- Email: [email protected]
Thioether-stapled macrocyclic inhibitors of the EH domain of EHD1
Bioorganic & Medicinal Chemistry 26 (2018) 1206–1211
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Thioether-stapled macrocyclic inhibitors of the EH domain of EHD1 Alissa J. Kamens, Kaley M. Mientkiewicz, Robyn J. Eisert, Jenna A. Walz, Charles R. Mace, Joshua A. Kritzer ⇑ Department of Chemistry, Tufts University, 62 Talbot Ave., Medford, MA 02155, United States
a r t i c l e
i n f o
Article history: Received 10 July 2017 Revised 31 August 2017 Accepted 6 September 2017 Available online 18 September 2017
a b s t r a c t Recycling of receptors from the endosomal recycling compartment to the plasma membrane is a critical cellular process, and recycling is particularly important for maintaining invasiveness in solid tumors. In this work, we continue our efforts to inhibit EHD1, a critical adaptor protein involved in receptor recycling. We applied a diversity-oriented macrocyclization approach to produce cyclic peptides with varied conformations, but that each contain a motif that binds to the EH domain of EHD1. Screening these uncovered several new inhibitors for EHD1’s EH domain, the most potent of which bound with a Kd of 3.1 lM. Several of the most potent inhibitors were tested in a cellular assay that measures extent of vesicle recycling. Inhibiting EHD1 could potentially slow cancer invasiveness and metastasis, and these cyclic peptides represent the most potent inhibitors of EHD1 to date. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Endocytosis is critical for normal cellular function, as is the recycling of endocytosed receptors back to the plasma membrane. While ‘‘fast recycling” pathways recycle material from the endosome directly to the membrane, ‘‘slow recycling” is a more regulated pathway that sorts material through a larger endocytic recycling compartment (ERC).1 Cancer invasion and metastasis depend on the slow recycling of growth factor receptors and components of cell adhesion complexes.2 Thus, blocking or slowing recycling from the ERC has been suggested as an attractive strategy for impeding invasion and metastasis. There are some known molecules that block slow recycling pathways, but they are mainly lysosomotropic agents and other molecules that impair vesicle trafficking in general.3–6 No pharmacological inhibitors specific to this pathway are currently known. The Eps15-homology-domain-containing (EHD) family of proteins is critical for sorting to and trafficking from the ERC.7,8 EHD1 in particular is required for export of integrins and other receptors from the ERC, where it assists in vesicle budding, vesicle elongation and membrane fission.9,10 Genetic inhibition of EHD1 function, including inactivation of its EH domain, results in impaired recycling of membrane receptors and decreased cancer invasiveness.11–14 Thus, EHD1 could be an excellent target for selectively blocking receptor recycling. EHD10 s membrane-remodeling functions are dependent on its dimerization and ATPase activities, and its localization at tubular regions of the ERC is dependent on the interactions of its Eps15-homology (EH) ⇑ Corresponding author. E-mail address: [email protected] (J.A. Kritzer). https://doi.org/10.1016/j.bmc.2017.09.007 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
domain.7–12 This suggests that an inhibitor of the EHD1 EH domain would prevent EHD1 localization at the ERC and impair recycling of integrins and other receptors. Protein-protein interactions can be challenging targets, but inhibitors of the EHD1-EH domain would be valuable for investigating the roles of EHD1 in vesicle trafficking, and for exploring new avenues of cancer therapy. In our prior work, we designed EHD1-EH inhibitors by incorporating the YNPFEE motif, derived from a known ligand, into a series of head-to-tail cyclic peptides.15 The highest-affinity inhibitor that we discovered, cNPF1, had a Kd of 9.9 lM. The increased affinity of this cyclic peptide over linear analogs was attributed to structural pre-organization of the recognition motif, and was driven primarily by improved binding enthalpy. We were inspired by recent work that used a ‘‘diversity-oriented stapling” approach to vary the conformation of a macrocyclization linker.16–19 In this work, we use diversity-oriented stapling to more broadly explore cyclic peptides that inhibit EHD1-EH, resulting in inhibitors with improved affinity and with selectivity for EHD1-EH over another EH domain from the endocytosis regulator Epidermal Growth Factor Receptor Substrate 15 (Eps15). 2. Results and discussion 2.1. Application of diversity-oriented macrocyclization to peptides with the EHD1 binding motif To explore EHD1-inhibiting peptides with a greater variety of macrocycle structures, we adapted robust and modular peptide modification strategies originally described by Timmerman et al.20 By flanking a peptide sequence with thiols, one can synthesize
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Scheme 1. Dithiol bis-alkylation to produce thioether-stapled peptides. Cyclization reactions are shown for the selected thioether-stapled peptides listed in Table 1. Reactions were run using 0.5 mM of the indicated linear peptide with 1.2 equivalents of the indicated linkers, in 50:50 acetonitrile:aqueous buffer (20 mM ammonium carbonate, pH = 8.0) for 2 h. Reactions were quenched with trifluoroacetic acid prior to lyophilization and HPLC purification. The NPF motif is shown in red, and the thiolcontaining residues (cysteine and cysteamine) and linkers are shown in blue.
