Thrombospondin-1 Mimetic Agonist Peptides ... - ACS Publications

Article pubs.acs.org/jmc

Thrombospondin‑1 Mimetic Agonist Peptides Induce Selective Death in Tumor Cells: Design, Synthesis, and Structure−Activity Relationship Studies Thomas Denèfle,†,‡,§ Héloise Boullet,†,‡ Linda Herbi,∥,⊥,# Clara Newton,†,‡,§ Ana-Carolina Martinez-Torres,∥,⊥,# Alexandre Guez,†,‡ Elodie Pramil,†,‡,∥,⊥,# Claire Quiney,∥,⊥,# Marilyne Pourcelot,†,‡,§ Mikail D. Levasseur,†,‡,§ Eva Lardé,†,‡,§ Roba Moumné,†,‡ François-Xavier Ogi,○ Pascal Grondin,◆ Hélène Merle-Beral,∥,⊥,∇ Olivier Lequin,†,‡ Santos A. Susin,∥,⊥,# and Philippe Karoyan*,†,‡,§ †

Sorbonne Universités, UPMC Université Paris 06, Ecole Normale Supérieure, CNRS, Laboratoire des Biomolécules, 75005 Paris, France ‡ Département de Chimie, Ecole Normale Supérieure, PSL Research University, UPMC Université Paris 06, CNRS, Laboratoire des Biomolécules, 75005 Paris, France § UPMC Université Paris 06, Laboratoire des BioMolécules, Site GSK, 25-27 Avenue du Québec, 91140 Les Ulis, France ∥ Cell Death and Drug Resistance in Lymphoproliferative Disorders Team, Centre de Recherche des Cordeliers, INSERM UMRS, 1138, Paris, France ⊥ Sorbonne Universités, UPMC Université Paris 06, UMRS 1138, Paris, France # Université Paris Descartes, Sorbonne Paris Cité, UMRS 1138, Paris, France ∇ AP-HP, GH Pitié-Salpêtrière, Service d’Hématologie Biologique, 75013 Paris, France ○ NanoTemper Technologies GmbH, Floessergasse 4, 81369 Munich, Germany ◆ GSK, 25 Avenue du Québec, 91140 Les Ulis, France S Supporting Information *

ABSTRACT: Thrombospondin-1 (TSP-1) is a glycoprotein considered as a key actor within the tumor microenvironment. Its binding to CD47, a cell surface receptor, triggers programmed cell death. Previous studies allowed the identification of 4N1K decapeptide derived from the TSP-1/ CD47 binding epitope. Here, we demonstrate that this peptide is able to induce selective apoptosis of various cancer cell lines while sparing normal cells. A structure−activity relationship study led to the design of the first serum stable TSP-1 mimetic agonist peptide able to trigger selective programmed cell death (PCD) of at least lung, breast, and colorectal cancer cells. Altogether, these results will be of valuable interest for further investigation in the design of potent CD47 agonist peptides, opening new perspectives for the development of original anticancer therapies.

■

INTRODUCTION

activations that result in a large variety of PCD types (Figure 1).3 An imbalance in the anti- and proapoptotic protein interactions (Figure 2)4 will lead to failure in cell death homeostasis, failure resulting in many diseases with excess or defect in PCD, such as cancers, autoimmune diseases, and neurological and cardiovascular disorders.5 In cancer cells, the PCD blockage is mainly caused by either cytogenetic abnormalities leading to overexpression of antiapoptotic proteins or down-regulation of proapoptotic

Despite significant breakthroughs in the understanding, prevention, and treatments, cancers remain the most challenging diseases to target,1 lung, breast, and colorectal being the most frequent ones according to World Cancer Research Fund International (WCRFI).2 Cancer diseases are the results of uncontrolled cell proliferations that escape programmed cell death (PCD) regulation. Remarkable progress has been made over the past 20 years to unravel, at the molecular level, the mechanisms by which an abnormal cell can escape PCD. Two pathways, i.e., the intrinsic and the extrinsic pathways, can trigger PCD in normal cell. Both pathways involve a cascade of protein−protein interactions (PPIs) and © 2016 American Chemical Society

Received: May 23, 2016 Published: August 15, 2016 8412

DOI: 10.1021/acs.jmedchem.6b00781 J. Med. Chem. 2016, 59, 8412−8421

Journal of Medicinal Chemistry

Article

Figure 1. Extrinsic and intrinsic PCD pathways. Intrinsic and the extrinsic pathways can trigger PCD in normal cell and are respectively mediated by death receptors of the TNF family and mitochondrion. Both pathways involve a cascade of protein−protein interactions (PPIs) and activations that result in a large variety of PCD types, from necroptosis to necrosis.

Figure 2. PCD pathways are regulated by many protein−protein interactions (PPIs), involving for example proapoptotic proteins of the Bcl-2 family, such as the BAK and BAX, and antiapoptotic proteins of the same family, such as Bcl-2 and Bcl-xL. The p53 protein is sequestrated by the Mdm2 in the cytoplasm. A DNA damage will activate the ATM kinase leading to the release of the phosphorylated p53 protein from the complex. The phosphorylated p53 will in turn activate PCD by interacting at least with proteins of the Bcl-2 family and IAP family.

allogeneic stem cell transplantation is the only curative option but is feasible in a highly selected group of patients only.14 Most of all, decoding the human protein−protein interactome network over recent years highlighted the limit of therapeutic agents designed to interact with a single pathway, inevitably resulting in the activation of compensatory mechanisms which allow tumor escape and restore disease progression.15 Regardless, we do believe that a peptide approach would be more relevant by addressing an extracellular target to answer their poor membrane permeability. Moreover, to overcome the compensatory mechanism, addressing a target located upstream of a signaling pathway and able to irreversibly trigger PCD might be more efficient. With this aim, many studies16 are focusing on the multifaceted functionalities of thrombospondin-1 (TSP-1)17 identified as one of the endogenous ligand of CD47.18 The TSP-1/CD47 complex is implicated in diverse biological functions,19 including the direct induction of PCD, and therapeutic targeting of the TSP-1

proteins. More than 50 000 chromosomal aberrations (gains or losses) have been reported to date.6 Because of the X-ray structure determination of some of the proteins involved in the PCD regulation, many compounds, including small molecules, peptides, and peptide mimics, have been designed to target intracellular protein−protein interactions (PPIs) and to restore PCD.7−11 Of note, both small molecules and peptide approaches remain ineffective to trigger PCD in the case of ataxia telangiectasia mutated (ATM) or tumor protein 53 (TP53) deletions that are frequent cytogenetic alterations associated with poor prognosis, especially in chronic lymphocytic leukemia (CLL),12 the most frequent form of adult leukemia in Western countries. If encouraging results have been obtained in patients refractory or in relapse with recent therapeutic approaches including the tyrosine kinase inhibitors idelalisib and ibrutinib, drug resistance and/or severe side effects are responsible for treatment failure and pejorative outcome.13 Until now, 8413



