N-Methylation of isoDGR Peptides: Discovery of a Selective Î±5Î²1

Article pubs.acs.org/jmc

Cite This: J. Med. Chem. 2018, 61, 2490−2499

N‑Methylation of isoDGR Peptides: Discovery of a Selective α5β1Integrin Ligand as a Potent Tumor Imaging Agent Tobias G. Kapp,†,# Francesco Saverio Di Leva,‡,# Johannes Notni,§ Andreas F. B. Rad̈ er,† Maximilian Fottner,† Florian Reichart,† Dominik Reich,§ Alexander Wurzer,§ Katja Steiger,∥ Ettore Novellino,‡ Udaya Kiran Marelli,⊥ Hans-Jürgen Wester,§ Luciana Marinelli,*,‡ and Horst Kessler*,† †

Institute for Advanced Study and Center of Integrated Protein Science, Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany ‡ Dipartimento di Farmacia, Università degli Studi di Napoli “Federico II”, Via D. Montesano 49, 80131 Naples, Italy § Lehrstuhl für Pharmazeutische Radiochemie, Technische Universität München, Walther-Meißner Straße 3, 85748 Garching, Germany ∥ Department of Pathology, Technische Universität München, Trogerstraße 18, 81675 München, Germany ⊥ Central NMR Facility and Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, 411008 Pune, India S Supporting Information *

ABSTRACT: Specific targeting of the integrin subtype α5β1 possesses high potential in cancer diagnosis and therapy. Through sequential N-methylation, we successfully converted the biselective α5β1/αvβ6 peptide c(phg-isoDGR-k) into a potent peptidic RGD binding α5β1 subtype selective ligand c(phg-isoDGR-(NMe)k). Nuclear magnetic resonance spectroscopy and molecular modeling clarified the molecular basis of its improved selectivity profile. To demonstrate its potential in vivo, c(phg-isoDGR-(NMe)k) was trimerized with the chelator TRAP and used as a positron-emission tomography tracer for monitoring α5β1 integrin expression in a M21 mouse xenograft.

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INTRODUCTION RGD binding integrins are αβ heterodimeric membrane adhesion receptors, which specifically recognize extracellular matrix (ECM) proteins featuring the tripeptide Arg-Gly-Asp (RGD) motif, such as fibrinogen (Fbg), fibronectin (Fn), vitronectin (Vn), and the latency associated peptide of TGF-β1 and -β3.1−3 Besides their role in cell adhesion, RGD binding integrins intensively cross-talk with cytokines and growth factors, such as the vascular endothelial growth factor (VEGF), to regulate key processes like cell migration, proliferation, and neo-angiogenesis.4−6 Under local hypoxia, these events can trigger the so-called angiogenic switch,7 which in turn promotes tumorigenesis, cancer growth, and metastasis.4−6 For these reasons, the RGD binding integrin subfamily has been represented as a major target for cancer diagnosis and therapy for a long time.5,6,8 Initially, medicinal chemists were attracted to the RGD binding subtypes αvβ3 and αvβ5 since these receptors are usually expressed at low levels in various tissues. However, they are highly up-regulated in many malignancies.6,9,10 Hence, several αvβ3/β5 ligands have been developed and clinically investigated for the treatment of various cancer types.5,6,11 The most prominent example is cilengitide, namely, © 2018 American Chemical Society

c(RGDf(NMe)V) (EMD121974), which reached the clinical phase III for the treatment of gliobastoma multiforme (GBM).9 However, clinical trials depicted that cilengitide cannot improve the overall survival rate in GBM patients who were treated with the standard chemoradiotherapy regimen.12 The failure of cilengitide raised doubts on the effective functionality of RGD binding integrins, particularly with regard to αvβ3 and αvβ5 in cancer and the real working mechanism of their exogenous ligands. This prompted further investigation, which depicted that the α5β1 integrin interacted with p53 in GBM to induce apoptosis, whereas αvβ3 did not have such an effect.10 In fact, cilengitide has more than one order of lower binding affinity for α5β1 than for αvβ3. Cilengitide has a half-life of 4 h in humans.13 Thus, its in vivo concentration might be too low for the desired antiangiogenic effects. In this regard, preclinical studies have previously demonstrated that αvβ3/β5 inhibitors, such as cilengitide, may have dose-dependent opposing effects in vivo. Indeed, low nanomolar concentrations of such drugs can paradoxically promote VEGF-mediated angiogenesis and Received: December 4, 2017 Published: February 28, 2018 2490

