Download Structural Requirements of Simple Organic

[CANCER RESEARCH 52, 6385-6389. November 15, 1992]
Advances in Brief
Structural Requirements of Simple Organic Cations for Recognition by
Multidrug-resistant Cells1
Marc Dellinger, Berton C. Pressman, Cesar Calderon-Higginson, Niramol Savaraj, Haim Tapiero,
Despina Kolonias, and Theodore J. Lampidis2
Museum d'Histoire Naturelle, Departement de Biophysiques, Paris, France ¡M.D.]; Department of Molecular and Cellular Pharmacology, University of ¡Miami,
School of Medicine, [B. C. P., C. C-H.J; Veterans Administration Hospital and Division of He matology/Oncology, University of Miami /A'. S.J; ICIG, Hôpital Paul
Brousse, Villejuif, France ¡H.TJ; and Department of Medical Oncology, University of Miami, School of Medicine, Miami, Florida 33136 [D. K., T. J. L.J
Abstract
We previously noted that a wide variety of drugs which are recognized
by multidrug-resistant cells (MDR+) are positively charged. However,
it remains unclear why and how such a large number of structurally
different compounds can be distinguished by MDR+ cells. The majority
of the diverse compounds subject to MDR are complex and thereby
complicate definitive structure/function characterization of the P-glycoprotein-mediated MDR mechanism. Using a series of simple aromatic
(alkypyridiniums) and nonaromatic (alkylguanidiniums) organic cations
differing in their lipophilicity by stepwise additions of single alkyl car
bons, we demonstrate by growth inhibition studies that a single aromatic
moiety and a critical degree of lipophilicity (log P > -1) are required for
recognition of these simple organic cations by MDR+ cells. Thus,
MDR •¿
cells are not cross-resistant to the nonaromatic guanidiniums
but do show cross-resistance to those aromatic pyridiniums with chain
lengths >four. Resistance ratios, as determined by comparison of 50%
inhibitory doses in MDR—versus MDR+ cells, increase as a function of
increasing chain lengths of these latter simple aromatic compounds.
Resistance to pyridinium analogues in MDR+ cells is reversible by
cotreatment with nontoxic doses of verapamil. Preliminary uptake data
with radioactive analogues further implicate the MDR mechanism of
lowered drug accumulation in accounting for resistance to the pyridin
ium homologues.
Utilization of these simple organic cations provides a rational basis
for better defining the physical chemical properties of more complex
compounds processed by the MDR mechanism and suggests a strategy
for designing chemotherapeutic agents with reduced susceptibility to
MDR.
Introduction
MDR3 describes a phenomenon in which a given cell or cell
line, when exposed to a single agent, either displays or becomes
cross-resistant to a large number of other diverse agents. Spe
cific genes have been identified (1, 2) which code for a A/r
180,000 glycoprotein (P-gp) found in the plasma membrane of
cells which exhibit MDR (2, 3). P-gp is thought to aid in low
ering intracellular drug accumulation and consequently to ren
der the cell resistant by functioning either as an active efflux
pump (2, 4) or by other mechanisms (5-7).
Received 7/29/92: accepted 10/1/92.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in accord
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by NIH Grants CA37109 and GM38920, a Veterans
Administration National Grant, and a grant from the Sylvester Comprehensive
Cancer Center.
2 To whom requests for reprints should be addressed, at Department of Cell
Biology and Anatomy, University of Miami, School of Medicine. Fox Cancer
Research Building, 1550 NW 10th Avenue. Miami, FL 33136.
3 The abbreviations used are: MDR, multidrug resistance; P-gp, P-glycoprotein;
III. Friend leukemia cells; alkylGu*. alkylguanidiniums; alkylPy*. alkylpyridiniums; octylPy*, octylpyridiniums.
The question of why and how such a large number of drugs,
which are relatively complex and differ widely from one another
in structure, are recognized by MDR+ cells remains unclear.
We have previously reported that the cationic charge of xanthene dyes was critical for their differential accumulation in
certain normal versus tumor epithelial cell lines (8-10). It was
subsequently shown that the normal epithelial cell line (CV-1)
that had reduced accumulation of these cationic xanthene dyes
expressed high amounts of the MDR-1 gene (11) and also ac
cumulated less of the cationic anthracycline, Adriamycin (12).
With the xanthene dyes, we found that zwitterionic analogues
showed less selectivity between MDR- and MDR+ cells than
the positively charged compounds (13). Several independent
studies with other types of compounds support the hypothesis
that cationic charge is an important feature for MDR recogni
tion (14-17). Beck and coworkers (17-19) in a series of papers
have suggested that a number of complex compounds which
interact (bind and or block) with Pg-p, and thereby modulate
MDR, share in common the physical chemical properties of
lipophilicity, cationic charge, and at least two planar aromatic
domains. These compounds, however, as well as the majority of
compounds found to be cross-resistant to MDR+ cells, are
complex in structure which makes interpretation of their MDR
structure/function activity difficult. Here, by means of a series
of simple aromatic and nonaromatic cations, which we have
synthesized (Fig. 1), differing in lipophilicity by systematic
additions of alkyl carbons, the specific molecular characteristics
necessary for recognition by the P-glycoprotein-mediated MDR
mechanism have been examined.
