小田切　優樹

熊本大学大学院医学薬学研究部薬物動態制御学分野

薬物と血清蛋白質との相互作用に関する分子機能的研究

薬物と血清蛋白質との相互作用に関する分子機能的研究

第19回日本薬物動態学会年会（金沢）2004.11.18

Protein Binding and Distribution of Drug

Tissue bound unbound

Unbound

BoundTarget siteDrug-Receptor

complex

Metablite

Absorption Excretion

Metabolism

Binding parameter, Binding Site

SpectrometrySpectrometrySpectrometry GeneticsGeneticsGenetics

OrganicChemistryOrganicOrganic

ChemistryChemistry

Short cut via gene

High-resolutionat atomic level

部位特異的変異法部位特異的変異法X-線結晶構造解析X-線結晶構造解析

Analysis throughCovalent bonds

光アフィニティラベル法光アフィニティラベル法

Approaches for structural biologyApproaches for structural biology

NH

O

CH 3

CH 2COOH

O

7-Anilino-4-Methylcoumarin-3-Acetic Acid (ACMA)

SO2NHCHCOOH

N(CH 3)2

C4H 9

Dansyl-DL-Norleuicine (DNSL)

NHCH(CH 2)3N(C2H 5)2

CH 3O

CH 3

Pamaquine (PQ)

NHCH(CH 2)3NH 2

CH 3O

CH 3

Primaquine (PR)

OOH O

COOH

Fluorescein (Flu)

N+

NH 2 C2H 5

NH 2

Br

Ethidium Bromide (EB)

N+

(CH 3)2N N(CH 3)2

(CH 2)11CH 3

Br

Acridine Orange-10-DodesylBromide (AODB)

S

N+

(CH 3)2N N(CH 3)2

Methylene Blue (MB)

N CH 3

Quinaldine (QD)

N COOH

Quinaldic Acid (QA)

NH

2(CH 3)N N(CH 3)2

Auramin (AO)

O O

OHCH

CH 2COCH 3

Warfarin (WF)

N+

C2H 5

CH

CH

N(CH 3)2I

2-(4-Dimethylaminostyryl)-n-ethylpyridinium Iodide (DASP)

N+

CH 3 CH

CH

N(CH 3)2I

4-(4-Diethylaminostyryl)-n

-ethylpyridinium Iodide (4-DASP)

Structure of Fluorescent Probes

Drug binding sites on HSAG.Sudlow. et al: The characterization of two specific drug binding sites on human serum albumin. Mol. Pharmacol. 11, 824-832. (1975)

Site III

Site II

Site I

O O

OH

CHCH 2COCH3

N(CH3)2

SO2NCH2COOH

CH3

O O

CH2COOH

HN

Z value（疎水性）

Depth

6782 74

14Å 16Å 19Å

Location of the ligand binding sites on HSALocation of the ligand binding sites on HSA

NC

Site I

Metal binding sitesCu++, Ni++

SH group containing ligandNOHg++

Cys34

Curry, S. et al. Nat. Struct. Biol. 5, 827-835

Site IIKetoprofen DiazepamMedium chain fatty acidL-thyroxineL-tryprophan

WarfarinPhenytoinFrosemideBilirubinCMPF

IA

IB

IIA

IIB

IIIA

IIIB

Arg410Arg410

Tyr411Tyr411

Type of mutationsType of mutations

Arg410Arg410 Tyr411Tyr411

N

NH2NH2

CH3 CH3

OH

OH

Wild-type R410A Wild-type Y411F Y411S Y411A

0

20

40

60

80

100un

boun

d K

P fr

actio

n (%

)

0

20

40

60

80

100

unbo

und

DZ

frac

tion

(%)

Ketoprofen (KP) と Diazepam (DZ) の各変異体への結合Ketoprofen (KP) と Diazepam (DZ) の各変異体への結合

The sample solutions contained 5µM KP or DZ and 10µM wild type or mutant HSAin 67mM sodium phosphate buffer (pH7.4).

