Efforts to Develop a High-Throughput Screening Tool Using Isoprenylcarboxyl Methyltransferase as a

Membrane Protein-based SensorDavid H. Thompson, Purdue-Chemistry

Current Drug Discovery Limitations forIntegral Membrane Protein (IMP) Targets

Platform technology for integral membrane protein-based sensors that are:• fast, reproducible & sensitive• compatible with high-throughput platforms (i.e., planar geometries, optical detection schemes)• capable of real-time read-out for kinetic & mechanistic studies• rugged• low false positive response rate• etc (patternable, controllable areal density of IMP, microfluidic/microelectronic interfacing….)

Need

Bakheet & Doig, Bioinformatics2009 25, 451

~60% of pharma targets are integral membrane proteins (IMP)

fewer than 200 IMP structures are known to the < 3Å resolutionnecessary for rational drug design approaches (i.e., < 0.5% PDB)

∴ screening methods are still required to discoveractive agents for IMP targets

issue is compounded by the fact that most IMP assays are:• slow• noisy• lack sensitivity

Iyengar et al., Mt. Sinai J Med2007 74, 27

Supported Membrane Sensor Concept

IMP Bolalipid & tether synthesis

Microfabrication

Membrane-Support Gap

BSi|

His6−Ni2+–NTA~10 nm

Si/SiO2

1 - 400 μ

5nm

A

Sensing method Bolalipid & tether design

APPLICATIONS→Platform technology for membrane protein-based sensors

→High throughput screening for drug discovery

→Mechanistic studies of IMP

μfabricated wells on nanoporous Al2O3(400 μm wells with 50 nm pores)

a

b

c

μfabricated wells on Si/SiO2(150 x 150 μm wells with 20μm electrodes)

K-Ras Signaling Pathway & MislocalizationSerial C-terminal PtM of eukaryotic CaaX proteins

GFP-K-Ras Localization in Mouse ES Cells

Bergo, Leung, Ambroziak, Otto, Casey, Young, J. Biol. Chem. 2000, 275, 17605-17610

SAH: S-Adenosyl homocysteine(product of Icmt enzymatic reaction)

SAM: S-Adenosyl methionine(substrate for Icmt)

GGTase+ GGPP

GGTase+ PPi

Geranylgeranyl Geranylgeranyl Geranylgeranyl

Geranylgeranyl

Hrycyna & Gibbs, BOMCL 2006 16, 4420-4423

Isoprenylcysteine Methyltransferase (ICMT): A Target for Discovery of anti-K-Ras Agents

mutations in the K-Ras oncogene are responsible for nearly 15% of all human cancersinhibition of Icmt results in the loss of transforming ability of K-Ras

∴ ICMT is a novel and attractive anti-cancer target

QD H M

EFQ

DE H E Y P

D

M

IRRNPL H E V T

TSY I L

GILL G I

FVGL F P

QI

R F K N

TIYY E L

FHFL S L

AIIF L N

F

AK

NY

PLK VH

S E S FL

LN

KG

N

SYMA

A H SFAI

L E CLVE

S F LFP

DL K I S

Y

QG

F

LI V L

LCGL V T

CLKT A L

YT

S

RT

I A M HT

AGH

I

SR

HP

SY

G L

GS

KT

FS

V

K K

V

T

VY

WS

K

LV

E D HS

H

F W W

LTA

L

SFK W L

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L

F

SL

PN

G I

LL

F

Q

D

N

LY K E

EVRIR

KE

IYEASFF

E

Y

GVG V K

I

IFPI

N-term

L1

L2

L3

L4

L5

C-term

ER Lumen

Cytosol

F

QQDD HH MM

EEFFQQ

DDEE HH EE YY PP

DD

MM

IIRRRRNNPPLL HH EE VV TT

TTSSYY II LL

GGIILLLL GG II

FFVVGGLL FF PP

QQII

RR FF KK NN

TTIIYYYY EE LL

FFHHFFLL SS LL

AAIIIIFF LL NN

FF

AAKK

NNYY

PPLLKK VVHH

SS EE SS FFLL

LLNN

KKGG

NN

SSYYMMAA

AA HH SSFFAAII

LL EE CCLLVVEE

SS FF LLFFPP

DDLL KK II SS

YY

QQGG

FF

LLII VV LL

LLCCGGLL VV TT

CCLLKKTT AA LL

YYTT

SS

RRTT

II AA MM HHTT

AAGGHH

II

SSRR

HHPP

SSYY

GG LL

GGSS

KKTT

FFSS

VV

KK KK

VV

TT

VVYY

WWSS

KK

LLVV

EE DD HHSS

HH

FF WW WW

LLTTAA

LL

SSFFKK WW LL

VVFFIIFF II VV

LL

FF

SSLL

PPN

G II

LLLL

FF

QQ

DD

NN

LLYY KK EE

EEVVRRIIRR

KKEE

IIYYEEAASSFFF

EE

YY

GGVVGG VV KK

II

IIFFPPII

N-term

L1

L2

L3

L4

L5

C-term

ER Lumen

Cytosol

F

Yeast ICMT Ste14p, 26 kD

myc3His10

His10myc3-Ste14p, 37 kD

diverse range of proteins embedded in membrane

His10

solubilization and purification by immobilized metal-affinity

chromatography (IMAC)

