Facile Double-Functionalization of Designed Ankyrin Repeat Proteins using Click and Thiol Chemistries

Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of “clickable” DARPins at about 30 mg per liter <i>E. coli</i> culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained <i>in vitro</i>. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications

Downregulation of SK-1 induces endoreduplication.

<p>(A) MDA-MB-231 wt cells and SK-1 kd cells were stained with propidium iodide and DNA content was measured by flow cytometry using a FACSCalibur flow cytometer and the Cell Quest software for data processing. (B) confocal microscopy of MDA-MB-231 wt and SK-1 kd cells upon actin (red) and nuclear staining (blue) using TRITC-labeled phalloidin (dilution 1∶1000) and DAPI, respectively. (C) calculation of the nuclear size of SK-1 kd cells relative to wt cells. (D) quantification of cells with nuclei in different size ranges (<10 µM, >10 µM, >15 µM). Data are means ± SD of three independent experiments; **p<0.01, ***p<0.001 compared to MDA-MB-231 wt cells values.</p

Downregulation of SK-1 compromises M phase arrest and sensitizes cells to taxol.

<p>(A) MDA-MB-231 wt and SK-1 kd cells were treated with various concentrations of taxol or DMSO as vehicle control for 24 h before apoptosis was measured by annexin V staining using flow cytometry. Data are means ± SD (n = 3); ***p<0.001 compared to DMSO treated cells. (B)<b>,</b> detection of phospho-histone H3(Ser10) as a marker of the mitotic index in MDA-MB-231 wt and SK-1 kd cells at different time points upon treatment with taxol (100 nM). Lysates were subjected to Western blotting using antibodies against total histone H3 (dilution 1∶2000) and phospho-histone H3(Ser10) (dilution 1∶1000). The Density of phospho-histone H3(Ser10) (pH3)/H3 ratio in taxol-treated MDA-MB-231 cells is presented as % of total H3. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); ***p<0.001 compared to wt cells. (C) MDA-MB-231 wt cells were treated with taxol (100 nM), the SK-1 inhibitor SK I II (10 µM), sphingosine (20 µM) or the various combinations for 48 h. DMSO was used as vehicle control. Cell viability was determined in colorimetric MTT assays. Data are means ± S.D. (n = 3); *p<0.05, ***p<0.001 compared to DMSO treated cells; ^#p<0.05, ^###p<0.001 compared to the respective taxol treated cells; ^¥¥¥p<0.001 compared to the respective SK I II treated cells.</p

Downregulation of SK-1 inhibits cdc2 activity and decreases Chk1 expression.

<p>(A) lysates from MDA-MB-231 wt, SK-1 kd, and SK-1 overexpressing cells (SK-1 ov) were subjected to Western blotting using antibodies against total cdc2 (dilution 1∶2000), phospho-cdc2(Tyr15) (dilution 1∶1000), total cyclin B1, and phospho-cyclin B1(Ser133) (dilution 1∶1000). The films were digitized and for each protein lane a density blot was measured. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); ***p<0.001 compared to wt cells (B) MDA-MB-231 wt cells were treated with the cdc2 inhibitor RO 3306 (9 µM) or DMSO as vehicle control for 24 h, stained with propidium iodide and DNA content was measured as described in Fig. 4. (C) Chk1 mRNA expression in MDA-MB-231 wt and SK-1 kd cells was measured by quantitative real-time PCR and data were normalized to 18S rRNA. ***p<0.001 compared to wt values. (D) Chk1 protein expression in MDA-MB-231 wt and SK-1 kd cells detected by Western blotting using antibodies against Chk1 (dilution 1∶500) or GAPDH (dilution 1∶2000) as loading control. The films were digitized and for each protein lane a density plot was measured. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); **p<0.01 compared to wt cells.</p

Downregulation of SK-1 results in sphingosine accumulation.