KP DZwild

-type

R410A

Y411A

R410A

/Y41

1AY41

1SY41

1F

wild-ty

peR41

0AY41

1AR41

0A/Y

411A

Y411S

Y411F

HSA変異体を利用した薬物結合部位予測HSA変異体を利用した薬物結合部位予測

e.g.) Site II Drug A(binding site unknown)

Binding experiment

Wild-type R410A/Y411A

Decreased binding to mutant

Yes No

Site II Other site

腎疾患

ネフローゼ症候群

肝疾患

糖尿病

心不全

心筋梗塞

外傷

炎症性疾患 1)

妊娠

尿毒症物質 遊離脂肪酸（透析時）

ビリルビン 胆汁酸

遊離脂肪酸

グリコシル化H SA カルバミル化H SA

グリコシル化H SA

薬物結合

酸性薬物

塩基性薬物

蛋白濃度

H SA A G P

内因性蛋白結合阻害物質の蓄積

遊離脂肪酸 その他？

1) 関節リウマチ、潰瘍性大腸炎、クローン病

各種病態下での血清蛋白結合の変動とその要因各種病態下での血清蛋白結合の変動とその要因

indoleacetic acid(IA)

CH2COOH

NHH

OSO3H

N

indoxyl sulfate

(IS)

(CMPF)3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid

CH3

OCH3(CH

2)2

COOH

(CH2) COOH2

CONHCH2COOH

hippuric acid(HA)

HSA bound uremic toxinsHSA bound uremic toxins

Ka = 9.1 x 105 (Site II) Ka = 2.1 x 105 (Site II)血中濃度；104.3 ± 49.4 (4.1 µM) 血中濃度；26.3 ± 11.7 (1.45 µM)

Ka = 0.1 x 105 (Site II)血中濃度；270.9 ± 141.2 (13.3 µM)

Ka = 130.5 x 105 (Site I)血中濃度；183.0 ± 38.0 (15.1 µM)

Drug-uremic toxin-fatty acid interaction

FR-uremic toxins FR-CMPF-oleate CMPF-oleate

0.00

0.01

0.02

0.03

0.04

0.05C

ontro

l

+ C

MP

F

+ IA

+ IS

+ H

A

Furo

sem

ide

free

fract

ion

a)

c, d) c, d)b, d)

a: P<0.001 vs Controlb: P<0.01 vs Controlc: P<0.05 vs Controld: P<0.01 vs + CMPF

0.00

0.02

0.04

0.06

0.08

Furo

sem

ide

free

fract

ion

+ C

MP

F

Con

trol

a)

[oleate]/[HSA] = 6

a: P<0.01 vs Control

0.00

0.02

0.04

0.06

0.08

2 4 6

[Oleate] / [HSA]

a)

a, b)

CM

PF

free

fract

ion

a: P<0.001 vs 2b: P<0.001 vs 4

Possible cascade displacement model in fatty acid-uremictoxin-drug system

FFAFFA

FFA

FFA

FFAhemodialysis

hemodialysis

Site ISite II

; site I bound drug

; site II bound drug

; CMPF

; site II bound uremic toxin

尿毒症物質の腎取り込みに関わるトランスポーター

Structure of nephron

近位尿細管

血管側 尿管側

OAT1

OAT3

Na+ Na+

dicarboxylate

MRP2ATPADP+Pi

上皮細胞

馬尿酸インドール酢酸

インドキシル硫酸CMPF

?

NPT1

MRP4ATPADP+Pi

側底膜 刷子縁膜

dicarboxylate

BCRPATPADP+Pi

OAT4

SR-A

AGE-albuminの体内動態特性

SR

PC

LEC

KC

SR

AGE-albumin

SR; スカベンジャー受容体PC; 肝実質細胞LEC; 肝内皮細胞KC; クッパー細胞

メサンギウム細胞

腎臓による取り込みにはメサンギウム細胞

のSR-Aを介したエンドサイトーシスが関与する．

肝臓による取り込み

には肝内皮細胞のス

カベンジャー受容体

を介したエンドサイ

トーシスが関与する．

AGE-albumin

AGE-albumin

Structural Characteristics of AGP

Structural Structural Characteristics of of AGPAGP

N5

147

15 38

54164

72

7585

183

: 糖鎖 : S-S 結合

分子量 : 40-45kDa

糖鎖含量 : 45%

β-sheet 構造

詳細な立体構造は不明

等電点：3.0（シアル酸含量:16%）

Binding of warfarin (A) and propranolol (B) to AGP

in the presence of the other ligand

0

0.1

0.2

0.3

0.4

0 0.1 0.2 0.3 0.4

0

0.1

0.2

0.3

0.4

0 0.01 0.02

(A)

