New 4-Aminoproline-Based Small Molecule Cyclopeptidomimetics as Potential Modulators of α4β1 Integrin

Integrin α4β1 belongs to the leukocyte integrin family and represents a therapeutic target of relevant interest given its primary role in mediating inflammation, autoimmune pathologies and cancer-related diseases. The focus of the present work is the design, synthesis and characterization of new peptidomimetic compounds that are potentially able to recognize α4β1 integrin and interfere with its function. To this aim, a collection of seven new cyclic peptidomimetics possessing both a 4-aminoproline (Amp) core scaffold grafted onto key α4β1-recognizing sequences and the (2-methylphenyl)ureido-phenylacetyl (MPUPA) appendage, was designed, with the support of molecular modeling studies. The new compounds were synthesized through SPPS procedures followed by in-solution cyclization maneuvers. The biological evaluation of the new cyclic ligands in cell adhesion assays on Jurkat cells revealed promising submicromolar agonist activity in one compound, namely, the c[Amp(MPUPA)Val-Asp-Leu] cyclopeptide. Further investigations will be necessary to complete the characterization of this class of compounds.


Introduction
Integrins constitute a major class of cell adhesion receptors in mammals and play a vital role in cell-cell and cell-extracellular environment communication by regulating crucial aspects of cellular functions, including migration, adhesion, differentiation, growth, and survival. They are expressed in almost all cell types with varied distribution pattern [1,2]. Given their fundamental contribution in human physiology, specific integrin dysregulation phenomena are linked to the pathogenesis of many disease states (including cancer, thrombosis, vascular diseases, autoimmune pathologies, osteoarthritis, osteoporosis), and this renders them attractive targets for biomedical research [3][4][5].
The integrin family comprises 24 different heterodimeric subtypes, classified according to the specific, non-covalent combination between α and β subunits. Among these, the α 4 β 1 and α 4 β 7 subtypes, as well as the β 2 integrin subclass, belong to the leukocyte-specific integrin family and are involved in the modulation of immune functions. In particular, the α 4 β 1 integrin, also known as very late antigen-4 (VLA-4), raised much attention due to its being constitutively expressed on the surface of lymphocytes and most leukocytes, and being involved in coordinating leukocyte homing in various tissues [6].
As a fruitful consequence of intense investigation on integrins, several integrin antagonists have been validated as drugs. For example, diverse small molecules and antibodies, including eptifibatide, tirofiban, and abciximab, which target the platelet-specific integrin α IIb β 3 , are effectively used as therapeutic agents in the treatment of acute coronary syndromes and prevention of myocardial infarct following coronary intervention [7]. On the other hand, the known roles of leukocyte-specific integrins in events such as inflammation and host defense has prompted parallel anti-integrin strategies, yielding effective therapeutic anti-inflammatory agents [8,9]. Indeed, targeting α 4 integrins has proven to be effective for the treatment of inflammatory diseases, including multiple sclerosis and Crohn's disease, with some monoclonal antibodies being approved for clinical practice [10,11].
Inflammatory responses are crucial for host defense and are subjected to a complex system of control, aiming to prevent tissue damage and dangerous consequences. Since many inflammatory diseases are characterized by an influx of lymphocytes and leukocytes in the inflamed tissue, there is a keen interest in finding and testing compounds that have the potential to modulate these processes [12]. In this context, integrin activation during the different steps of the leukocyte adhesion cascade is the result of a fine-tuned orchestra of activation pathways and local regulatory networks at the site of inflammation, whose malfunctioning may cause severe disease patterns. The diseases associated with α 4 β 1 (and α 4 β 7 ) integrins are mainly of inflammatory and autoimmune nature, implying a pathological accumulation of activated leukocytes in the affected tissues such as, for example, inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, asthma, multiple sclerosis, dry eye disease and allergic conjunctivitis [10,13]. Moreover, the strict correlation between inflammation and cancer is well established at present, and immunomodulation is recognized as a useful tool not only in the treatment of inflammatory and autoimmune pathologies, but also as an adjuvant in tumor therapy. It is known that chronic inflammatory states and tumor development are closely related and mutually supportive [14]. Indeed, during chronic inflammation, the release of chemokines and growth factors supports tumor development, while, on the other hand, the tumor state can induce the upregulation of immunosuppressive molecules and the dysregulated T-cell-mediated host responses. In addition, the α 4 β 1 integrin was demonstrated to play a pivotal role in tumor angiogenesis associated with chronic inflammation, a condition that may promote the angiogenetic switch in tumors [15]. Integrin α 4 β 1 is also involved in the recruitment of progenitor cells (multipotent cells derived from bone marrow stem cells), in the transendothelial tumor cell migration and, due to its overexpression in melanoma cells, α 4 β 1 is also considered a marker of metastatic risk [16,17].
