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Optimization and in vivo validation of peptide-vectors targeting the LDL-receptor Guillaume JACQUOT, Pascaline Lecorche, Jean-Daniel Malcor, Mathieu Laurencin, Maria Smirnova, Karine Varini, Cedric Malicet, Fanny Gassiot, Aude Faucon, Marion David, Nicolas Gaudin, Maxime Masse, Geraldine Ferracci, Vincent Dive, Salvatore Cisternino, and Michel Khrestchatisky Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00687 • Publication Date (Web): 22 Sep 2016 Downloaded from http://pubs.acs.org on September 30, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

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Optimization and in vivo validation of peptide-vectors targeting the LDL-receptor

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Guillaume Jacquot a*, Pascaline Lécorché a, Jean-Daniel Malcor a, Mathieu Laurencin

a,b

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Maria Smirnova c, Karine Varini e,a, Cédric Malicet a, Fanny Gassiot a, Aude Faucon a, Marion

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David a, Nicolas Gaudin e,a, Maxime Masse a, Géraldine Ferracci d, Vincent Dive b, Salvatore

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Cisternino c, Michel Khrestchatisky e*

,

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a

VECT-HORUS SAS, Faculté de Médecine secteur Nord, 51 Bd Pierre Dramard, CS80011,

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13344, Marseille Cedex 15, France.

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b

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CEA-DSV-iBiTecS, 91191 Gif/Yvette Cedex, France

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c

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Pharmacie, Université Paris Descartes, INSERM UMR S-1144, Paris 75006, France.

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d

Aix Marseille Univ, CNRS, CRN2M, Marseille, France.

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e

Aix Marseille Univ, CNRS, NICN, Marseille, France.

CEA-Saclay, Service d'Ingénierie Moléculaire de Protéines (SIMOPRO), Labex LERMIT,

Variabilité de réponse aux psychotropes, INSERM U1144, Paris, 75006, France ; Faculté de

17

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ABSTRACT

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Active targeting and delivery to pathophysiological organs of interest is of paramount

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importance to increase specific accumulation of therapeutic drugs or imaging agents while

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avoiding systemic side-effects. We recently developed a family of new peptide ligands of the

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human and rodent LDL-receptor (LDLR), an attractive cell-surface receptor with high uptake

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activity and local enrichment in several normal or pathological tissues (Malcor et al., J Med

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Chem 2010). Initial chemical optimization of the 15-mer, all natural amino acids compound

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1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation

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led to the cyclic 8-amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which

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specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ~3 hrs.

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Further introduction of non-natural amino acids led to the identification of compound

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60/VH4106 ([(D)-“Pen”M“Thz”RLRGC]c) that showed the highest KD value of 9 nM.

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However, this latter analogue displayed the lowest in vitro blood half-life (~1.9 hrs). In the

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present study, we designed a new set of peptide analogues, namely VH4127 to VH4131, with

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further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of

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previous and new analogues showed that the latter all displayed very high on-rates, in the 106

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s-1.M-1, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these

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new analogues showed increased blood half-lives in vitro, reaching ~7 and 10 hrs for VH4129

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and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both

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VH445 and the most balanced optimized analogue VH4127 ([cM“Thz”RLRG“Pen”]c),

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showing a KD of 18 nM and a blood half-life of ~4.3 hrs, that its higher on-rate correlated with

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a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis

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potential. Finally, intravenous injection of tritium-radiolabelled 3H-VH4127 in wild-type or

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ldlr -/- mice confirmed their active LDLR-targeting in vivo. Overall, this study extends our

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previous work towards a diversified portfolio of LDLR-targeted peptide-vectors with

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validated LDLR-targeting potential in vivo.

44 45 46 47

KEYWORDS

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peptide-vector; medicinal chemistry-based optimization; LDLR-targeting; tissue distribution;

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LDLR-knockout.