(B)

[warfarin]f (μM)

r pro

pran

olol

r war

farin

[propranolol]f (μM)

independent binding, competitive bindinganti-cooperative interaction,

P

BP BPA

PAKA

KB

KBA

KABB B

A

A

Basic ligandAcidic ligand

Steroidhormone

HN ORR

NNO

HH3C

H3CONHCH3

Ligands

UCN-01

UCN-02

Staurosporine

Ka (x 106 M-1)

288 ± 75

1.48 ± 0.11

11.3 ± 5.74

Katsuki M et al (2004) Pharm Res., 21:1648-1655

R

OH (β)

OH (α)

H

Fuse E et al (1999) Cancer Res., 59:1054-1060

t1/2 (hr)

CLtot (mL/h/kg)

Vdss (mL/kg)

855~1660

79.6~158

0.0407~0.102

Cancer patientsParameters

Native His Lys Trp Tyr0

50

100

Bou

nd (%

)

* *

*

*

*Statistically significant compared with native AGP; p<0.01.

Binding of UCNBinding of UCN--01 to AGP01 to AGP

Experimental process of Experimental process of photoaffinityphotoaffinitylabeling techniqueslabeling techniques

[3H]UCN-01

hv Tryptic digestion

AGP

Purification of peptidicfragments by rHPLC Amino acid analysis by

Edman degradation method

Photoaffinity labeling

Covalent bond

Separation of peptidicfragments by cHPLC

NN--terminal amino acid sequence analysis terminal amino acid sequence analysis by the by the EdmanEdman degradation methoddegradation method

1 2 3 4 5 6 7 8 9Number of cycle

0

0.5

1

1.5

2

2.5

3

Yiel

d of

PTH

-am

ino

acid

(pm

ol)

SD

V

V

Y

T

D

X

K

PITC: phenylisothiocyanatePTH : phenylthiohydantoin

QIPLCANLVP VPITNATLDQ ITGKWFYIAS

AFRNEEYNKS VQEIQATFFY FTPNKTEDTI

FLREYQTRQD QCIYNTTYLN VQRENGTISR

YVGGQEHFAH LLILRDTKTY MLAFDVNDEK

NWGLSVYADK PETTKEQLGE FYEALDCLRI

PKSDVVYTDW KKDKCEPLEK QHEKERKQEE

GES

10 20 30

40 50 60

70 80 90

100 110 120

130 140 150

160 170 180

Amino acid sequence of AGP

UCN-01

PhotolabelingPhotolabeling of wild type, W25A, of wild type, W25A, W122A and W160A with [W122A and W160A with [33H]UCNH]UCN--0101

*

43kDa Autoradiogram

Wild-type W25A W122A W160A0

50

100

150

200

Rad

ioac

tivity

, PSL

(a.u

.)

*Statistically significant compared with wild type; p<0.01.

80%ラベルペプチドの同定

S D V V T WDY K

UCN-01

160

部位特異的変異法部位特異的変異法

rAGP

ラベル部位の同定

W160A

光アフィニティラベル法光アフィニティラベル法

UCN-01AGP

Type I and II docking model of UCN-01 and AGP

Trp160Trp160

Type I

Type IIHydrophobic amino acids are shown in green.

Trp160Trp160

Type I Type II

Kopecky et al Biochem Biophys Res Commun 300, 41-6 (2003).

Amino acid residues around Trp160 that interactswith UCN-01 exhibited in type II docking model

UCN-01

Lys135Trp160

Lys161Pro131

Ile28

Tyr157Glu132 Leu138

Electrostatic interactionHydrogen bonding

0

0.25

0.5

500 550 600 650 700Wavelength (nm)

Fluo

resc

ence

inte

nsity

Fluorescence spectra of QRin the presence or serum protein

AGP (0.5 x 10-6 M)AGP (1.0 x 10-6 M)AGP (2.0 x 10-6 M)

HSA (2.0 x 10-6 M)

Binding of QR (%) to serum protein

AGP(2mg/100ml)

HSA(80mg/100ml)

γ-globulin(17mg/100ml)

23.0 3.4 1.2

N

CH2CH3

CH N(CH3)2+

I-

CH

Quinaldine red (QR)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 50 100 150 200 250