Article

Figure 3. Tumor cell death induced by 4N1K. (A) Cell death was measured in a flow cytometer by annexin-V and propidium iodide (PI) co-staining in the indicated cancer cell line treated for 2 h with the control peptide 4NGG (see ref 26) or 4N1K (300 μM). (B, Left) Cytotoxicity assessed in normal B cells (B cells) and B cells from CLL patients (CLL cells) treated as in (A) was recorded and expressed as a histogram. (B, Right) Cell death induced by 4NGG or 4N1K in CLL cells from individuals with functional TP53/ATM (drug reactive CLL patients), dysfunctional TP53 (del 17p), or dysfunctional ATM (del 11q) (both drug resistant CLL patients) was assessed by flow cytometry and expressed as a histogram. As a control for a typical drug treatment, cells were incubated 24 h with the topoisomerase II inhibitor etoposide (200 μM). Note that, in contrast to the results obtained with 4N1K, CLL cells with del 17p or del 11q are resistant to etoposide treatment. The data are presented as the mean ± sd (n ≥ 6 independent experiments).

receptor to treat liver cancer has recently been highlighted.20 The crystal structure of the complex has not been solved to date but a mechanistic calculation study of the TSP-1/CD47 complex has been reported21 and affinity chromatography allowed the identification of two epitope sequences within the C-terminal globular domain of TSP-1 containing the VVM motif, named 7N3 and 4N1.18 Recently, the 7N3 sequence (FIRVVMYEGKK) was used to explore the possibility that TSP-1 regulates VEGFR2 phosphorylation via CD47.22 Regarding the 4N1 sequence, it corresponds to a β-strand of eight residues (RFYVVMWK). Due to its poor solubility, the 4N1K (KRFYVVMWKK) peptide was designed by adding two lysine residues respectively at both ends of 4N1.18 A few studies have been reported about the ability of 4N1K to induce PCD.23 However, its pharmacological properties, including its affinity to CD47 receptor, metabolic stability, or structural behavior, have not been described so far. Here, we explore the ability of 4N1K to induce selective PCD in cancer cell lines while sparing normal cells. Considering its potential as a therapeutic approach to treat cancer, even from chronic lymphocytic leukemia (CLL) refractory patients with TP53 (del 17p) or ATM (del 11q) dysfunctions, we explored its serum stability and designed analogues resistant to proteases. In order to highlight the pharmacological properties of these peptides, we initiated a systematic structure−activity relationship (SAR) study with the development of a binding assay allowing assessment of the affinity of the 4N1K analogues to CD47. Conformational studies by CD and NMR were realized, and the data were compared with the X-ray structure of the 4N1 sequence in the context of the TSP-1 protein. We implemented an Ala-scan to identify the pharmacophores of 4N1K. Among the different

peptides designed, one peptide was identified for its increased stability in human serum, and its ability to induce PCD in lung, breast, CLL, and colorectal cancer cell lines was evaluated.

■

RESULTS AND DISCUSSION 4N1K has been reported to be associated with angiogenesis, cell survival, and invasion, and few studies reported the ability of 4N1K to induce apoptosis.24 Its pathological significance in patients human cancer tissues is not fully understood. Recently, it was shown that 4N1K expression was associated with cancer cell progression and survival in patients with urothelial carcinoma via the regulation of angiogenesis, apoptosis, and MMP-9 expression.25 Here, we demonstrate that this peptide is able to induce a 30 to 70% cell death rate for at least 17 cancer cell lines within 2 h only at 300 μM (Figure 3A). Noticeably, this peptide is able to spare normal B-lymphocytes obtained from healthy donors while efficiently inducing PCD in chronic lymphocytic leukemia (CLL) B cells, even those obtained from patients with a dysfunctional ATM or TP53 genes (Figure 3B), mutations that are associated with poor CLL prognosis. Altogether, these results highlight the therapeutic potential of this peptide to kill tumor cells. The major drawbacks of this approach remain the high peptide concentration (300 μM) used to induce the PCD that might result in CD47 independent effect and the poor metabolic stability of peptides that have to be solved to go further. As anticipated, 4N1K was fully degraded in human serum in less than 5 min. Thus, in order to improve the human serum stability, 4N1K analogues were designed with chemical modifications known to enhance peptide half-life, such as Nacetylation and C-carboxamide modifications of the peptide termini, judicious replacement of L-amino acids by their D8414



Article

Figure 4. Peptide designed in order to improve the human serum stability. Two peptide series were designed to improve the metabolic stability of 4N1K. The first series, composed of peptides 1 and 3, was derived from the TSP-1 epitope, 4N1, and the second series was derived from 4N1K, i.e., peptides 2 and 4.

amino acids counterparts or by β-homoamino acids (Figure 4). With this aim, two peptide series were synthesized, the first one including short peptides (eight residues) derived from the octapeptide 4N1, the TSP-1 epitope interacting with the CD47 receptor, the second one designed from the decapeptide 4N1K, and their metabolic stability was evaluated (Figure 5). While the poorly soluble 4N1 octapeptide is quickly degraded, simple N-terminus acetylation and C-terminus carboxamidation led to peptide 1 that appeared to be stable for 2 h in human serum, the exposure time chosen for PCD evaluation. The same approach to stabilize 4N1K and leading to peptide 2 was revealed to be unsuccessful. Indeed, almost all

peptide 2 was degraded in 2 h. This surprising result could be explained by the presence of furin, also known as PACE, a paired basic amino acids cleaving enzyme which is located at the plasma membrane of cells.27 This endoprotease cleaves protein precursors with narrow specificity following basic ArgXaa-Lys/Arg-Arg-like motifs28 that are present in peptide 2 after the chemicals modifications. Thus, in order to stabilize this peptide series, we considered that since the two N- and Ctermini lysine residues of 4N1K were introduced only in order to improve the epitope solubility and were not a priori related to the interaction of the peptide with the CD47 receptor, it could be possible to replace them by their D-analogues. This hypothesis led to the design of peptide 4 that appeared to be fully stable in human serum. Finally, peptide 3 was designed by replacing the N- and C-termini residues of 4N1 by their βhomoamino acids analogues, i.e., the β2-homoarginine and the β3-homolysine (synthesized as reported elsewhere)29 in place of respectively L-arginine and L-lysine. Indeed, the possible replacement of the N- and C-termini residues of angiotensin IV by the corresponding β-homoamino acids was previously demonstrated30 to maintain the amino acid side chain position and orientation and to improve the peptide serum stability, and this strategy appeared to be also successful for the epitope 4N1. Thus, three peptides designed here, i.e., 1, 3, and 4, were revealed to be much more stable than 4N1 and 4N1K, peptide 4 remaining intact for at least 2 h in human serum. Next, we quantified the solubility of the synthesized peptides, using peak integration of 1D 1H NMR spectra in aqueous solution. We observed that the parent peptide 4N1 shows very poor solubility, the solubilized peptide concentration being lower than 30 μM. In contrast, 4N1K, peptides 2 and 4 were soluble at concentrations above 1 mM. Furthermore, their 1D 1 H NMR spectra show sharp signals, proving the absence of significant peptide aggregation. In contrast, peptides 1 and 3 were poorly soluble, with solubility close to that of 4N1,

Figure 5. Peptide degradation assay. Four peptides (1, 2, 3, and 4) were designed in order to improve the human serum stability, which was determined by following peptide degradation after incubation at 37 °C for 2 h. 4NGG is the inactive analogue of 4N1K in which both valine of the VVM sequence have been mutated by two glycines.26 8415



