Stem Cell Bioengineering with Bioportides: Inhibition of Planarian Head Regeneration with Peptide Mimetics of Eyes Absent Proteins

Djeya1 (RKLAFRYRRIKELYNSYR) is a very effective cell penetrating peptide (CPP) that mimics the α5 helix of the highly conserved Eya domain (ED) of eyes absent (Eya) proteins. The objective of this study was to bioengineer analogues of Djeya1 that, following effective translocation into planarian tissues, would reduce the ability of neoblasts (totipotent stem cells) and their progeny to regenerate the anterior pole in decapitated S. mediterranea. As a strategy to increase the propensity for helix formation, molecular bioengineering of Djeya1 was achieved by the mono-substitution of the helicogenic aminoisobutyric acid (Aib) at three species-variable sites: 10, 13, and 16. CD analyses indicated that Djeya1 is highly helical, and that Aib-substitution had subtle influences upon the secondary structures of bioengineered analogues. Aib-substituted Djeya1 analogues are highly efficient CPPs, devoid of influence upon cell viability or proliferation. All three peptides increase the migration of PC-3 cells, a prostate cancer line that expresses high concentrations of Eya. Two peptides, [Aib13]Djeya1 and [Aib16]Djeya1, are bioportides which delay planarian head regeneration. As neoblasts are the only cell population capable of division in planaria, these data indicate that bioportide technologies could be utilised to directly manipulate other stem cells in situ, thus negating any requirement for genetic manipulation.


Introduction
Cellular and tissue permeability barriers are significant caveats for bioengineering strategies to develop bioactive agents and exploit novel intracellular drug modalities. As recently reviewed [1][2][3], cell penetrating peptides (CPPs), often polycationic linear sequences of 12-24 amino acids, are a versatile technology that can overcome the common biophysical constraint of ineffective intracellular access. Bioportides, CPPs with intrinsic bioactivities and so partially distinct from conventional inert vectors, accrete within eukaryotic cells to influence protein function and impact cell biology [4]. The molecular organisation of bioportides commonly includes mimetic sequences derived from functional protein domains to serve as selective modulators of intracellular protein-protein interactions (PPIs; [4][5][6]). Hence, bioportide technologies, presumably acting by a dominant-negative mechanism, enable both the understanding and discrete manipulation of intracellular signalling pathways that regulate cellular biology [5,6].
The triploblastic bilateral planarian Schmidtea mediterranea is a very common model organism employed to address fundamental cellular processes that include tissue regen-eration and the differentiation of neoblasts, relatively small pluripotent stem cells of mesenchymal origin [7,8]. Indeed, a total of 20-30% of S. mediterranea cells are neoblasts, the only planarian cell type capable of mitotic division [7,8]. A comprehensive S. mediterranea genomic database (SmedGD 2.0) confirmed that planaria are genetically more like vertebrates than both Drosophila melanogaster and Caenorhabditis elegans [9,10]. S. mediterranea is also a rigorous three-dimensional model to analyse the import of CPPs and bioportides into complex tissues presenting both physical and metabolic barriers [11].
In common with studies of arginine-rich peptides derived from both RNA-and DNAbinding proteins [12], planarian proteins that collectively control head remodelling and eye regeneration following decapitation are a viable source of cationic CPP vectors [11]. Djeya1 (RKLAFRYRRIKELYNSYR), an octadecapeptide sequence mimicking a highly conserved domain of eyes absent (Eya) proteins, is a particularly efficient and seemingly inert example of such a CPP vector sequence. Three days after head amputation, fluorescent Djeya1 effectively enters the unpigmented S. mediterranea blastema, a transient and heterogenous cell mass responsible for head morphogenesis, to penetrate deeper along the dorsal ventral axis [11]. Thus, CPPs such as Djeya1 provide the means to target bioactive agents to differentiate post-mitotic neoblast progeny [13] in addition to epithelial precursor cells or neoblast-derived mesenchymal cells.
Djeya1 mimics part of the α5 helix within the evolutionary conserved C-terminal Eya Domain (ED) of Eya proteins [14,15]. The ED domain acts as a transcriptional regulator known to bind proteins such as Dachshund and Sine Oculus [14,15]. Hence, the major objective of this study was to determine whether the bioengineering of Djeya1 analogues could provide rhegnylogically organised bioportides, CPPs in which the pharmacophores that enable cellular penetration and those essential for bioactivity are discontinuously organised. This is in contrast to a sychnologic organisation in which the pharmacophores for penetration and bioactivity are distinct. Moreover, given that Arg is the quantitatively dominant amino acid at PPI interfaces, there will clearly be some pharmacophores which are both cell penetrant and bioactive within a rhegnylogic organisation [4,5]. We hypothesized that analogues of Djeya1 with enhanced helicity would more effectively mimic ED to modulate PPIs and inhibit anterior pole and eye regeneration in S. mediterranea [11,[14][15][16].