DOI: 10.1021/acs.jmedchem.7b01752 J. Med. Chem. 2018, 61, 2490−2499

Journal of Medicinal Chemistry

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stimulate cancer growth, possibly through partial agonism.14 Overall, this evidence suggests that αvβ3 and αvβ5 play an ambiguous role in cancer development. A growing body of evidence depicts that other RGD binding integrins, such as α5β1, αvβ6, and αvβ8, are also unambiguously involved in tumor development, thus representing valuable targets for antiangiogenic drugs.15 In particular, the α5β1 receptor is strongly up-regulated during tumor angiogenesis, and its inhibition clearly results in arrest of cancer growth.6,16,17 Clinically, its expression has been associated with a more aggressive phenotype in high-grade gliomas and colon cancers.18,19 As mentioned above, α5β1 promotes tumor aggressiveness by interfering with the p53 pathway. Specifically, a negative crosstalk exists between the α5β1 integrin and the tumor suppressor p53, to impair p53 activation by chemotherapeutic drugs, while p53 specifically inhibits the expression of the α5 integrin subunit.20,21 In light of these recent advances, α5β1 is now considered one of the most prominent targets among RGD binding integrin receptors for cancer treatment, as well as for tumor diagnosis. Indeed, α5β1 selective ligands could be functionalized to afford molecular imaging probes, which would allow the in vivo monitoring of the expression pattern for this integrin subtype.22 Nonetheless, the lead identification of α5β1 selective inhibitors and their development are very challenging since the binding cavity, in comparison to other integrins of the RGD binding subfamily, in particular αvβ3 and αvβ6,23 only differs by a few single-point substitutions. In fact, no completely selective α5β1 RGD binding peptide has been described hitherto. The previously reported α5β1 selective peptidic ligand ATN-16124,25 does not bind to the RGD binding site and is not involved in cell adhesion.26 In an attempt to develop an RGD binding α5β1 selective ligand, we have recently turned our attention to cyclic isoDGR27−29 containing peptides. In contrast to classical RGD peptides, which often behave as partial agonists,30 isoDGR peptides have demonstrated the ability to show no agonistic effects.31 Thus, they represent a new opportunity for selectively targeting these receptors in vivo with reduced side effects. First, our efforts allowed us to identify c(G-isoDGR-G) as the lead integrin ligand.32 The subsequent mutation of the flanking glycine residues of this peptide, combined with a D-amino acid scan, led to the identification of c(phg-isoDGR-k) (1) as a dual ligand of the Fn recognizing integrins α5β1 and αvβ6.32,33 Here, in order to further enhance the potency and selectivity of 1 toward the α5β1 receptor, this peptide was subjected to a comprehensive N-methyl scan. In fact, the N-methylation of backbone amide bonds in cyclic peptides is a well-established approach for modulating their integrin binding affinity and subtype specificity,35 as has also been demonstrated in the case of cilengitide.9,34 In addition, N-methylation can significantly increase peptide stability against enzymatic degradation.35 Hence, we generated a library of the N-methylated derivatives of 1 (peptides 2−6), which were evaluated for their integrin binding affinity and selectivity profile. Nuclear magnetic resonance (NMR) spectroscopy and molecular modeling studies were performed in order to understand the binding mode of these newly synthesized peptides to the distinct integrin subtypes, which also provided general hints for the design of α5β1 specific ligands. Finally, imaging studies were performed in order to map the α5β1 expression in a human melanoma xenograft mouse model.

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RESULTS AND DISCUSSION Chemistry. Compounds 1−6. For the synthesis of peptides 1−6, the linear peptide sequences were synthesized on solid support by using the standard Fmoc-SPPS procedure. The cyclization position was chosen individually, dependent on the position of N-methylation in the corresponding sequence, in order to avoid head to tail-cyclization with a methylated Nterminus. The N-methylation procedure was performed using the on-resin methylation technique with methanol under Mitsunobu conditions.36 The mild and orthogonal cleavage from the solid support was performed by using a 20% HFIP solution in DCM. Subsequently, the Fmoc-deprotected linear peptides were head-to-tail cyclized by using DPPA/NaHCO3 in DMF. After the removal of the solvent, the cyclized peptides were fully deprotected in a mixture of trifluoroacetic acid (TFA)/H2O/triisopropylsilane (TIPS), respectively. After preparative high-performance liquid chromatography (HPLC) purification and lyophilization, 1−6 were obtained as TFA salts, respectively. Compound 7. For the synthesis of the pentynoic acid modified compound 7, the linear peptide H-Arg(Pbf)-(NMe)D-Lys(Dde)-D-Phg-isoAspOtBu)-Gly was synthesized on solid support by using the Fmoc protocol. After cyclization of this sequence and crude product isolation, the Dde protected D-Lys side chain was orthogonally deprotected by using hydrazine. Precipitation from the reaction mixture yielded the peptide c(DPhg-isoAsp(OtBu)-Gly-Arg(Pbf)-(NMe)-D-Lys) with acceptable purity. In the next step, this peptide was coupled to pentynoic acid by using the coupling reagent HATU with DIEA as base. After global deprotection, preparative HPLC purification, and lyophilization, 7 was obtained as a white TFA salt. Biological Evaluation. The binding affinities of compounds 2−6 toward the α5β1, αvβ6, and αvβ3 integrins were evaluated by a previously established ELISA-like assay, by using isolated integrins25,29 (Table 1). Each IC50 value extracted from Table 1. Evaluation of the Binding Affinities of 2−6 and Stem Peptide 1 for the α5β1, αvβ6, and αvβ3 Integrin Subtypes peptide sequence

compd

IC50 α5β1 [nM]

IC50 αvβ6 [nM]

IC50 αvβ3 [nM]

c(phg-isoD-G-R-k)30 c((NMe)phg-isoD-GR-k) c(phg-(NMe)isoD-GR-k) c(phg-isoD-(NMe)GR-k) c(phg-isoD-G-(NMe) R-k) c(phg-isoD-G-R(NMe)k)

1 2

8.7 ± 0.7 32 ± 4

19 ± 2 215 ± 33

>10000 >10000

3

>10000

>10000

>10000

4

>10000

>10000

>10000

5

>10000

>10000

>10000

6

2.9 ± 0.3

250 ± 52

>10000

a fitted curve was correlated to the reference affinity of an internal standard contained on the same well plate, respectively. For αvβ3 and α5β1, cilengitide was used as standard (0.54 and 15.4 nM, respectively), and for αvβ6, RTDlin (30 nM) was employed as reference. The evaluation of the compounds revealed the following results: first, the N-methylation of any amino acid of the tripeptide integrin binding motif isoDGR (peptides 3−5) completely inhibited binding to any investigated integrin 2491



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Figure 1. (A) Stereo view of the NMR derived conformation of peptide 6 (nonpolar hydrogens other than N-methyl have been omitted for clarity). The peptide has a βII-like turn structure around Arg-(NMe)-D-Lys. (B) Structural formula of peptide 6.

