Optimization and in Vivo Validation of Peptide ... - ACS Publications

Subscriber access provided by La Trobe University Library

Article

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.

Page 1 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

1

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

2 3

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

a,b

4

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

5

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

6

Cisternino c, Michel Khrestchatisky e*

,

7 8 9

a

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

10

13344, Marseille Cedex 15, France.

11

b

12

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

13

c

14

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

15

d

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

16

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

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

18

ABSTRACT

19

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

20

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

21

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

22

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

23

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

24

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

25

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

26

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

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

validated LDLR-targeting potential in vivo.

44 45 46 47

KEYWORDS

48

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

49

LDLR-knockout.


Page 2 of 34

Page 3 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

INTRODUCTION

51

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

52

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

53

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

54

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

55

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

56

distribution. Specific targeting systems showing considerable promise include i)

57

immunoconjugates whereby the targeting ligand is either a conventional monoclonal antibody

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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

67

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

68

11

69

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

70

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

71

tissue penetration that endows them with interesting pharmacokinetic profiles when compared

72

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

73

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

74

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

75

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

76

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

77

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

78

clinical settings in the near future20, 21.

79

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

80

the development of innovative active drug-targeting strategies1,

81

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

82

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

83

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


22-29

. These include i) high


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

84

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

85

upregulation in several human cancers.

86

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

87

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

88

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

89

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

90

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

91

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

92

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

93

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

94

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

95

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

96

displaying improved biochemical parameters, including increased apparent LDLR-binding

97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

intermediate compound 22/VH445 analogue.

108

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

109

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

110

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

111

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

112

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

113

crucial when developing peptides10,

114

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

115

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

116

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

117

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

20,

this parameter was also assessed in vitro. When


Page 4 of 34

Page 5 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

118


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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

119

Page 6 of 34

EXPERIMENTAL SECTION

120 121

Cell lines, culture and reagents

122

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

123

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

124

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

125

laboratories,

126

ATATATAAGCTTCGAGGACACAGCAGGTCGTGAT

127

TTAATTGTCGACCACGCCACGTCATCCTCCAGACT. Both constructs were fully

128

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

129

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

130

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

131

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

132

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

133

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

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

Mountain

View,

USA)

using

the

following

primers and

148 149

Peptide synthesis

150

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

151

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

152

N,Ndimethylformamide;

Fmoc,

9-fluorenylmethoxycarbonyl; 6

ACS Paragon Plus Environment

HATU,

1-

Page 7 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


153

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

154

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

155

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

156

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

157

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

158

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

159

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

160

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

161

quality available.

162

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

163

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

164

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

165

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

166

100% B in 4 min.

167

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

168

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

169

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

170

Flow rate was 20 mL/min.

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

3-oxid


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

187

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

188

lyophilized.

189

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

190

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

191

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

193

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

195

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

196

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

197

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

198

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

199

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

204

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

206

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

208

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

209

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

211

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

212

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

213

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

214

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

215

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

217

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

219

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

Page 8 of 34

Page 9 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


221 222

Surface Plasmon Resonance

223

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

232

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

233

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

234

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

236

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

240

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



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

255

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

268

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-

276

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

277

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

278

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

279

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

280

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

281

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

282

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

283 284

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

285

hLDLR-dsRed2 cells

286

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

287

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

288

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

Page 10 of 34

Page 11 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

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

298

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

299

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

300

affinity of the measured substrate.

301 302

Immunoblotting

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

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

311

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

312

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

313

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

314

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

315

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

316

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

317

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

318

Cambridge, UK).

319 320

Immunohistochemistry

321

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

322

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


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

323

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

324

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

325

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

326

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

327

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

328

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

329

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

330

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

331

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

332

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

333 334

Radiolabelling of VH4127

335

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

336

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

337

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

338

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

339

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

340

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

341

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

342

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

343

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

344 345

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

346

3

347

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

348

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

349

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

350

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

351

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

352

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

353

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

354

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

355

proximal intestine weighed before tissue solubilisation with Solvable®. Tritium

356

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


Page 12 of 34

Page 13 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


357

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

358

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

359

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

360

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

361 362

Statistical analysis

363

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

364

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

365

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

366

* p

Optimization and in Vivo Validation of Peptide ... - ACS Publications

Recommend Documents