Anterior Pole Regeneration and Eye Development Assay
Adult planaria, 4-6 mm in length, and selected for experimentation were starved 5 days prior to amputation. Transverse amputation of planarian heads, post-auricle and pre-pharynx, induced blastema formation in the trunk section, leading to eye regeneration and head remodeling [11].
Post amputation, planaria were immediately treated with exogenously added Djeya1 analogues in 35 mm diameter × 10 mm depth culture dishes (VWR International Ltd., Lutterworth, UK) to a final volume of 4 mL PAM and maintained at 20 • C. PAM, containing Djeya1 analogues, was replenished 4 days post amputation. Animals were allowed to regenerate for 7-8 days post amputation, whilst observations were performed daily to assess any morphological variations. To document the influence of bioportides upon head morphogenesis, living specimens were observed with a Swift SM series stereo microscope

Biological Characterization of Aib-Substituted Djeya1 Analogues
In the absence of a transformed neoblast cell line or a readily isolated primary culture, U373MG astrocytic tumour cells were employed as a robust model to determine CPP internalization and discount any unwanted cytotoxic effects [20,27]. Transformed cell lines known to overexpress Eya proteins were used to quantify the impact of Djeya1 analogues on cell migration (PC-3 prostate cancer cells [28]) and proliferation (U251 glioblastoma cells [29,30]). . Both media were supplemented with foetal bovine serum (FBS) 10% (v/v), penicillin (100 U mL −1 ), and streptomycin (100 µg mL −1 ), and all cell lines were maintained in a humidified atmosphere of 5% CO 2 at 37 • C.

Qualitative Peptide Uptake Analyses
Live confocal cell imaging analyses were employed to avoid fixation artefacts and establish the intracellular distributions of tetramethylrhodamine (TAMRA)-conjugated Djeya1 analogues [22]. U373MG cells were maintained as above, transferred to 35 mm sterile glass base dishes (NuncTM, Fisher Scientific, Loughborough, UK), and grown to ∼75% confluence. Cells were washed in phenol red-free DMEM prior to treatment with (TAMRA)-conjugated Djeya1 analogues diluted to a final concentration of 5 µM in phenol red free media. Treated cells were maintained at 37 • C in a humidified atmosphere of 5% CO 2 for the designated time periods. For the 1 h incubations, cells were also treated with CellMaskTM (5 µg mL −1 ; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) for an additional 5 min prior to observation using a Zeiss LSM 880 microscope equipped with live cell imaging chamber (Zeiss, Cambridge, UK). A total of 5 h incubations were also observed using a photomultiplier for transmitted light (T-PMT).

Quantitative Peptide Uptake Analyses
U373MG and U251 cells were maintained as above and then transferred to 6-well plates and grown to 80% confluence. Cells were washed and maintained in phenol redfree DMEM and subsequently treated with TAMRA-conjugated Djeya1 analogues at final concentrations of 1 µM, 2.5 µM, and 5 µM for 1 h in culture conditions as above. Cells were washed four times, detached with 300 µL of 1% (w/v) trypsin (without phenol red) at 37 • C, collected by centrifugation, and lysed in 300 µL 0.1 M NaOH for 2 h on ice. A total of 250 µL of each sample cell lysate was transferred to a black 96-well plate and analysed using a Thermo Fischer Scientific (Loughborough, UK) Fluoroskan Ascent FL fluorescence spectrophotometer (λAbs 544 nm/λEm 590 nm).
PC-3 prostate cancer cells were seeded in supplemented RPMI-1640 (as described above) into 96-well plates (1.0 × 10 4 cells/well) and maintained in a humidified atmosphere of 5% CO 2 at 37 • C for 24 h. Thereafter, cells were treated with 3 µM, 10 µM, and 25 µM of Djeya1 analogues in RPMI-1640 medium without FBS for 24 h, whilst maintaining culture conditions. Untreated (medium alone) cells were used as a control representing 100% viability. For the final hour of incubation, 10 µL of PrestoBlue TM Cell Viability reagent, resazurin-based reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to each well. A total of 100 µL of culture medium from each well was transferred to a black bottomed 96-well plate and the fluorescence at λ abs = 560 nm and λ em = 590 nm was measured using a microplate reader (Tecan Infinite ® 200 PROseries, Mannedorf, Switzerland). Cellular viability was expressed as a percentage of those cells treated with vehicle (medium) alone. Three independent experiments with five replicates for each condition were performed.

