were recorded using an EnVision^TM plate reader (PerkinElmer Life Sciences). In the primary screen, 79,000 compounds of a scaffold-based library were tested at a final concentration of 40 μm. Reordered or synthesized compounds were tested in dose-response experiments using an 11-fold 1:1 serial dilution starting at 200 μm. The serially diluted compounds were also tested in a deselection assay with a non-related protein pair, GST-Titin and His-tagged MBP-CARP (20 nm each), to evaluate the specificity of compounds inhibiting the interaction of GST-NΔ173 Tec and His-tagged FGF2. Finally, GST-His-Biotin, a fusion protein containing both an N-terminal GST tag and a C-terminal His tag, was used at 20 nm as an additional technical control. To generate dose-response curves and corresponding IC₅₀ values (Fig. 5; 13-fold 1:2 serial dilution starting at 200 μm), compounds were reordered from an independent batch of synthesis (compound 6, catalog no. Z51196782; compound 14, catalog no. Z15516480; compound 21, catalog no. Z17100166; compound 18, catalog no. BJ01-04; compound 19, catalog no. BJ01-05 (all compounds from Enamine)). Compound powders were dissolved in DMSO with a stock concentration of 10 mm, incubated for 10 min at 37 °C, and further treated by sonication for 5 min at room temperature. Compound stock solutions were stored at −20 °C.

Biochemical Reconstitution of Tec Kinase-mediated Tyrosine Phosphorylation of FGF2 and STAP1 (Figs. 6 and 7)

A Tec kinase in vitro phosphorylation assay was established to test the inhibitory potential and selectivity of the small molecule inhibitors reported in this study (Figs. 6 and 7). Tyrosine phosphorylation of FGF2 and STAP1, another established substrate of Tec kinase (48, 49), was tested in a final volume of 100 μl (60 mm HEPES, pH 7.5, 3 mm MgCl₂, 20 μm ATP, 3 μm sodium orthovanadate, 1.2 mm DTT, 50 ng/μl PEG 20000, 0.05% (w/v) Tween 20, 0.001% BSA). GST-NΔ173Tec (60 nm) was incubated in the absence (1% DMSO mock control) or presence of each compound (50 μm in 1% DMSO). Where indicated, reactions contained a 250 nm concentration of either FGF2 or STAP1 (Hölzel Diagnostika, catalog no. GWB-P0486E-100). Following incubation at 30 °C for 45 min (corresponding to the midpoint of linear product formation), the reaction was terminated using SDS sample buffer and incubation for 4 min at 95 °C. Proteins were separated by SDS-PAGE (BisTris 4–12% acrylamide gradient) and transferred to PVDF membranes. Phosphorylation levels of GST-NΔ173 Tec (autophosphorylation), FGF2, and STAP1 were quantified by a Western analysis using affinity-purified, polyclonal anti-phosphotyrosine antibodies (rabbit) raised against a FGF2-derived synthetic peptide containing a phosphorylated tyrosine residue (ANRpYLAMKED, where pY represents phosphotyrosine). This antibody was found to recognize both tyrosine-phosphorylated FGF2 and autophosphorylated Tec kinase. Phosphorylation levels of STAP1 were monitored using a monoclonal anti-phosphotyrosine antibody (clone 4G10®, catalog no. 05-321, Merck Millipore). Appropriate fluorescent secondary antibodies (goat anti-rabbit IgG conjugated with Alexa Fluor® 680, catalog no. A-21076, Life Technologies, Inc.; goat anti-mouse IgG conjugated with Alexa Fluor® 680, catalog no. A-21057, Life Technologies) were used to quantify all antigens employing the LI-COR Odyssey infrared imaging platform. In Fig. 6B, an SDS gel corresponding to the Western analysis shown in Fig. 6A was stained with Coomassie InstantBlue (Expedeon) to control for protein amounts and purity. The PageRuler prestained protein ladder was used, containing marker proteins with the following molecular masses: 140, 115, 80, 65, 50, 40, 30, 25, 15, and 10 kDa (Thermo Scientific/Fermentas, catalog no. 26616).