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peptide macrocyles through a reaction with a,a0 -dibromoxylenes or other dibromomethyl-aryl linkers. This reaction is high-yielding in solution, and produces cyclized products without significant dimer formation even at moderate dilution. Most reactions reach completion in room temperature within 1 h, independent from peptide length or sequence.18–23 Variation of the thiol-containing groups and linkers allows a diversity-oriented macrocyclization approach in which small libraries of cyclic peptides share a constant recognition epitope, but vary the conformation of that epitope within the macrocycle. To apply this strategy to EHD1-EH inhibitors, we produced linear peptides 1–5 and bis-alkylated them with a variety of linkers (Scheme 1 and Table S1). Peptides 1–5 all contain the core NPFE motif, but vary the presence of an N-terminal tyrosine and a second C-terminal glutamate, since prior work was unclear as to whether these residues were absolutely required for EDH1-EH recognition.24,25 Peptides 1–4 were synthesized on cysteamine 2chlorotrityl resin to afford a free thiol on the C-terminus, and were capped on the N-terminus using 4-pentynoic acid. Peptide 5 included a C-terminal cysteine rather than a cysteamine, and was synthesized on Wang resin to afford a carboxylic acid at the C-terminus. The linear peptides were synthesized using standard Fmoc solid phase peptide synthesis and they were cyclized as shown in Scheme 1. After synthesis and purification, we used our previously described fluorescence polarization (FP) competitive binding assay to evaluate these initial designs (Fig. S1),15 and selected a subset for further analysis.
2.2. Inhibitory potencies of bis-alkylated cyclic peptides Results for competition FP assays for selected bis-alkylated cyclic peptides are shown in Fig. 1 and summarized in Table 1. These results were compared to cNPF1, the highest-affinity EHD1-EH inhibitor reported to date.15 Cyclic peptide 1-PX had an IC50 of 27.1 ± 1.7 lM, which was a twofold improvement over cNPF1. 1MX, by contrast, had poorer potency than cNPF1, highlighting the importance of linker shape on inhibitory potency. 3-OX had an IC50 of 76.5 ± 4.6 lM. While this is significantly poorer than cNPF1, 3-OX is notable as the most potent inhibitor of this series with a single charged residue. We consistently observed that peptides with two carboxylates had stronger inhibition, as suggested by previous data.15,24,25 4-PX had an IC50 similar to that of cNPF1, while inhibition by 4-MX and 4-OX were weaker than cNPF1. Cyclic peptides derived from 5 had one glutamic acid and a free C-terminus, maintaining two carboxylates overall. 5-OX inhibited slightly better than cNPF1, with an IC50 of 34.0 ± 3.8 lM. 2.3. Direct binding of bis-alkylated cyclic peptides to EHD1-EH Next, we prepared fluorescein-labeled analogs of the peptides with the highest inhibitory potencies, in order to perform direct FP binding assays with EHD1-EH. The experiments were first conducted under low-salt (15 mM NaCl) conditions (Fig. S3, Table S2). These produced binding curves that saturated at high concentration, which provided upper bound parameters that could be
Fig. 1. Competitive inhibition data for EHD1-EH for selected cyclic peptides. (a) Competition fluorescence polarization data for cNPF1 (black, circle), 1-MX (red, square), 1-PX (green, diamond), and 3-OX (light blue, triangle). (b) Competition fluorescence polarization data for 4-OX (orange, inverted triangle), 4-MX (dark blue, circle), 4-PX (pink, diamond), 5-OX (dark green, triangle). Assays were performed at room temperature in 25 mM MOPS pH 6.8, 1 mM CaCl2, 15 mM NaCl, using 20 lM EHD1-EH and 10 nM cNPF1Flu. Error bars represent the standard error of the mean of three technical replicates, and three independent trials were performed for each experiment (see Fig. S2 for additional replicates). IC50 curve fits reflect values reported in Table 1.
Table 1 Summary of competitive binding data for EHD1-EH for selected thioether-stapled cyclic peptides. Linker specifies the chemical group attached to the two cysteines via thioether bonds, where OX denotes ortho-xylene, MX denotes meta-xylene, and PX denotes para-xylene. IC50 values represent averages and the standard error of the mean from curve fits to three independent trials, shown in Figs. 1 and S2. Name
cNPF1 1-MX 1-PX 3-OX 4-OX 4-MX 4-PX 5-OX
IC50 (lM)
peptide structure Sequence
N-term
C-term
Linker
YNPFEEGG CYNPFEE CYNPFEE CNPFE CNPFEE CNPFEE CNPFEE CNPFEC
backbone cyclic 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl acetyl
cysteamine cysteamine cysteamine cysteamine cysteamine cysteamine carboxylate
MX PX OX OX MX PX OX
55.5 ± 2.2 68.8 ± 2.7 27.1 ± 1.7 76.5 ± 4.6 62.8 ± 0.5 88.1 ± 1.5 38.9 ± 7.6 34.0 ± 3.8
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Fig. 2. Direct binding of selected cyclic peptides to EHD1-EH. (a) Fluorescence polarization binding data for cNPF1flu (black, circles), Flu-1-PX (green, diamonds), Flu-3-OX (light blue, triangles), and Flu-4-OX (orange, inverted triangles). (b) Fluorescence polarization binding data for Flu-5-OX (dark green, triangles), Flu-6-OX (blue, open circles), and Flu-7-OX (black, open squares). Assays were performed at room temperature in 25 mM MOPS pH 6.8, 1 mM CaCl2, 150 mM NaCl, using 20 lM EHD1-EH and 10 nM fluorescein-labeled probe. Error bars represent the standard error of the mean of three technical replicates, and three independent trials were performed for each experiment (see Fig. S4 for additional replicates). Kd curve fits reflect values reported in Table 2.