<p>(A) MDA-MB-231, NCI-H358, and HCT 116 cells were transduced with lentiviral SK-1 shRNA to downregulate SK-1 expression (SK-1 kd) or left untreated (wild-type, wt). MDA-MB-231 cells transduced with empty virus or transfected with the SK-1 cDNA for overexpression (SK-1 ov) are shown for comparison. Cell lysates were prepared and SK-1 protein expression was detected by Western blotting using antibodies against SK-1 (dilution 1∶1000) or GAPDH (dilution 1∶2000) as loading control. The two SK-1 splice variants SK-1a and SK-1b run at 43 and 51 kDa, respectively. The films were digitized and for each protein lane a density blot was measured. Each value in the graph represents the mean ± SD band density for each group (<i>n = </i>3); ***p<0.001 compared to wt cells. (B) cellular sphingosine was determined in the genetically modified SK-1 kd carcinoma cell lines, in the respective wt cells, and additional MDA-MB-231 control cells as described above (empty virus transfected, SK-1 ov). Lipid extractions were performed and sphingosine was quantified by mass spectrometry. Data are means ± SD (<i>n = </i>3); *p<0.05, **p<0.01, ***p<0.001 compared to wt cells. (C) endogenous sphingosine in the wt carcinoma cell lines upon treatment with the SK-1 inhibitor SK I II (10 µM) or DMSO as vehicle control for 24 h. Quantification was done as above. Data are means ± SD (n = 3); *p<0.05, **p<0.01 compared to DMSO treated cells.</p

Downregulation of SK-1 increases sphingosine which inhibits PKC.

<p>(A) lysates were prepared from MDA-MB-231, NCI-H358, and HCT 116 wt and SK-1 kd cells, and analyzed for PKC activity by Western blotting using antibodies against various p(Ser) PKC substrates (dilution 1∶1000) or GAPDH (dilution 1∶2000) as loading control. MDA-MB-231 cells treated with sphingosine or DMSO vehicle control were analyzed for comparison. (B) cells were treated with the PKC inhibitors CGP 41-251 (300 nM) or Ro 31-8220 (1 µM) or with DMSO as control for 24 h before collection of lysates or quiescent MDA-MB-231 wt cells were pretreated for 4 h with the PKC inhibitors Ro 31-8220 (1 µM), CGP 41-251 (300 nM) or DMSO as control. Thereafter, cells were stimulated with the PKC activator TPA (200 nM) for 15 min. and lysates were analyzed for p(Ser) PKC substrates as described above. (C) quiescent MDA-MB-231 wt and SK-1 kd cells were stimulated with the PKC activator TPA (200 nM) for 15 min, treatment of SK-1 kd cells with lactacystin (20 µM for 24 h) was used to control for protein degradation. Lysates were analyzed for p(Ser) PKC substrates as described above. (D) MDA-MB-231 wt and SK-1 overexpressing (SK-1 ov) cells were analyzed for p(Ser) PKC substrates as described above.</p

Inhibition of PKC decreases colony formation of carcinoma cells.

<p>MDA-MB-231 wt cells were seeded in 60 mm diameter dishes at a density of 700 cells per dish in cell culture medium. After 24 h cells were treated with various concentrations of the PKC inhibitors CGP 41-251, Ro 31-8220 or with sphingosine, DMSO was used as vehicle control. Colony formation of SK-1 kd cells (untreated) incubated under identical conditions is shown for comparison. Cells were incubated for another 14 d before colonies were stained with 2% crystal violet and counted using a ColCountTM (Mammalian Cell Colony Counter, Oxford Optronix). Only colonies containing more than 50 cells were evaluated. Data are means ± S.D. (n = 3); *p<0.05, **p<0.01, ***p<0.001 compared to the DMSO control values.</p

Novel Prodrug-Like Fusion Toxin with Protease-Sensitive Bioorthogonal PEGylation for Tumor Targeting

