Conjugation with eight-arm PEG markedly ... - ACS Publications

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Conjugation with eight-arm PEG markedly improves the in vitro activity and prolongs the blood circulation of staphylokinase Fangbing Qi, Chunyang Hu, Weili Yu, and Tao Hu Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00770 • Publication Date (Web): 03 Jan 2018 Downloaded from http://pubs.acs.org on January 3, 2018

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Bioconjugate Chemistry 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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Bioconjugate Chemistry

Conjugation with eight-arm PEG markedly improves the in vitro activity and prolongs the blood circulation of staphylokinase

Fangbing Qi 1, 2, Chunyang Hu1, Weili Yu 1, *, Tao Hu 1, *

1

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering,

Chinese Academy of Sciences, Beijing 100190, China 2

University of Chinese Academy of Sciences, Beijing 100190, China

Running title: Polymerized staphylokinase

* To whom the correspondence should be addressed.

Tao Hu, State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences No. 1 Bei-Er-Jie Street, Haidian District, Beijing 100190, China.

E-mail: [email protected]. Tel: +86-10-62555217. Fax: +86-10-62551813.

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ACS Paragon Plus Environment


Table of contents

Pharmacokinetics 16

SAK2-PEG

SAK (µ g/ml)

SAK1-PEG

SAK SAK1-PEG SAK2-PEG SAKp-PEG

12 8 4 0 0

SAKp-PEG

20

40

60

Time after injection (h)

Thermal stability

Bioactivity 75

120

60

90 45

o

Tm ( C)

Relative bioactivity (%)

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

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60

30 15

30

0

0

K SA

K SA

G G G -PE -PE -PE K1 K2 Kp SA SA SA

G G G -PE -PE -PE K1 K2 Kp SA SA SA

2


80

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ABSTRACT Staphylokinase (SAK) is a profibrinolytic protein and can be used for therapy of acute myocardial infarction and coronary thrombosis. However, SAK suffers from a short serum half-life time (~6 min) that limits its clinical application. PEGylation prolongs the half-life time of SAK, whereas significantly decreases the bioactivity of SAK for the steric shielding effect of PEG. To improve the bioactivity and prolong the half-life time of SAK, 8-arm PEG maleimide (8-arm PEG) was used for conjugation of multiple SAK molecules in one entity. C terminus of SAK was engineered with cysteine residue, followed by reaction with the maleimide moieties of 8-arm PEG to obtain the conjugate (SAKp-PEG). Conjugation with 8-arm PEG retained the secondary structure of SAK, slightly perturbed the tertiary structure of SAK and essentially maintained its in vitro bioactivity by the multivalence of SAK. Conjugation with 8-arm PEG increased the hydrodynamic volume and thus significantly prolonged the half-life time of SAK. SAKp-PEG elicited a 1.4-fold increase in the SAK-specific IgG titers as compared with SAK, and rendered no apparent toxicity to the cardiac, liver and renal functions of mice. Thus, multiple conjugation of a protein with 8-arm PEG was an effective strategy to develop a long-acting protein drug with improved bioactivity and prolonged blood circulation.

Key words: Staphylokinase, long acting, polyethylene glycol, conjugation, pharmacokinetics

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Bioconjugate Chemistry 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

INTRODUCTION Staphylokinase (SAK) is a profibrinolytic protein produced by Staphylococcus aureus.1 SAK activates transformation of plasminogen into plasmin to catalyze the fibrin hydrolysis on the thrombus surface.2 The efforts to treat acute myocardial infarction have focused on derivatives of tissue-type plasminogen activator (t-PA) that can be administered by bolus injection. SAK is equipotent to t-PA for coronary artery recanalization and is significantly fibrin-selective.3 Thus, SAK can be potentially used for the therapy of acute myocardial infarction or coronary thrombosis.4 However, the short serum half-life time of SAK significantly limits its clinical application. For example, frequent subcutaneous injection of SAK increases the distress and the burden of patients. Thus, the strategies to prolong the serum half-life time of SAK are highly desired for the clinical application of SAK. PEGylation, covalent conjugation of polyethylene glycol (PEG) with proteins, has been considered one of successful approaches to prolong the serum half-life time of proteins.5-6 PEGylation also increases the stability and decreases the immunogenicity, the proteolysis and the renal excretion of proteins.7-8 However, PEGylation suffers from the opposing effect of increasing the serum half-life time and decreasing the bioactivity. For example, conjugation with high Mw (e.g., 40 kDa) PEG increases the serum half-life time of proteins (e.g., interferon-α2b, IFN-α2b). However, it significantly decreases the bioactivity of proteins by interfering with the interaction of the proteins and receptors. This is primarily due to the steric shielding effect of PEG on the receptor binding sites of proteins.9 Moreover, high Mw PEG may be accumulated in the liver that results in the macromolecular syndrome. Some strategies have been developed to circumvent the above-mentioned opposing effect on the PEGylated proteins. Site-specific PEGylation at the site far

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from the bioactive domain of a protein is a feasible approach to decrease the steric shielding effect of PEG on this domain, thereby improving the bioactivity of PEGylated proteins.10 Dimerization or polymerization is another strategy to increase the in vivo serum half-life by doubling the Mw of proteins. Dimerization can increase the binding affinity of proteins to their receptors by the bivalence of proteins, and thus improve the bioactivity of proteins.