Table 2 Summary of direct binding data for EHD1-EH selected thioether-stapled cyclic peptides. K* denotes lysine acylated with NHS-fluorescein, X denotes penicillamine, and U denotes 1-naphthylalanine. Kd values represent averages and the standard error of the mean from curve fits to three independent trials, shown in Figs. 2, S3 and S4. Name
cNPF1Flu Flu-1-PX Flu-3-OX Flu-4-PX Flu-5-OX Flu-6-OX Flu-7-OX
Peptide structure Sequence
N-term
C-term
Linker
YNPFEEGK* CYNPFEE CNPFE CNPFEE CNPFEC CNPFEX CNPUEC
backbone cyclic 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl
cysteamine cysteamine cysteamine cysteamine cysteamine cysteamine
PX OX PX OX OX OX
applied to curve fits for data at physiological salt conditions (which did not always saturate). Then, direct binding assays were repeated under physiological salt conditions (150 mM NaCl). Fig. 2 shows binding data from FP experiments at physiological salt, and Table 2 summarizes the results. Overall, the results from direct binding assays followed trends that closely matched the trends observed in competition assays. Flu-1-PX and Flu-4-PX had Kd values of 6.0 ± 0.2 lM and 8.1 ± 0.2 lM, respectively. Both of these peptides had higher affinity than cNPF1Flu, which had a measured Kd of 16.8 lM under the same conditions. Despite only having one carboxylate, Flu-3-OX also bound EHD1-EH more tightly than cNPF1Flu, with a Kd of 14.6 ± 0.7 lM. Flu-5-OX had a Kd of 7.3 ± 0.7 lM, among the strongest binders we had yet produced. Based on a preliminary docking model of 5OX and EHD1-EH, we explored the effects of two individual substitutions. Peptide Flu-6-OX was analogous to Flu-5-OX, but had a penicillamine in place of the C-terminal cysteine. Peptide Flu-7OX replaced the phenylalanine within Flu-5-OX with 1-naphthylalanine. Flu-6-OX and Flu-7-OX improved affinity further, with Flu-6-OX being the most potent EHD1-EH ligand reported to date (Kd = 3.1 ± 0.1 lM). 2.4. Selectivity of cyclic peptides among EH domains Different classes of EH-domain-containing proteins play different roles in vesicle trafficking. As an initial test of the selectivity of our cyclic peptide EHD1-EH ligands, we tested those with the highest EHD1-EH affinity in an FP direct binding assay with an EH domain of an unrelated EH domain-containing protein, Eps15 (Fig. S5).23 Very little binding was detected under the experimental
Kd (lM) 15 mM NaCl
Kd (lM) 150 mM NaCl
3.0 ± 0.2 1.1 ± 0.01 4.5 ± 0.1 1.8 ± 0.1 1.6 ± 0.3 0.6 ± 0.004 0.87 ± 0.05
15.1 ± 1.4 6.0 ± 0.2 14.6 ± 0.7 8.1 ± 0.2 7.3 ± 0.7 3.1 ± 0.1 5.4 ± 0.2
conditions, corresponding to IC50 values well above 100 lM. Thus, these new EHD1-EH ligands were at least 15-fold selective for EHD1-EH over an EH domain of Eps15.
2.5. Measuring inhibition of recycling in cell culture EH domain inhibitors with micromolar potency are promising starting points for developing cellular probes that impair recycling. To monitor recycling in cell culture, we used the trafficking of transferrin as a model system. We developed a convenient pulsechase assay to monitor the recycling of fluorescently-labeled transferrin, and corroborated the results with microscopy (Fig. S6). Our protocols were derived from previous experiments used to monitor the importance of EHD1 knockdown in recycling and vesicle trafficking in HeLa cells.11–13 Cells were first pulsed with fluorescently labeled transferrin (Tf-488) to allow the fluorescently-labeled protein to be taken up by endocytosis and saturate vesicle trafficking pathways. When testing potential inhibitors, we included them during the pulse step to allow for more time for inhibitors to penetrate the cell and perturb trafficking. After the 60-min pulse, cells were chased with unlabeled transferrin for 30 min. Then cells were washed with cold phosphate-buffered saline (PBS) to halt trafficking, trypsinized, and fixed for subsequent analysis by flow cytometry. As a positive control, we performed the pulse-chase experiment with 50 lM primaquine, a small molecule known to non-specifically inhibit vesicle recycling.26–28 Primaquine treatment resulted in increased signal following the chase, consistent with impaired recycling of internalized Tf-488 (Fig. 3a). Next, we tested cyclic peptide EHD1-EH inhibitors by including 50 lM peptide during both the pulse and the chase. None of the molecules cNPF1, 1-PX
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Fig. 3. Monitoring endocytic recycling using flow cytometry. Cells were pulsed with Tf-488, then chased with unlabeled transferrin in order to monitor the rate of disappearance of Tf-488 over the course of 30 min, consistent with slow recycling. (a) Cells not treated with Tf-488 during the pulse show background autofluorescence (gray histogram). Cells exposed to Tf-488 and then a brief a 3-min chase with unlabeled transferrin (blue histogram) represent the maximum amount of internalized Tf-488. Cells that were pulsed and chased for 30 mins with unlabeled transferrin (red histogram) represent the basal level of recycling, while the cells with an identical pulse-chase but with 50 lM primaquine (green histogram) show inhibition of recycling. (b) The purple, pink, and black histograms represent cells treated with 5 lM Tat-6-OX, Tat-6scr-OX, and Tat-7-OX, respectively.