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INTRODUCTION

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Target-specific delivery of imaging agents or therapeutic drugs, especially highly toxic

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anticancer agents, is of paramount importance to increase accumulation at the

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pathophysiological site while reducing systemic side-effects. The conjugation of imaging

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agents or therapeutics to ligands that specifically target cell surface markers enriched in

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tissues of interest is the main noninvasive approach to attain better selectivity in tissue

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distribution. Specific targeting systems showing considerable promise include i)

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immunoconjugates whereby the targeting ligand is either a conventional monoclonal antibody

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(antibody-drug conjugates, ADCs), a bioengineered fragment thereof or a camelid VHH

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fragment (“nanobodies”); ii) small molecule-drug conjugates (SMDCs) comprising a small

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endogenous molecule as targeting moiety (e.g., the vitamin folic acid, carbohydrates); and iii)

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peptide-drug conjugates (PDCs) whereby the targeting ligand is a small endogenous or

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synthetic peptide (e.g., bombesin, somatostatin, LHRH or RGD-, NGR- or IBR-derived

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peptides)1-7. Depending on the imaging agent or therapeutic drug and their anticipated clinical

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indications, route of administration and dosing schedule, one targeting approach may be

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preferable to another. Despite their poor plasma stability unless chemically optimized,

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peptides smaller than 5 kDa combine many of the advantages of both small organic drugs and

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monoclonal antibodies and thereby have become a credible alternative as targeting ligands3, 8-

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11

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high target specificity, possible exploitation of non-natural amino acids to increase their

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binding affinity and in vivo stability, conjugation versatility, low immunogenicity and high

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tissue penetration that endows them with interesting pharmacokinetic profiles when compared

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to antibodies. Owing to these advantages, peptides have become increasingly popular in the

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last decade as targeting ligands for imaging agents9, 12, 13, anticancer small organic drugs14, 15,

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anticancer peptides16 and proteins17-19. As exemplified by one of the most clinically advanced

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PDC, namely ANG1005 which consists of three paclitaxel molecules covalently attached to

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the Angiopep-2 peptide that targets the low-density lipoprotein receptor-related protein-1

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(LRP-1), PDCs will undoubtedly respond to the high demand in targeted delivery systems in

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clinical settings in the near future20, 21.

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For several reasons the LDL cell surface receptor (LDLR) represents an attractive target for

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the development of innovative active drug-targeting strategies1,

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uptake and recycling activity thereby providing an effective cellular entry route for drugs

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acting at the intracellular level; ii) enrichment in some normal tissues where it contributes to

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major physiological processes, such as cholesterol metabolism in the liver, synthesis of major

. Besides their rather low production cost and easy characterization, small peptides display

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. These include i) high

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steroid hormones in the adrenal cortex and gonads, rapid cell growth in duodenal crypts; iii)

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upregulation in several human cancers.

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In this context, we recently described a family of constrained cyclic peptides, isolated by

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phage display biopanning, as new ligands of the human LDLR (hLDLR). They were shown to

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undergo specific receptor-mediated endocytosis in cells expressing either the human or the

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rodent form of the LDLR, with no binding competition with LDL, suggesting that these

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peptides bind to a domain distinct from that of LDL binding30. We performed an initial

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medicinal chemistry-based optimization from the cyclic 15-mer hit peptide VH411,

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previously referred to as compound 1 (DSGL[CMPRLRGC]cDPR)30 (Supplemental Table

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1). Using truncations, alanine-scanning, D-scanning and an extensive structure-activity

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relationship (SAR) investigation (displacement of the reference S-tagged VH411 by new

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analogs on CHO cells expressing hLDLR), we identified several optimized peptides

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displaying improved biochemical parameters, including increased apparent LDLR-binding

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affinity and in vitro blood stability. These include compound 19/VH434 ([CMPRLRGC]c),

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the minimal all-natural 8 amino acids analogue showing a higher apparent LDLR-binding

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affinity than the initial hit peptide 1/VH411, and compound 22/VH445 ([cMPRLRGC]c),

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comprising an N-terminal D-Cys leading to both a further improvement of the apparent

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LDLR-binding affinity and a 2-fold increase in mouse blood half-life (~3 hrs)30. The same

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SAR approach was subsequently used in a second round of optimization where each amino

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acid was replaced by structurally related non-natural amino acids30. This work led to the

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identification of new analogues with further improved apparent LDLR-binding affinity

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(Supplemental Table 1, compounds 40 to 78), which peaked with compound 60/VH4106

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([(D)-“Pen”M“Thz”RLRGC]c). However, no analogue showed a higher stability than the

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intermediate compound 22/VH445 analogue.