11

For example, Liu et al. prepared the SAK

dimers (dSAK) by site-specific dimerization of SAK at its C-terminus, followed by N-terminal PEGylation of dSAK.12 PEG-dSAK showed higher bioactivity and binding affinity to plasminogen than N-terminally PEGylated SAK (PEG-SAK). A dock-and-lock method was adapted to form PEGylated IFN-α2b dimers, which showed higher antiviral activity and longer half-life time than the PEGylated IFN-α2b in mice.13 In contrast, polymerization is expected to be more effective to increase the bioactivity by the multivalence of proteins and prolong the half-life time by multiplying the Mw of proteins. However, the short linkers among the multiple proteins may render a strong steric shielding effect of one protein on the other ones. A long linker (e.g., PEG linker) is desired to elongate the distance of the proteins and minimize the steric shielding effect of proteins. A recombinant SAK was used in the present study, which lacked the first 10 amino acids of native SAK and was connected with Gly-Gly-Cys at its C-terminus. A conjugate was prepared by conjugation of 8-arm PEG maleimide with multiple SAK molecules at the C-terminal Cys residue of SAK, where the C-terminus of SAK was far from its bioactive domain. SAK1-PEG and SAK2-PEG were prepared by conjugation of one PEG with one and two SAK molecules, respectively. Structural property, thermal stability, bioactivity and the pharmacokinetic parameters of SAKp-PEG were investigated in details. The immunogenicity and the toxicity of

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SAKp-PEG were measured to evaluate its potential side effect. SAK1-PEG and SAK2-PEG served as the controls for SAKp-PEG.

RESULTS Purification of the SAK samples. As indicated by MALDI-TOF analysis, SAK had an Mw of 14639.5 Da (the inset, Figure 1a). SAK1-PEG, SAK2-PEG and SAKp-PEG were loaded on a Superdex 200 column (1.6 cm×60 cm), respectively. As shown in Figure 1a, a single elution peak corresponding to SAK1-PEG was observed at 89.7 ml (Peak 1). Peak 1 was fractionated as the arrow indicated. Two elution peaks were observed when the reaction mixture containing SAK2-PEG was loaded (Figure 1b). The elution peak at 82.1 ml (Peak 2) was fractionated and further purified by a Mini S column (0.46 cm×5 cm). As shown in Figure 1c, three major peaks were observed and the peak corresponding to SAK2-PEG (Peak 3) was fractionated. As shown in Figure 1d, two major peaks were observed when the reaction mixture containing SAKp-PEG was loaded. The peak at 67.0 ml (Peak 4) corresponded to SAKp-PEG and was fractionated as the arrow indicated. Peak 5 corresponded to the mixture of SAK1-PEG and SAK2-PEG, which was then discarded.

a 8000

Intensity

30

20

14639.5

6000 4000 2000 0

5000 10000 15000 20000 25000

Mass (m/z)

10

b

30

1

Absorbance at 280 nm

40


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

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SAK

20

2

10

0

0 0

30

60

90

120

0

150

30

60

90

Volume (ml)

Volume (ml)

6


120

150

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40

c

100

3

40

80

30

60

20

40

10

20

0

0 0

10

20

30

40



50

Buffer B (%)

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


d

5

20

10

0 0

50

4

30

30

60

90

120

150

Voulme (ml)

Volume (ml)

Figure 1. Purification of the SAK samples. SAK1-PEG (a), SAK2-PEG (b) and SAKp-PEG (d) were purified by a Superdex 200 column (1.6 cm×60 cm). The column was equilibrated by 20 mM sodium phosphate buffer (pH 7.4) at a flow rate of 2.0 ml/min. The fractions corresponding to SAK2-PEG were purified by a Mini S column (0.46 cm×5 cm) (c). The column was equilibrated by 50 mM NaAc-HAc buffer (pH 5.0, Buffer A) and eluted by a linear salt gradient of 0-1 M NaCl in Buffer A at a flow rate of 0.7 ml/min.