In this complex scenario, the possibility to interfere with integrin activity is of great interest and α 4 integrins have become a target for fine modulation by interaction with small-molecule ligands, based on the emerging idea that antagonist ligands may interfere in leukocyte primary functions while, on the other hand, agonist ligands can serve to promote some useful integrin functions. Enhancement of cell adhesion, for example, impairing cell detachment, may prevent tumor cell migration and metastasis processes, or may induce progenitor cell retention for stem cell therapy [18].
The natural ligands of α 4 integrins comprise the vascular adhesion molecule-1 (VCAM-1) and the alternatively spliced connecting segment 1 (CS1) region of fibronectin (FN). In particular, FN is recognized through the Leu-Asp-Val (LDV) binding epitope [19], while VCAM-1 interacts with its receptor via the homologous and essentially isosteric binding sequence Gln-Ile-Asp-Ser-Pro-Leu (QIDSPL) [20]. The discovery that these short amino acidic sequences are minimal recognition motifs has prompted the research of smallmolecule peptidomimetics resembling the natural binding epitope and fitting into the groove at the α and β subunit interface [10,21]. Figure 1 collects some notable results in the discovery of linear peptidomimetic ligands, reminiscent of the LDV sequence and targeting the α 4 β 1 receptor.
BIO1211 is based on the peptide sequence Leu-Asp-Val-Pro (LDVP) substituted at the amino terminus with the 4-[(N-2-MethylPhenyl)Ureido]PhenylAcetyl group (MPUPA). The introduction of this last moiety was demonstrated to produce a substantial increase in both potency and enzymatic stability as compared to the LDV peptide precursor [23]; for this reason, BIO1211 is commonly used as a reference compound in many studies aiming to developing new α 4 β 1 ligands. The in vitro efficacy and potency of this compound were also confirmed in vivo: when administered as an aerosol, it showed prophylactic efficacy in a sheep model of allergic bronchoconstriction, electing this nonsteroidal compound as the first small-molecule α 4 β 1 antagonist to enter clinical trials. However, the residual peptide nature of BIO1211 caused a certain enzymatic instability. To overcome this behavior, a number of bioactive peptidomimetics have been prepared (Figure 1), which share common structural features, including an aromatic cap at the N-terminus, a suitable spacer, and a carboxylic group mimetic of the Asp residue, with BIO1211 [10]. Compound LLP2A (2, Figure 1), proposed by Peng et al. in 2006 [24], was identified in a competitive cell-based screening under a high concentration of soluble BIO1211. It showed an exceptionally high affinity toward α 4 β 1 receptor (IC 50 = 2 pM, Jurkat cell assay) without any effect on the cell proliferation and survival of α 4 β 1 -positive cells. For this reason, it was differently functionalized with NIR-fluorescence probes, or labelled with radionuclides ( 111 In, 64 Cu, 99m Tc and 18 F) to image several different tumors, including melanoma [25,26]. Recently, due to its high binding affinity to integrin α 4 β 1 , which is highly expressed on mesenchymal stem cells (MSCs) and regulates MSC homing, adhesion, migration and differentiation, LLP2A has also been exploited for tissue engineering and regenerative medicine applications [27].  [13,18,22,24]. The MPUPA moiety is depicted in blue.
