Identification of ligand-binding regions of P-glycoprotein by activated-pharmacophore photoaffinity labeling and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

Article abstract of DOI:10.1124/mol.61.3.637

Energy dependent efflux pumps confer resistance to anticancer, antimicrobial, and antiparasitic drugs. P-glycoprotein (Pgp, ABCB1) mediates resistance to a broad spectrum of antitumor drugs. Compounds that themselves are nontoxic to cells have been shown to act as inhibitors of Pgp. The mechanism of binding and transport of low-molecular-mass ligands by Pgp is still incompletely understood. This study introduces a series of propafenone-related photoaffinity ligands, which combine high specificity and selectivity for Pgp with high labeling efficiency. Molecules are intrinsically photoactivatable in the arylcarbonyl group, which represents a pharmacophoric substructure for this group of ligand molecules. A detailed study of the structure-activity relationship for this type of photoligand is presented. In subsequent experiments, these ligands were used to characterize the drug-binding domain of propafenone-type analogs. Matrix-assisted laser desorption/ionization - time-of-flight (MALDI-TOF) mass spectrometry shows that propafenone-type ligands preferentially label fragments assigned to putative transmembrane segments 3, 5, 6, 8, 10, 11, and 12. Labeled fragments are also identified in a highly charged region of 15 amino acids in the second cytoplasmic loop. This region corresponds to the so-called EAA-like motif, which has been proposed to play a role in the interaction between transmembrane domain and nucleotide binding domain of peroxisomal ATP-binding cassette transporters. In addition, a region in cytoplasmic loop 3 and between TM12 and the N terminus of the Walker A sequence of NBD2 are labeled by the ligands. Therefore, a number of confined protein regions contribute to the drug-binding domain of propafenone-type analogs.

Full text of DOI:10.1124/mol.61.3.637

0026-895X/02/6103-637–648$3.00
MOLECULAR PHARMACOLOGY
Copyright © 2002 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 61:637–648, 2002
Vol. 61, No. 3
1241/964057
Printed in U.S.A.
Identification of Ligand-Binding Regions of P-Glycoprotein by
Activated-Pharmacophore Photoaffinity Labeling and Matrix-
Assisted Laser Desorption/Ionization–Time-of-Flight Mass
Spectrometry
GERHARD F. ECKER, EDINA CSASZAR, STEPHAN KOPP, BRIGITTE PLAGENS, WOLFGANG HOLZER,
WOLFGANG ERNST, and PETER CHIBA
Institutes of Medical Chemistry (P.C., S.K.), Pharmaceutical Chemistry (G.E., B.P., W.H.), Applied Microbiology (W.E.), and the Mass
Spectrometry Unit (E.C.), University of Vienna, Austria
Received July 26, 2001; accepted November 16, 2001
This article is available online at http://molpharm.aspetjournals.org
ABSTRACT
Energy dependent efflux pumps confer resistance to anticancer, trix-assisted laser desorption/ionization—time-of-flight (MALDI-TOF)
antimicrobial, and antiparasitic drugs. P-glycoprotein (Pgp, ABCB1) mass spectrometry shows that propafenone-type ligands preferen-
mediates resistance to a broad spectrum of antitumor drugs. Com- tially label fragments assigned to putative transmembrane segments
pounds that themselves are nontoxic to cells have been shown to act 3, 5, 6, 8, 10, 11, and 12. Labeled fragments are also identified in a
as inhibitors of Pgp. The mechanism of binding and transport of highly charged region of 15 amino acids in the second cytoplasmic
low-molecular-mass ligands by Pgp is still incompletely understood. loop. This region corresponds to the so-called EAA-like motif, which
This study introduces a series of propafenone-related photoaffinity has been proposed to play a role in the interaction between trans-
ligands, which combine high specificity and selectivity for Pgp with membrane domain and nucleotide binding domain of peroxisomal
high labeling efficiency. Molecules are intrinsically photoactivatable in ATP-binding cassette transporters. In addition, a region in cytoplas-
the arylcarbonyl group, which represents a pharmacophoric sub- mic loop 3 and between TM12 and the N terminus of the Walker A
structure for this group of ligand molecules. A detailed study of the sequence of NBD2 are labeled by the ligands. Therefore, a number of
structure-activity relationship for this type of photoligand is pre- confined protein regions contribute to the drug-binding domain of
sented. In subsequent experiments, these ligands were used to char- propafenone-type analogs.
acterize the drug-binding domain of propafenone-type analogs. Ma-
Expression of drug-efflux pumps is one important mecha- (Pgp). Pgp is a 170-kDa protein of human origin that is
nism for target cells to protect themselves from the lethal
action of therapeutically administered drugs. During the
past decade, a number of efflux systems in bacteria, fungi,
parasites, and mammalian cells were identified. A common
property of these efflux pump systems, many of which share
a high degree of sequence homology, is broad substrate spec-
ificity. The phenotype arising as a consequence of the expres-
sion of these efflux pumps has therefore been termed multi-
drug resistance (MDR). Because advanced drug design paved
the way for the circumvention of a number of drug-specific
resistance mechanisms, this type of broad-spectrum resis-
tance is also increasingly observed in a clinical setting. The
most extensively studied drug transporter is P-glycoprotein
constitutively expressed in a variety of human tissues
(Gottesman and Pastan, 1993). Overexpression is observed in
resistant human tumors of different origin. P-glycoprotein is
the functional ortholog of microbial ABC transporters. These
include LmrA, an efflux transporter conferring multidrug
resistance in Lactococcus lactis (van Veen et al., 1996), the
yeast transporters snq2p (Servos et al., 1993) and pdr5p
(Balzi et al., 1994; Bissinger and Kuchler, 1994) and the
Candida albicans drug resistance proteins cdr1p (Prasad et
al., 1995) and cdr2p (Sanglard et al. 1997).
One concept for the circumvention of MDR is the simulta-
neous administration of cytotoxic drugs and inhibitors of
toxin efflux pumps. These modulators of multiresistance are
of interest to the pharmacologist because they are able to
reverse MDR in tumor cells (reviewed in Krishna and Mayer,
This work was supported by a grant from the Austrian Science Fund (FWF)
(Grant 13851) and the Austrian National Bank (Grant 8525).
ABBREVIATIONS: MDR, multidrug resistance; Pgp, P-glycoprotein; ABC, ATP-binding cassette; QSAR, quantitative structure activity relation-
ship; MS, mass spectrometry; PM, plasma membrane; CHCA, ␣-cyano-4-hydroxycinnamic acid; MALDI-TOF, matrix-assisted laser desorption/
ionization–time-of-flight; TFA, trifluoroacetic acid; PA, propafenone analog; CL, cytoplasmic loop; ECL, extracytoplasmic loop; AA, amino acid;
TM, transmembrane segment; NBD, nucleotide binding domain.
637