and 5-OX altered trafficking of Tf-488 (Fig. S7). Due to their size and negative charge, we presumed that these peptides were incapable of entering the cell either passively or via an endocytic route. To promote cell penetration, we conjugated the Tat cell-penetrating sequence to some of our higher-affinity ligands.29 We tested Tat-6-OX and Tat-7-OX at 5 lM (Fig. 3b). We also tested a scrambled version of Tat-6-OX, Tat-6scr-OX, as an additional control. None of the peptides had any effect on Tf-488 recycling. 3. Conclusions In this work, we report continued progress in the development of EHD1 inhibitors. We used an FP competition assay to screen a small library of conformationally diverse cyclic peptides. We discovered multiple peptides with single-digit micromolar affinity for EHD1-EH under physiological salt conditions. Flu-6-OX had a Kd of 3.1 lM, which makes it the most potent EHD1-EH ligand reported to date. Existing inhibitors of other vesicle-trafficking proteins have similar affinities for their targets,3,30–32 raising the possibility of using these cyclic peptides as cellular inhibitors of long-loop recycling. However, none of the cyclic peptides had measurable effects on slow recycling pathways even when conjugated to the cell-penetrating peptide Tat. This could be due to poor cell penetration, but other possible explanations include intracellular mislocalization, off-target binding, or incomplete understanding of the effects of inhibiting EHD1-EH. Current efforts are focused on improving the cell penetration of EHD1 inhibitors to produce molecules that inhibit slow recycling in live cells. Ultimately, such molecules will be valuable probes for understanding the roles of EHD1 in cancer cell invasion and metastasis. 4. Experimental 4.1. Protein preparation The EH domains of EHD1 (EHD1-EH) and Eps15 (Eps15-EH2) were prepared as described previously.15,23,24 The concentration of each protein was determined by absorbance at 280 nm, with an extinction coefficient of 13980 M1cm1. 4.2. Peptide synthesis and purification Materials were purchased from EMD Biosciences, Anaspec, Advanced Chemtech, and Creosalis. Linear peptides were synthesized by standard Fmoc solid-phase peptide synthesis, and purified
by reversed-phase HPLC on either a C8 or a C18 column.18 The Tatlinked peptides were synthesized on 2-Chlorotrityl resin using standard Fmoc chemistry on a Tribute-UV IR peptide synthesizer from Gyros Protein Technologies. 4.3. Bis-alkylation of thiol-containing peptides Linear peptides were dissolved in reaction buffer (50% acetonitrile, 50% 20 mM ammonium carbonate aqueous buffer, pH = 8.0) to a concentration of 0.5 mM as determined by weight, or by absorbance at 280 nm for tyrosine-containing peptides. Reactions were bubbled with argon for 20 min, then 1.2 equivalents of the desired linker was dissolved in acetonitrile and added dropwise over the course of 1 min. The reactions were quenched after 2 h with the addition of trifluoroacetic acid until the reaction had a pH of 4.0. The quenched mixture was lyophilized prior to purification by reversed-phase HPLC on either a C8 or a C18 column. Purity greater than 95% was measured by reinjection on an analytical C18 column, and identity was verified by MALDI-MS. Concentrations of cyclic peptides lacking tyrosine were determined by amino acid analysis. 4.4. Fluorescence polarization competitive binding assay Peptides were tested under low-salt conditions (25 mM MOPS pH 6.8, 1 mM CaCl2, 15 mM NaCl) as described.15 Inhibitors were added to 20 lM EHD1-EH and incubated at room temperature for 30 min in 384-well black plates (Corning). Then the cNPF1Flu fluorescent probe was added to a final concentration of 10 nM in 30 lL, and incubated at room temperature. Fluorescence polarization was measured on a Tecan F200 Pro 1 h after the addition of the probe, and repeat readings were taken at later time points to verify that binding had reached equilibrium. IC50 curve fits were calculated with Kaleidagraph Synergy Software. Three technical replicates were performed in each experiment, and three independent experiments were performed for each peptide. 4.5. Fluorescence polarization direct binding assay Fluorescein-labeled peptides were tested for direct binding under low-salt and physiological salt conditions (25 mM MOPS pH 6.8, 1 mM CaCl2, 1.5% DMSO, 0.1% Tween-20, and 15 or 150 nM NaCl). Each probe was incubated at a final concentration of 10 nM at room temperature with varying concentrations of EHD1-EH in 30 lL in 384-well black plates. Fluorescence
A.J. Kamens et al. / Bioorganic & Medicinal Chemistry 26 (2018) 1206–1211
polarization was measured on a Tecan F200 Pro 1 h after the addition of the probe, and repeat readings were taken at later time points to verify that binding had reached equilibrium. Kd curve fits were calculated with Kaleidagraph Synergy Software. Three technical replicates were performed in each experiment, and three independent experiments were performed.
4.6. Cell-Based transferrin recycling assay protocol All serum and media were purchased from Life Technologies. HeLa cells (ATCC CCL-2) were seeded in twelve-well plates and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% Fetal Bovine Serum at 37 °C with an atmosphere of 5% CO2. Once the cells reached 80% confluence, they were starved for 30 min in DMEM with 0.5% w/v bovine serum albumin (BSA). The cells were then subjected to a 60-min pulse incubation with fluorescently labeled transferrin (25 lg/mL, TF-488, from Life Technologies) in DMEM with 0.5% w/v BSA. When testing inhibitors, they were included in the pulse step. Following the pulse, a 30-min chase was conducted in 10% FBS in DMEM with unlabeled transferrin at a concentration of 2 mg/mL. Inhibitors were also included at their respective concentrations, and the final DMSO concentration of each well was 0.2% v/v. 50 lM primaquine was used as a positive control. The cells were washed with cold PBS, trypsinized and fixed for subsequent analysis by flow cytometry. 10,000 live cells were counted for each sample using a Millipore-Sigma Guava easyCyte 6HT-2L benchtop flow cytometer.