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These results prompted us to analyze the individual impact of single amino acid substitutions

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on both the LDLR-binding affinity and in vitro blood stability of these peptides. This led to

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the design of new analogues, namely VH4127 to VH4131, comprising the most beneficial

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substitution combinations. We performed a detailed analysis of their association and

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dissociation constants towards the hLDLR. Because metabolic stability in circulating blood is

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crucial when developing peptides10,

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compared to VH445, the analogue showing the most balanced profile, namely VH4127,

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displayed improved specific binding to cell surface LDLR and endocytosis potential. Most

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importantly, intravenous injection of tritium-labeled VH4127 led to a preferential

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accumulation of the peptide and/or its metabolites in LDLR-enriched tissues in wild-type

20,

this parameter was also assessed in vitro. When

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Molecular Pharmaceutics

(WT) mice compared to ldlr -/- (LDLR-knockout) mice.

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EXPERIMENTAL SECTION

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Cell lines, culture and reagents

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The phLDLR-EGFP and phLDLR-dsRed2 plasmids were generated by cloning into the

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pEGFP-N1 and pDsRed2-N1 vectors (Clontech) respectively the full length coding region for

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hLDLR following RT-PCR amplification from human brain total RNA (Clontech

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laboratories,

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ATATATAAGCTTCGAGGACACAGCAGGTCGTGAT

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TTAATTGTCGACCACGCCACGTCATCCTCCAGACT. Both constructs were fully

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sequenced and used to transfect Chinese Hamster Ovary cells (CHO-K1, ATCC number

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CCL-61TM) using JetPei (Polyplus Transfection, France) according to manufacturer’s

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instructions. 48 hours after transfection, cells were seeded in culture medium (Nutrient

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Mix/F12 (Ham) Glutamax medium supplemented with 10% fetal calf serum Life

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Technologies (Saint Aubin, France), containing 800 µg/mL Geneticin (Life Technologies) as

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selection agent. Individual fluorescent cells were seeded in 96-well plates. Isolated clones

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expressing GFP or DsRed2 fused to the C-termini of hLDLR were selected, amplified and

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validated for the expression of the fusion proteins of interest. The validated stable cell lines

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were named CHO-hLDLR-EGFP and CHO-hLDLR-dsRed2. Cells were cultured in Nutrient

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Mix/F12 (Ham) Glutamax medium supplemented with 10% fetal calf serum, 100µg/mL

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streptomycin, 100 U/mL penicillin and 500µg/mL Geneticin in a humidified 5% CO2

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atmosphere at 37 °C. For immunofluorescence studies, cells were seeded two days before the

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experiments onto glass coverslips in 24-well plates at 5.104 cells/well.

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Nutrient Mix/F12 (Ham) Glutamax (cell culture medium), fetal calf serum, Ca2+-free and

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Mg2+-free phosphate buffer saline (D-PBS), Penicillin/Streptomycin, Geneticin 50µg/mL,

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Hoechst (#33258) and Prolong Gold anti-fade reagent were purchased from Life Technologies

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(Saint Aubin, France). Low endotoxin bovine serum albumin (BSA) (K45-011-500g) was

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purchased from PAA (Les Mureaux, France). The goat anti-S-Tag antibody (ab19321) was

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purchased from Abcam (Cambridge, USA). The secondary antibody donkey anti-goat-A647

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(715-605-151) was purchased from Jackson Immunoresearch (Suffolk, UK).

Mountain

View,

USA)

using

the

following

primers and

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Peptide synthesis

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Abbreviations - AcOH, acetic acid; DCM, dichloromethane; EDT, 1-2-ethanedithiol; Eq,

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equivalent; ESI, electrospray ionization; DIPEA, N,N-diisopropylethylamine; DMF,

152

N,Ndimethylformamide;

Fmoc,

9-fluorenylmethoxycarbonyl; 6

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HATU,

1-

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[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium

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hexafluorophosphate; MeCN, acetonitrile; “Pen”, Penicillamine; “Pip”, Pipecolic acid; RP-

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HPLC, reverse-phase high performance liquid chromatography; SPPS, solid phase peptide

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synthesis; TFA, trifluoroacetic acid; “Thz”, Thiazolidine; TIS, triisopropylsilane.

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Chemicals - Fmoc-protected amino acids, TFA, TIS, EDT, HATU, Rink Amide AM resin (on

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polystyrene-1% DVB) were purchased from Iris Biotech (Marktredwitz, Germany). Piperidin,

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solvents for peptide synthesis and HPLC were purchased from Analytic Lab (St Mathieu De

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Tréviers, France). All other chemicals were purchased from Sigma-Aldrich at the highest

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quality available.