According to a common trend in bioactive peptide research, the introduction of cyclic scaffolds, including proline derivatives and other five-membered heterocycles, has been exploited by many researchers as amide bond isosteres and conformational restraints in the design and synthesis of peptidomimetic integrin ligands [28]. Along this line, the insertion of a D-configured β2-proline scaffold into a peptidomimetic structure led to the development of compound DS-70 (3, Figure 1), which demonstrated a high binding affinity for human α 4 β 1 integrin and potent antagonist activity of α 4 -mediated cell adhesion.
Additionally, it was successfully tested in a guinea pig preclinical model of allergic conjunctivitis [13]. Lastly, compound THI0019 (4, Figure 1) [18] was the first α 4 β 1 agonist designed and synthesized starting from a potent α 4 β 1 antagonist as a template [29]. THI0019 was generated by introducing two structural modifications into a previously identified α 4 β 1 antagonist. As a result, THI0019 enhanced the rolling, spreading, adhesion, and migration of endothelial progenitor cells in vitro in a α 4 β 1 -dependent fashion; the authors suggested that compound 4 could temporarily occupy the ligand binding pocket, inducing a small conformational change in the receptor that favors agonist displacement and binding of natural ligand, thus opening opportunities for stem cell therapy [18].
Despite the relevant results obtained in the preclinical evaluation of these molecules as targeting motifs in the construction of imaging probes, potential treatments in ocular diseases, or innovative materials for regenerative medicine, there is still ample room for the development of new and structurally varied binders, which may enrich the pool of existing α 4 β 1 ligands.
The Amp scaffold is a new-to-nature, yet nature-reminiscent small-molecular entity, which can be grafted onto the peptide sequence of interest and impart proper ligand conformation [30], while conferring stability toward enzymatic degradation. Moreover, the Amp nucleus possesses a N α -proline site free for covalent bonding to useful functional units; indeed, the Amp-based cyclopeptide cores were covalently conjugated to either fluorescent tags, chelating units, or established therapeutic drugs to obtain hybrid dualactive structures and nanoparticles [33][34][35][36][37][38][39][40].
In the present study, the Amp scaffold was selected as the core unit for building up a new class of cyclic small-molecule peptidomimetics by linking it to proper pharmacophoric groups, aiming to target the α 4 β 1 integrin receptor. To explore this possibility, we designed and synthesized a small collection of cyclic aminoproline-based peptidomimetics of general formula c[Amp(MPUPA)Xaa-Xbb-Xcc-Xdd] 7 (Figure 2), in which the Amp scaffold was grafted onto suitable peptide sequences (LDV motif and analogues) and functionalized at the N α -proline site with the well-known α 4 -integrin targeting MPUPA moiety.
In this work, we report the molecular modelling-driven design, the synthesis, and the chemical characterization of a collection of seven tetra-and pentacyclopeptidomimetics of type 7, as well as the evaluation of their binding competence towards the α 4 β 1 integrin receptor by cell adhesion assays using Jurkat cells in the presence of VCAM-1, with the aim to preliminarily assess their ability to bind α 4 β 1 integrin and possibly serve as modulators of integrin function.

Design of Novel α 4 β 1 Ligands
The study of the interactions between ligands and their biological targets greatly benefits from the availability of ligand-receptor crystallographic insights; since no X-ray analyses exist to date on the crystal structure of the α 4 β 1 receptor, or of the same receptor in complex with its small-molecule ligands, the design of a new class of cyclic Amp-based peptidomimetics required the generation and validation of a α 4 β 1 receptor model by molecular modelling studies [41].