Acknowledgments This project was funded in part by NSF-1507456.
A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2017.09.007.
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Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Thioether-stapled macrocyclic inhibitors of the EH domain of EHD1 Alissa J. Kamens, Kaley M. Mientkiewicz, Robyn J. Eisert, Jenna A. Walz, Charles R. Mace, Joshua A. Kritzer ⇑ Department of Chemistry, Tufts University, 62 Talbot Ave., Medford, MA 02155, United States
a r t i c l e
i n f o
Article history: Received 10 July 2017 Revised 31 August 2017 Accepted 6 September 2017 Available online 18 September 2017
a b s t r a c t Recycling of receptors from the endosomal recycling compartment to the plasma membrane is a critical cellular process, and recycling is particularly important for maintaining invasiveness in solid tumors. In this work, we continue our efforts to inhibit EHD1, a critical adaptor protein involved in receptor recycling. We applied a diversity-oriented macrocyclization approach to produce cyclic peptides with varied conformations, but that each contain a motif that binds to the EH domain of EHD1. Screening these uncovered several new inhibitors for EHD1’s EH domain, the most potent of which bound with a Kd of 3.1 lM. Several of the most potent inhibitors were tested in a cellular assay that measures extent of vesicle recycling. Inhibiting EHD1 could potentially slow cancer invasiveness and metastasis, and these cyclic peptides represent the most potent inhibitors of EHD1 to date. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Endocytosis is critical for normal cellular function, as is the recycling of endocytosed receptors back to the plasma membrane. While ‘‘fast recycling” pathways recycle material from the endosome directly to the membrane, ‘‘slow recycling” is a more regulated pathway that sorts material through a larger endocytic recycling compartment (ERC).1 Cancer invasion and metastasis depend on the slow recycling of growth factor receptors and components of cell adhesion complexes.2 Thus, blocking or slowing recycling from the ERC has been suggested as an attractive strategy for impeding invasion and metastasis. There are some known molecules that block slow recycling pathways, but they are mainly lysosomotropic agents and other molecules that impair vesicle trafficking in general.3–6 No pharmacological inhibitors specific to this pathway are currently known. The Eps15-homology-domain-containing (EHD) family of proteins is critical for sorting to and trafficking from the ERC.7,8 EHD1 in particular is required for export of integrins and other receptors from the ERC, where it assists in vesicle budding, vesicle elongation and membrane fission.9,10 Genetic inhibition of EHD1 function, including inactivation of its EH domain, results in impaired recycling of membrane receptors and decreased cancer invasiveness.11–14 Thus, EHD1 could be an excellent target for selectively blocking receptor recycling. EHD10 s membrane-remodeling functions are dependent on its dimerization and ATPase activities, and its localization at tubular regions of the ERC is dependent on the interactions of its Eps15-homology (EH) ⇑ Corresponding author. E-mail address: [email protected] (J.A. Kritzer). https://doi.org/10.1016/j.bmc.2017.09.007 0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
domain.7–12 This suggests that an inhibitor of the EHD1 EH domain would prevent EHD1 localization at the ERC and impair recycling of integrins and other receptors. Protein-protein interactions can be challenging targets, but inhibitors of the EHD1-EH domain would be valuable for investigating the roles of EHD1 in vesicle trafficking, and for exploring new avenues of cancer therapy. In our prior work, we designed EHD1-EH inhibitors by incorporating the YNPFEE motif, derived from a known ligand, into a series of head-to-tail cyclic peptides.15 The highest-affinity inhibitor that we discovered, cNPF1, had a Kd of 9.9 lM. The increased affinity of this cyclic peptide over linear analogs was attributed to structural pre-organization of the recognition motif, and was driven primarily by improved binding enthalpy. We were inspired by recent work that used a ‘‘diversity-oriented stapling” approach to vary the conformation of a macrocyclization linker.16–19 In this work, we use diversity-oriented stapling to more broadly explore cyclic peptides that inhibit EHD1-EH, resulting in inhibitors with improved affinity and with selectivity for EHD1-EH over another EH domain from the endocytosis regulator Epidermal Growth Factor Receptor Substrate 15 (Eps15). 2. Results and discussion 2.1. Application of diversity-oriented macrocyclization to peptides with the EHD1 binding motif To explore EHD1-inhibiting peptides with a greater variety of macrocycle structures, we adapted robust and modular peptide modification strategies originally described by Timmerman et al.20 By flanking a peptide sequence with thiols, one can synthesize
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Scheme 1. Dithiol bis-alkylation to produce thioether-stapled peptides. Cyclization reactions are shown for the selected thioether-stapled peptides listed in Table 1. Reactions were run using 0.5 mM of the indicated linear peptide with 1.2 equivalents of the indicated linkers, in 50:50 acetonitrile:aqueous buffer (20 mM ammonium carbonate, pH = 8.0) for 2 h. Reactions were quenched with trifluoroacetic acid prior to lyophilization and HPLC purification. The NPF motif is shown in red, and the thiolcontaining residues (cysteine and cysteamine) and linkers are shown in blue.