Article

FYVV segment tend to be largely unstructured, as demonstrated by average J values and analysis of Hα-HN NOE intensities. It is likely that the small PII content inferred from the CD analysis arises from local conformations of the residues that do not belong to the FYVV segment. The conformational analysis shows that the investigated peptides are flexible in aqueous solution but that the four residues of the FYVV segment have a propensity to populate the extended region of Ramachandran diagram. Therefore, the conformational space of the free peptide remains close to the epitope β-strand conformation observed in the context of the full TSP-1 protein. The intrinsic backbone preferences of the FYVV segment can be ascribed to its hydrophobic character with the presence of aromatic and aliphatic residues and to steric effects involving the two β-branched Val residues. The next point we studied was the influence of the chemical modifications introduced in the peptides on their biological properties in terms of affinity and activity. Noticeably, the 4N1K−CD47 interaction which was originally established by Gao and Frazier on a chromatography assessment has never been quantified in terms of affinity measurements (Kd). Thus, we decided to develop a binding assay that could help us in a rational design approach correlating peptide chemical modifications and pharmacological properties. Our target, CD47, is a membrane receptor, and since Brown and co-workers33 reported that normal ligand binding and signaling by CD47 requires a long-range disulfide bond between the extracellular and the membrane-spanning domains, we developed a binding experiment using MEC-1 cell membrane preparation in order to keep the receptor integrity (see Supporting Information). Two techniques were initially evaluated in parallel, i.e., microscale thermophoresis (MST) and OctetRed (OR). MST is a powerful recent technology (Nanotemper Technologies) which detects changes in the hydration shell of molecules and measures biomolecule interactions under close-to-native conditions: immobilization-free and in bioliquids of choice. Alternatively, the technology employed by the OR platform is based on the principles of optical interferometry and is related to surface plasmon resonance (SPR). The technology allows the real-time label-free detection power of biolayer interferometry. However, compared with MST, this technique is limited by the required immobilization of the sample leading to its peculiar orientation. The major drawback using both techniques was encountered in analyzing poorly soluble samples. Nevertheless and since the 4N1K/ CD47 interaction has never been quantified, both techniques proved to be useful and the same overall trends were observed by MST and OR, with a 10-fold affinity improvement measured by MST, the difference being attributed to the limitation described above for OR. The Kd values are given in Table 2 (MST and OR) and Table 3 (OR) (see Supporting Information). In parallel to the solubility and affinity assessments, the ability of the peptides to induce cell death was evaluated by annexin-V and propidium iodide (PI) colabeling on MEC-1 tumor B cells treated with the peptides at 200 μM for 2 h (Table 2) as previously reported.34 As could be expected, the 4NGG peptide in which two key valine residues of the VVM sequence (previously stated as the pharmacophore of the CD47 binding epitope of TSP-1)35 have been replaced by two glycine residues was ineffective in binding to MEC-1 membrane preparation and in inducing MEC-1 cytotoxicity. No pertinent data were obtained with 4N1 and

indicating that the presence of a single cationic residue at both extremities of the peptide is not sufficient to confer significant aqueous solubility. The conformational propensity of each peptide was then examined by CD spectroscopy (see CD spectra in Supporting Information). Far-UV CD spectra of the different peptides are dominated by a random coil signature, as inferred from the strong negative band around 195 nm. Deconvolution of the spectra leads to random coil contributions ranging from 80% to 90%. Peptides exhibit no helical propensity, and a small β-sheet content can be observed only for the least soluble 4N1 and peptide 1, suggesting that the β-structure arises from aggregated species. Interestingly, most peptides exhibit a small fraction of PII conformation, as suggested by the weak positive band around 220−225 nm. Overall, CD spectroscopy indicates that these short linear peptides tend to be largely unstructured in aqueous solution. NMR spectroscopy was then used to probe the local conformations of individual amino acids, based on the analysis of NMR parameters such as 1Hα and 13Cα chemical shifts deviations (CSD), 3JHN−Hα coupling constants, and NOEs. The chemical shift deviations of 1Hα and 13Cα resonances are calculated as the differences between observed chemical shifts and corresponding values in random coil conformation for each amino acid. Significant CSDs can be used to detect the presence of helices, sheets, or turns. The CSDs exhibit values close to zero for the different analogues (data not shown), confirming that these peptides do not adopt regular secondary structures in solution, as shown above by CD spectroscopy. 3 JHN−Hα coupling constants were also analyzed, as this parameter depends on the ϕ angle value via a Karplus relationship. Interestingly, the 3JHN−Hα values (Table 1) shows Table 1. Comparison of Experimental NMR 3JHN−Hα Coupling Constants with Jcoil and Values Inferred from X-ray Structure coupling constant (Hz) a

residue

Jcoil

Arg2 Phe3 Tyr4 Val5 Val6 Met7 Trp8 Lys9 Lys10

6.92 7.35 7.32 7.55 7.55 6.97 7.01 6.92 6.92

4N1K

4

2

4NGG

X-rayb

7.0 7.9 7.9 8.1 7.8 7.4 7.0 7.4 7.3

br s 7.4 7.8 8.1 7.9 7.3 nd 7.5 7.9

7.2 7.9 7.8 8.0 7.6 7.1 6.8 7.3 6.6

br s 8.0 7.8 (Gly) (Gly) 7.3 nd nd nd

9.1 4.6 8.9 9.2 9.8 9.5 9.4 9.6

a

Average coupling constants from the coil library taken from ref 31. Values calculated on 1UX6 crystal structure using the Karplus relationship from ref 32.

b

significant deviation from random coil values that typically fall in the 6−7.5 Hz range. In particular, the four residues FYVV in 4N1K, peptides 2 and 4 are characterized by large 3JHN−Hα values around 8 Hz, which are higher than corresponding values in the coil library.23 Thus, these residues have a strong propensity to populate the extended β region of the Ramachandran diagram which is associated with large J values in comparison with other regions (PII, αR, αL). The observation of strong sequential Hαi-HNi+1 NOEs also suggests that extended conformations are significantly populated. In contrast, residues on either side of 8416



Article

Table 2. Structure Characterization, Affinity, and Activity of the Peptides Designed To Improve Stability

a

peptide

sequence

Kd a (μM) (OR)

Kd a (μM) (MST)

MEC-1 cell death (%),a 2 h

4N1 1 3 4N1K 4NGG 2 4

RFYVVMWK Ac-RFYVVMWK-NH2 (β2hR)FYVVMW(β3hK) KRFYVVMWKK KRFYGGMWKK Ac-KRFYVVMWKK-NH2 kRFYVVMWKk

NPD 1.5 ± 0.59 NPD 19 ± 1.6 NPD 40 ± 24 43 ± 21

10 ± 0.8 0.771 ± 0.07 NPD 2.28 ± 0.22 NPD 1.2 ± 0.16 3.04 ± 0.25

13 27 NPD 23 2 30 63

Reported Kd values and % of PCD are an average of at least three independent experiments. NPD: nonpertinent data.