638
Ecker et al.
2000) and microbial systems (Lomovskaya and Watkins, alogs were defined and corresponded to those observed for
2001) in vitro. the parental compounds. These ligand molecules were sub-
A characterization of the interaction of toxin efflux pumps sequently used as photoprobes, which led to the identification
with low-molecular-mass inhibitors has been pursued by of protein regions contributing to the binding-domain of
photoaffinity labeling (for comprehensive reviews on the sub- propafenone analogs.
ject, see Beck and Qian, 1992; Dey et al., 1998; Greenberger,
1998; Safa, 1998), site-directed mutagenesis (Loo and Clarke,
1998, 2000, 2001a,b), ligand-based design (see the recent
Materials and Methods
Design and Synthesis of Compounds. Synthesis of compounds
review by Wiese and Pajeva, 2001, and references therein),
GPV05 to GPV180 was achieved as described previously (Chiba et al.
and Pgp-ATPase activity measurements (reviewed in Senior
1995, 1996; Tmej et al., 1998). Benzophenone derivatives were syn-
et al., 1998).
thesized in analogy to the procedures for GPV317 and GPV319 as
outlined in Scheme 1 (Tmej et al., 1998). Thus, a corresponding
Cysteine scanning and site-directed mutagenesis identi-
fied amino acid residues in transmembrane segments, which
are likely to be involved in drug binding (Ma et al., 1997; Dey
et al., 1999; Loo and Clarke, 2000, 2001b). Electron cryo-
microscopy of two-dimensional crystals has led to structural
resolution of hamster Pgp at approximately 10 Å (Rosenberg
et al., 2001). Despite this progress, the interaction of ligand
molecules with Pgp is still inadequately defined on a molec-
ular basis. We thus pursued an approach combining ligand-
based design in conjunction with quantitative structure ac-
tivity relationship (QSAR) studies to characterize the
interaction of low-molecular-mass ligands with Pgp in
greater detail.
On the basis of studies conducted on a compound library of
substances that are structurally related to propafenone, com-
mon structural and physicochemical parameters of this class
of inhibitors have been defined and pharmacophoric sub-
structures have been identified (Chiba et al., 1996; Ecker et
al., 1996, 1999).
Photoaffinity labeling represents one possible experimen-
tal strategy to characterize those regions of the protein mol-
ecule in which an interaction with low-molecular-mass li-
gands takes place. Similar to certain steroids, propafenone
and its analogs are intrinsically photoactivatable, whereby
activation takes place in the arylcarbonyl substructure. We
previously identified the carbonyl group as a pharmacophore
with respect to Pgp (Chiba et al., 1996). This group acts as a
hydrogen bond acceptor, which is likely to interact with a
hydrogen bond donor group in the protein (Ecker et al., 1999).
Formation of the biradical species is favoured, when the
carbonyl group is part of a benzophenone substructure.
Therefore, a series of propafenone-analogous molecules con-
taining a benzophenone substructure were synthesized (Fig.
1). Quantitative structure activity relationships of these an-
2-hydroxy-benzophenone (1) is reacted with epichlorohydrin to give
the epoxide 2. Subsequent nucleophilic ring opening with piperidine
yields the desired aminoalcohols (B59, BP11). Thieno-derivative
BP01 was synthesized in an analogous manner. In the case of the
pyrazine analog B47, the corresponding 2-chloro derivative was used
instead of the hydroxy-derivative. In this case, formation of the
epoxide was achieved with 2,3-epoxypropanol. The desoxy-analog
BP23 was obtained via reaction of BP59 with hydroxylamine hydro-
chloride followed by hydrogenation of the oxime 3.
The phenolic precursor for the 5-methyl-analogs GPV442 and
GPV443 was synthesized via acylation of 4-methylphenol (4) fol-
lowed by Fries rearrangement of the phenylester 5 (Scheme 2). The
permanently charged ammonium derivative GPV708 was obtained
via methylation of the corresponding diethylamino-derivative (Chiba
1995). IR, MS, NMR spectroscopy and elemental analysis confirmed
the structure of all novel compounds. 1H-NMR spectra are given in
Table 1. Structures are represented in Table 2.
Calculation of logP Values. The logP values were calculated
using the software package ChemOffice (option: best method; Cam-
bridgeSoft Corporation, Cambridge, MA). Calculated logP values are
given in Table 2.
Cell Lines. The human T-lymphoblast cell line CCRF-CEM and
the multidrug resistant lines CCRF vcr1000 and CCRF adr5000
were provided by V. Gekeler (Byk Gulden, Konstanz, Germany). The
resistant lines were obtained by stepwise selection in vincristine- or
daunorubicin-containing medium (Boer et al., 1996). Cells were kept
under standard culture conditions (RPMI 1640 medium supple-
mented with 10% fetal calf serum). The Pgp-expressing resistant cell
lines were cultured in the presence of 1000 ng/ml vincristine or 5000
ng/ml daunorubicin, respectively. One week before the experiments,
cells were transferred into medium without selective agents or an-
tibiotics. Insect Sf9 cells were obtained from the American Type
Culture Collection (Manassas, VA).
Daunorubicin Efflux Studies. Daunorubicin efflux studies
were performed as described previously (Chiba et al., 1996). Briefly,
cells were pelleted, the supernatant was removed by aspiration, and
cells were resuspended at a density of 1 ϫ 106/ml in RPMI 1640
medium containing 3 ␮M daunomycin. Cell suspensions were incu-
Scheme 1. Synthesis of benzophenone derivatives. a, NaOH/epichloro-
hydrine; b, piperidine/MeOH; c, H2NOH-HCl/EtOH; d, H2/Pd-C/EtOH-
MeOH (or Na/1-propanol).
Fig. 1. General chemical structure of propafenone-type analogs and cor-
responding benzophenone photoaffinity ligands