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Analytical and purification methods - Reaction progress and purity monitoring were carried

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out on a Dionex UltiMate® 3000 system equipped with a C18 KinetexTM (5 µm, 150 mm x 4.6

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mm). Detection was done at 214 nm. Elution system was composed of H2O/0.1%TFA

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(solution A) and MeCN/0.1%TFA (solution B). Flow rate was 2 mL/min with a gradient of 0-

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100% B in 4 min.

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Crude products were purified by RP-HPLC on a Dionex UltiMate® 3000 system equipped

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with a C18 LunaTM (5 µm, 100 mm x 21.2 mm). Detection was done at 214 nm. Elution

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system was composed of H2O/0.1%TFA (solution A) and MeCN/0.1%TFA (solution B).

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Flow rate was 20 mL/min.

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SPPS - Peptides were synthesized at 100 µmol or 250 µmol scale. Peptides were assembled

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on the resin by automated Fmoc synthesis protocols run on a Liberty-12-channel synthesizer

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(CEM µWaves). Each coupling step involved Fmoc removal followed by a coupling run.

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Briefly, Fmoc groups were removed with piperidine/DMF (20%) using a short (40 W, 75 ºC,

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30 s) followed by a long cycle (35 W, 70 ºC, 180 s). After DMF washings, coupling was

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carried out with a 0.2 M solution of Fmoc-amino acid (5eq in the 100µmol scale or 4eq in the

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250µmol scale), in the presence of 0.5 M HATU (5eq in the 100µmol scale or 4eq in the

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250µmol scale) and 2 M DIPEA (10eq in the 100µmol scale or 8eq in the 250µmol scale) at

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23 W, 70ºC for 10 min except for Fmoc-Cys(Trt)-OH whose coupling temperature was

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limited to 50°C. Fmoc-Arg(Pbf), Fmoc-Met-OH and Fmoc-Cys(Trt)-OH required an

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additional coupling step. After removal of the last Fmoc protecting group, the N-deblocked

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peptide resins were N-propionylated or N-acetylated using a solution of propionic or acetic

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anhydride in DCM (50%, 2*5min). The resin-bound peptide was then side-chain deprotected

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and cleaved from the resin using TFA/H2O/TIS/EDT (94:2:2:2, 2 h) with mild orbital shaking.

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After filtration of the mixtures, the peptides were precipitated by addition of cold diethyl

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ether, centrifuged at (3220g; 5 min) and the supernatants were discarded. The crude peptides 7 ACS Paragon Plus Environment

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were then dissolved in MeCN/H2O in the presence of 0.1%TFA (0% to 50% of MeCN) and

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lyophilized.

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Peptide cyclisation - Disulfide bridges were obtained by intramolecular cyclisation from two

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thiol functions of two suitably protected Cys or Pen without initial purification. Crude solid

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peptides were dissolved in AcOH (0.5%, 0.5mg/mL). Solutions were adjusted to pH 7-8 using

192

an aqueous solution of (NH4)2CO3 (2M). An aqueous solution of K3Fe(CN)6 (3.25 g/L) was

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added slowly to the peptide mixture until persistence of a slight yellow color. Monitoring of

194

the cyclisation reaction was performed by RP-HPLC. After completion of the reaction the

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mixture was filtered over a PVDF membrane (0.45µm) and purified by preparative HPLC

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using the following gradient: 0%B for 3min, 0%B to x1%B in 5min, then in the purification

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gradient x1%B to x2%B in 30min (see Supplemental Table 2). The homogeneity and

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identity of synthetic peptides were assessed by analytical RP-HPLC. Peptides were

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satisfactorily checked for identity by ESI mass spectrometry on LCQ Fleet (ThermoFisher)

200

used in positive mode. Retention times, yields, and mass of the peptides are given in

201

Supplemental Table 2.

202 203

FRET assay on CHO-hLDLR-dsRed2 cells

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A FRETWorks S-Tag assay (Merk, Darmstadt, Germany) was used to determine the relative

205

LDLR-binding and endocytosis of new peptide analogues compared to a reference peptide

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when incubated on CHO-hLDLR-dsRed2 cells. This assay is based on the interaction of a

207

reference S-Tag peptide with an enzymatically inactive ribonuclease S protein. This resulted

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in the reconstitution of a fully functional ribonuclease S-enzyme, which cleaves a short

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chimeric ribo/deoxyribo nucleotide with a fluorophore on the 5’end and a quencher on the

210

3’end. Cleavage of this substrate by the reconstituted ribonuclease decouples the quencher