Figure 2. (A) Binding mode of 6 (green sticks) at the α5β1 integrin as predicted by MD calculations. The α5 and β1 subunits are depicted as light blue and yellow surfaces, respectively. Receptor amino acid side chains that are important for ligand binding are represented as sticks. The metal ion at the MIDAS is shown as a purple sphere. (B) Superposition between the MD predicted 6/α5β1 complex (receptor not shown) and the crystal structures of the α5β140 and αvβ6 integrins.41 The peptide is shown as green sticks. The α5β1 and αvβ6 receptors are represented as gray and red surfaces, respectively. In both integrin subtypes, residues that are important to selectivity are highlighted as sticks and transparent surfaces. The metal ion at MIDAS is shown as a purple sphere. The PDB IDs for α5β1 and αvβ6 are 4WK4 (ref 40) and 4UM9 (ref 41), respectively.

subtype. Conversely, peptides 2 and 6 containing N-methylated 1 5 D-Phg and D-Lys , respectively, exhibited significant integrin binding affinity. In particular, 2 showed a slightly improved α5β1/αvβ6 selectivity ratio, in comparison to the lead peptide 1. However, the absolute value for the α5β1 binding affinity also decreased. More interestingly, in peptide 6, the α5β1 binding affinity was improved by a factor of 3, from 8.7 to 2.9 nM, while the αvβ6 binding affinity decreased from 19 to 250 nM. Thus, the α5β1/αvβ6 selectivity ratio strongly improved from 3, for peptide 1, to 86 for peptide 6. As a conclusion, by the Nmethylation of D-Lys5, the α5β1/αvβ6 biselective integrin ligand 1 could be turned into a highly potent and selective peptidic α5β1 ligand 6. To the best of our knowledge, this is the most potent peptidic α5β1 specific ligand described to date (see Supporting Information). Remarkably, the binding affinity and selectivity profile of this peptide is comparable to that of the α5β1 selective peptidomimetic compound 44b,37 which was recently discovered and functionalized,38 and is the bioactive part of the molecular imaging agent 68 GaAquibeprin.39 Nuclear Magnetic Resonance Experiments. NMR studies of 6 were carried out in DMSO-d6. Compound 6 exhibited a single set of 1H chemical shift resonances (Table S1) that support a single predominant conformation. The conformation of 6 (Figure 1) was calculated by a distance geometry (DG) program by using NOE derived distances as

restraints (see Experimental Section). The peptide conformation has a βII-like turn around Arg-(NMe)-D-Lys with dihedral angles (ϕ,ψ) = (−46°, −178.3°) and (95°, −25.1°) for Arg and (NMe)-D-Lys, respectively, containing a hydrogen bond between D-Phg-NH and Gly-CO. This is also supported by the poor solvent accessibility of D-Phg-NH, which is indicated by its low chemical shift temperature coefficient of −2 ppb/K (Δδ/ΔT, calculated for the backbone NHs over a temperature range of 295 to 315 K, shown in Table S2). Molecular Modeling. To elucidate the molecular basis of the integrin activity and selectivity of our newly synthesized isoDGR cyclopeptides, computational studies were carried out on the most promising compound 6. In particular, we performed molecular dynamics (MD) simulations on the 6/ α5β140 complex (see Experimental Section). The MD predicted that the binding pose is highly stable, as revealed by the low r.m.s.d. of the peptide Cα carbons with the main ligand/receptor interactions conserved throughout the entire simulation (Figure S1). In this pose (Figure 2A), 6 binds to α5β1 according to the canonical interaction pattern of RGD binding integrin ligands.15 In particular, the Arg4 guanidinium group makes a salt-bridge with the (α5)-D227 side chain and a hydrogen bond with the backbone CO of the (α5)-Y186. On the other side, the isoAsp2 carboxylate group chelates the Mg2+ ion at the metal-ion dependent adhesion site (MIDAS) and contacts the backbone NH of (β1)-N218 through a water 2492