Photoaffinity Labeling of P-Glycoprotein
639
three consecutive washes in 100% fetal bovine serum at 37°C. Rho-
damine 123 was added to give a final concentration of 0.52 ␮M and
cells were incubated at 37°C for 30 min, after which time a steady
state of fluorochrome uptake was reached. Cells were chilled in ice
water and washed once with ice-cold RPMI1640 medium. Mean
fluorescence units per cell were determined by use of the attractor
software in a FACSCalibur flow cytometer (BD Biosciences).
Radiolabeling of Pgp with [3H]GPV51 and Gel Electro-
phoretic Separation Conditions. Plasma membrane (PM) prepa-
rations of Sf9 cells transfected with the baculovirus construct con-
taining the his-tagged mdr1 gene (Germann et al., 1990) were
prepared by nitrogen cavitation and subsequent discontinuous su-
crose-gradient centrifugation as described previously (Schmid et al.,
1999). PM vesicles were taken up in phosphate-buffered saline and
preincubated with [3H]GPV51 (5 ␮Ci; final concentration, 2.75 ␮M)
for 30 min at ambient temperature. Subsequently, samples were
irradiated under conditions identical to those described above. Un-
labeled GPV51 or GPV90 was added at 100-fold molar excess. After
irradiation, samples were centrifuged at 50,000g for 30 min at 4°C.
Protein pellets were taken up in 1ϫ SDS/sample buffer, loaded on
7.5% polyacrylamide gels, and run at 35 mA for 60 min using a
Hoefer Mighty Small II SE250 unit (Amersham Biosciences, Vienna,
Austria). Gels were fixed in methanol/glacial acetic acid/water (50:
10:40) for 30 min, washed overnight in double-distilled water, and
soaked in Amplify (Amersham Biosciences) for 30 min. Gels were
dried for 2 h in a vacuum gel dryer (Bio-Rad, Vienna, Austria) at
80°C and subjected to fluorography using an Amersham enhanced
chemiluminescence Hyperfilm.
Scheme 2. Synthesis of 5-methyl precursors. a, 4-Iodobenzoic acid chlo-
ride/pyridine; b, AlCl3/160°C.
bated at 37°C for 30 min. After this time, a steady state of dauno-
rubicin accumulation was reached. Tubes were chilled on ice and
cells were pelleted at 500g. Cells were washed once in RPMI 1640
medium to remove extracellular daunorubicin. Subsequently, cells
were resuspended in medium prewarmed to 37°C, containing either
no modulator or chemosensitizer at various concentrations ranging
from 3 nM to 500 ␮M, depending on solubility and expected potency
of the modifier. Generally, eight serial dilutions were tested for each
modulator. After 1, 2, 3, and 4 min, aliquots of the incubation mix-
ture were drawn and pipetted into 4 volumes of ice-cold stop solution
(RPMI 1640 medium containing verapamil at a final concentration of
100 ␮M). Parental CCRF-CEM cells were used to correct for simple
membrane diffusion, which was less than 3% of the efflux rate
observed in resistant cells. Samples drawn at the respective time
points were kept in an ice water bath and measured within an hour
on a FACSCalibur (BD Biosciences, Heidelberg, Germany) flow cy-
tometer as described. Dose-response curves were fitted to the data
points using nonlinear least-squares and EC50 values were calcu-
lated as described previously (Chiba et al., 1996). EC50 values of
individual compounds are given in Table 2 and represent the average
and S.D. of at least triplicate determinations.
Chemicals for In-Gel Digestion and Mass Spectrometry.
High-quality water for the in-gel digestion and the mass spectromet-
ric experiments was prepared using a Milli-Q water purification
system (Millipore, Bedford, MA). Ammonium hydrogen carbonate
was obtained from FLUKA (Sigma-Aldrich, Vienna, Austria), dithio-
threitol for the reduction of proteins before in-gel digestion was
Irradiation Inactivation of Pgp. Whole cells were preincubated
with ligand for 5 min at room temperature, placed on ice, and
irradiated intermittently (six times for 30 s each) with a 500-W
mercury lamp (Lot-Oriel, Darmstadt, Germany) in the presence and
absence of the photoactivatable benzophenone analog GPV51. A
1-mm glass plate was placed in the light path to filter most of the UV purchased from Serva (Novex, San Diego, CA), iodoacetamide was
light with wavelengths below 300 nm. The nonphotoactivatable an- supplied by Sigma-Aldrich, sequencing grade trypsin and chymo-
alog GPV90 was included in duplicate samples at 100-fold molar trypsin was obtained from Roche Diagnostics GmbH (Mannheim,
excess for each individual concentration of GPV51 to prove the spec- Germany). Acetone was supplied by AppliChem (Darmstadt, Germa-
ificity of the reaction. Subsequently, unbound ligand was removed by ny). High-performance liquid chromatography–grade acetonitrile,
TABLE 1
NMR spectra of newly synthesized compounds
Compound
BP001
Formula
1H-NMR; chloroform-d, ␦ (ppm)
C
19H23NO3S 1.41 (m, 2H, Pip H-4), 1.52 (m, 4H, Pip H-3, H-5), 2.06 (m, 2H, -CH2-Pip), 2.19 and 2.39 (both m, 4H, Pip H-2, H-6),
2.94 (broad, 1H, OH), 3.74 (m, 1H, -CH-OH), 3.99 (d, 2H, -O-CH2-), 6.87 (A, X-System, 1H, Th H-4), 7.42 (t, 2H, Ph
3
H-3, H-5), 7.50 (t, 1H, Ph H-4), 7.56 (A, X-System, 1H, Th H-5, J(H-4, H-5) ϭ 5.5 Hz), 7.76 (d, 2H, Ph H-2, H-6)
B59
C
21H25NO3 1.39 (m, 2H, Pip H-4), 1.50 (m, 4H, Pip H-3, H-5), 2.02 (m, 2H, -CH2-Pip), 2.34 and 2.17 (both m, 4H, Pip H-2, H-6),
3.43 (s, 1H, OH), 3.69 (m, 1H, -CH-OH), 3.87 (q, 1H, -O-CH2-), 3.95 (q, 1H, -O-CH2-), 6.98 (d, 1H, Ph H-3), 7.05 (t,
1H, Ph H-5), 7.42 (m, 2H, PhЈ H-3, H-5), 7.43 (m, 1H, Ph H-6), 7.45 (m, 1H, Ph H-4), 7.53 (t, 1H, PhЈ H-4), 7.77 (d,
2H, PhЈ H-2, H-6)
19H23N3O3 1.43 (m, 2H, Pip H-4), 1.57 (m, 4H, Pip H-3, H-5), 2.36 (m, 2H, -CH2-Pip), 2.54 and 2.35 (both m, 4H, Pip H-2, H-6),
4.02 (m, 1H, -CH-OH), 4.40 (m, 2H, -O-CH2-), 7.46 (t, 2H, Ph H-3, H-5), 7.60 (t, 1H, Ph H-4), 7.86 (d, 2H, Ph H-2,
H-6), 8.24 (,,sЉ, 2H, Pyraz H-5, H-6)
21H27NO2 1.47 (m, 2H, Pip H-4), 1.61 (m, 4H, Pip H-3, H-5), 2.38 (m, 2H, -CH2-Pip), 2.57 and 2.36 (both m, 4H, Pip H-2, H-6),
3.56 (broad, 1H, OH), 3.92 (q, 1H, -O-CH2-), 4.00 (m, 1H, -O-CH2-), 4.00 (s, 2H, Ph-CH2-Ph), 4.03 (m, 1H, -CH-OH),
6.87 (m, 1H, Ph H-3), 6.91 (m, 1H, Ph H-5), 7.12 (m, 1H, Ph H-6), 7.18 (m, 1H, PhЈ H-4), 7.19 (m, 2H, PhЈ H-2, H-
6), 7.20 (m, 1H, Ph H-4), 7.26 (m, 2H, PhЈ H-3, H-5)
22H27NO4 1.39 (m, 2H, Pip H-4), 1.50 (m, 4H, Pip H-3, H-5), 2.01 (m, 2H, -CH2-Pip), 2.34 and 2.17 (both m, 4H, Pip H-2, H-6),
3.33 (broad, 1H, OH), 3.66 (m, 1H, -CH-OH), 3.85 (s, 3H, -OCH3), 3.91 and 3.84 (q and m, 2H, -O-CH2-), 6.50 (d,
B047
C
BP023
C
BP011
C
4
3
1H, Ph H-3), 6.57 (dd, 1H, Ph H-5, J(H-3, H-5) ϭ 2.2 Hz), 7.40 (m, 2H, PhЈ H-3, H-5), 7.46 (m, 1H, Ph H-6, J(H-5, H-6)
ϭ 8.5 Hz), 7.51 (t, 1H, PhЈ H-4), 7.73 (d, 2H, PhЈ H-2, H-6)
GPV442
C
20H25NO3 0.87 (t, 3H, 3J ϭ 7.3 Hz, -CH2-CH3), 1.46 (sx, 2H, 3J ϭ 7.3 Hz, -CH2-CH3), 2.30 (s, 3H, -CH3), 2.39-2.51 (m, 4H, -
CH2N-CH2-), 3.4 (br, 2H, OH, NH), 3.78-3.91 (m, 3H, O-CH2-CH-), 6.87 (d, J ϭ 8.4 Hz, Ph H-3), 7.20 (s, 1H, Ph H-
6), 7.25 (d, 1H, J ϭ 8.4 Hz, Ph H-4), 7.43 (t, 2H, J ϭ 7.6 Hz, PhЈ H-3, H-5), 7.55 (t, 1H, J ϭ 7.5 Hz, PhЈ H-4), 7.77
(t, 2H, J ϭ 7.4 Hz, PhЈ H-2, H-6)
GPV443
GPV708
C
C
20H24NO3I 0.90 (t, 3H, 3J ϭ 7.3 Hz, -CH2-CH3), 1.50 (sx, 2H, 3J ϭ 7.3 Hz, -CH2-CH3), 2.29 (s, 3H, -CH3), 2.37-2.54 (m, 4H, -CH2-
N-CH2), 3.83-3.93 (m, 5H, O-CH2, CH-OH, OH, NH), 6.86 (d, 1H, J ϭ 8.4 Hz, Ph H-3), 7.17 (s, 1H, Ph H-6), 7.25 (d,
1H, J ϭ 8.4 Hz, Ph H-4), 7.47 (d, 2H, J ϭ 8.4 Hz, PhЈ H-3, H-5), 7.78 (d, 2H, J ϭ 8.4 Hz, PhЈ H-2, H-6)
21H28NO3I 1.32 (t, 6H, J ϭ 7.2 Hz, 2 ϫ -CH2-CH3), 3.16 (s, 3H, Nϩ-CH3), 3.37-3.64 (m, 6H, -CH2-Nϩ(-CH2)2), 4.03-4.11 (m, 1H, J
ϭ O-CHa), 4.31 (dd, 1H, J ϭ 4.3/10.8 Hz, O-CHb), 4.6-4.75 (m, 1H, CH(OH)), 7.05 (d, 1H, Ph H-3), 7.11 (t, 1H, Ph
H-5), 7.38 (m, 1H, Ph H-6),), 7.48-68 (m, 4H, PhЈ H-3, H-4, H-5, Ph H-4), 7.85 (d, 2H, PhЈ H-2, H-6)