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from the fluorophore which becomes fluorescent (excitation 492 nm, emission 520-535 nm)

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and can be quantified in a Beckton Dickinson DU800 spectrophotometer. This FRET assay

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was used to test the potential for new peptide analogues (10 µM) to inhibit the binding and

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endocytosis during 1 h at 37°C of the VH445-S-Tag or VH4106-S-Tag reference peptides (10

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µM) on CHO-hLDLR-dsRed2 cells (8.105 cells per well in 6-well plates)30. Each condition

216

was tested in triplicate. At the end of the incubation period, cells were extensively washed in

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D-PBS and then lysed in PBS 0.1% Triton X100 containing 1/400 protease inhibitor cocktail

218

(Sigma-Aldrich, Saint-Quentin Fallavierville, France). 20 µL of cell extracts were mixed with

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180 µl of reaction mix (1X Fret assay buffer, FRET ArUAA substrate and S-Tag Grade S-

220

Protein). All the reactions were processed according to manufacturer’s instructions. 8 ACS Paragon Plus Environment

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221 222

Surface Plasmon Resonance

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Recombinant hLDLR and mLDLR (His-tagged) were purchased from Sino Biological

224

(Beijing. China). Interaction of ligands with LDLR was tested at 25°C using a Biacore T200

225

apparatus (GE Healthcare) and HBS (50 mM HEPES-NaOH pH 7.4, 150 mM NaCl, 50 µM

226

EDTA, 0.005% Tween-20) as running buffer. The His-tagged LDLR was immobilized on a

227

NiHC sensor chip (Xantec, Dusseldorf, Germany) at a density between 35-60 fmol/mm2. A

228

control flowcell without LDLR was used as reference. The single-cycle kinetic method was

229

used to measure the kinetic parameters (on-rate/association constant and off-rate/dissociation

230

constant) and calculate the equilibrium dissociation affinity constant KD (off-rate/on-rate) of

231

the different ligands with LDLR. Ligands were diluted in running buffer and injected

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sequentially 2 min at 30 µl/min using increasing concentrations (50 to 800 nM for VH411,

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VH434, VH445 and VH445-S-Tag and 10 to 160 nM for VH4106, VH4106-S-Tag, VH4127,

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VH4127-S-Tag, VH4128, VH4129, VH4130 and VH4131). Blank run injections of running

235

buffer were performed in the same conditions before ligand injections. Double-subtracted

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sensorgrams were globally fitted with the 1:1 Langmuir binding model from Biacore T200

237

Evaluation version 2.0. Data are representative of two to nine independent experiments.

238 239

In vitro blood stability

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The in vitro blood stability of VH445 analogues was determined as described in Malcor et al.

241

30

242

quantification in mouse plasma using LC-MS/MS. A volume of 1500 µL of freshly prepared

243

Lithium Heparin Swiss (CD-1) mouse blood (mixed blood from an adequate number of mice)

244

was spiked with each compound at the nominal concentration of 2 µM and subsequently

245

incubated at 4°C or 37°C for the indicated times. At prescribed times, at least 250 µL of blood

246

was centrifuged (5 min, 2500 rpm, 10 °C). A volume of 100 µL of the supernatant was

247

collected and 10 µL of formic acid and 300 µL of acetonitrile were added for acidification

248

and protein precipitation, respectively. After centrifugation, the supernatant was analysed

249

using a Shimadzu LC equipment coupled to an API 4000 triple quadrupole mass spectrometer

250

(Applied Biosystems). Mass transitions were selected as follows: VH434 494.8/494.8 kDa,

251

VH445 495.4/104.1 kDa, VH4106 518.2/70.1 kDa, VH4127 518.4/463.9 kDa, VH4128

252

525.4/70.2 kDa, VH4129 509.2/84.2 kDa, VH4130 516.4/84.1 kDa, VH4131 523.3/84.2 kDa.

253

The lower limit of quantification (LLOQ) was set for all peptides at 25-50 nM. As the

254

degradation kinetics of all compounds tested were best described by an exponential regression

. Briefly, an analytical method was developed for each peptide for their detection and

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curve (i.e. first-order reaction kinetics), in vitro half-lives (t½) were estimated using GraphPad

256

Prism software v5.03 on the basis of the following equation: C(t) = (C0-Plateau).e-kt + Plateau

257

(one-phase decay, assuming a Plateau constant value of 0), with t½ = ln2/k.