Cellular Proliferation
Cellular proliferation was determined using measurements of cell viability and employed the MTT conversion assay as described above. U251 cells were cultured as previously described, grown to anastomose in 96-well plates, and treated from 4-72 h with Djeya1 analogues (3 µM, 10 µM, 25 µM) in DMEM supplemented with FBS 10% (v/v) and maintained in a humidified atmosphere of 5% CO 2 at 37 • C. Cell viability was expressed as absorbance minus background at 540 nm. Cells treated with medium alone were also included at each designated time point and acted as a comparator when constructing growth curves.

Cell Migration Assays
PC-3 cells (1.00 × 10 5 ) were seeded in supplemented RPMI-1640 (as described above) in 24-well plates and incubated for 24 h at 37 • C in a humidified atmosphere of 5% CO 2 . Confluent cells monolayers were wounded by scratching lines with a 200 µL pipette tip. Cells were washed in phosphate-buffered saline (PBS) and incubated with fresh medium without FBS containing 3 µM or 25 µM of Djeya1 analogues for 48 h. Untreated cells were also included in the assay. Photographs were taken under ×40 magnification for EVOS TM M5000 imaging (Thermo Fisher Scientific, Waltham, MA, USA), immediately after wound incision and after 48 h. Results were expressed as a percentage of wound closure, relative to the time at 0 h. Three independent replicates of each condition were performed.

Graphical Representations and Statistical Analyses
These were performed using GraphPad Prism 9 software. For cytotoxicity and cellular proliferation assays, statistical analyses of changes in cellular viability employed an unpaired, 2-tailed, non-parametric Mann-Whitney test. Statistical analyses of changes in wound closure used a paired, 2-tailed, non-parametric Wilcoxon matched pairs signed rank test.