: AGP-QR system

: AGP-QR-androstanedione system

: SRID method

Standard curves for measurement of AGPFl

uore

scen

ce in

tens

ity

: AGP-QR-androstanedione-HSA-γ-globulinsystem

AGP Concn. (mg/100ml)Effect of androstanedione of serum

protein on binding percentage for QR-AGP

systemsAGP-QR system

AGP-QR-androstanedione

system

AGP-QR-androstanedione-

HSA-γ-globulin system

Binding (%) 23.0 5.0 24.0

Relationship of the AGP concentrations determined

by SRID method and by QR method

0

100

200

300

0 100 200 300

Fluorescence probe method AGP concn. (mg/100ml)

SRID

met

hod

AG

P co

ncn.

(mg/

100m

l) y=1.03x-7.66 (r=0.93)

Comparison of QR method and SRID methodQR method SRID method

Measuring timeOperating procedureDetection rangeOperation costCoefficient of variation

Short (1hr)Simple5-500 mg/100mlInexpensive< 3%

Long (50hr)Complex12-199 mg/100mlExpensive< 10%

Proposed model for AGP-mediated drug transport through interaction with biomembrane

Blood Tissue

AGP

Membrane

Drug

β-sheet structure

AGP

α-helix structure

生体膜との相互作用による構造転移（β→α）

構造転移による薬物放出

AGPのα-helix構造→生体膜との相互作用の指標

1

10

100

6 7 8 9 10 11 12 13

- [θ] x 10-3 at 222nm (deg・cm-2・dmol-1)( α-Helix content)

- [θ] x 10-3 at 222nm (deg・cm-2・dmol-1)( α-Helix content)

AG

P bo

und

to m

embr

ane

(%)

AG

P bo

und

to m

embr

ane

(%)

α-Helixα-Helix

α-Helix Formation of AGP through Binding to Membrane

α-Helix Formation of AGP through Binding to Membrane

α-Helix formationα-Helix formation

Native state(β-sheet form)

Binding to MembraneBinding to Membrane

λmax = λmax(PG非存在下)ー λmax(PG存在下)

親水的環境

疎水的環境

Trp (W25, W122, W160) Microenvironment of AGP in the Presence of PG-membrane

Trp (W25, W122, W160) Microenvironment of AGP in the Presence of PG-membrane

WT

Trp

mic

roen

viro

nmen

t (λm

ax)

-10

0

10W122A

Trp122

W122 膜外部

W25A

W25 膜内部

Trp25

W160A

W160 膜表面近傍

Trp160

60

70

80

90

100

6 7 8 9 10 11 12 13

- [θ] at 222nm x 10-3(deg・cm-2・dmol-1)( α-Helix content )

- [θ] at 222nm x 10-3(deg・cm-2・dmol-1)( α-Helix content )

Prog

este

rone

bou

nd to

AG

P (%

)Correlation between Binding

Capacity and α-Helix Contents of AGP

Correlation between Binding Capacity and α-Helix Contents of

AGP生体膜との相互作用によ

るα-Helix構造の形成生体膜との相互作用によ

るα-Helix構造の形成

薬物結合能の低下

(結合型薬物の遊離)

Ligand Binding vs α-Helix Contents (pH7.4 ～ 4.5)

非結合型薬物濃度の上昇　　→組織へ

非結合型薬物濃度の上昇　　→組織へ

Proposed model for interaction of AGP with membraneProposed model for interaction of AGP with membrane

Membrane

Native state(β-sheet form)

Binding to Membrane(Electrostatic Interaction)

構造転移 (β→ α)

Trp25:内部、 Trp122:外部、 Trp160:表面近傍

Ligand Release

Trp122Trp25

Trp160

Mild Acidic ConditionMild Acidic Condition

結合部位のトポロジー解析・分光学的手法

・構造活性相関解析

・光アフィニティラベル法

・部位特異的変異法

・ドッキングシュミレーション

病態時での蛋白結合

・蛋白質の量的変動

・蛋白質の質的変動

（コンフォメーション変化，翻訳後修飾）

・内因性物質の蓄積

（脂肪酸，尿毒症物質）

蛋白介在性組織取り込み

・生体膜との相互作用に伴う構造転移

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