The work started from the atomic coordinates of the single α 4 and β 1 domains, which were available from the α 4 β 7 integrin complex (PDB code: 3V4V) [42] and the α 5 β 1 integrin complex (PDB code: 3VI4) [43], respectively. In fact, these integrins possess a high degree of structural conservation, a large, solvent-exposed ligand-binding site at the α/β interface, and a divalent cation (Mg 2+ ) at the metal ion-dependent adhesion site (MIDAS), which may be involved in a coordinated bond with a carboxylate group of the ligand. Using the α 5 β 1 integrin complex as a template, the α 4 subunit was aligned with α 5 bound to β 1 ; then, the α 5 subunit was removed, giving a preliminary α 4 β 1 complex. The integrin complex thus obtained was refined and optimized by a minimization protocol ( Figure 3) and then subjected to a validation procedure through docking studies. To this aim, eight known α 4 β 1 integrin antagonists were selected, namely compound BIO1211 (1, Figure 1), compounds 8a, 8b, 9, 10a, 10b, 11a, and 11b ( Figure 4), along with one novel Amp-based cyclic candidate (compound c[Amp(MPUPA)-Leu-Asp-Val-Gly] 12, Figure 4). This small collection of known peptidic and peptidomimetic structures showed a certain level of molecular diversity and inhibitory potencies towards α 4 β 1 integrin, ranging from micromolar to low nanomolar values [44][45][46]. In particular, the low-nanomolar cyclic peptides 8a, 8b, and 9, containing the Cys-Asp-Pro-Cys or Cys-Ser-Pro-Cys core structures, and their spirocyclic analogues 10a and 10b, constrained by a disulfide (or a thioether) bridge, were conceived to mimic the essential α 4 β 1 IDS or LDV binding sequences [44,45]. Compound 11a represents one of the linear analogues of BIO1211 obtained by a retro-sequence strategy and containing a dehydro-βproline ring which, similarly to compounds 8-10, showed a potent inhibitory activity of α 4 β 1 /VCAM interaction with IC 50 in the nanomolar range [46], accompanied by a superior enzymatic stability respect to cyclic peptides 8-10. Figure 5 shows the binding poses of compounds BIO1211 and 11a within the α 4 β 1 binding site, as well as their overlapping structures. The analysis of these binding poses revealed that both compounds can interact with Mg 2+ cation in the β subunit and share a similar disposition within the binding pocket of the receptor, with the common functional groups interacting with the same amino acid residues in the α subunit. Notably: (i) the ureido group within MPUPA of both compounds establishes a bidentate interaction with Glu124 (E124) residue, (ii) the terminal aromatic ring of the ureido group establishes a cation-π interaction with Lys156 (K156) residue, (iii) the isopropyl group of 11a adopts a spatial orientation similar to the leucine side chain of BIO1211. This last observation would explain the experimental evidence showing that compound 11b (the enantiomer of 11a) is considerably less active on α 4 β 1 [46]. Furthermore, BIO1211 establishes a Hbond with Tyr187 (Y187), a crucial interaction, as highlighted by reported mutagenesis studies [47]. The rationalization of the binding poses of the selected compounds proved to be in agreement with the SAR studies reported in the literature [44,46], supporting the reliability of the developed receptor model. The validated model was used in the subsequent docking studies, where the same experimental protocol was applied, to identify the binding modes of Amp-bearing α 4 β 1ligands, and to predict possible structural modifications improving affinity toward the α 4 β 1 integrin. To this end, the docking procedure was used to evaluate c[Amp(MPUPA)Leu-Asp-Val-Gly] (12) as a new potential α 4 β 1 ligand. In Figure 6, the binding pose of compound 12 is shown and compared to that of BIO1211. From the analysis of the docking poses, we noticed that cyclopeptide 12 (pink sticks) would be able to establish some comparable interactions to BIO1211 (purple sticks) in the binding pocket of the α 4 β 1 receptor model. Compound 12 seems to be particularly able to (i) chelate the divalent cation (Mg 2+ ) through the carboxylate group of the Asp residue, (ii) interact with the amino acidic residues Tyr187, Lys156 and Glu124 in a similar way as BIO1211; (iii) its Val residue seems to assume the favorable spatial orientation that was observed for BIO1211, and (iv) the MPUPA moiety of both compounds occupies the same region.