258 259

Binding and uptake of VH445 analogues on CHO-hLDLR-EGFP cells

260

For cell surface binding experiments, sub-confluent cells were washed three times on the day

261

of the experiment with cold (4°C) D-PBS. Cells were then incubated 30 min at 4°C with 250

262

µL of each peptide diluted in cell culture medium at a final concentration of 0.1 µM

263

containing 1% (w:v) low endotoxin BSA (incubation medium). At the end of the incubation

264

period, cells were washed three times with cold D-PBS, fixed with PBS/paraformaldehyde

265

4% (PFA) for 10 min at room temperature and processed for immunocytochemistry.

266

For uptake experiments, cells were incubated with 0.1 µM of each peptide in the incubation

267

medium for 1 hr at 37°C. At the end of the incubation period, cells were washed three times

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with D-PBS, fixed with PBS/ PFA for 10 min at room temperature and processed for

269

immunocytochemistry.

270

For immunocytochemistry, coverslips were washed three times in PBS and incubated for 5

271

min at room temperature in saturation/permeabilization buffer containing 3% (w:v) BSA and

272

0.1% Triton X-100 in PBS. After three washes with PBS, cells were incubated for 30 min at

273

room temperature in saturation buffer containing 3% (w:v) BSA in PBS. An anti-S-Tag

274

primary antibody diluted 1/1000e in saturation buffer was then added for 1 hr at room

275

temperature. After three washes with PBS, a specific secondary antibody (donkey anti-goat-

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A647 diluted 1/800e in saturation buffer) was added for 1 hr at room temperature together

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with Hoechst (1/1000e) for nuclei staining. After three washes with PBS, coverslips were

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mounted in Prolong Gold anti-fade and were observed using a confocal microscope LSM700

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(Zeiss) with Zen 2012 software. Images were obtained using a 63x Plan Apochom oil

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immersion objective. Images were analyzed using ImageJ software. Quantification of cell

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surface binding of S-Tagged peptides was performed by measuring the mean fluorescence of

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20-30 cells, individualized using the freehand selection tool.

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Evaluation of the Michaelis-Menten Km uptake parameters of VH445 analogues on CHO-

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hLDLR-dsRed2 cells

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The constant Km represents the concentration at the half-maximal velocity of the

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uptake/endocytosis for hLDLR experiments and was measured with VH445-S-Tag and

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VH4127-S-Tag peptides by incubating the S-Tag peptide at the selected concentration on 10 ACS Paragon Plus Environment

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confluent CHO-hLDLR-dsRed2 cells for 1 hr at 37°C (8.105 cells per well in 6-well plates).

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Cells were then extensively washed with PBS 1X before being scraped, centrifuged and lysed

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in 80µL PBS 1X 0.1% Triton X100. A FRET assay was then used as described above to

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quantify the S-tagged peptides bound to the cells. Each condition was performed on six

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independent wells. The signal intensity obtained with blank cells was subtracted from each

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individual value. The non-specific binding was obtained from the linear regression of

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saturating concentration values. Individual specific signal intensities were then obtained by

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subtracting the non-specific signal from the total signal. A nonlinear regression was used to

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estimate the Km value using GraphPad Prism software v5.03 with a Hill coefficient of 1.

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Although Km for a transport system is an apparent parameter resulting from diverse processes

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(e.g. binding, translocation…), its value may represent a rough estimation of the binding

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affinity of the measured substrate.

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Immunoblotting

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In order to assess membrane expression level of LDLR in mouse tissues, wild-type and ldlr -/-

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C57BL/6 mice (The Jackson Laboratory, Stock No. 2207) were deeply anesthetized with a

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cocktail of ketamine (100 mg/kg)/xylazine (10 mg/kg) and transcardially perfused with 0.9%

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NaCl before organs were removed. Membrane preparations were then extracted using the

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ProteoExtract Subcellular Proteome Extraction Kit (Merck, Darmstadt, Germany), according

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to manufacturer’s instructions. Total protein content was then quantified using the BioRad DC

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Protein Assay (Bio-Rad, Hercules, CA, USA). 20 µg of tissue membrane proteins were next

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separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on a 4-

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12% polyacrylamide gel (Bolt, Life Technologies) and transferred onto nitrocellulose

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membranes using the iBlot 2 device (Life Technologies). LDLR was detected with a goat

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anti-murine LDLR (R&D systems, Minneapolis, MN, USA) diluted at 1/800 in blocking

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buffer (TBS-Tween 0.1% non-fat milk 5%). Membranes were then incubated with a

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peroxidase-conjugated donkey anti-goat secondary antibody (Jackson Immunoresearch,

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Baltimore, MD, USA). Finally, proteins were detected using a chemiluminescence kit (Roche

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Diagnostics) and revealed with the Genesis program of G:Box chemi system (Syngene,

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Cambridge, UK).