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bridge. Interestingly, the Gly3 backbone CO also participates in metal coordination, which further stabilizes the ligand-binding mode. With regard to flanking residues, D-Phg1 extends toward the wide and lipophilic region defined by the specificitydetermining loop (SDL) of α5β1, where it can establish stacking interactions with the (α5)-W157 and (α5)-F187 aromatic rings as well as hydrophobic contacts with the (β1)L219 side chain. Additional lipophilic contacts are formed by the backbone N-methyl group of D-Lys5 with the (α5)-F187 phenyl ring. However, the side chain of the latter peptide residue does not establish specific contacts with α5β1. In fact, it is fully solvent exposed, which indicates that it might be available for functionalization (see next paragraph). The predicted binding mode is consistent with the low nanomolar IC50 exhibited by 6 at the α5β1 integrin. It allows us to explain why 6 and the lead peptide 1 show similar potency toward this receptor. Indeed, the binding modes of the two peptides are rather similar and only differ in the stacking partner of the DPhg1 ring, which in the case of 1 is the (β1)-Y127 side chain33 instead of (α5)-W157 and/or (α5)-F187. The MD predicted 6/α5β1 binding mode also allows clarifying the reasons for the lower affinity exhibited by this peptide toward αvβ6, in comparison to α5β1, which mainly resides in single-point substitutions that distinguish the SDL binding region of these two integrin subtypes. In fact, a superposition between the 6/ α5β1 complex and the αvβ6 crystal structure (PDB code: 4UM9)41 would suggest that, in the latter receptor, due to the (α5)-W157/(α6)-D148 substitution, the peptide D-Phg1 side chain can establish a lower number of hydrophobic interactions, in comparison to α5β1 (Figure 2B). Similarly, the lower αvβ6 potency of 6 with respect to the lead peptide 1 might be ascribed to a decreased number of lipophilic contacts established by the D-Phg1 side chain of the former peptide in the SDL region of the receptor. Here, as reported in ref 33, the phenyl ring of 1 can establish numerous van der Waals interactions with the side chains of residues such as (β6)-A117, (β6)-I174, (β6)-A208, and (β6)-I210. Finally, a superposition between the 6/α5β1 complex and the αvβ3 X-ray structure (PDB code: 1L5G)42 indicates that the SDL region of the latter receptor might not accommodate the D-Phg1 phenyl ring of 6, which is similar to what was previously described for 1.33 This is due to the steric hindrance of the bulky (β3)-Y166 and (β3)R214 side chains that replace the smaller (β1)-S171 and (β1)L219 of α5β1, respectively. This would in turn explain the inactivity of our novel isoDGR peptides toward αvβ3 (Figure S2). Overall, these outcomes allowed us to better define strategies for designing α5β1 specific RGD or isoDGR cyclopeptides. Here, we demonstrated that the spatial orientation of the aromatic flanking residue can help to gain selectivity, not only against αvβ3 but also against αvβ6. In fact, in both α5β1 and αvβ6 a wide and lipophilic cleft opens below the SDL, which can be occupied by properly oriented aromatic rings. However, while in αvβ6 hydrophobic residues (i.e., (β6)A117, (β6)-I174, (β6)-A208, and (β6)-I210) mostly belong to the β subunit, in α5β1 they (i.e., (α5)-W157 and (β1)-L219) are located at the α/β interface (Figure 2B). Therefore, different subsites of the RGD binding cavity can be targeted by reorienting the peptide aromatic ring and fine-tuning ligand affinity and selectivity toward the distinct integrin subtypes. Trimerization and in Vivo Imaging. For molecular imaging agents based on small peptidic integrin ligands, such as the αvβ3-integrin ligand c(RGDfK), multimerization has been found to greatly improve target affinities and in vivo

performance. In other words, they improve the target-tonontarget contrast.43 Prompted by the molecular modeling outcomes, which indicate that the (NMe)-D-Lys5 side chain of peptide 6 is not directly involved in the binding to α5β1, this moiety was selected for chelator functionalization. Thus, we employed a click-chemistry based strategy toward symmetrical trimeric chelator conjugates;44 the lysine side chain of 6 was first decorated with the pentynoic acid affording compound 7 and was subsequently coupled to a tris(azide) functionalized triazacyclononane-triphosphinate (TRAP)45 chelator scaffold via CuAAC (see Experimental Section). By the above assumption, the functionalization of 6 on the lysine side chain with pentynoic acid did not significantly influence the α5β1 binding affinity (2.9 nM vs 4.3 nM). The resulting TRAP trimer 8 showed a remarkable 36-fold enhancement in α5β1integrin affinity, in comparison to the pentynoic acid-functionalized monomer 7 (0.12 vs 4.3 nM, respectively). In light of its potential application as selective targeting agent for molecular imaging, it is even more important to highlight that, in the binding assay, trimerization increased the α5β1/αvβ6 selectivity ratio from 86 (6) to 375 for the Ga-complexed trimer (8) (Table 2). Table 2. Evaluation of the Binding Affinities of 7 and GaTRAP Trimer 8 in Comparison to the Unfunctionalized Peptide 6 for the α5β1, αvβ6, and αvβ3 Integrin Subtypes peptide sequence c(phg-isoD-G-R(NMe)k) pentynoic acid conjugate of 6 Ga-TRAP-[7]3 a

compd

IC50 α5β1 [nM]

IC50 αvβ6 [nM]

IC50 αvβ3 [nM]

6

2.9 ± 0.3

250 ± 52

>10000

7

4.3 ± 0.5

n.d.a

n.d.a

Ga-8

0.12 ± 0.02

45 ± 15

672 ± 123

n.d.: not determined.

Labeling with the positron emitter 68Ga (T1/2 = 68 min) afforded the corresponding positron emission tomography (PET) radiopharmaceutical 68Ga-8, which was used for the in vivo mapping of the α5β1-integrin expression in a mouse bearing a subcutaneous M21 (human melanoma) xenograft. Figure 3 shows that molar activity (as inversely proportional to the molar amount of tracer) had a strong influence on imaging results. High molar activity (i.e., low molar amount) did not only affect the elevated uptake in the α5β1-integrin positive tumor but also cited low-level target expressions, such as the cartilage and particularly the knee joints for which a low-grade physiological expression has been described previously.46 By applying lower molar activity, saturation of areas with low receptor expression densities was achieved.47 This resulted in improved tumor-to-background contrast.

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CONCLUSION Recently, α5β1-integrin has emerged as an attractive target among the RGD binding integrin receptors for tumor diagnosis and therapy. Consequently, the demand for α5β1 specific ligands is strongly increasing. However, due to the structural similarity of the relevant RGD recognizing integrin subtypes, this represents a challenging task. In the pursuit of α5β1 selective ligands, we were able to identify the cyclic isoDGR pentapeptide c(phg-isoDGR-k) (1) as a α5β1 and αvβ6 biselective integrin ligand.33 In this study, a new library of isoDGR peptides (2−6) as potential α5β1 binders was 2493