His10

AFM Analysis of Ste14p in Supported Membranes

Shan Zou, Akiko Murakawa, Linda Johnston

Lipid Feature Height(nm)

Supported Membrane

DLPC 0.9‐2.5 continuous bilayer

DMPC 0.6‐1.0 continuous bilayer

POPC 0.7‐1.8 continuous bilayer

DSPC 0.3‐0.5 bilayer patches

80% 20%

Dodecylmaltoside-Mediated His10-Ste14pReconstitution Produces Liposomes with 80% of

the Catalytic Domains on the Outer Surface

His-Ste14p in POPC on mica

DLPC

DSPC

DLPCDSPC

>100 nN~50 nN

<20 nN

Strategies to Orient Immobilize Membrane Proteins Within a Supported Membrane on PEG Modified Glass

Jong-Mok Kim/Elias Franses/Lukas Tamm

water gap thickness varies with PEG MW

supported membrane

SiO2

water gap thickness varies with PEG surface concentration

supported membrane

SiO2

I. Szleifer

POPC on bare Si: 1.83 nm ± 0.29 nmPOPC on C18-PEG4000-Si(OEt)3 modified Si: 16.9 nm ± 0.84 nm

FLIC Microscopy

EllipsometryPEG3400-NTA on Si: 16.9 nm ± 3.3 nmmPEG5000 on Si: 13.7 nm ± 2.9 nm

NTA

Bilayer Membrane

PEG Linker

Glass Surface C18-PEG-APTES NTA-PEG-APTES

NTA-PEG-Si(OEt)3 Stearyl ether-PEG-Si(OEt)3

Kd for His10-GFP-Ste14p on Ni2+:NTA-PEG Surfaces

A. Murakawa

Supported Membrane Sensor Fabrication

Binding & Release Behavior of His10-Ste14p on PEG-NTA Determined by SPR

PEG3400

A. Murakawa

Binding & Release of his-GFP-Ste14p on PEG Substrates

Preparation of his-Ste14p Supported Membrane by Surface-mediated Reconstitution

Natural Archae & Synthetic Bolalipids

POPC

Halophilic Bacteria and Methanogens

Thermophilic Bacterial Bolalipids

OH

O

OO

O

OH

OH

O

O

OH

O

O

OH

O

O

OH

OH

HO

HO

HO

Bolalipid(membrane-spanning chain)

Bilayer Membrane delamination

200 nm

FFEM of Bilayer Membrane

200 nm

FFEM of Bolalipid Membrane

C20BAS

C32phytBAS

C20BAS Characterization

W. Febo-Ayala, D.P. Holland, S. Bradley, D.H. Thompson, Langmuir 2007 23, 6276

~40 Å

D PFG-NMR = 1.8 x 10-8 cm2/s

28 Å

D PFG-NMR = 1.9 x 10-8 cm2/s

POPCC20BAS

C20BAS Lateral Diffusion Rate is Similar to Monopolar Lipids by FRAP & PFG-NMR

Tm = 17°C for C20BAS Membranes

0102030405060

-20 0 20 40 60 80

chem

ical

shi

ft an

isot

ropy

(p

pm)

T (°C)

31P NMR Chemical Shift Anisotropy

for C20BAS

W. Febo-Ayala, D.P. Holland, S. Bradley, D.H. Thompson, Langmuir 2007 23, 6276

Time (min)

102030405060708090

100

0

NaClHBSTris

% C

alce

in R

elea

se

600 1200 3000 3600 42000 1800 2400

7:3 C20BAS:Chol Vesicles Retain a Chemical Gradient

200 nmCryo-TEM of 7:3

C20BAS:Chol

A. Patwardhan

D.P. Holland, A.V. Struts, M.F. Brown, D.H. Thompson, J. Am. Chem. Soc. 2008 130, 4584; Biophys. J. 2009 97, 2700-2709

C20BAS Membranes are Highly Ordered

C20BAS

DLPC

S. Morera-Félix, K. Solka, W. Febo-Ayala, C. A. Hrycyna, D. H. Thompson, Biochemistry 2006 45, 14683-14694

Spec

ific

Act

ivity

of S

te14

p (x

10

-3pm

ol/m

in/m

g)Functional Assay of Ste14p in Bolalipid

Membrane Vesicles of Varying Composition (Bolalipid:E. Coli Lipid Mixtures)