Starting from compound 12, six additional cyclic Amp-based cyclopeptide derivatives were designed, namely, compounds 13-18 (Figure 7), to be launched in the synthesis program. The design was rooted in the following considerations: (i) substitution of the Glu residue for Asp could further favor the interaction of the carboxylate group of the side chain with the divalent cation of MIDAS (e.g., compound 12 vs. 13, 14 vs. 15, 16 vs. 17), (ii) restriction of the cyclopeptide ring via Gly depletion could provide insights about the influence of ring size and constrain on binding affinity (e.g., pentapeptide compounds 12-13 vs. tetrapeptide analogues 14-18); (iii) exploitation of retro-sequences could expand exploration of the pharmacophoric space (e.g., VDL-based compound 16 vs. LDV counterpart 14, and VEL-based compound 17 vs. LEV counterpart 15), (iv) substitution of the RGD sequence for LDV would generate derivative 18, which could likely be used as a negative control in α 4 β 1 -directed biological assays.

Synthesis of Novel α 4 β 1 Ligands
The synthesis of the designed compounds 12-18 began with the chemoselective in-solution N α deprotection of commercially available Fmoc-4-amino-1-Boc-pyrrolidine-2-carboxylic acid (19) to 20 (Scheme 1) and subsequent functionalization with the 4-[(N-2-methylphenyl)ureido]phenylacetyl (MPUPA) moiety 21, to provide the N-Fmoc-Amp(MPUPA)-OH scaffold 22 (79% yield) to be used in the following SPPS procedures. The MPUPA moiety 21 was instead synthesized, with good yield, starting from the commercially available precursors, o-tolyl isocyanate and 4-aminophenylacetic acid, following literature procedure [22]. The synthesis of compound 22 entailed the preliminary activation of the carboxylic function within MPUPA unit 21 by means of HATU/HOAt/collidine coupling system in dry DMF, followed by the addition of 20. For the synthesis of the linear precursors of targeted cyclopeptides 12-18, the Fmocbased SPPS strategy was adopted, followed by in-solution cyclization and deprotection protocols (Scheme 2). All the linear peptide sequences were prepared, starting from the proper acid-labile chlorotrityl chloride resin preloaded with one of the three different starting amino acid residues, Asp(tBu), Glu(tBu) or Gly. Within all the designed peptide sequences, the aminoproline scaffold played a critical role; in fact, in all instances, this unit was in a central position within the linear peptides, creating a local constraint that would likely pre-organize the terminal chains toward the final macrocyclization step. The synthesis of the designed sequences required the stepwise addition of Fmoc-protected amino acids to the growing peptides, with alternating coupling steps (in the presence of HATU/HOAt/collidine) and Fmoc-removal procedures (by using 20% piperidine/DMF solution); then, the linear peptide sequences were readily cleaved from the resin using the conventional AcOH/TFE/DCM mixture. The crude linear peptides 23-29 were obtained in yields ranging from 78% to 99% for the entire solid phase sequences. The linear peptides 23-29 were then subjected to delicate, in-solution, head-to-tail cyclization. The cyclization reactions were carried out under diluted conditions (1-3 mM) in a solution of dry DCM/DMF solvent mixture, in a 15:1 ratio. The crude cyclized peptides were purified by automated flash chromatography, furnishing the protected cyclized peptides 30-36 with yields ranging from 43% to 88%. Finally, side-chain deprotection of the cyclic peptides was carried out under acidic conditions (TFA/TIS/H 2 O 95:2.5:2.5). Compounds 12-18 were recovered as TFA salts after RP-HPLC purification, in yields ranging from 23% to 63%, with overall yields ranging from 21% to 44%. Target compounds 12-18 were fully characterized by high-resolution ESI mass spectrometry as well as various NMR techniques.