Site-Directed Bioengineering of the Djeya1 CPP
The entire ED of Eya proteins is exceptionally well conserved to provide a unique tyrosine phosphatase activity within a multi-domain protein classified as a haloacid dehalogenase [13][14][15]33]. The transcriptional activity of Eya proteins, dependent upon PPIs notably with Dachshund and SIX family members, appears to be regulated by the dephosphorylation activity of the ED [14,15,33,34]. The CPP Djeya1 (Table 1; [11]), identical in planarians Dugesia juponica and S. mediterranea, mimics helix 5 of the ED conserved within plants, fungi, and animals, a region that could participate in PPIs required for trancriptional activity [33,34]. Within a consensus sequence of this helical domain, three variable positions (10,13,16) were selected for Aib-substitution as a bioengineering strategy to promote helicity. Very conservative changes are indicated at positions 10 (I/V) and 13 (L/I) with more chemical variability at site 16 (S/T/A/Q). We did not introduce Aib at position 18 (R/K) as the loss of cationic charge could impact on the uptake efficacy of the modified octadecapeptide. The sequences of these peptides and others used in this study are presented in Table 2.  The synthesis and purification of Djeya1, Tat (denotes the Tat peptide, Tat 48−60 ), C105Y, and mitoparan have been described elsewhere [11]. All peptides were synthesized as Cterminal amides.  16 ]Djeya1 displayed spectra typical of α-helical and random coil structure (Figure 1a). Figure 1a shows two minima at 220 nm and 207 nm, which is typical of α-helical peptides, and a negative band at 195 nm, which is characteristic of a random coil structure. The low intensities of these bands indicate a mixture of these structures, which is confirmed by further analysis in  A key determinant in the membrane interaction of these peptides likely involves the adoption of secondary structures in the anisotropic environment of the interface [35]. This phenomenon is often investigated using TFE, a membrane-mimicking solvent with an α-helix-enhancing effect [36,37]. Figure 1b indicates that in a 50% (v/v) TFE/PBS (pH 7.4) mixture, all Djeya1 analogues adopted conformations characterized by two minima near 205 and 225 nm, respectively, and a maximum at 193 nm, which is typical of α-helical peptides [38]. Further analysis of these CD spectra showed that enhanced α-helicity was observed for Djeya1, [Aib 10 ]Djeya1, and [Aib 16 ]Djeya1 (45.50 ± 4.94%, 51.50 ± 0.07 and 46.00 ± 1.41%; Table 3b) with the remaining structural contributions to the peptide provided by random coil and β-type architectures. However, the presence of 50% TFE/PBS (v/v) mixture did not enhance the α-helical structure of [Aib 13 ]Djeya1.
It is widely accepted that peptides, including CPPs, adopt and enhance an α-helical structure in the presence of a lipid. Therefore, the structure of the peptides used here (Table 3c,d) were analysed in the presence of lipid vesicles at a lipid:peptide ratio of 100 to 1. Figure 1c shows that in the presence of the zwitterionic lipid DMPC, peptides underwent conformational change and adopted an α-helical structure, which ranged from 42% to 45% (Table 3c). In the presence of the anionic lipid DMPS, Figure 1d shows that Djeya1 and [Aib 10 ]Djeya1 displayed 47% and 63% α-helicity, respectively ( mixture, all Djeya1 analogues adopted conformations characterized by two minima near 205 and 225 nm, respectively, and a maximum at 193 nm, which is typical of α-helical peptides [38]. Further analysis of these CD spectra showed that enhanced α-helicity was observed for Djeya1, [Aib 10 ]Djeya1, and [Aib 16 ]Djeya1 (45.50 ± 4.94%, 51.50 ± 0.07 and 46.00 ± 1.41%; Table 3b) with the remaining structural contributions to the peptide provided by random coil and β-type architectures. However, the presence of 50% TFE/PBS (v/v) mixture did not enhance the α-helical structure of [Aib 13 ]Djeya1.  treated had achieved full regeneration and 33% remained unregenerated and in stasis. Further observations included an absence of generalized necrosis without excessive mucous production as a general indicator of animal stress. In these assays both Djeya1 and [Aib 10 ]Djeya1 were inactive.

Aib-Substituted Djeya1 Analogues Are Efficient CPPs
Red fluorescent TAMRA-conjugated peptides were utilised to compare the efficacy of cellular uptake. Comparative investigations utilised Tat and C105Y CPPs [11] as positive controls.

Qualitative Peptide Uptake Analyses
Confocal analyses compared the intracellular distribution of peptides in U373MG cells [11]. Observations after 1 h of exogenous peptide application (Figure 3a) revealed a similar distribution of Djeya1 analogues, which were, in part, perinuclear, and which were enhanced compared with both Tat and C105Y controls. Even after an extended period of 5 h (Figure 3b), Djeya1 analogues were absent from the cell nucleus.

Aib-Substituted Djeya1 Analogues Are Efficient CPPs
Red fluorescent TAMRA-conjugated peptides were utilised to compare the efficacy of cellular uptake. Comparative investigations utilised Tat and C105Y CPPs [11] as positive controls.

Qualitative Peptide Uptake Analyses
Confocal analyses compared the intracellular distribution of peptides in U373MG cells [11]. Observations after 1 h of exogenous peptide application (Figure 3a) revealed a similar distribution of Djeya1 analogues, which were, in part, perinuclear, and which were enhanced compared with both Tat and C105Y controls. Even after an extended period of 5 h (Figure 3b

Quantitative Peptide Uptake Analyses
These studies (Figure 4) compared the intracellular uptake of Aib-substituted Djeya1 analogues into U373MG cells and U251 cells, the latter of which is reported to overexpress Eya proteins. All three Aib-substituted Djeya1 analogues are highly efficient CPPs, broadly comparable with Djeya1 and C105Y but superior to Tat, though they displayed a different rank order of uptake efficacy in the two cell lines: U373MG [Aib 16 (Figure 4b) compared with U373MG cells (Figure 4a).

Quantitative Peptide Uptake Analyses
These studies (Figure 4) compared the intracellular uptake of Aib-substituted Djeya1 analogues into U373MG cells and U251 cells, the latter of which is reported to overexpress Eya proteins. All three Aib-substituted Djeya1 analogues are highly efficient CPPs, broadly comparable with Djeya1 and C105Y but superior to Tat, though they displayed a different rank order of uptake efficacy in the two cell lines: U373MG [Aib 16 (Figure 4b) compared with U373MG cells (Figure 4a).