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triazacyclononane-1,4,7-triacetic acid) was purchased from CheMatech (Dijon, France). Analytical and preparative HPLC were performed on Shimadzu gradient systems with a SPD-20A dual wavelength UV/vis detector (220 nm, 254 nm). Eluents were purified water (from Millipore system, A) and acetonitrile (J. T. Baker Ultra Gradient HPLC grade, supplemented with 5% H2O, B), each containing 0.1% trifluoroacetic acid. Analytical HPLC was done on a Nucleosil 100−5 C18 column (125 × 4.6 mm), flow 1.0 mL/min. Preparative HPLC purification was done on a Multospher 100 RP 18−5 μ column (250 × 10 mm), flow 5.0 mL/min, with eluents as above. Mass spectra (ESI) were measured on a 500-MS Ion Trap spectrometer (Varian, by Agilent Technologies) (Figure S4). pH values were measured with a SevenEasy pH-meter (Mettler Toledo, Gießen, Germany). Peptide Synthesis: General Procedures. Loading of TCP-Resin. Peptide synthesis was carried out using 2-chlortritylchlorid-resin (TCP-resin) (0.9 mmol/g) following standard Fmoc-strategy. FmocXaa-OH (1.2 equiv) was attached to the TCP-resin with DIEA (2.5 equiv) in anhydrous CH2Cl2 (10 mL/g resin) at room temperature for 2 h. The remaining trityl chloride groups were capped by addition of a solution of MeOH, DIEA (5:1; v/v; 1.0 mL/g resin) for 15 min. The resin was filtered and washed with CH 2 Cl 2 (5×) and Nmethylpyrrolidon (NMP; 3×). The loading capacity was estimated to 0.9 mmol/g (100%). On-Resin Fmoc Deprotection. The resin-bound Fmoc peptide was treated with 20% (v/v) piperidine in NMP for 10 min and a second time for 5 min. The resin was washed with NMP (5×). Standard Amino Acid Coupling. A solution of Fmoc-Xaa-OH (2.0 equiv), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorphosphate (HATU) (2.0 equiv), 1-hydroxy-7-azabenzotriazole (HOAt; 2.0 equiv), and DIEA (5.0 equiv) in NMP (10 mL/g resin) was added to the resin-bound free amine peptide and shaken for 60 min at room temperature and washed with NMP (5×). On-Resin N-Methylation. The linear, Fmoc-deprotected peptide is washed with CH2Cl2 (3×) incubated with a solution of 2nitrobenzenesulfonyl chloride (o-NBS-Cl, 4.0 equiv) and 2,4,6Collidine (10 equiv) in CH2Cl2 for 20 min at room temperature. The resin is washed with CH2Cl2 (3×) and THF abs. (5×). A solution containing PPh3 (5.0 equiv) and MeOH abs. (10 equiv) in THF abs. is added to the resin. DIAD (5.0 equiv) in a small amount of THF abs. is added stepwise to the resin, and the solution is incubated for 15 min and washed with THF (5×) and NMP (5×). For o-NBS deprotection, the resin-bound o-NBS-peptides were stirred in a solution of mercaptoethanol (10 equiv) and DBU (5.0 equiv) in NMP (10 mL/g resin) for 5 min. The deprotection procedure was repeated one more time, and the resin was washed with NMP (5×). Cleavage of Linear Peptides from the Resin. For complete cleavage from the resin the peptides were treated three times with a solution of CH2Cl2 and hexafluoroisopropanol (HFIP; 4:1; v/v) at room temperature for half an hour, and the solvent evaporated under reduced pressure. Cyclization with DPPA. To a solution of peptide in DMF (1 mM peptide concentration) and NaHCO3 (5.0 equiv), diphenylphosphoryl azide (DPPA; 3.0 equiv) was added at room temperature and stirred overnight or until no linear peptide could be observed by ESI-MS. The solvent was evaporated in vacuo, and the crude material was directly used for the next step. Removal of Acid Labile Side Chain Protecting Groups. Cyclized peptides were stirred in a solution of TFA, water, and TIPS (95:2.5:2.5; v/v/v) at room temperature for 1 h or until no more protected peptide could be observed by ESI-MS and precipitated in diethyl ether. The sediment was washed with diethyl ether (2×). Dde Deprotection in Solution. The orthogonal deprotection of Dde was performed using a solution of hydrazine hydrate (2 vol %) in DMF for 30 min at room temperature. The progress of the reaction was monitored by ESI-MS. After completion of the reaction, the peptide was precipitated with sat. aq. NaCl solution and washed with HPLC grade water (2×). Coupling of Pentynoic Acid in Solution. The free amine was dissolved in DMF (∼0.1 mol/L), pentynoic acid (1.2 equiv), HATU (1.2 equiv), and DIEA (3.0 equiv) were added, and the solution was

Figure 3. PET images (maximum intensity projections, 75 min p.i.) of a SCID mouse bearing an M21 (α5β1-integrin expressing human melanoma) xenograft on its right shoulder, as indicated by the arrows. The images show two independent scans with different total molar amounts of 68Ga-8. The images are normalized to the tumor (note the different %ID/mL scales) and highlight the reduction of uptake in areas with low target expression for lower molar activities. Complete blockade with excess unlabeled (40 nmol) proved α5β1-integrin specificity of tumor uptake (see Supporting Information, Figure S5).

generated through the sequential N-methylation of peptide 1. Biological evaluation demonstrated that one of these compounds, namely, c(phg-isoDGR-(NMe)k) (6), represented one of the most potent and selective peptide ligands of the α5β1 receptor to date.24,26 The molecular basis for the improved α5β1 affinity and selectivity profile of 6, with respect to lead structure 1 was subsequently revealed by NMR spectroscopy and computational studies, which provided detailed hints for the design of future α5β1 ligands. In the next step, 6 was conjugated to pentynoic acid on the lysine side chain, and trimerized by using the bifunctional chelator TRAP as a scaffold. This conjugate 8 was labeled with the positron emitter 68Ga in order to afford the PET tracer 68Ga-8, which could be successfully deployed in order to monitor the α5β1 expression in human melanoma xenografted mice. This proof of concept experiment demonstrated the applicability of 68Ga-8 as a potential imaging agent for the in vivo imaging of α5β1 expressing tumors.