study, peptide 4 appeared to be the most efficient peptide in inducing MEC-1 cell death: compared with 4N1K, this peptide is stable in human serum for at least 2 h and it is able to induce 63% of cell death in 2 h at 200 μM, concentration for which only 23% of cell death is observed with 4N1K. When used at 50 μM, compound 4 has the same potency as 4N1K used at 200 μM to induce cell death (see Figure 6, around 25−30%): peptide 4 is thus much more potent than 4N1K, and this improvement in efficiency can be explained by its higher serum stability compared with 4N1K or peptide 2. With this tool in hand, we started an Ala-scan study in order to highlight the pharmacophores of peptide 4 (Table 3). Except for the N- and C-termini of D-lysine that were introduced to enhance both peptide serum stability and solubility and were not related to the interaction with the receptor, each residue of the peptide 4 sequence was replaced by an alanine and the influence of these chemical modifications was evaluated by the ability of the modified peptides to bind to MEC-1 membrane preparation and to induce cell death. The binding data reported in Table 3 can be grouped into three peptide families: the first family is composed of peptides with slightly improved affinities, i.e., R2A and K9A for which the basic residues R and K were replaced by an alanine. In these cases, the improvement in binding affinity might be correlated to a deleterious effect of the positive charges in these positions. Both peptides remained twice more potent than 4N1K, and the slight decreased potency to induce cell death, if significant and compared with peptide 4, might be explained by a lower solubility and higher hydrophobicity as suggested by the LCMS retention time (see Supporting Information). The second family is composed of peptides for which the Ala-scan drastically impacted the binding potencies highlighting the pharmacophores that are F3, Y4, V5 and to a somewhat lesser extent V6 and W8. Indeed, this peptide family can be

Table 3. Structure Characterization, Affinity, and Activity of the Peptides Designed for the Identification of Pharmacophores peptide

structure

Kd a (μM) (OR)

MEC-1 cell death (%),a 2 h, [peptides] = 200 μM

4 4N1K R2A F3A Y4A V5A V6A M7A W8A K9A M7Nleb

kRFYVVMWKk KRFYVVMWKK k(A)FYVVMWKk kR(A)YVVMWKk kRF(A)VVMWKk kRFY(A)VMWKk kRFYV(A)MWKk kRFYVV(A)WKk kRFYVVM(A)Kk kRFYVVMW(A)k kRFYVV(Nle)WKk

43 ± 21 19 ± 1.6 6.2 ± 1.9 NPD NPD NPD NPD 42 ± 21 NPD 27 ± 21 18 ± 0.6

63 23 44 6 0 12 30 6 35 46 47

a

Reported Kd values and % cell death are an average of two or more measurements. NPD: nonpertinent data. bNle: norleucine.

peptide 3 that remained the less soluble compounds as described above. Regarding the soluble analogues (peptides 2 and 4), the affinity is in a similar range and similar to that of 4N1K (around 20 μM), and this might be reasonably explained by considering that these peptides share the same pharmacophores (supposed to date to be the VVM motif), the same core sequence, and only the N- and C-termini ends have been modified. The slight affinity improvement observed with peptide 1 must be interpreted with caution because of its lower solubility (see tR in Supporting Information) but might be explained by the fact that the N-termini ammonium and/or carboxylate function could be deleterious for binding, whereas a slight decrease in activity might be the consequence of peptide poor solubility. Among all the compounds designed in our

Figure 6. Peptide 4 induces cell death in lung, breast, and colorectal cancer cell lines. Left: Cell viability was measured in a flow cytometer by annexin-V and propidium iodide (PI) co-staining in the indicated cancer cell line treated for 2 h at different peptide 4 concentrations. The data refer to annexin-V/PI co-positive staining and are presented as the mean ± SEM. Right: Representative cytofluorometric panels of an annexin-V/PI costaining performed in the indicated cancer cell line untreated or treated with peptide 4 (200 μM, 2 h). The percentages refer to the annexin-V or annexin-V/PI-positive staining. 8417



■

subdivided into two subfamilies: a first one highlighting the residues crucial for both affinity and activity, composed of F3, Y4, V5, and a second one highlighting residues involved in binding but with less incidence on the activity, i.e., V6 and W8. The last “group” is composed of one peptide, i.e., M7A. The Ala-scan on the methionine residue led to a peptide retaining the affinity for CD47 but with no potency to induce cell death, suggesting a potential role of methionine in the agonist/ antagonist imbalance ability. The contribution of the sulfur atom was evaluated with the design of an analogue in which norleucine has replaced methionine. The corresponding peptide fully recovered its potency regarding affinity and PCD induction, suggesting that the sulfur atom is not crucial for the potencies of the analogues. This is an important point if one considers that sulfur oxidation might occur in solution leading to sulfoxide or sulfone derivatives, modifying the peptide properties as described elsewhere.36 Altogether, the results obtained here suggest that the peptides pharmacophores for CD47 are not restricted to the VVM motif but also encompass the F3-Y4-V5 sequence, Y4 being the most crucial residue of the sequence, since its replacement by an alanine led to complete loss of binding affinity and ability to induce PCD. Moreover, the sulfur atom is not required and methionine can be replaced by chemically more stable amino acids. These data will be of valuable interest in the design of new peptide mimics of peptide 4. Finally, peptide 4 was evaluated for its ability to induce cell death in cancer cells by testing its cytotoxicity in three cancer cell lines that are the most frequent ones according to WCRFI, i.e., lung, breast, and colorectal cancers (Figure 6). As depicted in Figure 6, a concentration dependent cell death was observed, peptide 4 being able to trigger PCD at 50 μM inducing a 25% (A-549 cell line) to 38% (HCT116 cell line) rate of cell death within 2 h and reaching 80% of cell death at 200 μM for the colorectal cancer cell lines. The type of PCD induced by peptide 4 was characterized by a double annexinV/ PI positive staining. Therefore, as currently defined in the apoptotic field,37 peptide-4-mediated PCD in tumor cells corresponds to a typical “programmed necrotic PCD pathway”.

