Efforts to Develop a High-Throughput Screening Tool Using...
Transcript of Efforts to Develop a High-Throughput Screening Tool Using...
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
VFIF I V
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
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
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