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EXPERIMENTAL SECTION

General Experimental. Peptide Synthesis. All reagents and solvents were obtained from commercial suppliers and used without further purification. Analytical HPLC-ESI-MS was performed on a Hewlett-Packard Series HP 1100 equipped with a Finnigan LCQ mass spectrometer using a YMC-Hydrosphere C18 column (12 nm pore size, 3 μm particle size, 125 mm × 2.1 mm), flow 0.55 mL/min, or YMC-Octyl C8 column (20 nm pore size, 5 μm particle size, 250 mm × 2.1 mm), flow 0.35 mL/min, and H2O (0.1% v/v formic acid)/ MeCN (0.1% v/v formic acid) as eluents. All final compounds were analyzed via analytical HPLC to confirm a purity of ≥95% (220 nm). Semipreparative HPLC was performed using a Beckmann instrument (system gold, solvent delivery module 126, UV detector 166), an YMC ODS-A column (20 × 250 mm, 5 μm), flow rate 8 mL/min, linear gradients of H2O (0.1% v/v TFA) and MeCN (0.1% v/v TFA). Trimerization of 7 (Compound 8). Unless otherwise noted, all reagents and solvents were of analytical grade. TRAP(azide)3 was synthesized as described previously (Scheme S1).42 NOTA (1,4,72494



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stirred overnight or until no residual free amine could be observed by ESI-MS. After removal of the solvent in vacuo the residue was taken up in EtOAc and washed with sat. aq. NH4Cl solution, sat. aq. NaHCO3 solution, and with sat. aq. NaCl solution, respectively. After drying the organic phase with Na2SO4, the solvent was removed in vacuo yielding the crude peptide conjugate. Trimerization of 7 (Compound 8). TRAP(azide)3·TFA (3.1 mg, 3.3 μmol, 1.0 equiv) was dissolved in water (100 μL) and combined with a solution of 7 (6.8 mg, 9.9 μmol, 3.0 equiv) in a 1:1 (v/v) mixture of tBuOH and water (200 μL). Subsequently, aq. sodium ascorbate (0.5 M, 50 equiv), followed by aq. Cu(OAc)2·H2O (0.05 M, 1.2 equiv) was added. A brown precipitate was formed, which dissolved after stirring, resulting in a clear green solution. For demetalation, aq. 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA, 8 mM, 12 equiv) was added, and the pH was adjusted to 2.2 with 1 N aq. HCl. After 1 h at 60 °C, the mixture was directly subjected to preparative HPLC purification (Table 3). After lyophilization of the eluate, 8·TFA (1.1 mg, 0.4 μmol, 11%) was obtained as a colorless solid.

incubation for 1 h at r.t., the plate was washed three times with PBS-T. Then, secondary peroxidase-labeled antibody (4) (100 μL/well) was added to the plate and incubated for 1 h at r.t. After washing the plate three times with PBS-T, the plate was developed by quick addition of SeramunBlau (50 μL per well, Seramun Diagnostic GmbH, Heidesee, Germany) and incubated for 1 min at r.t. in the dark. The reaction was stopped with 3 M H2SO4 (50 μL/well), and the absorbance was measured at 450 nm with a plate reader (POLARstar Galaxy, BMG Labtechnologies). The IC50 of each compound was tested in duplicate, and the resulting inhibition curves were analyzed using OriginPro 7.5G software. The inflection point describes the IC50 value. All determined IC50 were referenced to the activity of the internal standard.

α5β1:

(1) 0.5 μg/mL, human fibronectin, Sigma-Aldrich (2) 2.0 μg/mL, human α5β1-integrin, R&D (3) 1.0 μg/mL, mouse antihuman CD49e, BD Biosciences (4) 2.0 μg/mL, antimouse IgG-POD, Sigma-Aldrich αvβ6: (1) 0.4 μg/mL, LAP (TGF-β), R&D (2) 0.5 μg/mL, human αvβ6-Integrin, R&D (3) 1:500 dilution, antiαv mouse antihuman MAB1978, Millipore (4) 2.0 μg/mL, antimouse IgG-POD, Sigma-Aldrich αvβ3: (1) 1.0 μg/mL, human vitronectin; Millipore (2) 2.0 μg/mL, human αvβ3-integrin, R&D (3) 2.0 μg/mL, mouse antihuman CD51/61, BD Biosciences (4) 1.0 μg/mL, antimouse IgG-POD, Sigma-Aldrich NMR Spectroscopy. For NMR spectroscopic studies, approximately 2 mg of the compound was dissolved in 125 μL of DMSO-d6. The required NMR spectra were recorded at 300 K on Bruker 500 MHz spectrometer equipped with TXI cryoprobe. 1H-1D, TOCSY, ROESY, HSQC, and HMBC NMR experiments have been acquired. To estimate the solvent shielding or hydrogen bonding strengths of NH protons, temperature dependency of the NH chemical shifts was studied by acquiring 1H-1D spectra from 295 to 315 K in steps of 5 K increments. Mixing times of 80 and 200 ms were used for TOCSY and ROESY experiments, respectively. HSQC spectra were recorded with a direct proton carbon coupling constant of 140 Hz, and HMBC spectra with a long-range 1H−13C coupling constant of 7 Hz. For HSQC spectra, a 13C composite pulse decoupling was utilized. Eight thousand (except HSQC: 1k) data points were recorded in the direct dimension, 384 (512) (heteronuclear spectra) in the indirect dimension. For all spectra, a 1.5 s relaxation delay was used after every transient. Exponential/square sine window functions were used for apodization of the spectra. Proton−Proton Internuclear Distances for Structure Calculation. ROE cross peaks in corresponding ROESY spectra recorded in various solvents were integrated by using the box method in SPARKY software. These integrated volumes of ROE cross peaks were converted to proton−proton internuclear distances by linear approximation method. Thus, calculated distances were then relaxed by ±10 to generate upper and lower distance bounds to account for experimental and simulation uncertainties (Table S3). Distance Geometry (DG) Calculations. Metric matrix DG calculations were carried out with a home-written distance geometry program utilizing random metrization. The above calculated experimental distance restraints, which are more restrictive than the geometric distance bounds (holonomic restraints), were used to create the final distance matrix. Fifty structures were calculated for each system. The structures were then verified and checked for violations, if any, with respect to the given experimental distance restraint inputs. The structures that best satisfied the distance inputs were then taken forward for docking analysis. Molecular Modeling. First, a manual docking of the 6 NMR conformation into the headpiece of the α5β1 integrin crystallized in complex with a cyclic RGD peptide (PDB code: 4WK4)40 was accomplished. Specifically, we superimposed the Cα carbons of isoAsp2