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Immunohistochemistry

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C57BL/6 mice wild-type (WT) and ldlr -/- (The Jackson Laboratory, Stock No. 2207) were

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deeply anesthetized as above and perfused with 50 mL of 0.9% NaCl followed by 50 mL of 11 ACS Paragon Plus Environment

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PBS 1X- 4% paraformaldehyde (PFA). Organs were then removed and snap frozen in cold

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isopentane. Sections (14 µm thick) were obtained using a Cryostat (Leica CM-3050-S) and

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stored at -80°C. Organ sections were first permeabilized and binding blocked for 2 hrs at

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room temperature using a solution of PBS 1X, 0.1% Triton X-100 and 3% Bovine Serum

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Albumin (BSA). Sections were then incubated overnight at 4°C with an anti-murine LDLR

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primary antibody (R&D Systems) 1/200, followed by Alexa 488-donkey anti-goat (1/800)

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(Life Technologies) for 2 hrs at room temperature. Nuclei were stained with Hoechst (0.5

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µg/mL, Life Technologies). Sections were mounted using Prolong Gold Anti-fade (Life

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Technologies) reagent on Superfrost glass slides. Images were taken and processed using a

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confocal microscope (LSM 700) and Zen software (Zeiss, Jena, Germany).

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Radiolabelling of VH4127

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Radioactive peptides with high specific radioactivity (RAS, up to 100 Ci/mmol) were

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prepared by acylation of the N-ter amine function by tritiated N-propionyl-succinimide

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(NPS). The tritiation reaction of the VH4127 peptide (see Supplemental Table 2) in the N-ter

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position by propionylation was carried out in DMF (1 mg peptide in 100 µl to 450 µl

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according to solubility) by adding 0.1 equivalent of tritiated NPS for 5 min at room

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temperature, then 0.9 equivalent of cold NPS (non-tritiated) for 1 hr, followed by a new

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equivalent of cold NPS for 5 hr. The reaction medium was then left at 4 °C overnight and

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purified the following day using HPLC. The specific activity (SA) for Pr(3H)-VH4127-NH2

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(3H-VH4127) was calculated to 32 Ci/mmol.

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In vivo tissue distribution of VH4127 in wild-type or ldlr -/- mice

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3

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anesthetized C57BL/6 WT (Elevage Janvier, Le Genest-Saint-Isle, France) and ldlr -/- male

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mice (The Jackson Laboratory, Bar Harbor, ME, USA; Stock No. 2207). For anaesthesia, a

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combination of ketamine (140 mg/kg) and xylazine (8 mg/kg) was injected intraperitoneally

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(10 µL/g of b.w). After 10 min, mice were sacrificed by cardiac puncture and blood samples

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collected in glass tubes containing heparin lithium anticoagulant. The plasma was

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immediately recovered after centrifugation. Mice were transcardially rinsed with 20 mL 0.9%

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NaCl . The liver, adrenals and kidneys were collected, weighed and solubilised in scintillation

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glass vials with Solvable® (Perkin Elmer). Intestinal content was washed with 0.9% NaCl and

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proximal intestine weighed before tissue solubilisation with Solvable®. Tritium

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disintegrations per minute (dpm) were counted in solubilised tissues with a Tri-Carb 2810TR

H-VH4127 was injected intravenously at a dose of 10 µCi/mice (200 µL) into the tail vein of

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counter (Perkin Elmer) after addition of the scintillation cocktail Ultima-Gold XR® (Perkin

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Elmer). These mice experiments complied with the ethical rules of the European directive

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(210/63/EU) for experimentation with laboratory animals and they were approved by the

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ethics review committee of Paris Descartes University (approval n°12-184/12-2012).

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Statistical analysis

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Statistical analyses were performed using Excel® software. Error bars represent standard

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deviations (SD) for in vitro experiments and standard error of the mean (s.e.m.) for in vivo

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tissue distribution studies. p values were generated by Student’s t test and denoted as follows:

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* p