640
Ecker et al.
TABLE 2
Chemical structure and calculated logP and EC50 values for daunorubicin efflux inhibition of propafenone analogs and related compounds.
methanol, and isopropanol were purchased from Sigma-Aldrich, tri-
Silver Staining and In-Gel Digestion. The silver staining was
fluoroacetic acid was obtained from Pierce (Rockford, IL), and formic carried out according to the method of Shevchenko et al. (1996) and
acid was supplied by Merck (Merck KgaA; Darmstadt, Germany). the in-gel digestion was executed without destaining of the gel as
The ␣-cyano-4-hydroxycinnamic acid (CHCA) matrix for the MALDI- described in Durauer et al. (2000).
TOF MS measurements was acquired from Bruker Daltonik GmbH
MALDI-TOF Mass Spectrometry. The mass spectrometer used
(Bremen, Germany) and was used without further purification. Ni- in this work was a REFLEX III (Bruker Daltonik GmbH) MALDI-
trocellulose was purchased from Bio-Rad (Hercules, CA). Protein and TOF instrument, equipped with a standard nitrogen laser (337 nm).
peptide standards used to calibrate the MALDI mass spectrometer The spectra were recorded in reflectron mode, positive ionization,
were acquired from Applied Biosystems (Foster City, CA).
and with 25-kV acceleration voltage. The laser power was varied on

Photoaffinity Labeling of P-Glycoprotein
641
TABLE 2
Continued.
1 Chiba et al. (1996).
2 Chiba et al. (1995).
3 Tmej et al. (1998).
a relative scale of 0 to 100 and was kept at the threshold value to
matrix [saturated solution in 0.1% TFA-acetonitrile 50:50 (v/v)] di-
obtain appropriate signal intensity. The calibration of the instru- rectly onto the sample slide. Each spectrum was produced by accu-
ment was done externally with [MϩH]ϩ ions of des-Arg-bradykinin, mulating data from 90 to 120 consecutive laser shots. Monoisotopic
angiotensin I, Glu-fibrinopeptide B, and adrenocorticotropin (clips mass values are reported in Table 3. Spectra were interpreted with
1–17 and 18–39). The samples were prepared with a 75:25 (v/v) the aid of the Mascot (Matrix Science Ltd, London, UK) or MS-Fit
mixture of CHCA matrix (saturated solution in acetone) and nitro- (Clauser et al., 1999) software using the NR database (NCBI Re-
cellulose [10 mg/ml solution in acetone-isopropanol 50:50 (v/v)] solu- sources, National Institutes of Health, Bethesda, MD).
tions. One microliter of the mixture was placed onto the sample slide
and allowed to dry at room temperature. Part of the aliquot (0.5 ␮l)
of the in-gel digestion was mixed with 0.5 ␮l 0.1% TFA on this thin
layer of matrix crystals and was dried by the application of vacuum.
Samples were washed with ice-cold 0.1% TFA. Hydrophobic peptides
were purified and concentrated on Poros 20 R1 material (Applied
Biosystems) loaded into GeLoader tips (Eppendorf-Netheler-Hinz-
GmbH, Hamburg, Germany). The chromatography material was
Results
QSAR of Photoligands. The arylcarbonyl substructure is
common to PAs and benzophenones. This allowed the design
of benzophenone analogs, which share the pharmacophoric
carbonyl group with propafenone and are photoactivatable
conditioned with 0.1% TFA and the peptides were eluted with CHCA upon irradiation at a wavelength of 360 nm. Figure 2 shows

642
Ecker et al.
TABLE 3
Ligand-modified peptide fragments identified by MALDI-TOF mass spectometry
m/z (mi) [M ϩ H]ϩ
Mass
Accuracy
Modif. Start End
E
Ligand
AA Sequence
Submitted
Matched
ppm
TM3
194 208
M-ox 194 200
194 200
194 201
M-ox 194 204
195 200
pGlu 195 201
M-ox 195 201
195 201
195 204
M-ox 195 204
pGlu
FQSMATFFTGFIVGFTRGW
1174.610 1174.546
1173.599 1173.581
1412.744 1412.640
1641.865 1641.782
1127.550 1127.503
1141.562 1141.525
1174.610 1174.546
1173.632 1173.581
1505.787 1505.699
1462.691 1462.657
54
15
74
51
42
32
54
43
58
23
C
C
C
C
C
C
C
C
C
C
GPV51 FQSMATFFTGFIVGFTRGW
GPV708 FQSMATFFTGFIVGFTRGW
GPV319 FQSMATFFTGFIVGFTRGW
GPV708 FQSMATFFTGFIVGFTRGW
GPV319 FQSMATFFTGFIVGFTRGW
GPV51 FQSMATFFTGFIVGFTRGW
GPV51 FQSMATFFTGFIVGFTRGW
GPV708 FQSMATFFTGFIVGFTRGW
BP11
FQSMATFFTGFIVGFTRGW
GPV51 FQSMATFFTGFIVGFTRGW
1446.660 1446.662
1446.720 1446.662
1462.726 1462.657
Ϫ1
40
47
pGlu 195 204
pGlu 195 204
M-ox 195 204
pGlu
C
C
C
GPV51 FQSMATFFTGFIVGFTRGW
GPV442 FQSMATFFTGFIVGFTRGW
GPV442 FQSMATFFTGFIVGFTRGW
1477.785 1477.687
1920.940 1920.909
66
16
M-ox 195 204
pGlu
C
C
GPV708 FQSMATFFTGFIVGFTRGW
M-ox 195 208
pGlu
BP11
FQSMATFFTGFIVGFTRGW
1969.900 1969.925
1821.988 1821.916
1240.691 1240.684
1067.600 1067.578
Ϫ13
40
6
pGlu 195 208
201 212
202 212
202 208
202 208
205 208
205 208
272 291
272 286
273 279
273 279
273 285
273 285
280 285
287 291
304 316
304 307
304 310
304 310
304 314
305 312
305 312
305 314
306 312
306 312
306 314
306 314
306 316
308 314
308 315
308 316
311 316
327 343
327 336
327 335
333 343
336 343
337 343
337 339
755 784
755 759
755 760
755 762
756 760
756 762
756 762
758 767
760 767
760 767
763 767
763 770
763 770
763 771
763 771
763 775
768 771
C
C
C
C
C
C
C
GPV319 FQSMATFFTGFIVGFTRGW
GPV319 FQSMATFFTGFIVGFTRGW
GPV51 FQSMATFFTGFIVGFTRGW
GPV51 FQSMATFFTGFIVGFTRGW
GPV319 FQSMATFFTGFIVGFTRGW
GPV317 FQSMATFFTGFIVGFTRGW
GPV442 FQSMATFFTGFIVGFTRGW
KELERYNKNLEEAKRIGIKK
21
9
878.469
866.520
762.414
878.461
866.471
762.441
57
Ϫ35
CL2
2354.093 2354.230
1320.590 1320.680
1278.689 1278.670
1978.010 1978.044
1072.542 1072.553
1134.658 1134.573
Ϫ58
Ϫ68
15
T
T
T
T
T
T
T
GPV319 KELERYNKNLEEAKRIGIKK
BP11
KELERYNKNLEEAKRIGIKK
GPV51 KELERYNKNLEEAKRIGIKK
GPV708 KELERYNKNLEEAKRIGIKK
Ϫ17
Ϫ10
75
BP11
KELERYNKNLEEAKRIGIKK
GPV317 KELERYNKNLEEAKRIGIKK
GPV317 KELERYNKNLEEAKRIGIKK
LLIYASYALAFWY
989.570
TM5
890.499
989.608
Ϫ38
890.524
Ϫ28
35
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
BP11
BP11
LLIYASYALAFWY
LLIYASYALAFWY
1211.699 1211.656
1276.675 1276.666
1571.770 1571.873
1282.670 1282.694
1240.691 1240.684
1500.807 1500.799
1127.596 1127.599
1127.640 1127.599
1345.668 1345.705
1694.817 1694.872
1345.710 1345.705
1173.660 1173.588
1297.738 1297.647
1418.731 1418.700
1201.679 1201.598
7
GPV319 LLIYASYALAFWY
GPV51 LLIYASYALAFWY
Ϫ66
Ϫ19
6
BP11
LLIYASYALAFWY
GPV51 LLIYASYALAFWY
BP11 LLIYASYALAFWY
5
Ϫ3
36
GPV442 LLIYASYALAFWY
GPV51 LLIYASYALAFWY
GPV442 LLIYASYALAFWY
GPV51 LLIYASYALAFWY
GPV51 LLIYASYALAFWY
GPV317 LLIYASYALAFWY
Ϫ27
Ϫ32
4
61
70
BP11
LLIYASYALAFWY
22
GPV51 LLIYASYALAFWY
GPV317 LLIYASYALAFWY
SIGQVLTVFFSVLIGAF
67
TM6
1479.831 1479.810
1290.642 1290.732
1634.804 1634.867
1222.719 1222.672
1033.577 1033.594
14
Ϫ70
Ϫ39
38
Ϫ16
27
C
C
C
C
C
C
BP11
SIGQVLTVFFSVLIGAF
GPV51 SIGQVLTVFFSVLIGAF
GPV319 SIGQVLTVFFSVLIGAF
BP11
SIGQVLTVFFSVLIGAF
GPV51 SIGQVLTVFFSVLIGAF
GPV317 SIGQVLTVFFSVLIGAF
749.433
TM8
995.486
749.413
FSLLFLALGIISFITFFLQGFTFGKAGEIL
FSLLFLALGIISFITFFLQGFTFGKAGEIL
995.545
Ϫ59
Ϫ77
Ϫ3
Ϫ45
Ϫ3
Ϫ77
Ϫ54
20
Ϫ41
Ϫ74
33
Ϫ67
5
Ϫ37
Ϫ51
Ϫ32
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
BP11
1066.537 1066.619
1292.747 1292.751
GPV51 FSLLFLALGIISFITFFLQGFTFGKAGEIL
BP11
FSLLFLALGIISFITFFLQGFTFGKAGEIL
919.510
919.551
GPV442 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV442 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV51 FSLLFLALGIISFITFFLQGFTFGKAGEIL
1103.669 1103.672
1066.537 1066.619
1462.777 1462.856
1202.728 1202.704
1160.646 1160.694
BP11
BP11
FSLLFLALGIISFITFFLQGFTFGKAGEIL
FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV442 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV317 FSLLFLALGIISFITFFLQGFTFGKAGEIL
967.446
967.518
1266.741 1266.699
1239.636 1239.719
1413.774 1413.767
1475.732 1475.787
1920.922 1921.020
BP11
FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV708 FSLLFLALGIISFITFFLQGFTFGKAGEIL
BP11
FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV317 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV317 FSLLFLALGIISFITFFLQGFTFGKAGEIL
896.448
896.477
BP11
FSLLFLALGIISFITFFLQGFTFGKAGEIL