Highly potent biotoxins like Pseudomonas exotoxin A (ETA) are attractive payloads for tumor targeting. However, despite replacement of the natural cell-binding domain of ETA by tumor-selective antibodies or alternative binding proteins like designed ankyrin repeat proteins (DARPins) the therapeutic window of such fusion toxins is still limited by target-independent cellular uptake, resulting in toxicity in normal tissues. Furthermore, the strong immunogenicity of the bacterial toxin precludes repeated administration in most patients. Site-specific modification to convert ETA into a prodrug-like toxin which is reactivated specifically in the tumor, and at the same time has a longer circulation half-life and is less immunogenic, is therefore appealing. To engineer a prodrug-like fusion toxin consisting of the anti-EpCAM DARPin Ec1 and a domain I-deleted variant of ETA (ETA″), we used strain-promoted azide alkyne cycloaddition for bioorthogonal conjugation of linear or branched polyethylene glycol (PEG) polymers at defined positions within the toxin moiety. Reversibility of the shielding was provided by a designed peptide linker containing the cleavage site for the rhinovirus 3C model protease. We identified two distinct sites, one within the catalytic domain and one close to the C-terminal KDEL sequence of Ec1-ETA″, simultaneous PEGylation of which resulted in up to 1000-fold lower cytotoxicity in EpCAM-positive tumor cells. Importantly, the potency of the fusion toxin was fully restored by proteolytic unveiling. Upon systemic administration in mice, PEGylated Ec1-ETA″ was much better tolerated than Ec1-ETA″; it showed a longer circulation half-life and an almost 10-fold increased area under the curve (AUC). Our strategy of engineering prodrug-like fusion toxins by bioorthogonal veiling opens new possibilities for targeting tumors with more specificity and efficacy

Biochemical characterization of StSPL.

<p>(A) Purity of purified WT StSPL. The molecular weight marker is shown in lane 1, the pooled fractions after size-exclusion chromatography were detected by Coomassie staining of the gel (lane 2) and by Western blotting with an antibody recognizing the C-terminal His-tag (lane 3). (B) Schematic representation of the StSPL dimer. Subunit A is depicted in grey, whereas subunit B is in black. A phosphate ion found in the active site of both subunits is depicted as a red dot, while the cofactor (PLP) is denoted by a blue hexagon. (C) Spectrophotometric activity assay of WT StSPL. The red curve represents the visible spectrum of the native protein before addition of substrate, corrected by the dilution factor. The black curves depict the visible spectra at regular intervals (1 min, 2, 4, 6, 8, 10, 12, 15, and 30 min) after addition of S1P. The transient peaks at 420 and 403 nm appearing upon addition of substrate correlate with protein activity. (D) Mass spectrometric activity assay of WT StSPL. The left panel depicts the reaction mixture measured just after mixing protein and substrate. The m/z 163.07 and 380.26 peaks correspond to the end product phosphoethanolamine and the substrate S1P, respectively. The right panel shows the reaction mixture after 75 min incubation at 20°C. No peak corresponding to S1P was detectable above background level.</p

Effect of StSPL on S1P-stimulated MAPK phosphorylation, cell proliferation and migration of endothelial cells.

<p>(A) Quiescent EA.hy 926 human endothelial cells were treated for 10 min with either vehicle (Co) or S1P (1 µM) in the absence or presence of WT StSPL (StSPL; 10 µg/ml) or the K311A mutant (10 µg/ml). Cell lysates were prepared and separated by SDS-PAGE, transferred to nitrocellulose and subjected to Western blotting using antibodies against phospho-p42/p44 (dilution of 1∶1000, upper panel) and total p42/p44-MAPK (dilution each 1∶6000, lower panel). Data are representative of four independent experiments. (B) Quiescent cells were treated for 28 h with either vehicle (-) or S1P (1 µM), which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL) or the K311A mutant, in the presence of [³H]thymidine. Incorporated radioactivity was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022436#s4" target="_blank">Materials and Methods</a> section. Results are expressed as cpm/well of incorporated [³H]thymidine and are means ±S.D. (n = 4). ***p<0.001 considered statistically significant when compared to the vehicle treated control values; ^##p<0.01, ^###p<0.001 when compared to the S1P-treated values by one-way ANOVA analysis and Bonferroni post test. (C) Quiescent cells were treated for 14 h with DMEM (Co) or S1P (1 µM) which had been pretreated for 30 min at 37°C with either vehicle (-), WT StSPL (StSPL) or the K311A mutant. Thereafter, migrated cells were analysed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022436#s4" target="_blank">Materials and Methods</a> section. Results are expressed as migrated cells per counted field and are means ±S.D. (n = 3). ***p<0.001 considered statistically significant when compared to the vehicle treated control values; ^###p<0.001 when compared to the S1P-treated values.</p