Article

EXPERIMENTAL SECTION

1. Synthesis and Characterization of the Peptides. General Methods. Chemicals. All commercial chemicals and solvents were reagent grade and were used without further purification unless otherwise specified. All reactions except those in aqueous media were carried out with the use of standard techniques for the exclusion of moisture. All reactions were performed under argon or nitrogen in oven-dried glassware using anhydrous solvents and standard syringe techniques. β2-Homolysine and homoarginine were synthesized as previously reported.39 Peptide synthesis transformations and washes were performed at 20 °C. All Fmoc carbamate protected amino acid derivatives, HATU, HOAt, HBTU, HOBt, Fmoc-Rink amide (200− 400 mesh, loading 0.62 mmol/g), and 2-CTC resin (100−200 mesh, loading 1.6 mmol/g) were purchased from Iris Biotech (Marktredwitz, Germany). Reagents such as DIEA, piperidine, DMF, IPA, Ac2O, MeOH, TFA, and TIS were obtained from Sigma-Aldrich (St. Louis, MO, USA). Molecular weights of compounds were calculated using ChemBioDraw Ultra 12. All final products were of >95% purity unless otherwise indicated (determined by analytical reverse phase LC−MS). Analytical data are given in the Supporting Information. Analytics. Two methods were conducted for LC−MS analysis. Method A. Analytical HPLC was conducted on a X-Select CSH C18 XP column (30 mm × 4.6 mm i.d., 2.5 μm), eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient: 0−3.2 min, 0−50% B; 3.2−4 min, 100% B. Flow rate was 1.8 mL/min at 40 °C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionization [ES+ to give (MH)+ molecular ions] or electrospray negative ionization [ES− to give (MH)− molecular ions] modes. The cone voltage was 20 V. Method B. Analytical HPLC was conducted on a X-Select CSH C18 XP column (30 mm × 4.6 mm i.d., 2.5 μm), eluting with 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B), using the following elution gradient: 0−3.2 min, 5−100% B; 3.2−4 min, 100% B. Flow rate was 1.8 mL/min at 40 °C. The mass spectra (MS) were recorded on a Waters ZQ mass spectrometer using electrospray positive ionization [ES+ to give (MH)+ molecular ions] or electrospray negative ionization [ES− to give (MH)− molecular ions] modes. The cone voltage was 20 V. Purification. Preparative scale purification of peptides was performed by reverse phase HPLC on a Waters system consisting of a quaternary gradient module (Water 2535) and a dual wavelength UV/visible absorbance detector (Waters 2489), piloted by Empower Pro 3 software using the following columns: preparative MachereyNagel column (Nucleodur HTec, C18, 250 mm × 16 mm i.d., 5 μm, 110 Å) and preparative Higgins analytical column (Proto 200, C18, 150 mm × 20 mm i.d., 5 μm, 200 Å) at a flow rate of 14 mL/min and 20 mL/min, respectively. Small-scale crudes (95%) of the corresponding pooled fractions was checked by LC−MS using method A. Manual Loading of the First Amino Acid. Solid-phase peptide syntheses were performed in polypropylene Torviq syringes (10 or 20 mL) fitted with a polyethylene porous disk at the bottom and closed with an appropriate piston. Solvent and soluble reagents were removed through back and forth movements. The 2-CTC resin was previously swelled in strictly anhydrous DCM (distilled) for 2 h. Side chain

■

CONCLUSIONS We have here demonstrated that the 4N1K peptide (KRFYVVMWKK) is able to induce selective cell death in tumor cells while sparing normal cells, thus underlying its therapeutic potential. Aware of the drawbacks of a peptidic approach, we initiated a systematic SAR study, gathering the development of a binding assay, structural studies by CD and NMR, chemical syntheses, and metabolic stability studies. All of them lead to the design of peptide 4, a 4N1K analogue, and to the identification of its pharmacophores. This peptide is to date the first serum stable agonist ligand of CD47, and its potency in triggering cell death was evaluated on cancer cells including CLL cells from patients refractory to all treatments. Of importance, the use of newly approved drug to treat CLL (i.e., idelalisib) has been recently restricted because of severe side effects highlighting the relevance of developing new strategies to target cancer diseases. Overall, the work realized and the results obtained in this study will be of valuable interest for further investigation in the design of peptide 4 mimics, we termed PKHB1,38 opening new perspectives for the development of original anticancer therapies. 8418



Article

peptides 4N1, peptide 1, and peptide 3, only partial 1H assignments could be obtained (Tables S5−S7). 3JHN−Hα coupling constants were measured on 1D 1H WATERGATE experiments. The chemical shift deviations of Hα protons and Cα carbons were calculated as the differences between observed chemical shifts and random coil values reported in water. The conformational analysis of thrombospondin-1 was carried out on the crystal structure of a C-terminal fragment of human thrombospondin-1 (PDB entry 1UX6)41 using Pymol (Schrö dinger) and Procheck42 programs. Predictions of NMR chemical shifts on the X-ray structure were carried out using SHIFTX2 program.43 5. Biological Assays. Binding affinity measurements by microscale thermophoresis (MST)44 were realized on a Monolith NT115 system (Nanotemper Technologies, Munich, Germany) for 4NGG, 4N1K, and peptides 1, 2, 3, and 4 (see Supporting Information). The measurement method is based on the directed movement of molecules along a temperature gradient, an effect termed “thermophoresis”. A local temperature difference ΔT leads to a local change in molecule concentration (depletion or enrichment), quantified by the Soret coefficient ST: chot/ccold = exp(−STΔT). MEC-1 cell membrane preparation was labeled using the Nanotemper NT-647 labeling kit as described elsewhere.45 The labeled preparation was eluted with PBS and stored at 4 °C. A stock solution of each peptide was prepared in DMSO (5 mM) and then diluted with PBS. The assay details and the results are given in the Supporting Information. Binding affinity measurements by biolayer interferometry were realized on an Octet RED96 system (Pall FortéBio Corp., Menlo Park, CA) for all the peptides described here. This system monitors interference of light reflected from two sources (an internal reflection surface and the liquid/solid interface of a fiber optic sensor) to measure the rate of binding of molecules to the biosensor surface. MEC-1 cell membrane preparations were biotinylated with the EZLink NHS-PEG4-biotin kit from Thermo-Scientific. Biotinylated membranes were then loaded onto SuperStreptavidin (SSA) biosensors (Pall FortéBio Corp.) at empirically determined concentrations. All affinity measurements were carried out in assay buffer (PBS with 0.2% bovine serum albumin (BSA) and 1% DMSO) at 30 °C. Assay details are described in the Supporting Information. Flow Cytometry. Cell death was assessed by flow cytometry using annexin-V-APC (0.1 μg/mL; BD Biosciences) and propidium iodide (PI, 0.5 μg/mL) double labeling. Data were obtained in a FACSCanto II (BD Biosciences) and analyzed with the FlowJo software. Cell Death Induction. To induce PCD, 1 × 106 cells/mL were treated for 2 h with the indicated peptide at concentrations ranging from 50 to 400 μM. After authorized consent forms were obtained, peripheral blood was collected from healthy volunteers (B cells) and from CLL patients diagnosed according to classical morphological and immunophenotypic criteria. Pitie-Salpetriere Hospital Institutional Ethic Committee approved this study. Mononuclear cells were purified from blood samples using a standard Ficoll-Hypaque gradient, and B cells were positively selected by magnetic beads coupled to anti-CD19 monoclonal antibody (Miltenyi Biotech). Alternatively, PCD induction by 4NGG, 4N1K, and PKHB1 was tested in a panel of cancer cell lines (ATCC) described in Supporting Information. B-Lymphocytes, tumor cell lines, and MEC-1 cells (an established CLL cell line with dysfunctional TP53 that was used in the peptide screening assays) were cultured in complete medium (RPMI 1640 supplemented with 10% fetal calf serum, 2 mM L-glutamine, and 100 U/mL penicillin−streptomycin). Statistical Analysis. Results are displayed as the mean ± SD and were statistically analyzed by the Mann−Whitney test using the GraphPad Prism software. Concerning CLL cells, our assessment included an equal number of Binet stage A and Binet Sstage B/C patients.