SEC analysis. The SAK samples and the PEG reagents were analyzed by an analytical Superdex 200 column (1 cm×30 cm). SAK was eluted as a single and symmetric peak at 17.7 ml (Figure 2a). In contrast, SAK1-PEG was eluted as a single peak at 13.3 ml that was left-shifted as compared with that of SAK. This suggested that conjugation with PEG increase the hydrodynamic volume of SAK. The elution peak of SAK2-PEG (12.7 ml) was left-shifted as compared with that of SAK1-PEG. The elution peak of SAKp-PEG was at 10.6 ml, indicating the largest hydrodynamic volume of SAKp-PEG among the four samples. The apparent Mw of SAKp-PEG was calculated to be ~94 kDa, indicating that ~5.6 CT molecules (~15 kDa) were conjugated with one 8-arm PEG (10 kDa). Two-arm PEG and mPEG were both eluted as a single peak at 15.8 ml, which 7



was left-shifted as compared with that of SAK (17.7 ml). Although the Mw of PEG (10 kDa) was lower than that of SAK (~15 kDa), PEG could bind water to form a hydrated layer and thus showed a larger hydrodynamic volume than SAK. It was known that star-shaped polymers exhibited a smaller hydrodynamic radius compared with linear polymers of the same molecular weight.14 Thus, 8-arm PEG was eluted as a single peak at 16.6 ml, which was right-shifted as compared with mPEG (15.8 ml). The peaks corresponding to the free PEGs were not observed for the three SAK samples, indicating no contamination of free PEGs with the three SAK samples.

SDS-PAGE analysis. As shown in Figure 2b, all the SAK samples showed a single electrophoresis band, indicating their high purity. SAK1-PEG contained one PEG (10 kDa) and one SAK (~15 kDa), corresponding to an apparent Mw of ~32 kDa. The aberrant band migration was hindered by the conjugated PEG. Similarly, SAK2-PEG contained one PEG (10 kDa) and two SAKs (~30 kDa), corresponding to an apparent Mw of ~50 kDa. SAKp-PEG show a wide band migration, corresponding to the apparent Mw value of 110-140 kDa. The high apparent Mw of SAKp-PEG was partially due to the aberrant band migration of PEG. SAKp-PEG and 8-arm PEG were calculated to contain ~1.5 and ~7.5 maleimide groups, respectively. Thus, one 8-arm PEG was conjugated with ~6.0 SAK molecules, which was close to the result of SEC analysis (~5.6 SAK molecules per PEG). The iodine stain of SAK1-PEG, SAK2-PEG and SAKp-PEG (Lanes 3-5) indicated the absence of free PEG and the presence of PEG in the three conjugates.

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a

b kDa

SAKp-PEG


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


SAK2-PEG

Coomassie blue stain 1

2

3

4

Iodine stain 5

3

4

5

94.0 66.2

SAK1-PEG

43.0 SAK

31.0

8-arm PEG 2-arm PEG

20.1

mPEG

14.4

0

5

10

15

20

25

Volume (ml)

Figure 2. Characterization of the SAK samples. SEC analysis (a) was performed on an analytical Superdex 200 column (1 cm×30 cm). The column was eluted with 20 mM sodium phosphate buffer (pH 7.4) at a flow rate of 0.5 ml/min. SDS-PAGE analysis (b) was conducted using a 15% polyacrylamide gel. The gel was stained by Coomassie blue for protein detection and by iodine for PEG detection. Lanes 1-5 were standard marker, SAK, SAK1-PEG, SAK2-PEG and SAKp-PEG, respectively.

DLS analysis. The molecular radii of the SAK samples were measured by dynamic light scattering (DLS). As shown in Figure 3a, the molecular radius of SAK1-PEG (4.35 nm) was higher than that of SAK (2.64 nm). This reflected that conjugation with PEG could enhance the molecular radius of SAK. In contrast, the molecular radii of SAK2-PEG and SAKp-PEG were 5.85 nm and 9.86 nm, respectively. Thus, SAKp-PEG showed the largest radius among the four SAK samples, due to the synergistic effect of SAK conjugation and PEG on the molecular radius of SAK. CD analysis. The second and tertiary structures of the SAK samples were investigated by circular dichroism (CD) spectroscopy. As shown in Figure 3b, the far-UV CD spectrum of SAK (190-260 nm) exhibited a characteristic strong band at 208 nm, which reflected a rich β-sheet content of SAK. The far-UV CD spectra of