Table 3. Analytical Data of the Cyclic Peptides peptide sequence

compd

mass calculated

mass measured

RP-HPLC tR [min]

c(phg-isoD-G-R-k)30 c((NMe)phg-isoD-GR-k) c(phg-(NMe)isoD-GR-k) c(phg-isoD-(NMe)GR-k) c(phg-isoD-G-(NMe) R-k) c(phg-isoD-G-R(NMe)k) Pentynoic acid conjugate of 6 TRAP[7]3

1 2

589.3 603.3

590.425,a 604.4a

5.9925,b 5.11b

3

603.3

604.4a

5.32b

4

603.3

604.4a

4.98b

5

603.3

604.4a

5.24b

6

603.3

604.4a

5.09b

7

683.3

684.4a

3.63c

8

2877.1

1439.8d [M + 2H+]

7.5e

a Finnigan LCQ mass. bHewlett-Packard Series HP 1100, gradient 0− 100%, 30 min. cHewlett-Packard Series HP 1100, gradient 10−90%, 15 min. d500-MS Ion Trap spectrometer. eNucleosil 100−5, gradient 10−90%, 15 min.

Peptide Synthesis: Analytical Data. Integrin Binding Assay. The activity and selectivity of integrin ligands were determined by a solid-phase binding assay according to the previously reported protocol25,29 using coated extracellular matrix proteins and soluble integrins. The following compounds were used as internal standards: Cilengitide, c(RGDf(NMe)V) (αvβ3, 0.54 nM; αvβ5, 8 nM; α5β1, 15.4 nM), linear peptide RTDLDSLRT (RTDlin) (αvβ6, 33 nM; αvβ8, 100 nM), and tirofiban (αIIbβ3, 1.2 nM). Flat-bottom 96-well ELISA plates (BRAND, Wertheim, Germany) were coated overnight at 4 °C with the ECM-protein (1) (100 μL per well) in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6). Each well was then washed with PBS-T-buffer (phosphate-buffered saline/Tween20, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 0.01% Tween20, pH 7.4; 3 × 200 μL) and blocked for 1 h at room temperature with TS-B-buffer (Tris-saline/BSA buffer; 150 μL/well; 20 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, pH 7.5, 1% BSA). In the meantime, a dilution series of the compound and internal standard is prepared in an extra plate, starting from 20 μM to 6.4 nM in 1:5 dilution steps. After washing the assay plate three times with PBS-T (200 μL), 50 μL of the dilution series was transferred to each well from B−G. Well A was filled with 100 μL of TSB solution (blank), and well H was filled with 50 μL of TS-B-buffer. Fifty microliters of a solution of human integrin (2) in TS-B-buffer was transferred to wells H−B and incubated for 1 h at r.t. The plate was washed three times with PBS-T buffer, and then primary antibody (3) (100 μL per well) was added to the plate. After 2495