0

2000

4000

6000

8000

10000

12000

0:100 10:90 20:80 50:50 75:25 100:0

C20BAS

C32-phy

bolalipid:E. coli polar lipid

C32phytBAS

C20BAS

A. Patwardhan & D. H. Thompson, Org. Lett. 1999 1, 241-24428Å

C20BAS

N-His10myc3C

40Å

C32-phytBAS

CN-His10myc3

b c dE. coli polar lipid

C20BAS

C32phytBAS

Nile Blue anti-myc-FITC

merged phasecontrast

Conceptual Diagram of Interferometric Detection

G. Acharya, C.-L. Chang, D. Holland, D. H. Thompson, C. Savran, Angew. Chem. Int. Ed. 2008 47, 1051-1053

SAH solution SAH capture byanti-SAH-magnetic

microbeads

N

S

Magnetic isolationand rinsing of microbeads

Laserdiode

anti-SAH-magneticmicrobeads

microcontact printed aptamer-Au surface

Preparation of Stamp

PDMS-StampMaster

Inking Stamping Patterned Surface

Stamping

biotinylated BSA

magnetic μsphere-antibody conjugate

biotinbiotinylated aptamer

conjugate

SAH

homocysteine antibodyrecognition site

adenosine aptamerrecognition site

adenosine recognition by 39-mer aptamer sequence5' CGG AUG AGA CGC UUG GCG UGU GCU GUG GAG AGU CAU CCG 3'

Kd = 5 x 10-8 M

Optical Microscopy of μCP Au Surfaces After Exposure to SAH-Immunocaptured Beads

G. Acharya, C.-L. Chang, D. Holland, D. H. Thompson, C. Savran, Angew. Chem. Int. Ed. 2008 47, 1051-1053

Calibration Curve for S-Adenosyl Homocysteine Detection Using SAH-Immunocaptured Beads on μCP Au Surfaces

G. Acharya, C.-L. Chang, D. Holland, D. H. Thompson, C. Savran, Angew. Chem. Int. Ed. 2008 47, 1051-1053

Controls

Scale bar = 30 μm

biot

in

biot

in

biot

in

H

magnetic μsphere-antibody conjugate

biot

in

biotinylated aptamerconjugate

SAH

G. Acharya, C.-L. Chang, D. Holland, D. H. Thompson, C. Savran, Angew. Chem. Int. Ed. 2008 47, 1051-1053

High-Throughput Platform for Interferometric Detection

Streptavidin-immobilized disc

transfer magnetically-captured SAHproduced in multiple assay reactions

SAH

biotin

prime disc with

biotinylatedadenosine aptamer

assay readout viainterferometric

detection device

www.quadraspec.com

Conclusions

• NTA-PEG-modified surfaces are capable of efficient his10-Ste14p capture and his10-Ste14p orientation during supported membrane formation

• C20BAS bolalipid forms monolayer membranes that have similar permeability, melting transition, and lateral diffusion as conventional monopolar lipids

• C32phytBAS bolalipid vesicles retains ≥ 75% Ste14p activity, however, it is lost in C20BAS vesicles that have significant hydrophobic mismatch

• The membrane-support gap can be controlled through appropriate choice of grafted PEG MW

• Interferometric sandwich assays provide an attractive method for SAH detection

Financial SupportNIH CA112427

Purdue Center for Cancer Research

Akiko Murakawa

Jong-MokKim

DavidHolland

CollaboratorsMichael F. Brown

Elias FransesChris HrycynaLinda Johnston

Gil U. Lee

Horia PetracheS. Scott Saavedra

Cagri SavranIgal SzleiferLukas Tamm

WilmaFebo-Ayala

Seok-HeeHyun

PatrickPalafox

S. Morera-Félix, K. Solka, W. Febo-Ayala, C. A. Hrycyna, D. H. Thompson, Biochemistry 2006 45, 14683-14694

Spec

ific

Act

ivity

of S

te14

p (x

10

-3pm

ol/m

in/m

g)Functional Assay of Ste14p in Bolalipid

Membrane Vesicles of Varying Composition (Bolalipid:E. Coli Lipid Mixtures)

0

2000

4000

6000

8000

10000

12000

0:100 10:90 20:80 50:50 75:25 100:0

C20BAS

C32-phy

bolalipid:E. coli polar lipid

C32phytBAS

C20BAS

A. Patwardhan & D. H. Thompson, Org. Lett. 1999 1, 241-24428Å

C20BAS

N-His10myc3C

40Å

C32-phytBAS

CN-His10myc3

b c dE. coli polar lipid

C20BAS

C32phytBAS

Nile Blue anti-myc-FITC

merged phasecontrast

b c dE. coli polar lipid

C20BAS

C32phytBAS

Nile Blue anti-myc-FITC

merged phasecontrast

G. Longo, D. H. Thompson, I. Szleifer, Biophys. J. 2007 93, 2809

Mean Field Theory ofC20BAS:Monopolar Lipid Phases

I

II

IIIIVI

II

III

IV