Cytotoxicity of Djeya1 Analogues
A negative influence of peptides upon the viability of neoblasts could underlie their ability to inhibit head morphogenesis in S. mediterranea ( Figure 2). As indicated in Figure  5, Djeya1 analogues had no influence upon the viability of U373MG cells following exposure to peptides at concentrations of 0.1-30 µM for 4 h. Treatment of PC-3 cells with Djeya1 analogues for a longer time of 24 h similarly induced no significant changes in cellular viability (Supplementary Materials; Figure S1).

Cytotoxicity of Djeya1 Analogues
A negative influence of peptides upon the viability of neoblasts could underlie their ability to inhibit head morphogenesis in S. mediterranea ( Figure 2). As indicated in Figure 5

Impact of Djeya1 Analogues upon U251 Cellular Proliferation
Planarian neoblasts replenish lost organs and tissues by proliferation within hours of any injury [7,8,39,40]. Many wound-induced transcriptional changes necessary for these homeostatic events are regulated by the activity of extracellular signal-regulated kinase (ERK) [29,40]. U251 has been reported to have a high endogenous expression of Eya2, and siRNA knockdown of this protein decreases the proliferation and invasion of these cells, whilst Eya2 positively regulates p42/44 MAPK activity [29]. We have previously reported [11] that p42/44 inhibition prevents anterior pole regeneration in S. mediterranea. Thus, in these experiments, we compared the impact of Aib-substituted Djeya1 analogues upon the proliferation of U251 cells employing the p42/44 MAPK inhibitor U1026 as a positive control. As revealed in Figure 6, bioengineered Djeya1 analogues had minimal impact upon cellular proliferation and, whilst statistically significant data were recorded at some higher peptide concentrations, this influence was independent of concentration.  [11,31]. Data points are mean ± SEM from two experiments performed in triplicate. Statistical analyses employed a non-parametric Mann-Whitney test to compare changes in viability to that of MitP at 30 µM, (*** p = 0.0001, ** p = 0.0022), GraphPad Prism 9 software.

Impact of Djeya1 Analogues upon U251 Cellular Proliferation
Planarian neoblasts replenish lost organs and tissues by proliferation within hours of any injury [7,8,39,40]. Many wound-induced transcriptional changes necessary for these homeostatic events are regulated by the activity of extracellular signal-regulated kinase (ERK) [29,40]. U251 has been reported to have a high endogenous expression of Eya2, and siRNA knockdown of this protein decreases the proliferation and invasion of these cells, whilst Eya2 positively regulates p42/44 MAPK activity [29]. We have previously reported [11] that p42/44 inhibition prevents anterior pole regeneration in S. mediterranea. Thus, in these experiments, we compared the impact of Aib-substituted Djeya1 analogues upon the proliferation of U251 cells employing the p42/44 MAPK inhibitor U1026 as a positive control. As revealed in Figure 6, bioengineered Djeya1 analogues had minimal impact upon cellular proliferation and, whilst statistically significant data were recorded at some higher peptide concentrations, this influence was independent of concentration.

Aib-Substituted Djeya1 Analogues Enhance Cell Migration
Stem cell migration is considered a fundamental process of tissue maintenance in metazoans and of particular importance following tissue loss and wounding. Migrating neoblasts and their progeny adopt distinctive behaviours to selectively regenerate appropriate missing tissues in planaria and form the regeneration blastema where differentiation is completed [40,41]. Thus, we hypothesized that bioportides influencing head morphogenesis in S. mediterranea might adversely inhibit cellular migration in PC-3 cells that express high levels of Eya. Contrary to these expectations, all Aib-substituted Djeya analogues enhanced cellular migration, measured in wound closure assays, at concentrations of both 3 µM and 25 µM (Figure 7). Further details of these assays are provided in Supplementary Materials ( Figure S2). MTT conversion is expressed as Abs@540 nm minus background, and data points are mean ± SEM from two experiments performed in sextuplicate. A total of 4 h and 72 h, with one experiment performed in sextuplicate. Control denotes cells treated with medium alone. Statistical analyses comparing significant differences in cell viability to the untreated control were performed at 48 h and 72 h using the unpaired, 2-tailed, non-parametric Mann-Whitney test, (* p < 0.05, ** p < 0.005, *** p = 0.0001, **** p < 0.0001), using GraphPad Prism 9 software.