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peptide macrocyles through a reaction with a,a0 -dibromoxylenes or other dibromomethyl-aryl linkers. This reaction is high-yielding in solution, and produces cyclized products without significant dimer formation even at moderate dilution. Most reactions reach completion in room temperature within 1 h, independent from peptide length or sequence.18–23 Variation of the thiol-containing groups and linkers allows a diversity-oriented macrocyclization approach in which small libraries of cyclic peptides share a constant recognition epitope, but vary the conformation of that epitope within the macrocycle. To apply this strategy to EHD1-EH inhibitors, we produced linear peptides 1–5 and bis-alkylated them with a variety of linkers (Scheme 1 and Table S1). Peptides 1–5 all contain the core NPFE motif, but vary the presence of an N-terminal tyrosine and a second C-terminal glutamate, since prior work was unclear as to whether these residues were absolutely required for EDH1-EH recognition.24,25 Peptides 1–4 were synthesized on cysteamine 2chlorotrityl resin to afford a free thiol on the C-terminus, and were capped on the N-terminus using 4-pentynoic acid. Peptide 5 included a C-terminal cysteine rather than a cysteamine, and was synthesized on Wang resin to afford a carboxylic acid at the C-terminus. The linear peptides were synthesized using standard Fmoc solid phase peptide synthesis and they were cyclized as shown in Scheme 1. After synthesis and purification, we used our previously described fluorescence polarization (FP) competitive binding assay to evaluate these initial designs (Fig. S1),15 and selected a subset for further analysis.
2.2. Inhibitory potencies of bis-alkylated cyclic peptides Results for competition FP assays for selected bis-alkylated cyclic peptides are shown in Fig. 1 and summarized in Table 1. These results were compared to cNPF1, the highest-affinity EHD1-EH inhibitor reported to date.15 Cyclic peptide 1-PX had an IC50 of 27.1 ± 1.7 lM, which was a twofold improvement over cNPF1. 1MX, by contrast, had poorer potency than cNPF1, highlighting the importance of linker shape on inhibitory potency. 3-OX had an IC50 of 76.5 ± 4.6 lM. While this is significantly poorer than cNPF1, 3-OX is notable as the most potent inhibitor of this series with a single charged residue. We consistently observed that peptides with two carboxylates had stronger inhibition, as suggested by previous data.15,24,25 4-PX had an IC50 similar to that of cNPF1, while inhibition by 4-MX and 4-OX were weaker than cNPF1. Cyclic peptides derived from 5 had one glutamic acid and a free C-terminus, maintaining two carboxylates overall. 5-OX inhibited slightly better than cNPF1, with an IC50 of 34.0 ± 3.8 lM. 2.3. Direct binding of bis-alkylated cyclic peptides to EHD1-EH Next, we prepared fluorescein-labeled analogs of the peptides with the highest inhibitory potencies, in order to perform direct FP binding assays with EHD1-EH. The experiments were first conducted under low-salt (15 mM NaCl) conditions (Fig. S3, Table S2). These produced binding curves that saturated at high concentration, which provided upper bound parameters that could be
Fig. 1. Competitive inhibition data for EHD1-EH for selected cyclic peptides. (a) Competition fluorescence polarization data for cNPF1 (black, circle), 1-MX (red, square), 1-PX (green, diamond), and 3-OX (light blue, triangle). (b) Competition fluorescence polarization data for 4-OX (orange, inverted triangle), 4-MX (dark blue, circle), 4-PX (pink, diamond), 5-OX (dark green, triangle). Assays were performed at room temperature in 25 mM MOPS pH 6.8, 1 mM CaCl2, 15 mM NaCl, using 20 lM EHD1-EH and 10 nM cNPF1Flu. Error bars represent the standard error of the mean of three technical replicates, and three independent trials were performed for each experiment (see Fig. S2 for additional replicates). IC50 curve fits reflect values reported in Table 1.
Table 1 Summary of competitive binding data for EHD1-EH for selected thioether-stapled cyclic peptides. Linker specifies the chemical group attached to the two cysteines via thioether bonds, where OX denotes ortho-xylene, MX denotes meta-xylene, and PX denotes para-xylene. IC50 values represent averages and the standard error of the mean from curve fits to three independent trials, shown in Figs. 1 and S2. Name
cNPF1 1-MX 1-PX 3-OX 4-OX 4-MX 4-PX 5-OX
IC50 (lM)
peptide structure Sequence
N-term
C-term
Linker
YNPFEEGG CYNPFEE CYNPFEE CNPFE CNPFEE CNPFEE CNPFEE CNPFEC
backbone cyclic 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl acetyl
cysteamine cysteamine cysteamine cysteamine cysteamine cysteamine carboxylate
MX PX OX OX MX PX OX
55.5 ± 2.2 68.8 ± 2.7 27.1 ± 1.7 76.5 ± 4.6 62.8 ± 0.5 88.1 ± 1.5 38.9 ± 7.6 34.0 ± 3.8
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Fig. 2. Direct binding of selected cyclic peptides to EHD1-EH. (a) Fluorescence polarization binding data for cNPF1flu (black, circles), Flu-1-PX (green, diamonds), Flu-3-OX (light blue, triangles), and Flu-4-OX (orange, inverted triangles). (b) Fluorescence polarization binding data for Flu-5-OX (dark green, triangles), Flu-6-OX (blue, open circles), and Flu-7-OX (black, open squares). Assays were performed at room temperature in 25 mM MOPS pH 6.8, 1 mM CaCl2, 150 mM NaCl, using 20 lM EHD1-EH and 10 nM fluorescein-labeled probe. Error bars represent the standard error of the mean of three technical replicates, and three independent trials were performed for each experiment (see Fig. S4 for additional replicates). Kd curve fits reflect values reported in Table 2.