Photoaffinity Labeling of P-Glycoprotein
643
TABLE 3
Continued
m/z (mi) [M ϩ H]ϩ
Mass
Accuracy
Modif.
Start End
E
Ligand
AA Sequence
Submitted
Matched
ppm
Ϫ72
Ϫ22
14
967.481
980.487
967.551
980.509
768 772
771 775
771 777
771 784
772 775
773 777
778 784
789 798
789 793
789 793
789 793
789 797
790 798
790 798
791 797
852 862
852 861
856 859
856 859
856 860
856 861
856 862
856 862
858 861
858 862
939 984
C
C
C
C
C
C
C
GPV51 FSLLFLALGIISFITFFLQGFTFGKAGEIL
BP11 FSLLFLALGIISFITFFLQGFTFGKAGEIL
1290.663 1290.645
1854.949 1855.001
GPV317 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV51 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV442 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GPV442 FSLLFLALGIISFITFFLQGFTFGKAGEIL
GP319 FSLLFLALGIISFITFFLQGFTFGKAGEIL
RYMVFRSMLR
Ϫ28
19
791.446
909.504
791.431
909.436
75
57
1118.567 1118.503
CL3
1084.622 1084.550
1042.570 1042.540
1042.524 1042.540
1545.756 1545.793
1649.821 1649.823
1561.780 1561.788
1225.643 1225.663
66
29
T
T
T
T
T
T
C
BP11
RYMVFRSMLR
GPV442 RYMVFRSMLR
GPV51 RYMVFRSMLR
GPV51 RYMVFRSMLR
GPV317 RYMVFRSMLR
GPV51 RYMVFRSMLR
GPV708 RYMVFRSMLR
IYGWQLTLLLL
Ϫ15
Ϫ24
Ϫ1
M-ox
M-ox
Ϫ5
Ϫ16
2 M-ox
TM10
1650.797 1650.919
784.453
801.442
956.609
Ϫ74
9
Ϫ39
44
Ϫ64
Ϫ21
Ϫ67
Ϫ14
Ϫ22
C
C
C
C
C
C
C
C
C
GPV317 IYGWQLTLLLL
GPV442 IYGWQLTLLLL
GPV51 IYGWQLTLLLL
784.446
801.473
956.567
BP11
GPV708 IYGWQLTLLLL
BP11 IYGWQLTLLLL
IYGWQLTLLLL
1025.578 1025.644
1182.710 1182.735
1230.694 1230.776
GPV319 IYGWQLTLLLL
GPV442 IYGWQLTLLLL
GPV317 IYGWQLTLLLL
786.487
786.498
1003.590 1003.612
TM11/12
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
TM11 ECL6 TM12
ECL6
764.428
764.420
10
40
939 942
939 950
939 951
939 951
939 951
939 951
939 951
939 953
943 950
943 951
943 953
945 950
945 951
945 957
951 953
951 959
951 962
960 971
960 971
963 968
963 971
963 975
963 975
964 971
964 976
964 976
964 971
964 971
969 975
969 975
969 976
969 976
969 976
969 976
976 978
976 983
976 978
976 983
979 994
979 994
984 994
1018 1037
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV319 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV442 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV442 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
1859.902 1859.828
2010.016 2009.886
1870.990 1870.877
1887.027 1886.871
1870.881 1870.877
1886.867 1886.871
2162.869 2162.982
2 M-ox
2 M-ox
65
60
83
M-ox
M-ox
2
Ϫ2
Ϫ52
66
BP11
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
978.472
978.407
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
1452.753 1452.655
1760.847 1760.755
1118.572 1118.506
1338.646 1338.579
1935.685 1935.797
67
52
M-ox
BP11
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
59
2 M-ox
M-ox
GPV708 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV708 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV442 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
50
Ϫ58
64
2 M-ox
847.446
847.392
1496.788 1496.719
1772.856 1772.811
1767.856 1767.919
1783.850 1783.914
1049.567 1049.636
1372.713 1372.767
1870.896 1870.999
1886.904 1886.994
1379.606 1379.708
1870.922 1870.999
1886.879 1886.994
1366.692 1366.703
1382.718 1382.698
1167.625 1167.561
1167.639 1167.561
1384.770 1384.675
1280.683 1280.645
1296.723 1296.640
1996.184 1996.133
46
25
Ϫ36
Ϫ36
Ϫ66
Ϫ39
Ϫ55
Ϫ48
Ϫ74
Ϫ41
Ϫ61
Ϫ8
14
M-ox
BP11
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV442 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
BP11
BP11
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
M-ox
M-ox
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
BP11
BP11
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSPEDVLLVFSAVVFGAMAVGQVSSF
M-ox
M-ox
GPV319 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV319 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV442 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV442 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV442 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV708 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV319 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV51 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
55
67
69
30
64
M-ox
26
705.409
1312.625 1312.724
705.409 705.419
1208.654 1208.694
1925.866 1925.968
2006.906 2006.937
1500.745 1500.709
705.419
Ϫ14
Ϫ75
Ϫ14
Ϫ33
Ϫ53
Ϫ15
24
BP11
GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV319 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
GPV317 GITFSFTQAMMYFSYAGCFRFGAYLVAHKLMSFEDVLLVFSAVVFGAMAVGQVSSF
IDSYSTEGLMPNTLEGNVTF
Table continued on next page.
the correlation of calculated logP values and the logarithm of licity). All data points are located within the 95% confidence
the Pgp-inhibitory potency for a series of ortho-acylphe- interval of the linear regression line (dotted lines in Fig. 2).
noxypropanolamines, with R being methyl, ethyl, phenyl, The high value of the correlation coefficient (r2 ϭ 0.997)
phenylethyl, or naphthylethyl (in order of increasing lipophi- demonstrates that R influences pharmacological activity