Biological Evaluation
To investigate the ability of the newly synthesized cyclopeptidomimetics 12-18 to recognize and bind α 4 β 1 integrin, cell adhesion assays were performed on VCAM-1. The compounds were evaluated for their ability to interfere with α 4 β 1 integrin-mediated cell adhesion by using Jurkat cells, which are known to constitutively express this integrin [46,[48][49][50][51]. Compound BIO1211 (1) was included as a reference antagonist ligand, which is able to significantly reduce Jurkat cell adhesion to VCAM-1. The results of cell adhesion assays are summarized in Table 1.  Under the adopted experimental conditions, most of the synthesized compounds were unable to compete with VCAM-1 for the binding to the α 4 β 1 receptor expressed on Jurkat cells and no effect was detected on the impairing or promoting of cell adhesion (anti-adhesive or pro-adhesive effect) at the tested concentrations (ranging from 0.1 nM to 100 µM). The new cyclopeptidomimetic 16, instead, was able to modulate α 4 β 1 integrin-mediated cell adhesion, with an interesting potency in the submicromolar range; in particular, it behaved as an agonist, as it was able to increase Jurkat cell adhesion to VCAM-1 as compared to the control. More specifically, this compound, which features a constrained cyclotetrapeptide ring containing the retro-sequence Val-Asp-Leu, showed a dose-dependent enhancement in cell adhesion with an EC 50 of 0.37 µM, and, for this reason, it was referred to as an agonist.
In an attempt to rationalize our results, two additional experiments were envisaged to evaluate the possible competition for VCAM-1 binding site between Amp-based cyclotetrapeptide 16 and BIO1211, which is described as a potent noncovalent antagonist of α 4 β 1 /VCAM-1 interaction in both receptor-binding studies and cell adhesion assays [22]. In the first set of experiments, Jurkat cells were pre-incubated with BIO1211 (1 µM) for 30 min and then incubated with compound 16 (100 µM), before being plated in VCAM-1 coated wells. As expected, BIO1211, acting as an antagonist, significantly decreased Jurkat cell adhesion to VCAM-1. Moreover, compound 16 was not able to modify the reduced cell adhesion induced by BIO1211 (Figure 8a). Similarly, when Jurkat cells were pre-incubated with compound 16, BIO1211 did not modify the increased cell adhesion. When Jurkat cells were pre-incubated with BIO1211 and then with compound 16, the latter was not able to modify the reduction in adhesion induced by BIO1211. Similarly, BIO1211 was not able to revert the effect induced by pre-incubation with compound 16. (b). Even in absence of VCAM-1, compound 16 was able to induce Jurkat cell adhesion; BIO1211 (10 nM-100 mM) was not able to reduce the increment of cell adhesion induced by compound 16. Control cells were not pre-incubated with any compound. Jurkat cells plated in wells coated with 10 µg/mL bovine serum albumin (BSA) were considered as negative control. Each value is the mean ± SD from four separate experiments carried out in quadruplicate. **** p < 0.0001 vs. BSA-coated wells; #### p < 0.0001 vs. control (Newman-Keuls test after ANOVA).
In a second set of experiments, the ability of compound 16 to increase cell adhesion was tested in the absence of VCAM-1. Wells were coated by passive adsorption with compound 16 or BSA (both at 10 µg/mL) as a negative control and Jurkat cell adhesion was measured (Figure 8b). Compound 16 produced a significant adhesion of Jurkat cells, even in the presence of different concentrations of BIO1211.
With the present data, a more detailed analysis of the structure-activity relationship within our ligand collection would be unwise.

Discussion
Two main aspects emerge from the experimental results given above, which may deserve comment: first, a discrepancy was observed between the computational-driven design and the experimental results, and second, an agonist behavior emerged in one candidate instead of the "expected" antagonist activity of the modeled structures.
Regarding the first point, it has to be underlined that, lacking sound structural details of the α 4 β 1 integrin, a reliable model for the design of potential α 4 β 1 ligands remains elusive, although several molecular modeling studies, computational screenings and 3D models have been reported to date [52][53][54][55]. Additionally, the high degree of conformational flexibility featuring the targeted receptor was not considered in this study, and this could have played a decisive role in decreasing the predictability potential of the molecular modeling studies.