Aib-Substituted Djeya1 Analogues Enhance Cell Migration
Stem cell migration is considered a fundamental process of tissue maintenance in metazoans and of particular importance following tissue loss and wounding. Migrating neoblasts and their progeny adopt distinctive behaviours to selectively regenerate appropriate missing tissues in planaria and form the regeneration blastema where differentiation is completed [40,41]. Thus, we hypothesized that bioportides influencing head morphogenesis in S. mediterranea might adversely inhibit cellular migration in PC-3 cells that express high levels of Eya. Contrary to these expectations, all Aib-substituted Djeya analogues enhanced cellular migration, measured in wound closure assays, at concentrations of both 3 µM and 25 µM (Figure 7). Further details of these assays are provided in Supplementary Materials ( Figure S2).

Discussion
The molecular bioengineering of CPPs and bioportides can enhance intracellular uptake and confer or increase biological activity [4][5][6]42]. As recently reviewed [43], biophysical methodologies, including circular dichroism, have impacted the understanding of the structural determinants of CPP trafficking and the molecular mechanisms of bioportides. Numerous studies of the secondary structures of CPPs have highlighted the possibility that a helical conformation, perhaps induced by contact with phospholipid membranes, may support the passage of CPPs into cells [43,44]. One or more cationic alpha helices, particularly those containing statistically enriched Arg residues, are commonly located at protein-protein interaction (PPI) sites. Cation-π interactions, which stabilize these PPIs [45], commonly involve interactions between arginine and tyrosine residues. Hence, we hypothesized that Aib-substitution might induce helicity within Djeya1 to further increase penetration efficacy [11]. We also anticipated that the structural constraints imposed by Aib-substitution would enable bioengineered Djeya1 analogues, by a dominant-negative mechanism, to modulate PPIs within the ED domain. Interference with EYA functions as