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SAK1-PEG, SAK2-PEG and SAKp-PEG were almost superimposed on that of SAK. As shown in Table 1, the α-helix, β-sheet and β-turn contents of SAK1-PEG, SAK2-PEG and SAKp-PEG were comparable to those of SAK. Thus, the secondary structure of SAK was essentially not altered upon conjugation with 8-arm PEG. The near-UV CD spectrum of SAK arises from the contributions from the environments of Phe, Trp and Tyr residues.15 Thus, the near-UV CD spectra (250-320 nm) was used to measure the tertiary structure of SAK. As shown in Figure 3c, the spectrum of SAK showed a clear fine structure with two positive peaks at 290 nm and 305 nm attributed to Trp signals. A shoulder around 250-265 nm was attributed to Phe signals. The CD spectrum of SAK1-PEG was at a slightly upper position as compared to that of SAK in this region. Moreover, the CD spectrum of SAKp-PEG was at a slightly upper position as compared to those of SAK1-PEG and SAK2-PEG. Thus, conjugation with 8-arm PEG slightly perturbed the tertiary structure of SAK. DSC analysis. Differential scanning calorimetry (DSC) is a reliable method to directly measure the heat associated conformational changes, such as unfolding. The temperature of protein unfolding (Tm) is commonly used as an indicator of protein stability. 16 As shown in Table 1, the Tm of SAK1-PEG was higher than that of SAK. This suggested that the thermal stability of SAK was increased by the thermally stable PEG, due to its strong hydrogen bonding and hydrophobic interaction with SAK. In contrast, SAK2-PEG showed a Tm value lower than SAK1-PEG and higher than SAKp-PEG. Because one PEG was conjugated with two SAKs for SAK2-PEG and multiple SAKs for SAKp-PEG, the protection effect of PEG on each SAK molecule of SAKp-PEG was the lowest among the three SAK samples. Although the thermal stability of SAKp-PEG was decreased by this factor, it was still higher than that of SAK.

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Table 1 The secondary structure and the thermodynamic property of the SAK samples a Sample

a

α-Helix

β-Sheet

β-Turn

Random

Tm b

(%)

(%)

(%)

(%)

(oC)

SAK

15.3

38.5

8.6

37.5

58.4

SAK1-PEG

15.1

39.1

8.4

37.4

69.9

SAK2-PEG

15.2

39.0

8.3

37.5

67.6

SAKp-PEG

14.8

39.0

8.8

37.1

64.2

The secondary structures of the SAK samples were obtained by the structure fitting

software that has been built-in to the JASCO J-810 spectrometer. b

The temperature at the maximum point of the heat capacity curve.

Intrinsic fluorescence analysis. Intrinsic fluorescence of a protein is largely due to its Trp and Tyr residues. As shown in Figure 3d, the emission fluorescence intensity of SAK1-PEG is slightly higher than that of SAK, along with unnoticeable shift in the maximum wavelength of 362 nm. As compared with SAK1-PEG, SAK2-PEG and SAKp-PEG both showed slightly higher fluorescence intensity with the maximum wavelength of 362 nm. Thus, conjugation with 8-arm PEG slightly perturbed the Trp and Tyr environment of SAK. 12

9

a

6

b


9

Ellipticity

Molecular radius (nm)

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


6

3

3 0 -3 -6 -9

0

SAK

SAK1-PEG SAK2-PEG SAKp-PEG

190

200

210

220

230

240

Wavelength (nm)

11


250

260


400

1.0

d

c

0.0 -0.5 SAK SAK1-PEG SAK2-PEG SAKp-PEG

-1.0 -1.5 -2.0 250

260

270

280

290

300

310

Fluorescence intensity

0.5

Ellipticity

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

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320

300

200


100 300

320

Wavelength (nm)

340

360

380

400

Wavelength (nm)

Figure 3. Structural analysis of the SAK samples. The molecular radius (a) was measured by dynamic light scattering. The CD spectra were recorded at 25 oC with a 0.2 cm light path cuvette in the far-UV region (b) and the near-UV region (c). The intrinsic fluorescence emission spectra (d) were recorded from 300 nm to 400 nm at 25 oC using an excitation wavelength of 280 nm.

In vitro bioactivity. In vitro bioactivities of the SAK samples were estimated by the fibrinolytic assay. The relative bioactivity of SAK was set as 100%. As shown in Figure 4a, the relative activity of SAK1-PEG was 51.9%. Presumably, the large hydration shell of PEG around SAK could sterically shield the bioactive domain of SAK and retard the interaction between SAK and fibrinogen, thereby decreasing the bioactivity of SAK. In contrast, the relative bioactivities of SAK2-PEG and SAKp-PEG were 71.2% and 90.7%, respectively. This indicated that conjugation with 8-arm PEG could essentially maintain the bioactivity of SAK. Immunogenicity. The SAK-specific IgG titers elicited by the SAK samples were measured by ELISA assay. As shown in Figure 4b, SAK could elicit a certain SAK-specific IgG titer in the mice. The SAK1-PEG group showed lower IgG titers than the SAK group (P

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