Quantification of FGF2 Tyrosine Phosphorylation in Cells (Fig. 8)

CHO cells expressing FGF2-GFP in a doxycycline-dependent manner were incubated in the presence of active (compounds 6, 14, and 21) and inactive (compounds 18 and 19) compounds (50 μm in 0.5% DMSO) as well as under mock conditions (0.5% DMSO). Incubation times were identical to those in experiments quantifying FGF2 secretion and cell viability (see below; Figs. 9 and 10). Cells were lysed in a detergent-containing buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% (w/v) Nonidet P-40, 1 mm sodium vanadate, 0.1% 2-mercaptoethanol, 1× EDTA-free protease inhibitor mixture from Roche Applied Science, 1× phospho-stop mixture from Roche Applied Science). Following lysis using sonication, a clear supernatant was obtained by low speed centrifugation, removing aggregated material. GFP trap magnetic beads (Chromotek; GFP-Trap®_MA) were added to affinity-purify FGF2-GFP according to standard procedures. Beads were treated with SDS sample buffer followed by SDS-PAGE and Western blotting. Signals were quantified using the LI-COR imaging platform. Antigens were detected using anti-phosphotyrosine antibodies (4G10; Millipore) and affinity-purified anti-FGF2 antibodies (26, 50).

Quantification of FGF2 Secretion Using Cell Surface Biotinylation (Fig. 9)

Secreted FGF2 remains bound to heparan sulfates on cell surfaces without release into cell culture supernatants (8, 50). This allows for biochemical detection of the secreted population based upon cell surface biotinylation experiments (8, 23, 26, 51). CHO cells stably expressing FGF2-GFP in a doxycycline-dependent manner were cultivated in the presence of compounds 6, 14, 21, 18, and 19 (see Figs. 4 (58) and 5) at 10, 25, and 50 μm in 0.5% DMSO (final concentration) as well as under mock conditions. Following incubation at 37 °C for 24 h, in the continued presence of compounds, doxycycline was added to induce expression of FGF2-GFP. After another 16 h of cultivation at 37 °C, cells were treated with a membrane-impermeable biotinylation reagent, EZ-Link Sulfo-NHS-SS-Biotin (sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate, catalog no. 21331; Pierce), targeting primary amines on cell surface proteins. Following quenching and detergent-mediated cell lysis, biotinylated material and non-biotinylated material were separated from each other using streptavidin beads (UltraLink immobilized streptavidin, Pierce). Aliquots from the total cell lysate (corresponding to input) and aliquots of the purified biotinylated fractions (corresponding to the secreted population) were subjected to SDS-PAGE and Western analysis. Affinity-purified anti-GFP antibodies (50) and monoclonal anti-GAPDH antibodies (Lifetech-Ambion) were used to detect FGF2-GFP and GAPDH, respectively. GAPDH was used as a control protein localized exclusively to the intracellular space. Using appropriate fluorescent secondary antibodies, FGF2-GFP and GAPDH were quantified using the LI-COR Odyssey imaging platform. FGF2 secretion under mock conditions (0.5% DMSO) was defined as 100% secretion efficiency.

Quantitative Analysis of Cell Proliferation (Fig. 10)

To detect potential pleiotropic effects on cell viability of small molecule inhibitors, we monitored cell proliferation in the absence and presence of the compounds introduced in Fig. 4 (58). CHO cells constitutively expressing mCherry fused to a nuclear localization signal (56, 57) were cultivated under conditions equivalent to those in the experiments shown in Fig. 9. Cell proliferation was quantified in real time, employing an IncuCyte® Zoom live cell imaging microscope (Essen BioScience), providing an absolute quantification of fluorescent cell nuclei with kinetic resolution (34). In this way, cell proliferation was monitored for 72 h (with measurements conducted every 2 h) under the conditions indicated (Fig. 10).