644
Ecker et al.
TABLE 3
Continued
m/z (mi) [M ϩ H]ϩ
Mass
Modif. Start End
E
Ligand
BP11
AA Sequence
Accuracy
Submitted
Matched
ppm
Ϫ22
Ϫ74
Ϫ53
Ϫ60
18
866.396
824.344
866.415
824.405
1018 1021
1018 1021
1018 1026
1018 1026
1018 1026
1018 1031
1022 1031
1022 1031
1022 1031
1027 1031
1027 1037
1027 1037
C
C
C
C
C
C
C
C
C
C
C
C
IDSYSTEGLMPNTLEGNVTF
GPV51 IDSYSTEGLMPNTLEGNVTF
1353.715 1353.643
1311.554 1311.633
1311.657 1311.633
1925.866 1925.905
1447.740 1447.699
1389.686 1389.694
1389.667 1389.694
1025.536 1025.481
1656.730 1656.778
1564.807 1564.788
BP11
IDSYSTEGLMPNTLEGNVTF
GPV51 IDSYSTEGLMPNTLEGNVTF
GPV442 IDSYSTEGLMPNTLEGNVTF
Ϫ20
28
BP11
BP11
IDSYSTEGLMPNTLEGNVTF
IDSYSTEGLMPNTLEGNVTF
Ϫ6
GPV 51 IDSYSTEGLMPNTLEGNVTF
GPV442 IDSYSTEGLMPNTLEGNVTF
GPV319 IDSYSTEGLMPNTLEGNVTF
GPV319 IDSYSTEGLMPNTLEGNVTF
GPV708 IDSYSTEGLMPNTLEGNVTF
Ϫ19
54
Ϫ29
12
solely by its contribution to overall lipophilicity of the mole- Three of these six compounds were varied on the nitrogen
cules. Thus, replacement of the phenylethyl group, which is atom (diethylamine (GPV51), piperidine (B59), and p-fluoro-
characteristic for PAs, by phenyl, which renders the com- phenylpiperazine (GPV319)). In two compounds (BP01, B47),
pound a benzophenone, is not critical with regard to the the central aromatic ring was replaced by the bioisosteric
interaction of the carbonyl group.
structures thiophene or pyrazine, respectively. Compound
In a next step, the phenylpropiophenone core structure GPV443, which carries an iodo atom attached to the benzo-
was therefore replaced by benzophenone. Subsequently, the phenone core, was designed as a potential radioligand. This
central aromatic ring system, the carbonyl group or substitu- modification did not lead to a change in the log potency/logP
ents of the nitrogen atom were varied to prove that the ratio.
structure-activity relationship pattern is retained for this
GPV317 showed higher activity than predicted on basis of
subclass of compounds. Figure 3 shows a plot of calculated its logP value, which was due to the previously demonstrated
logP values versus log potency values for daunorubicin efflux beneficial effect of the hydroxyphenylpiperidine group (Tmej
inhibition. The solid line represents the linear relationship et al., 1998). Based on the favorable minus sigma effect of the
for a series of PAs, as established previously (Chiba et al., methoxy group, which increases the hydrogen bond acceptor
1996). Six benzophenones with a broad variation in lipophi- strength of the carbonyl oxygen, BP11 was also located above
licity (logP 1.78–4.87) were located within or close to the 95% the correlation line. This is in agreement with results ob-
confidence interval represented by the dotted lines in Fig. 3.
Fig. 3. Plot of calculated logP versus log potency for a series of benzo-
Fig. 2. Plot of calculated logP versus log potency for a series of o-
acylphenoxypropanolamines with different substituent R2 (compare Ta-
ble 2). Data points represent, in order of increasing lipophilicity, com-
pounds GPV17 (methyl), GPV12 (ethyl), B59 (phenyl), GPV05
(phenylethyl), and GPV180 (naphtylethyl). The linear regression is
shown as a solid line. Dotted lines represent the 95% confidence interval.
Error bars are given for at least triplicate determinations. If not shown,
error bars are located within symbols.
phenone derivatives (E). The solid line represents the correlation line
previously established for a series of PAs (cf. Chiba et al., 1996. Linear
regression line and 95% confidence interval for substances 1c, 1d, 1e, 1g,
1h, 1i, 1k, and 1l). BP11 and GPV317 (ࡗ) are compounds containing a
benzophenone structure of which the corresponding phenylpropiophe-
none analogs have previously been shown to be located above the corre-
lation line. BP23 (F) is the compound lacking a carbonyl oxygen. If not
shown, error bars are located within symbols.