The in vitro biological evaluation showed that, among the seven candidates, compound c[Amp(MPUPA)Val-Asp-Leu] (16) exhibited a low-micromolar (0.37 µM) agonist activity in Jurkat cell adhesion assay. The fact that the rational design based on antagonist ligands consigned an agonist product must not come as a surprise, as this was testified by notable precedents even in the field of α 4 β 1 ligands [18,24]. It has been demonstrated that even small structural variations in the integrin ligand core can cause the shift from antagonist to agonist behavior [18,49].
Competition experiments involving compound 16 and BIO1211 revealed that the respective agonist and antagonist behavior were not reciprocally modified. These results seem to exclude the competition between compound 16 and BIO1211 for the same binding site, and the agonist activity of 16 could be ascribed to an interaction of this compound in a different region of the receptor. This behavior has already been observed for other ligands of this integrin; for example, for known compound LLP2A (2, Figure 1), whose binding site on α 4 β 1 integrin receptor was claimed to be different, and close to (or only partially overlapping) with the binding site of VCAM-1 [24].
Finally, in contrast to RGD-dependent integrins, the binding regions of α 4 integrins (in particular the α 4 β 7 binding site) have been described as long and wide crevices, open at both ends and capable of the lengthwise accommodation of differently shaped binders [42]. This fact could explain that compounds 16 and BIO1211 do not seem to share the same binding region and could provide a reason for the difficulty encountered in the rational design.  [43] and α 4 β 7 (PDB code: 3V4V) [42] crystal structures were used for generation of the α 4 β 1 complex: the α 4 subunit was obtained by the α 4 β 7 complex, while the β 1 subunit was derived from α 5 β 1 receptor. Using the α 5 β 1 integrin complex as a template, the α 4 subunit was aligned with α 5 bound to β 1 (α 5 β 1 ), then the α 5 subunit was removed, giving a preliminary α 4 β 1 complex. The complex was prepared by using the Protein Preparation Wizard tool of Maestro 9.1 (https://www.schrodinger.com/; accessed on 9 November 2018) minimized by a multi-step protocol in which the harmonic restraints were gradually scaled. The complex obtained was used for the following docking studies.

Ligand Docking Calculations
All docking studies were carried out using the same experimental protocol. The structures of the different antagonists were prepared from the fragment-building tool available in Maestro 9.1 and the geometries were optimized using the force field OPLS-2005 [56]. The docking grid was centered on the Mg 2+ atom and a grid size of 12 × 12 × 12 Å was used. Docking studies were performed using Glide as software, the SP method and the enhanced sampling method for conformational exploration of the different ligands. The remaining docking parameters were used as default.  (29). Resin swelling. The desired resin (1 equiv) was swollen in a solid phase reaction vessel with dry DMF (2 mL) under mechanical stirring; after 40 min the solvent was drained and the resin was washed with DCM (2×) and DMF (2×). Peptide coupling. A preformed solution of Fmoc-AA-OH (1.5 equiv) in dry DMF (2 mL) was treated with HATU (2 equiv), HOAt (2 equiv) and 2,4,6-collidine (2 equiv), and stirred for 10 min before adding to the resin. The mixture was shaken at room temperature for 5 h. Completion of the reaction was checked by the Kaiser test. The solution was drained and the resin was washed several times with DMF (2×), iPrOH, (2×), Et 2 O (2×), DCM (2×). The resin was then treated with 20% piperidine in DMF (2 mL) and the mixture was stirred for 30 min (Fmoc cleavage). The solution was drained and the resin was washed with DMF (2×), iPrOH, (3×), Et 2 O (2×), DCM (2×). The couplings of the further amino acids, in the proper sequence, were carried out under the same conditions. Resin cleavage. After coupling of the last Fmoc-AA-OH, the resin was treated with 2 mL of the cleavage mixture DCM/TFE/glacial AcOH (3:1:1) and kept under mechanical stirring for 20 min at room temperature. The solution was recovered and the resin was carefully washed with DCM (2×). This protocol was repeated twice. The combined solution was evaporated under reduced pressure affording the desired linear peptide, which was used in the following synthetic step without further purification.