protected Fmoc-Aa-OH (0.30 mmol, 1 equiv) was coupled to 2-CTC resin (400 mg, loading 1.6 mmol/g) in the presence of DIEA (1.2 mmol, 4 equiv) in DCM (4 mL). The unreacted sites on the resin were capped by washing with a mixture of DCM/MeOH/DIEA (17:2:1) repeated 3 times. Thus, loading was reduced to 0.80 mmol/g for optimal peptide growth. In the case of Rink amide resin, swelling in DCM was done similarly in 2 h (500 mg, loading 0.62 mmol/g). However, the first coupling was directly performed with protected Fmoc-Aa-OH (1.2 mmol, 4 equiv), HBTU (1.2 mmol, 4 equiv), HOBt (1.2 mmol, 4 equiv), and DIEA (2.4 mmol, 8 equiv) without loading decrease. Manual Solid Phase Peptide Synthesis. In all syntheses the scale was 0.30 mmol. Fmoc group was split off by treatment with piperidine/DMF (1:4) (1 × 1 min, 1 × 10 min). Washing steps between deprotection and coupling were carried out with DMF (3 × 1 min), IPA (3 × 1 min), and DMF (3 × 1 min). Activation step was carried out with Fmoc-Aa-OH (1.2 mmol, 4 equiv), HBTU (1.2 mmol, 4 equiv) as coupling agent, HOBt (1.2 mmol, 4 equiv) as auxiliary nucleophile, and DIEA (2.4 mmol, 8 equiv) as base. The activated amino acid is then transferred to the resin where the coupling was performed for 1−18 h. Supported coupling reactions were monitored by classical Kaiser test (solution kit from Sigma-Aldrich). When elongation of the peptide chain was completed, a washing step with MeOH was added after final N-terminal Fmoc removal in order to totally shrink the resin under vacuum. Final Side Chain Deprotection and Cleavage from the Resin. The crude peptides were treated with the following cleavage cocktail: TFA/H2O/TIS (95/2.5/2.5, 10 mL). The syringes were shaken for 3 h and then precipitated 3 times using cooled Et2O (3 × 30 mL), recovered after centrifugation (3 × 5 min, 7800 rpm). Diethyl ether was removed (3 times), and then the peptide pellets were dried (under nitrogen flow). The resulting crude peptide was dissolved in aqueous 0.1% (v/v) TFA. Purification was conducted on reversed-phase HPLC Prep C18 column, eluting with 0.1% TFA in water (solvent A) and 0.1% TFA in acetonitrile (solvent B) as described earlier. 2. Stability Studies in Human Serum. To a mixture of 250 μL of human serum and 750 μL of RPMI 1640 was added 20 μL of the peptide DMSO stock solution at 10 mg/mL. The mixture was incubated at 37 °C. Aliquots of 100 μL were removed from the medium at different times, mixed with 100 μL of acetonitrile and incubated at 4 °C for at least 15 min to precipitate all the serum proteins. After centrifugation at 12 000 rpm for 2 min, an amount of 50 μL of the supernatant was transferred to an injection vial and analyzed by HPLC with a linear gradient of MeCN in water (5−95% + 0.1% TFA). The relative concentrations of the remaining soluble peptides were calculated by integration of the absorbance at 220 nm as a function of the retention time (peak area). 3. CD Spectroscopy. CD spectra were recorded on a Jasco 815 spectropolarimeter (Jasco Inc., Easton, MD) over the wavelength range 190−260 nm, at 0.2 nm intervals and 10 nm·min−1 scan speed. Peptides were dissolved at 50 mM concentration in a 10 mM sodium phosphate buffer at pH 7.4, using 0.1 cm path length quartz cells (Hellma, Mullheim, Germany). CD spectra were deconvoluted with CDFriend program in order to estimate the peptide secondary structure content. 4. NMR Conformational Analysis. NMR experiments were recorded on a 500 MHz Bruker Avance III spectrometer (Wissembourg, France) equipped with a TCI cryoprobe. NMR samples were prepared in 50 mM sodium succinate buffer, pH 5, using 2 mM peptide concentration. Sodium 2,2-dimethyl-2-silapentane-d6-5sulfonate (Sigma-Aldrich) was added at 0.1 mM concentration for chemical shift calibration and assessment of the solubilized peptide concentration. NMR experiments were processed with TOPSPIN 2.1 software (Bruker). Spectra were analyzed with Sparky program.40 1H, 13 C, and 15N resonances were assigned from the analysis of 2D 1H−1H TOCSY (DIPSI2 isotropic scheme of 70 ms duration), 2D 1H−1H ROESY (300 ms mixing time), 2D 1H−13C HSQC, 2D 1H−15N HSQC, and 2D 1H−13C HMBC spectra. The complete 1H, 13C, and 15 N NMR assignments of peptides 4N1K, peptide 2, peptide 4, and 4NGG are provided in Tables S1−S4. For the three poorly soluble 8419



■

Article

ASSOCIATED CONTENT

factor receptor 2; MST, microscale thermophoresis; OR, OctetRed

S Supporting Information *

■

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00781. Additional experimental details concerning (1) peptides characterization, (2) CD spectra, (3) NMR tables, (4) binding affinity measurements by biolayer interferometry (BI) and microscale thermophoresis (MST), (5) cancer cell lines used in this manuscript, and (6) molecular formula strings (PDF)