Table 2 Summary of direct binding data for EHD1-EH selected thioether-stapled cyclic peptides. K* denotes lysine acylated with NHS-fluorescein, X denotes penicillamine, and U denotes 1-naphthylalanine. Kd values represent averages and the standard error of the mean from curve fits to three independent trials, shown in Figs. 2, S3 and S4. Name
cNPF1Flu Flu-1-PX Flu-3-OX Flu-4-PX Flu-5-OX Flu-6-OX Flu-7-OX
Peptide structure Sequence
N-term
C-term
Linker
YNPFEEGK* CYNPFEE CNPFE CNPFEE CNPFEC CNPFEX CNPUEC
backbone cyclic 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl 4-pentynyl
cysteamine cysteamine cysteamine cysteamine cysteamine cysteamine
PX OX PX OX OX OX
applied to curve fits for data at physiological salt conditions (which did not always saturate). Then, direct binding assays were repeated under physiological salt conditions (150 mM NaCl). Fig. 2 shows binding data from FP experiments at physiological salt, and Table 2 summarizes the results. Overall, the results from direct binding assays followed trends that closely matched the trends observed in competition assays. Flu-1-PX and Flu-4-PX had Kd values of 6.0 ± 0.2 lM and 8.1 ± 0.2 lM, respectively. Both of these peptides had higher affinity than cNPF1Flu, which had a measured Kd of 16.8 lM under the same conditions. Despite only having one carboxylate, Flu-3-OX also bound EHD1-EH more tightly than cNPF1Flu, with a Kd of 14.6 ± 0.7 lM. Flu-5-OX had a Kd of 7.3 ± 0.7 lM, among the strongest binders we had yet produced. Based on a preliminary docking model of 5OX and EHD1-EH, we explored the effects of two individual substitutions. Peptide Flu-6-OX was analogous to Flu-5-OX, but had a penicillamine in place of the C-terminal cysteine. Peptide Flu-7OX replaced the phenylalanine within Flu-5-OX with 1-naphthylalanine. Flu-6-OX and Flu-7-OX improved affinity further, with Flu-6-OX being the most potent EHD1-EH ligand reported to date (Kd = 3.1 ± 0.1 lM). 2.4. Selectivity of cyclic peptides among EH domains Different classes of EH-domain-containing proteins play different roles in vesicle trafficking. As an initial test of the selectivity of our cyclic peptide EHD1-EH ligands, we tested those with the highest EHD1-EH affinity in an FP direct binding assay with an EH domain of an unrelated EH domain-containing protein, Eps15 (Fig. S5).23 Very little binding was detected under the experimental
Kd (lM) 15 mM NaCl
Kd (lM) 150 mM NaCl
3.0 ± 0.2 1.1 ± 0.01 4.5 ± 0.1 1.8 ± 0.1 1.6 ± 0.3 0.6 ± 0.004 0.87 ± 0.05
15.1 ± 1.4 6.0 ± 0.2 14.6 ± 0.7 8.1 ± 0.2 7.3 ± 0.7 3.1 ± 0.1 5.4 ± 0.2
conditions, corresponding to IC50 values well above 100 lM. Thus, these new EHD1-EH ligands were at least 15-fold selective for EHD1-EH over an EH domain of Eps15.
2.5. Measuring inhibition of recycling in cell culture EH domain inhibitors with micromolar potency are promising starting points for developing cellular probes that impair recycling. To monitor recycling in cell culture, we used the trafficking of transferrin as a model system. We developed a convenient pulsechase assay to monitor the recycling of fluorescently-labeled transferrin, and corroborated the results with microscopy (Fig. S6). Our protocols were derived from previous experiments used to monitor the importance of EHD1 knockdown in recycling and vesicle trafficking in HeLa cells.11–13 Cells were first pulsed with fluorescently labeled transferrin (Tf-488) to allow the fluorescently-labeled protein to be taken up by endocytosis and saturate vesicle trafficking pathways. When testing potential inhibitors, we included them during the pulse step to allow for more time for inhibitors to penetrate the cell and perturb trafficking. After the 60-min pulse, cells were chased with unlabeled transferrin for 30 min. Then cells were washed with cold phosphate-buffered saline (PBS) to halt trafficking, trypsinized, and fixed for subsequent analysis by flow cytometry. As a positive control, we performed the pulse-chase experiment with 50 lM primaquine, a small molecule known to non-specifically inhibit vesicle recycling.26–28 Primaquine treatment resulted in increased signal following the chase, consistent with impaired recycling of internalized Tf-488 (Fig. 3a). Next, we tested cyclic peptide EHD1-EH inhibitors by including 50 lM peptide during both the pulse and the chase. None of the molecules cNPF1, 1-PX
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Fig. 3. Monitoring endocytic recycling using flow cytometry. Cells were pulsed with Tf-488, then chased with unlabeled transferrin in order to monitor the rate of disappearance of Tf-488 over the course of 30 min, consistent with slow recycling. (a) Cells not treated with Tf-488 during the pulse show background autofluorescence (gray histogram). Cells exposed to Tf-488 and then a brief a 3-min chase with unlabeled transferrin (blue histogram) represent the maximum amount of internalized Tf-488. Cells that were pulsed and chased for 30 mins with unlabeled transferrin (red histogram) represent the basal level of recycling, while the cells with an identical pulse-chase but with 50 lM primaquine (green histogram) show inhibition of recycling. (b) The purple, pink, and black histograms represent cells treated with 5 lM Tat-6-OX, Tat-6scr-OX, and Tat-7-OX, respectively.