Discussion
The molecular bioengineering of CPPs and bioportides can enhance intracellular uptake and confer or increase biological activity [4][5][6]42]. As recently reviewed [43], biophysical methodologies, including circular dichroism, have impacted the understanding of the structural determinants of CPP trafficking and the molecular mechanisms of bioportides. Numerous studies of the secondary structures of CPPs have highlighted the possibility that a helical conformation, perhaps induced by contact with phospholipid membranes, may support the passage of CPPs into cells [43,44]. One or more cationic alpha helices, particularly those containing statistically enriched Arg residues, are commonly located at protein-protein interaction (PPI) sites. Cation-π interactions, which stabilize these PPIs [45], commonly involve interactions between arginine and tyrosine residues. Hence, we hypothesized that Aib-substitution might induce helicity within Djeya1 to further increase penetration efficacy [11]. We also anticipated that the structural constraints imposed by Aib-substitution would enable bioengineered Djeya1 analogues, by a dominant-negative mechanism, to modulate PPIs within the ED domain. Interference with EYA functions as a transcriptional regulator would likely manifest as inefficient head morphogenesis in decapitated S. mediterranea [15,33,34].
This study identified two bioportides, [Aib 13 ]Djeya1 and [Aib 16 ]Djeya1, which delay head regeneration in the S. mediterranea model; [Aib 10 ]Djeya1, in common with Djeya1 [11], is an effective CPP, but unable to influence planarian morphogenesis. Djeya1, in common with Aib-substituted analogues, adopts significant α-helical structure in aqueous solution that is only marginally influenced by a change to 50% (v/v) TFE and in the presence of a lipid environment. It is noteworthy that the mitochondriotoxic bioportide mitoparan, a peptide that accretes within mitochondria to promote intrinsic apoptosis, includes Aib to replace Ala at position-10 of mastoparan, a known α-helix adopting peptide [32]. Thus, the molecular bioengineering of CPPs by Aib-substitution can produce significant variations in bioactivities that are not necessarily the result of gross changes in peptide secondary structure.
The bioactivities of [Aib 13 ]Djeya1 and [Aib 16 ]Djeya1 cannot be readily explained by increased uptake into cells. Both qualitative and quantitative analyses show that the uptake of all Aib-substituted Djeya1 analogues are generally comparable to that of the parent CPP and better than other CPPs, including Tat and C105Y. It is, however, notable that a different rank order of penetrative efficacy was evident between U373MG and U251 cell lines, which in part may be attributable to the U251 cell line overexpressing the EYA protein [29]. We have previously proposed [22] that the propensity for cellular penetration and intracellular accumulation of CPPs involves two distinct processes: the first is translocation across the plasma membrane (hydrophobicity and cationic charge are significant factors), and the second is accretion at intracellular loci. Thus, the increased uptake of [Aib 13 ]Djeya1 within U251 cells could reflect, in part, enhanced accretion at intracellular loci, particularly since [Aib 10 ] Djeya1, also with a conservative aliphatic side chain substitution for Aib, showed a reduced uptake efficacy compared to [Aib 13 ]Djeya1.
It would be intriguing to establish the mechanism of uptake for these novel Aibsubstituted Djeya1 analogues. Following live confocal cell imaging, all analogues showed a clear punctate intracellular distribution--data which suggest an endocytotic mechanism of intracellular uptake. Further investigations, including the co-labelling with specific markers of endocytosis or micropinocytosis, in addition to quantitative uptake analyses performed at both 4 • C and 37 • C, could define a mechanism of cellular uptake. Additionally, all analogues demonstrated a distinct absence of cytotoxicity even at a peptide concentration of 30 µM. Thus, these peptides do not induce gross perturbations of the plasma membrane to induce Ca 2+ -dependent necrosis.
Efforts to define a molecular mechanism of action of both [Aib 13 ]Djeya1 and [Aib 16 ]Djeya are hampered both by a lack of accessible planarian neoblast cultures and knowledge of a defined function for the α5 helix of ED that they mimic. However, we are confident that our investigations can exclude peptide-induced neoblast death as a biological explanation for the impact of [Aib 13 ]Djeya1 and [Aib 16 ]Djeya1 on anterior pole remodeling. Neither would it seem likely that these bioportides adversely influence cellular proliferation, a process fundamental to this regenerative process [16][17][18]39,40]. The observation that all three Aib-substituted Djeya1 analogues moderately enhance cell migration (wound healing) is intriguing and worthy of further investigation. However, since [Aib 10 ]Djeya1 does not inhibit heard morphogenesis in S. mediterranea, it would appear unlikely that this positive influence upon cell migration underlies the mechanism of action of [Aib 13 ]Djeya1 and [Aib 16 ]Djeya1, bioportides that delay head regeneration.
In common with the dominant negative action of many other bioportides [4][5][6]42], both sychnologic and rhegnylogic in molecular organization, we propose that [Aib 13 ]Djeya1 and [Aib 16 ]Djeya1 modulate head regeneration by interfering with PPIs mediated by the ED of Eya proteins. Support for this hypothesis is provided by a study of the G393S mutation of ED, identified from a patient with cataracts and both renal and optic abnormalities [46]. In the human ED, this missense mutation site is located close to R 399 , the first residue of ED mimetic Djeya1 analogues. Further analyses of this mutation indicated this site to be critical for the interaction of ED with unknown proteins that bridge well characterized PPIs between Eya and SIX family proteins [46].
Considering that there are so many similarities between planarian proteins and those expressed in higher vertebrates [47], we anticipate that bioportides able to influence the distribution and function of Eya proteins may prove valuable for other studies of physiology and pathology. For example, mutations in the human EYA1 gene cause branchio-oto-renal (BOR) syndrome [46]. The multifunctional nature of Eya proteins also influences tumour progression through multiple mechanisms [48]. Moreover, it is probable that [Aib 13 ]Djeya1 and [Aib 16 ]Djeya1 could be utilized to further understand and directly influence human stem cells, thus negating the requirement for genetic manipulation.

Conclusions
With the aim of modulating planarian stem cell biology through targeting PPIs integral to anterior pole and eye regeneration, rational design of helicogenic peptides, corresponding to the α5 helix of the highly conserved ED domain, generated the bioportides [Aib 13 ]Djeya1 and [Aib 16 ]Djeya1. Thus far, manipulations of planarian stem cell biology have predominantly utilized siRNA interference. To the best of our knowledge, this is the first instance in which bioportide technologies have given a functional response in this three-dimensional model, a response which is unlikely to be a detrimental consequence of cytotoxicity, inhibition of cellular proliferation, or migration.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.