■

REFERENCES

(1) Biemar, F.; Foti, M. Global progress against cancerchallenges and opportunities. Cancer Biol. Med. 2013, 10, 183−186. (2) World Cancer Research Fund International (WCRFI). Worldwide data. http://www.wcrf.org/int/cancer-facts-figures/worldwidedata (accessed July 14, 2016). (3) Figure 1, designed by P. Karoyan with the corresponding proteins coordinates from RSCB PDB. (4) Figure 2, designed by P. Karoyan with the corresponding proteins coordinates from RSCB PDB. (5) Favaloro, B.; Allocati, N.; Graziano, V.; Di Ilio, C.; De Laurenzi, V. Role of apoptosis in disease. Aging 2012, 4, 330−349. (6) (a) Lobo, I. Chromosome abnormalities and cancer cytogenetics. Nat. Educ. 2008, 1 (1), 68. (b) Calin, G. A.; Croce, C. M. MicroRNAs and chromosomal abnormalities in cancer cells. Oncogene 2006, 25, 6202−6210. For more information see the following: http://cgap.nci. nih.gov/Chromosomes/Mitelman (accessed May, 20, 2016). (7) Secchiero, P.; di Iasio, M. G.; Gonelli, A.; Zauli, G. The MDM2 inhibitor nutlins as an innovative therapeutic tool for the treatment of haematological malignancies. Curr. Pharm. Des. 2008, 14, 2100−2110. (8) Gandhi, L.; Camidge, D. R.; de Oliveira, M. R.; Bonomi, P.; Gandara, D.; Khaira, D.; Hann, C. L.; McKeegan, E. M.; Litvinovich, E.; Hemken, P. M.; Dive, C.; Enschede, S. H.; Nolan, C.; Chiu, Y.-L.; Busman, T.; Xiong, H.; Krivoshik, A. P.; Humerickhouse, R. H.; Shapiro, G. I.; Rudin, C. M. Phase I study of Navitoclax (ABT-263), a novel Bcl-2 family inhibitor, in patients with small-cell lung cancer and other solid tumors. J. Clin. Oncol. 2011, 29, 909−916. (9) Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-H.; Qian, D.; Lu, J.; Qiu, S.; Bai, L.; Peng, Y.; Cai, Q.; Wang, S. Design of Small-Molecule Peptidic and Non-Peptidic Smac Mimetics. Acc. Chem. Res. 2008, 41, 1264−1277. (10) Murray, J. K.; Gellman, S. H. Targeting protein−protein interactions: lessons from p53/MDM2. Biopolymers 2007, 88, 657− 686. (11) Chang, Y. S.; Graves, B.; Guerlavais, V.; Tovar, C.; Packman, K.; To, K.-H.; Olson, K. A.; Kesavan, K.; Gangurde, P.; Mukherjee, A.; Baker, T.; Darlak, K.; Elkin, C.; Filipovic, Z.; Qureshi, F. Z.; Cai, H.; Berry, P.; Feyfant, E.; Shi, X. E.; Horstick, J.; Annis, D. A.; Manning, A. M.; Fotouhi, N.; Nash, H.; Vassilev, L. T.; Sawyer, T. L. Stapled αhelical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3445−3454. (12) Dohner, H.; Stilgenbauer, S.; Benner, A.; Leupolt, E.; Kröber, A.; Bullinger, L.; Döhner, K.; Bentz, M.; Lichter, P. Genomic aberrations and survival in chronic lymphocytic leukemia. N. Engl. J. Med. 2000, 343, 1910−1916. (13) Gilead. Direct Healthcare Professional Communication. https://www.hpra.ie/docs/default-source/default-document-library/ important-safety-information---zydelig-%28idelalisib%29.pdf?sfvrsn=0 (accessed May, 20, 2016). (14) Smolej, L.; Šimkovič, M. Practical approach to management of chronic lymphocytic leukemia. Arch. Med. Sci. 2016, 12, 448−456. (15) Wilson, F. H.; Johannessen, C. M.; Piccioni, F.; Tamayo, P.; Kim, J. W.; Van Allen, E. M.; Corsello, S. M.; Capelletti, M.; Calles, A.; Butaney, M.; Sharifnia, T.; Gabriel, S. B.; Mesirov, J. P.; Hahn, W. C.; Engelman, J. A.; Meyerson, M.; Root, D. E.; Jänne, P. A.; Garraway, L. A. A functional landscape of resistance to ALK inhibition in lung cancer. Cancer Cell 2015, 27, 397−408. (16) Henkin, J.; Volpert, O. V. Therapies using anti-angiogenic peptide mimetics of thrombospondin-1. Expert Opin. Ther. Targets 2011, 15, 1369−1386. (17) Jeanne, A.; Schneider, C.; Martiny, L.; Dedieu, S. Original insights on thrombospondin-1-related antireceptor strategies in cancer. Front. Pharmacol. 2015, 6, 1−13. (18) (a) Kosfeld, M. D.; Frazier, W. A. Identification of a new cell adhesion motif in two homologous peptides from the COOH-terminal

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +33144274469. Author Contributions

T.D., C.N., H.B., A.G., M.P., and P.K. performed all peptides syntheses. T.D., A.G., H.B., M.D.L., E.L., and P.K. performed the serum stability experiments. P.K. designed the binding experiments and performed them with P.G., F.-X.O., and T.D. O.L. performed the NMR studies. P.K., A.G., and O.L. performed and interpreted the CD experiments. L.H., A.C.M.-T., E.P., and C.Q. performed in vitro studies in normal and CLL B-lymphocytes, MEC-1 cells, and the panel of tumor cell lines. H.M.-B. and S.A.S. conceived and supervised cell death experimental work, interpreted the data, and helped to write the manuscript. P.K. conceived and supervised this project, designed the peptides and the experiments, interpreted all the data, and wrote the manuscript with the reviewing of the coauthors. Notes

The authors declare the following competing financial interest(s): The patent applications PCT/EP2013/061727 and PCT/EP2014/077335 included results from this paper. The authors declare that no other competing interests exist.

■

ACKNOWLEDGMENTS This work was supported by the Labex Michem (T.D. Ph.D. thesis grant), French Research Ministry (H.B. Ph.D. thesis grant), Fondation ARC, Ligue Contre le Cancer-Comité de Paris, Association Laurette Fugain, Fondation pour la Recherche Medicale, French National Research Agency (Grant ANR-12-EMMA-0045), and French National Cancer Institute (Grant INCa-5839). P.K. is grateful to Nanotemper Technologies for MST technical support, J. Kirilowsky, and A. Denis from GSK for hosting the LBM Team, SATT Lutech, and DGRTT from UPMC for helpful support (E.L. grant).

■

ABBREVIATIONS USED TSP-1, thrombospondin-1; CD47, cluster of differentiation 47; PCD, programmed cell death; WCRFI, World Cancer Research Fund International; PPI, protein−protein interaction; Bcl-2, Bcell lymphoma 2; BAK, B-cell lymphoma 2 homologous antagonist killer; BAX, B-cell lymphoma 2 associated X protein; Bcl-xL, B-cell lymphoma, extra large; p53 or TP53, tumor protein 53; DNA, deoxyribonucleic acid; ATM kinase, ataxia telangiectasia mutated kinase; IAP, integrin associated protein; CLL, chronic lymphocytic leukemia; Del17p, deletion of part of the short arm of chromosome 17; Del11q, deletion of the long arm on chromosome 11; VEGFR2, vascular endothelial growth 8420