and 5-OX altered trafficking of Tf-488 (Fig. S7). Due to their size and negative charge, we presumed that these peptides were incapable of entering the cell either passively or via an endocytic route. To promote cell penetration, we conjugated the Tat cell-penetrating sequence to some of our higher-affinity ligands.29 We tested Tat-6-OX and Tat-7-OX at 5 lM (Fig. 3b). We also tested a scrambled version of Tat-6-OX, Tat-6scr-OX, as an additional control. None of the peptides had any effect on Tf-488 recycling. 3. Conclusions In this work, we report continued progress in the development of EHD1 inhibitors. We used an FP competition assay to screen a small library of conformationally diverse cyclic peptides. We discovered multiple peptides with single-digit micromolar affinity for EHD1-EH under physiological salt conditions. Flu-6-OX had a Kd of 3.1 lM, which makes it the most potent EHD1-EH ligand reported to date. Existing inhibitors of other vesicle-trafficking proteins have similar affinities for their targets,3,30–32 raising the possibility of using these cyclic peptides as cellular inhibitors of long-loop recycling. However, none of the cyclic peptides had measurable effects on slow recycling pathways even when conjugated to the cell-penetrating peptide Tat. This could be due to poor cell penetration, but other possible explanations include intracellular mislocalization, off-target binding, or incomplete understanding of the effects of inhibiting EHD1-EH. Current efforts are focused on improving the cell penetration of EHD1 inhibitors to produce molecules that inhibit slow recycling in live cells. Ultimately, such molecules will be valuable probes for understanding the roles of EHD1 in cancer cell invasion and metastasis. 4. Experimental 4.1. Protein preparation The EH domains of EHD1 (EHD1-EH) and Eps15 (Eps15-EH2) were prepared as described previously.15,23,24 The concentration of each protein was determined by absorbance at 280 nm, with an extinction coefficient of 13980 M1cm1. 4.2. Peptide synthesis and purification Materials were purchased from EMD Biosciences, Anaspec, Advanced Chemtech, and Creosalis. Linear peptides were synthesized by standard Fmoc solid-phase peptide synthesis, and purified
by reversed-phase HPLC on either a C8 or a C18 column.18 The Tatlinked peptides were synthesized on 2-Chlorotrityl resin using standard Fmoc chemistry on a Tribute-UV IR peptide synthesizer from Gyros Protein Technologies. 4.3. Bis-alkylation of thiol-containing peptides Linear peptides were dissolved in reaction buffer (50% acetonitrile, 50% 20 mM ammonium carbonate aqueous buffer, pH = 8.0) to a concentration of 0.5 mM as determined by weight, or by absorbance at 280 nm for tyrosine-containing peptides. Reactions were bubbled with argon for 20 min, then 1.2 equivalents of the desired linker was dissolved in acetonitrile and added dropwise over the course of 1 min. The reactions were quenched after 2 h with the addition of trifluoroacetic acid until the reaction had a pH of 4.0. The quenched mixture was lyophilized prior to purification by reversed-phase HPLC on either a C8 or a C18 column. Purity greater than 95% was measured by reinjection on an analytical C18 column, and identity was verified by MALDI-MS. Concentrations of cyclic peptides lacking tyrosine were determined by amino acid analysis. 4.4. Fluorescence polarization competitive binding assay Peptides were tested under low-salt conditions (25 mM MOPS pH 6.8, 1 mM CaCl2, 15 mM NaCl) as described.15 Inhibitors were added to 20 lM EHD1-EH and incubated at room temperature for 30 min in 384-well black plates (Corning). Then the cNPF1Flu fluorescent probe was added to a final concentration of 10 nM in 30 lL, and incubated at room temperature. Fluorescence polarization was measured on a Tecan F200 Pro 1 h after the addition of the probe, and repeat readings were taken at later time points to verify that binding had reached equilibrium. IC50 curve fits were calculated with Kaleidagraph Synergy Software. Three technical replicates were performed in each experiment, and three independent experiments were performed for each peptide. 4.5. Fluorescence polarization direct binding assay Fluorescein-labeled peptides were tested for direct binding under low-salt and physiological salt conditions (25 mM MOPS pH 6.8, 1 mM CaCl2, 1.5% DMSO, 0.1% Tween-20, and 15 or 150 nM NaCl). Each probe was incubated at a final concentration of 10 nM at room temperature with varying concentrations of EHD1-EH in 30 lL in 384-well black plates. Fluorescence
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polarization was measured on a Tecan F200 Pro 1 h after the addition of the probe, and repeat readings were taken at later time points to verify that binding had reached equilibrium. Kd curve fits were calculated with Kaleidagraph Synergy Software. Three technical replicates were performed in each experiment, and three independent experiments were performed.
4.6. Cell-Based transferrin recycling assay protocol All serum and media were purchased from Life Technologies. HeLa cells (ATCC CCL-2) were seeded in twelve-well plates and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% Fetal Bovine Serum at 37 °C with an atmosphere of 5% CO2. Once the cells reached 80% confluence, they were starved for 30 min in DMEM with 0.5% w/v bovine serum albumin (BSA). The cells were then subjected to a 60-min pulse incubation with fluorescently labeled transferrin (25 lg/mL, TF-488, from Life Technologies) in DMEM with 0.5% w/v BSA. When testing inhibitors, they were included in the pulse step. Following the pulse, a 30-min chase was conducted in 10% FBS in DMEM with unlabeled transferrin at a concentration of 2 mg/mL. Inhibitors were also included at their respective concentrations, and the final DMSO concentration of each well was 0.2% v/v. 50 lM primaquine was used as a positive control. The cells were washed with cold PBS, trypsinized and fixed for subsequent analysis by flow cytometry. 10,000 live cells were counted for each sample using a Millipore-Sigma Guava easyCyte 6HT-2L benchtop flow cytometer.
Acknowledgments This project was funded in part by NSF-1507456.
A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2017.09.007.
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