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and Arg4 as well as the centroid of the D-Phg1 phenyl ring of 6 with those of the corresponding residues (Arg3, Asp5, Trp7) in the cocrystallized ligand, which was then removed from the complex. Subsequently, to mimic the typical interaction pattern of RGD integrin ligands, the side chains of isoAsp2 and Arg4 of 6 were properly optimized to interact with the metal ion at the MIDAS in the β1 subunit and with (α5)-D227, respectively. The cocrystallized Ca2+ ions in the receptor structure were then replaced with Mg2+ ions. The complex was then prepared for submission to MD simulations using the LEaP module of the Amber 2016 package.48 The f f14SB amber force field49 was used to parametrize both the protein and the peptide. Missing parameters for the D-Phg1, isoAsp2, and D-(NMe)Lys5 peptide residues were generated with Antechamber.50 Specifically, charges were computed using the restrained electrostatic potential (RESP) fitting procedure.51 The ESP was first calculated by means of the Gaussian09 package52 using a 6-31G* basis set at Hartree−Fock level of theory, and then the RESP charges were obtained by a two-stage fitting procedure using Antechamber.51 Missing bonds, angles, torsion, and improper torsion angle parameters were finally generated using the same program. The complex was then solvated in a 12.0 Å layered cubic water box using the TIP3P water model parameters.53 The addition of eight further Mg2+ ions ensured neutrality. The 12−6−4 LJ-type nonbonded model parameters developed by Li and Merz54 were employed to treat metals. Concerning the MD setup, a 10 Å cutoff (switched at 8 Å) was used for atom pair interactions. The longrange electrostatic interactions were computed by means of the particle mesh Ewald (PME) method using a 1.0 Å grid spacing in periodic boundary conditions. The RATTLE algorithm was applied to constrain bonds involving hydrogen atoms, thus allowing the use of a 2 fs integration time step interval. The system was minimized and heated up to 300 K using harmonic constraints, which were gradually released along the equilibration process. The Mg2+ ions and their coordination shells were further equilibrated for 10 ns. Production runs were then performed in the NPT ensemble, at 1 atm and 300 K. Radiochemistry. 68Ga labeling for rodent experiments was carried out as described previously.55 Briefly, employing a fully automated system (GallElut+, Scintomics GmbH, Germany), nonprocessed eluate of a 68Ge/68Ga-generator with SnO2 matrix (by IThemba LABORATORIES, SA; 1.25 mL, 1 M HCl, eluted 68 Ga activity approximately 1 GBq) was adjusted to pH 2 by adding HEPES buffer (400 μL of a 2.7 M solution, prepared from 14.4 g of HEPES and 12 mL of water) and used for labeling of 0.3 nmol of 8 for 5 min at 95 °C. Purification was done by SPE, using a SepPak C8 light cartridge. Molar activity of the product was approximately 2 GBq/nmol at the time of injection. Determination of radiochemical purity was done by radioTLC on Agilent ITLC-SG chromatography paper; eluent, 0.1 M trisodium citrate (product Rf = 0, noncomplexed 68Ga is eluted near the front). Octanol−water distribution coefficients at pH 7.4 (log D7.4) were determined by the shake-flask method as previously described.56 Approximately 1 MBq of 68Ga-8 was added to equal volumes (0.5 mL) of 1-octanol and phosphate-buffered saline (PBS) in Eppendorf cups, which were shaken vigorously for 2 min, the phases were separated by centrifugation at 11 500g for 15 min, and activities contained in aliquots of both phases (200 μL), respectively, were measured in a gamma counter. In Vivo Imaging. All animal experiments were approved by the local authorities and performed in accordance with current animal welfare regulations in Germany. Culture of M21 human melanoma cells and generation of respective xenografts in mice as well as micro-PET imaging were performed as previously described.45 Briefly, M21 human melanoma cells were cultivated in RPMI 1640 medium, supplemented with 10% FBS and 1% gentamicin (all from Biochrom AG, Berlin, Germany) at 37 °C in a humidified atmosphere containing 5% CO2. Tumor xenografts were generated by injecting approximately 1.5 × 107 cells, suspended in serum-free medium supplemented with Matrigel (Corning, #354262), into the right shoulder of 6 to 8 week old, female SCID mice (CB17, Charles River, Germany). When tumors had grown to a diameter of 6−8 mm (usually 2−3 weeks after inoculation), animals were subjected to PET studies. Approximately 20 MBq of 68Ga-8 was administered into the tail vein under isoflurane

anesthesia, then animals were allowed to wake up with access to food and water. PET was recorded 75 min p.i. for 20 min. Data were reconstructed as single frames employing a three-dimensional ordered subset expectation maximum (OSEM3D) algorithm.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b01752. NMR spectroscopy; molecular modeling; trimerization of 7; PET images (PDF) PDB coordinates of the 6/α5β1 MD predicted complex (PDB) Molecular formula strings (CSV)

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AUTHOR INFORMATION

Corresponding Authors

*(H.K.) Phone: +49 89 289 13300. E-mail: [email protected]. *(L.M.) Phone: +39 0816 78619. E-mail: [email protected]. ORCID

Johannes Notni: 0000-0002-3964-3391 Luciana Marinelli: 0000-0002-4084-8044 Horst Kessler: 0000-0002-7292-9789 Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS T.G.K. acknowledges the International Graduate School for Science and Engineering (IGSSE) of the Technische Universität München (TUM) for financial support. This work was supported by the Reinhart Koselleck Grant of the Deutsche Forschungsgemeinschaft (DFG KE 147/42-1) to H.K. and by CIPSM. The study was supported by PRIN2015 FCHJ8E. DFG grant NO822/4-1 awarded to (J.N.).

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ABBREVIATIONS USED CuAAC, copper(I)-catalyzed azide alkyne cycloaddition; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCM, dichloromethane; Dde, 2-acetyldimedone; DG, distance geometry; DIAD, diisopropyl azodicarboxylate; DIEA, diisopropyl ethyl amine; DMF, dimethylformamide; DPPA, diphenylphosphoryl azide; ECM, extracellular matrix; ELISA, enzyme-linked immunosorbant assay; Fbg, fibrinogen; Fn, fibronectin; Fmoc, fluorenylmethyloxycarbonyl; GBM, gliobastoma multiforme; HATU, O(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium-hexafluorphosphate; HFIP, hexafluoroisopropanol; HOAt, 1hydroxy-7-azabenzotriazole; HPLC, high-performance liquid chromatography; isoAsp, isoD, L-isoaspartate; LAP, latencyassociated peptide; MD, molecular dynamics; MIDAS, metalion dependent adhesion site; NMe, N-methyl; NMP, Nmethylpyrrolidon; NOE, nuclear Overhauser effect; NOTA, 1,4,7-triazacyclononane-1,4,7-triacetic acid; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PET, positron emission tomography; phg, D-phenylglycine; SCID, severe combined immunodeficient; SDL, specificity-determining loop; SPPS, solid phase peptide synthesis; TCP, chloro-(2′-chloro)trityl polystyrene; TFA, trifluoroacetic acid; TGF, transforming growth factor; THF, tetrahydrofuran; TIPS, triisopropylsilane; 2496



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TRAP, triazacyclononane-triphosphinate; VEGF, vascular endothelial growth factor; Vn, vitronectin

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N-Methylation of isoDGR Peptides: Discovery of a Selective Î±5Î²1

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