Photoaffinity Labeling of P-Glycoprotein
645
tained for the corresponding PAs (P. Chiba, G. Ecker, unpub- of 2.75 ␮M. This is in agreement with data obtained from
lished observations). Reductive removal of the carbonyl oxy- irradiation inactivation experiments (cf. Fig. 4).
gen led to compound BP23, which showed an approximately
Nonradioactive GPV51 competed with [3H]GPV51 binding
3.3-fold lower activity than predicted on basis of its lipophi- in a dose-dependent manner in both CCRF adr5000 T-lym-
licity. These data unequivocally demonstrate the importance phoblasts and baculovirus-transduced Sf9 insect cells (Fig. 5,
of the carbonyl group as a pharmacophoric substructure.
A and B, lanes 2–8). Competition was visible starting at 12.8
Irreversible Inhibition of Pgp by Irradiation in the ␮M and was almost complete at 32 ␮M. A similar picture was
Presence of GPV51. Fig. 4 shows that irradiation of Pgp in obtained when the nonphotoactivatable methoxy-derivative
the presence of GPV51 led to irreversible inactivation of Pgp GPV90 was used as the competitor (Fig. 5C).
function. A concentration-dependent increase in steady state
Identification of Ligand Modified Fragments by
fluorochrome accumulation was observed with increasing MALDI-TOF Mass Spectrometry. Six ligands (BP11,
concentration of GPV51 (Fig. 4, F). At each concentration of GPV51, GPV317, GPV319, GPV442, and GPV708) were used
GPV51, a 100-fold molar excess of the nonphotoactivatable in subsequent photoaffinity labeling studies. After irradia-
propafenone analog GPV90 prevented the inactivation of tion of PM vesicles in the presence of ligand, samples were
Pgp. This indicated high specificity of the photolabeling re- separated by polyacrylamide gel electrophoresis and protein
action (Fig. 4, ‚). A similar dose-dependent inactivation was bands were visualized by silver staining. The band corre-
observed with all other benzophenone analogs (data not sponding to Pgp was excised and digested with trypsin and
shown). Under identical irradiation conditions, Pgp was not chymotrypsin in separate reactions. Trypsin digests yielded
inactivated by phenylpropiophenones.
protein fragments that were derived from putative cytoplas-
Radiolabeling of Plasma Membrane Proteins by
[3H]GPV51 and Competition by Unlabeled GPV51 and
GPV90. Pgp was separated in 7.5% polyacrylamide gels as
described under Materials and Methods. The band corre-
sponding to Pgp was visualized by silver staining, excised,
and purity was assessed by MALDI-TOF MS fingerprinting.
For samples in which plasma membrane vesicles were irra-
diated in the presence of [3H]GPV51, one major band was
visible in fluorographs. By Western blotting, this band was
confirmed to correspond to Pgp (data not shown). In Pgp-
expressing CCRF T-lymphoblasts, the band had a molecular
mass of 170 kDa (Fig. 5A, lane 1), whereas in Sf9 insect cells,
in which the protein is only core glycosylated, Pgp migrated
at 140 kD (Fig. 5, B and C, lane 1). According to the specific
activity of [3H]GPV51 (20 Ci/mmol), given the number of
disintegrations per minute associated with the Pgp-band and
an estimated protein amount of 10 ng, the labeling efficiency
computed to be approximately 15% at a ligand concentration
Fig. 4. Irreversible inactivation of Pgp by UV-irradiation in presence of
the benzophenone GPV51. The steady-state accumulation levels of Rh123
are given as mean fluorescence units per cell at increasing concentration
of GPV51 (F) and in the simultaneous presence of GPV51 and the non-
photoactivatable analog GPV90 (‚). Symbols and error bars represent
the mean and S.D. of two independent experiments performed in dupli-
cate. Samples in which Pgp was blocked by the addition of 10 ␮M GPV31
served as controls (649 Ϯ 108 mean fluorescence units) for complete
inactivation.
Fig. 5. Fluorography of 7.5% polyacrylamide gels of plasma membrane
preparations of CCRF adr5000 cells (A) or Sf9-cells infected with the
baculovirus vector containing the mdr1 gene (B and C). The prominent
band visible in samples irradiated in the presence of [3H]GPV51 is that
corresponding to Pgp (arrows). In the presence of either unlabeled GPV51
(A and B) or GPV90 (C), intensity of the Pgp-band decreased. Concentra-
tions of competitor were 500 ␮M (lane 2), 200 ␮M (lane 3), 80 ␮M (lane 4),
32 ␮M (lane 5), 12.8 ␮M (lane 6), 5.1 ␮M (lane 7), and 2.1 ␮M (lane 8),
respectively. Lane 9 represents unirradiated control samples.

646
Ecker et al.
mic (CL) and extracytoplasmic loops (ECL). An almost com- covering the same region were also recovered. Three cys-
plete recovery of transmembrane segments was however ob- teine-containing fragments that were accessible to ligand
tained in chymotrypsin digested samples. The sequence modification were recovered for putative TM11. These cys-
coverage was approximately 50% for trypsin digests alone teines are chemically modified by carbamidomethylation
and nearly 60% for samples digested with chymotrypsin. during sample preparation. Correct mass-prediction for
When combining the sequence coverage obtained with either chemical modifications on one hand and varying (ligand-
enzyme alone, more than 80% of the Pgp protein sequence mass dependent) peak shifts on the other hand confirmed
was recovered. Identity of the protein fragments was sup- peptide identity.
ported by detection of complete and partial modifications of
amino acid residues introduced during sample processing
(compare Table 3). These were complete chemical modifica-
Discussion
tion of cysteine-residues (carbamidomethylation), partial ox-
We studied the interaction of Pgp with low-molecular-mass
idation of methionine residues and partial formation of pyro- ligands by designing a new class of photoactivatable mole-
glutamic acid in peptides containing N-terminal glutamines. In cules. PAs and benzophenones have in common an arylcar-
addition, use of a set of ligands resulted in shifts of peptide mass bonyl moiety. The carbonyl group was identified as a phar-
peaks to different composite masses, depending on the exact macophore and evidence for an interaction via hydrogen bond
mass of the respective ligand.
formation was obtained (Chiba et al., 1996). This allowed the
Unmatched fragment masses recovered in control samples direct use of a pharmacophoric substructure as a photoacti-
(irradiated in the absence of photoaffinity ligands) were ex- vatable group. Design of newly synthesized compounds was
cluded from further consideration. Monoisotopic peak masses guided by replacement of the phenylpropiophenone core
that did not correspond to unmodified Pgp fragments were structure by benzophenone. The advantages of benzophenone
then matched to the sum of the exact mass of the individual
ligand and the monoisotopic mass of protein fragments ob-
tained in in silico digests. Up to five missing enzyme cleavage
sites were considered. Each sample was measured repeat-
edly. Additional measurements were performed on samples
in which hydrophobic fragments were enriched by use of a
reversed phase material as described under Materials and
Methods. A representative mass spectrum is shown in Fig. 6.
Consensus labeling regions were defined by frequency dis-
tribution analysis, whereby the required number of ligands
binding within these regions was set to a minimum of four of
six. Results are summarized in Table 3. Submitted and
matched peptide masses, mass accuracy in parts per million,
position of start and end amino acid of the peptide fragment,
amino acid sequence, and ligand and putative location within
Pgp are specified. In addition, the enzyme used in digests is
indicated (C, chymotrypsin; T, trypsin). The following protein
regions were identified to contain covalently bound ligands:
putative TM3 (AA residues 194–208), TM5 (AA 304–316),
TM6 (AA 327–343), a region in putative TM8 extending
seven amino acids into the C-terminal cytoplasmic loop 3
(CL3) (AA 755–784), TM10 (AA852–862), and a sequence
stretching from AA 939 in TM11 to AA 994 in TM12 (includ-
ing the extracytoplasmic loop 6). In addition, CL2 was la-
beled in a region confined to AA 272 to 291. All fragments
recovered in the latter region showed an overlap with the
so-called EAA-like motif, which is located between amino
acids 278 and 294 (Shani et al., 1996). CL3 was labeled in the
region extending from AA 789 to 798. In addition, overlap-
ping fragments were found for the region spanning AA 1018
to 1037. With the exception of TM6 and TM12, in which
ligand-modified fragments were identified for only four of six
ligands, fragments covalently modified by either five or all
six of the ligand molecules were found for the other regions.
Multiple overlapping fragments were found within the spec-
ified regions (Table 3). In putative TM3 and TM11, as well as
in CL3 and ECL6, fragments containing oxidized methionine
residues were detected. Conversion of N-terminal glutamines
to pyroglutamic acid was detectable for TM3 and occurred
either alone or in combination with methionine oxidation.
Fig. 6. Positive ion MALDI mass spectrum of chymotryptic peptides
detected after the in-gel digestion of the modified (a) and control (b) Pgp.
Identified, unmodified Pgp-peptides (ء), as well as chymotrypsin autoly-
sis peptides (ࡗ) are labeled accordingly. Inset, presence of a modified
peptide (IDSYSTEGL; AA 1018–1026) in the sample treated with the
Because both modifications are partial, unmodified peptides ligand GPV51 (a) that is absent in controls (b).