General Procedure for Cyclization Reaction
Protected cyclic peptides 30-36 were prepared according to the following general procedure. A solution of linear peptide (1 equiv) and 2,4,6-collidine (3 equiv) in dry DCM/DMF solvent mixture (15:1 ratio) was prepared. The mixture was stirred under argon at room temperature and added dropwise to a solution of HATU (3 equiv) and HOAt (3 equiv) in dry DCM/DMF solvent mixture (15:1 ratio). The reaction mixture was degassed by argon/vacuum cycles (3×) and left to stir under argon at room temperature for 5 h. After reaction completion, the solution was concentrated under vacuum. The  Jurkat E6.1 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and were routinely cultured in RPMI-1640 (LifeTechnologies, Milan, Italy) supplemented with 10% FBS (fetal bovine serum, Life Technologies) and 2 mM glutamine. Cells were kept at 37 • C under 5% CO 2 humidified atmosphere. Jurkat cells are a widely used cell model to study potential agonist or antagonist ligands able to modulate integrin-mediated cell adhesion [46,[48][49][50][51]. Jurkat cells endogenously express α 4 β 1 integrin [13].

Cell Adhesion Assays
The assays were performed as previously described [51]. In brief, black 96 well plates were coated with VCAM-1 (2 µg/mL) overnight at 4 • C; then, non-specific hydrophobic binding sites were blocked with 1% BSA (bovine serum albumin, Sigma-Aldrich) in HBSS (Life Technologies) for 30 min at 37 • C. Jurkat cells were labelled with CellTracker green CMFDA (12.5 µM, Life Technolgies) and pre-incubated with various concentration (10 −4 -10 −10 M) of each new cyclopeptidomimetic or with the vehicle (DMSO) for 30 min at 37 • C. Afterwards, Jurkat cells were plated on VCAM-1-coated wells and incubated for 30 min at 37 • C. Then, wells were washed three times with 1% BSA in HBSS and Jurkat cells were lysed with 0.5% Triton X-100 in PBS for 30 min at 4 • C. Green fluorescence (Ex485 nm/Em 535 nm) was measured in an EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Experiments were performed in quadruplicate and repeated at least three times. The number of adherent cells was determined by comparison with a standard curve made with a known concentration of labelled Jurkat cells. Data analysis and IC 50 /EC 50 values were calculated using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA).
In another set of experiments, Jurkat cells were plated (500,000 cells/well) in 96-wells plate previously coated by passive absorption with VCAM-1 (2 µg/mL) or with compound 16 (10 µg/mL), the most effective compound under examination, or with BSA (10 µg/mL, as negative control). To investigate any potential ligand binding competition, 30 min before plating cells on VCAM-1-coated wells, BIO1211 (1 µM) was added to the cells pre-incubated with compound 16 (100 µM) or compound 16 was used to treat to the cells pre-incubated with BIO1211. Moreover, Jurkat cells pre-incubated with different concentrations of BIO1211 (100 mM-10 nM) were plated in compound 16-coated wells. The number of adherent cells was determined as described above.

Conclusions
The central role played by α 4 β 1 integrin in inflammatory and autoimmune pathologies and tumor-related diseases has been widely explored, so the search for potent and selective α 4 β 1 integrin binders has been and remains a topic of interest in current biomedical research.
The present study, addressing the synthesis of new potential α 4 β 1 integrin ligands, is rooted in this challenging field of research. Based on some initial computational suggestions, seven new cyclic peptidomimetics, all bearing a common aminoproline core scaffold and an MPUPA hexocyclic motif, were synthesized and structurally characterized. Preliminary in vitro biological evaluation revealed that one of these candidates, compound 16, featuring the constrained c(Amp-VDL) cyclotetrapeptide structure, showed a moderate ability to enhance Jurkat cell adhesion to VCAM-1, and further biological evidence pointed to the exclusion of competition with the known antagonist BIO1211 for the same receptor binding site.
Further biological investigations will be necessary for a complete characterization of the agonist behavior of our compounds, to assess integrin selectivity and possibly define structural requirements for agonist vs. antagonist activity, with the ultimate intention of contributing to the expanding knowledge in the field of small-molecule integrin ligands.