Article

binding domain of human thrombospondin. J. Biol. Chem. 1993, 268, 8808−8814. (b) Gao, A.-G.; Frazier, W. A. Identification of a receptor candidate for the carboxyl-terminal cell binding domain of thrombospondins. J. Biol. Chem. 1994, 269, 29650−29657. (c) Gao, A. G.; Lindberg, F. B.; Finn, M. B.; Blystone, S. D.; Brown, E. J.; Frazier, W. A. Integrin-associated protein is a receptor for the Cterminal domain of thrombospondin. J. Biol. Chem. 1996, 271, 21−24. (19) See for example the following: (a) Kaur, S.; Soto-Pantoja, D. R.; Stein, E. V.; Liu, C.; Elkahloun, A. G.; Pendrak, M. L.; Nicolae, A.; Singh, S. P.; Nie, Z.; Levens, D.; Isenberg, J. S.; Roberts, D. D. Thrombospondin-1 Signaling through CD47 Inhibits Self-renewal by Regulating c-Myc and Other Stem Cell Transcription Factors. Sci. Rep. 2013, 3, 1673−1711. (b) Miller, T. W.; Kaur, S.; Ivins-O’Keefe, K.; Roberts, D. D. Thrombospondin-1 is a CD47-dependent endogenous inhibitor of hydrogen sulfide signaling in T cell activation. Matrix Biol. 2013, 32, 316−324. (20) Roberts, D. D.; Kaur, S.; Soto-Pantoja, D. R. Therapeutic targeting of the thrombospondin-1 receptor CD47 to treat liver cancer. J. Cell Commun. Signal. 2015, 9, 101−102. (21) Floquet, N.; Dedieu, S.; Martiny, L.; Dauchez, M.; Perahia, D. Human thrombospondin’s (TSP-1) C-terminal domain opens to interact with the CD-47 receptor: A molecular modeling study. Arch. Biochem. Biophys. 2008, 478, 103−109. (22) Kaur, S.; Martin-Manso, G.; Pendrak, M. L.; Garfield, S. H.; Isenberg, J. S.; Roberts, D. D. Thrombospondin-1 inhibits VEGF receptor-2 signaling by disrupting its association with CD47. J. Biol. Chem. 2010, 285, 38923−38932. (23) Mateo, V.; Lagneaux, L.; Bron, D.; Biron, G.; Armant, M.; Delespesse, G.; Sarfati, M. CD47 ligation induces caspase-independent cell death in chronic lymphocytic leukemia. Nat. Med. 1999, 5, 1277− 1284. (24) Johansson, U.; Higginbottom, K.; Londei, M. CD47 ligation induces a rapid caspase- independent apoptosis-like cell death in human monocytes and dendritic cells. Scand. J. Immunol. 2004, 59, 40−49. (25) Miyata, Y.; Watanabe, S.; Kanetake, H.; Sakai, H. Thrombospondin-1-derived 4N1K peptide expression is negatively associated with malignant aggressiveness and prognosis in urothelial carcinoma of the upper urinary tract. BMC Cancer 2012, 12, 372. (26) Manna, P. P.; Frazier, W. A. The mechanism of CD47dependent killing of T cells: heterotrimeric Gi-dependent inhibition of protein kinase A. J. Immunol. 2003, 170, 3544−3553. (27) Teuchert, M.; Berghöfer, S.; Klenk, H.-D.; Garten, W. Recycling of furin from the plasma membrane. J. Biol. Chem. 1999, 274, 36781− 36789. (28) Henrich, S.; Cameron, A.; Bourenkov, G. P.; Kiefersauer, R.; Huber, R.; Lindberg, I.; Bode, W.; Than, M. E. The crystal structure of the proprotein processing proteinase furin explains its stringent specificity. Nat. Struct. Biol. 2003, 10, 520−526. (29) (a) Moumne, R.; Larregola, M.; Boutadla, Y.; Lavielle, S.; Karoyan, P. Aminomethylation of chiral silyl enol ethers: access to beta(2)-homotryptophane and beta(2)-homolysine derivatives. Tetrahedron Lett. 2008, 49, 4704−4707. (b) Moumne, R.; Denise, B.; Guitot, K.; Rudler, H.; Lavielle, S.; Karoyan, P. New scalable asymmetric aminomethylation reaction for the synthesis of beta(2)amino acids. Eur. J. Org. Chem. 2007, 2007 (12), 1912−1920. (30) Lukaszuk, A.; Demaegdt, H.; Szemenyei, E.; Toth, G.; Tymecka, D.; Misicka, A.; Karoyan, P.; Vanderheyden, P.; Vauquelin, G.; Tourwe, D. Beta-homo-amino acid scan of angiotensin IV. J. Med. Chem. 2008, 51, 2291−2296. (31) Avbelj, F.; Grdadolnik, S. G.; Grdadolnik, J.; Baldwin, R. L. Intrinsic backbone preferences are fully present in blocked amino acids. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 1272−1277. (32) Vuister, G. W.; Bax, A. Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNHa) coupling constants in 15N-enriched proteins. J. Am. Chem. Soc. 1993, 115, 7772−7777. (33) Rebres, R. A.; Vaz, L. E.; Green, J. M.; Brown, E. J. Normal ligand binding and signaling by CD47 (integrin-associated protein)

requires a long range disulfide bond between the extracellular and membrane-spanning domains. J. Biol. Chem. 2001, 276, 34607−34616. (34) Martinez-Torres, A.-C.; Quiney, C.; Attout, T.; Boullet, H.; Herbi, L.; Vela, L.; Barbier, S.; Chateau, D.; Chapiro, E.; Nguyen-Khac, F.; Davi, F.; Le Garff-Tavernier, M.; Moumné, R.; Sarfati, M.; Karoyan, P.; Merle-Béral, H.; Launay, P.; Susin, S. A. CD47 agonist peptides induce programmed cell death in refractory chronic lymphocytic leukemia B cells via PLCγ1 activation: evidence from mice and humans. PLoS Med. 2015, 12 (3), e1001796. (35) McDonald, J. F.; Dimitry, J. M.; Frazier, W. A. An amyloid-like C-terminal domain of thrombospondin-1 displays CD47 agonist activity requiring both VVM Motifs. Biochemistry 2003, 42, 10001− 10011. (36) Sagan, S.; Karoyan, P.; Chassaing, G.; Lavielle, S. Further delineation of the two binding sites associated with Tachykinin Neurokinin-1 receptors using [3-Prolinomethionine11]SP analogues. J. Biol. Chem. 1999, 274, 23770−23776. (37) Galluzzi, L.; Bravo-San Pedro, J. M.; Vitale, I.; Aaronson, S. A.; Abrams, J. M.; Adam, D.; Kroemer, G. Essential versus accessory aspects of cell death: recommendations of the NCCD. Cell Death Differ. 2015, 22 (1), 58−73. (38) Karoyan, P.; Launay, P.; Merle-Beral, H.; Susin, S. A. Method and pharmaceutical composition for use in the treatment of cancer. WO 2013182650 A1, 2013. (39) (a) Moumne, R.; Denise, B.; Parlier, A.; Lavielle, S.; Rudler, H.; Karoyan, P. One-pot synthesis of β-amino acid derivatives via addition of bis(O-silyl) ketene acetals on iminium salts. Tetrahedron Lett. 2007, 48, 8277−8280. (b) Moumne, R.; Denise, B.; Guitot, K.; Rudler, H.; Lavielle, S.; Karoyan, P. New scalable asymmetric aminomethylation reaction for the synthesis of β2-amino acids. Eur. J. Org. Chem. 2007, 2007 (12), 1912−1920. (40) Goddard, T. D.; Kneller, D. G. Sparky, version 3; University of California: San Francisco, CA; http://www.cgl.ucsf.edu/home/ sparky/ (accessed May, 20, 2016). (41) Kvansakul, M.; Adams, J. C.; Hohenester, E. Structure of a thrombospondin C-terminal fragment reveals a novel calcium core in the type 3 repeats. EMBO J. 2004, 23, 1223−1233. (42) Laskowski, R. A.; MacArthur, M. W.; Moss, D. S.; Thornton, J. M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 1993, 26, 283−291. (43) Han, B.; Liu, Y.; Ginzinger, S.; Wishart, D. SHIFTX2: significantly improved protein chemical shift prediction. J. Biomol. NMR 2011, 50, 43−57. (44) Jerabek-Willemsen, M.; Wienken, C. J.; Braun, D.; Baaske, P.; Duhr, S. Molecular interaction studies using microscale thermophoresis. Assay Drug Dev. Technol. 2011, 9, 342−353. (45) Monolith NT Protein Labeling Kit RED-NHS. User Manual. http://www.helsinki.fi/biosciences/corefacilities/ microscalethermophoresis/Protein_Labeling_Manual_V012-REDNHS.pdf (accessed May, 20, 2016).

8421


Thrombospondin-1 Mimetic Agonist Peptides ... - ACS Publications

Recommend Documents