Photoaffinity Labeling of P-Glycoprotein
647
photochemistry were subject to comprehensive reviews by man peroxisomal ABC transporters indicate the regions im-
Dorman and Prestwich (1994, 2000). portance for functionality. On basis of sequence conservation
To demonstrate a comparable structure-activity relation- and the results of mutational analysis, the EAA-like motif
ship pattern for both PAs and benzophenones, variations was suggested to mediate the interaction between TMD and
were introduced in the molecules on basis of previously de- NBD (Shani et al., 1996). Binding of substrate-type ligands
fined pharmacophoric key features of PAs. These included within this region might thus be required for coupling ATP-
modifications of the central aromatic ring system and the binding and/or hydrolysis to substrate translocation.
carbonyl group as well as variations in the vicinity of the
Additional support for an involvement of putative TM5, -6,
nitrogen atom. As shown under Results, an analogous QSAR -10, -11, and -12 comes from data using cysteine-scanning
pattern was obtained for benzophenones and phenylpropio- mutagenesis in combination with substrate protection assays
phenones (Figs. 2 and 3).
to characterize the drug binding domain of Pgp (Loo and
Irradiation of Pgp-expressing CCRF vcr1000 cells in the Clarke, 2000, 2001b). A number of amino acid residues,
presence of these benzophenone ligands led to irreversible, which these authors proposed to be oriented toward the drug-
dose-dependent inactivation of Pgp. This inactivation was binding domain shared by verapamil, vincristine, and colchi-
reversed by the presence of the nonphotoactivatable methoxy cine, are located within regions of Pgp, which have now been
analog GPV90. Results from both QSAR studies and irradi- identified as being accessible to covalent modification by
ation inactivation experiments indicate identical binding do- propafenone analog-type photoaffinity ligands. These include
mains for photoligands and propafenones.
When irradiated in the presence of tritiated GPV51, Pgp and T945 in TM11 and L975, V982, G984, and A985 in TM12.
represented the major radiolabeled plasma membrane pro- In conclusion, the present study demonstrates that the
I306 of putative TM5, L339 and A342 in TM6, as well as F942
tein in fluorographs. Radiolabeling was competed by unla- binding pattern for propafenone type analogs is distinct and
beled GPV51 as well as the methoxy-derivative GPV90. This is composed of regions that have previously been proposed to
was initial evidence for the specificity of the labeling reac- be involved in binding of other ligand molecules including
tion.
vinblastine, cyclosporins, iodoarylazidoprazosine, verapamil,
Irradiation of Pgp in the presence of a set of photoligands, and colchicin.
proteolytic in-gel digestion, and subsequent MALDI-TOF
The set of affinity ligands introduced in this study com-
mass spectrometry led to the identification of a number of bines several favorable properties. First, molecules are in-
covalently modified protein fragments. Regions of the mole- trinsically photoactivatable. Structural modification by in-
cule, which are accessible to covalent ligand modification, sertion of a photoactivatable group is therefore unnecessary.
include putative TM3, -5, -6, -8, -10, -11, and -12. In addition, Second, quantitative structure activity relationships have
confined regions in the second and third cytoplasmic loop as been well established for this group of compounds. Third,
well as ECL6 and a region spanning AA 1018 to 1037 were chymotrypsin digests yield a large number of protein frag-
found to bind the photoligands.
ments. However, use of a set of ligand molecules of different
Earlier affinity labeling studies used analogs of vinblas- molecular mass allows accurate identification of consensus
tine, the dihydropyridine azidopine, iodoarylazidoprazosine, binding regions.
forskolin and an iodinated derivative (iodoarylazidoprazos-
Finally, the quaternary compound GPV06 has previously
ine-forskolin), tamoxifen aziridine, as well as two benzophe- been demonstrated to be a substrate for Pgp (Schmid et al.,
none analogs of paclitaxel. Comprehensive reviews on the 1999). The analogous benzophenone GPV708 shows a label-
subject have been published (Beck and Qian 1992; Dey et al., ing pattern that corresponds to that of the other ligand mol-
1998; Greenberger, 1998; Safa, 1998). In combination with ecules. Therefore, this compound will provide a valuable tool
immunological mapping, these studies led to the identifica- for studying binding regions for substrate molecules at dif-
tion of photoaffinity drug-binding epitopes. The position of ferent stages of the catalytic cycle. Systematic ligand modi-
the drug-binding domain in the N-terminal half of Pgp was fication in combination with high-resolution mass spectrom-
proposed to span putative TM5 and TM6 (Greenberger, etry represents a powerful tool to obtain information on
1998). In the C-terminal half, ligand-binding regions have binding-domain organization and might thus link structural
been reported to extend from the C terminus of TM11 to the information at atomic resolution to functional aspects.
N terminus of the Walker A motif of NBD2 (Greenberger,
The ligand binding regions identified in this study contain
1998). Isenberg et al. (2001) recently used [125I]iodoarylazi- hydrogen bond donors, such as T199, S309, S766, T862,
doprazosine to obtain radioactively labeled peptide frag- T941, and Y950, that are highly conserved within mamma-
ments that were isolated and sequenced. The authors iden- lian P-glycoproteins. An interaction of Pgp with ligands via
tified amino acid regions 248 to 312 and 758 to 800 as being hydrogen bond formation has been demonstrated (Ecker et
involved in binding of this radioligand.
al., 1999; Seelig and Landwojtowicz, 2000). Upon addition of
Eight protein regions characterized in this study as con- ATP, a relative repositioning of putative TM6 and -12 has
tributing to the binding domain of propafenone analogs recently been shown (Loo and Clarke, 2001a). Furthermore,
[AA272–286 (CL2), AA304–316 (TM5), AA327–343 (TM6), repacking of the transmembrane domains of P-glycoprotein
AA755–784 (TM8), AA789–798 (CL3), AA958–975 (ECL6), during the transport ATPase cycle has been demonstrated by
AA976–994 (TM12), and AA1018–1037] fall within those electron cryomicroscopy (Rosenberg et al., 2001). We propose
protein regions specified above as binding structurally differ- that this movement relocates the ligand from the hydropho-
ent Pgp-photoligands.
bic membrane environment to the aqueous environment of
The region extending from AA 272 to AA 286 in CL2 the Pgp-chamber and that this relocation results in an in-
overlaps with a region known as the so-called EAA-like motif stantaneous cleavage of hydrogen bonds by water molecules.
(Shani and Valle, 1996). Disease-producing mutations in hu- The accompanying decrease in affinity might be the crucial

648
Ecker et al.
tion of three photobinding sites of iodoarylazidoprazosin in hamster P-
glycoprotein. Eur J Biochem 268:2629–2634
step leading to a release of the ligand-molecule from the
protein. We believe that the availability of this set of ligand
molecules will allow experimental testing of this hypothesis.
Krishna R and Mayer LD (2000) Multidrug resistance (MDR) in cancer Mechanism,
reversal using modulators of MDR and the role of MDR modulators in influencing
the pharmacokinetics of anticancer drugs. Eur J Pharmacol Sci 11:265–283.
Lomovskaya O and Watkins W (2001) Inhibition of efflux pumps as a novel approach
to combat drug resistance in bacteria. J Mol Microbiol Biotechnol 3:225–236.
Loo TW and Clarke DM (1998) Mutational analysis of human P-glycoprotein. Meth-
ods Enzymol 292:480–492.
Loo TW and Clarke DM (2000) Identification of residues within the drug-binding
domain of the human multidrug resistance P-glycoprotein by cysteine-scanning
mutagenesis and reaction with dibromobimane. J Biol Chem 275:39272–39278.
Loo TW and Clarke DM (2001a) Cross-linking of human multidrug resisntace P-
glycoprotein by the substrate Tris-(2-maleimidoethyl)amine, is altered by ATP
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31800–31805.
Acknowledgments
The baculovirus expression vector pVL941-MDR1/A was gener-
ously provided by Dr. M. M. Gottesman (National Cancer Institute,
Bethesda, MD). We thank Andrea Barta for stimulating discussion.
Dedicated to W. Fleishhacker and E. Kaiser.
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