Materials and Methods
Cell Lines. Previously, we reported that the MDR+ cells used here
were developed from parental MDR- FLC by exposure to stepwise
increases of Adriamycin and had the following typical MDR properties:
broad spectrum resistance to cytotoxic drugs (>2000-fold resistance to
Adriamycin as compared to MDR- cells); high levels of MDR-1 gene
expression; lowered plasma transmembrane potentials; and reduced
accumulation of Adriamycin, rhodamine 123, and other lipophilic cat
ions compared to their sensitive MDR—counterparts (20). Lower drug
accumulation in these MDR+ cells is reversed by the widely used MDR
modulator, verapamil. with concomitant reversal of resistance to these
agents (21).
Compounds. Alkylguanidiniums and alkylpyridiniums were synthe
sized as previously described (22). Verapamil was purchased from
Sigma (St. Louis, MO).
Growth Inhibition Assays. Growth inhibition assays were performed
by seeding exponentially growing sensitive (FLC) or resistant (ARN)
cells at 1.0 x IO5 cells/ml in Eagle's minimal essential medium sup
plemented with 10% fetal bovine serum at 37°Cin 10% CO2air and
treating each continuously with the indicated doses of guanidinium
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SIMPLE ORGANIC CATIONS AND MDR
and pyridinium analogues. At 72 h, cells excluding trypan blue
were counted, and survival curves were derived as previously described
(23).
Partition Coefficient Determinations. Partition coefficients for each
of the guanidinium and pyridinium analogues, expressed as log P, were
determined spectrophotometrically with the system: octanol-1 (highperformance liquid chromatography grade) versus l M Mg(ClO4)2. The
inclusion of the salt provides a lipophilic companion aniónfor the test
cation, so that the partition of the cation is a function of its intrinsic
lipophilicity.
Drug Uptake Assays. Exponentially growing MDR+ and MDRcells at 1.3 x 106/ml were treated, respectively, with either [l4C]guanidinium or l-4-[werA>'/-l4C]octylpyridinium for 60 min at 30°Cin
'•A
OUA
100
6a....
C2-GUA-Ht%¿A
,
O
tt
licity is progressively increased, by the addition of alkyl car
bons, the cytotoxic potency of these compounds increases ex
ponentially in both MDR- and MDR+ cells. Although these
nonaromatic compounds are positively charged, MDR+ cells
exhibit relatively little (<3-fold) or no cross-resistance to
them.
In contrast, we find that MDR+ cells are cross-resistant to
a series of aromatic cations, alkylPy+ (Fig. 1) of equivalent
lipophilicity to the alkylGu* which differ from them in that
U
o
'
NH~—C —¿
NHR
* +
Fig. I. Structures of guanidiniums and pyridiniums used in these studies. An
alogues differing by stepwise additions of single alkyl carbons were synthesized,
ranging in chain length from guanidinium to octylguanidinium. and from methylpyridinium to dodecylpyridinium.
C4-PYR0AA«
¿
•¿'£'
CS-P'YR-A
C3-GUA-
•¿*•m
*A.
•¿
A,
. .i.v
C4-GUA.\
'
10050GUANIDINIUMSSflA
100so0 C6-PYR-*8V•I
K
%
^AA
tt
C7-PYR*.'*'¡Õ1
100500100150A
$
O
se
o
C"""A"f..
their charge is delocalized by a single pyridine ring (Fig. 2).
Moreover, in addition to aromaticity, a critical chain length,
equivalent to a minimal degree of lipophilicity (log P > —¿1.00;
Table 1), is required before MDR+ cells show cross-resistance
to these simple aromatic compounds. To determine whether the
position of the alkyl chain is also critical for MDR recognition,
we synthesized l-methyl-4-octylPy+ and found that MDR+
cells show a similar level of cross-resistance to this compound
as to its yV-alkyl counterpart, l-octylPy+. Thus, the chain
length, i.e., degree of lipophilicity of the aromatic alkylPy+, but
NH
•¿
.«&.. .
o
u
lengths of C7, CIO and C12 in MDR+ cells is reversed by
cotreatment with nontoxic doses of verapamil, further support
the involvement of the MDR efflux mechanism for these com-
'
%û
....fe•
Results
Growth-inhibitory Activity of Alkylguanidiniums and Alkylpyridiniums in MDR+ and MDR- Cells. The inhibition of
growth after 72-h exposure to the series of nonaromatic cations,
alkylGu+ (Fig. 1), as shown in Fig 2, indicates that as lipophi
of MDR is its sensitivity to modulation by the calcium channel
antagonist, verapamil (25), and consequently, verapamil has
been used to verify P-gp-mediated MDR (4). Thus, the data in
Fig. 3, which show that cross-resistance to alkylPy"1"with chain
1
•¿
.¡ft
600100500100so0
5%CO2/95% air. At this time both compounds reach equilibrium in
MDR+ and MDR- cells. Intracellular uptake was determined accord
ing to the methods previously described (24).
not the position of the alkyl groups, determines the recognition
of cations by the P-gp-mediated MDR process.
Reversal of Cross-Resistance in MDR+ Cells to C7, CIO, and
C12 AlkylPy* Analogues by Verapamil. One of the hallmarks
C1-PYR
10050010050010050PYRIDINIUMS'
*
-
•¿V4'•s
AAAAA
so •¿â€¢C10-PYR
,AA.
DRUG
CONCE
NTRATION
.
(M)
Fig. 2. Seventy-two-h growth inhibition assays of guanidiniums (left) and pyridiniums (right), varying in lipophilicity by the stepwise addition of alkyl carbons
(GUA to C8-GUA and Cl-PYR to C-IOPYR) in MDR- (•)and MDR+ (A)
Friend leukemia cells. Note the lack of difference in growth inhibition between
MDR- and MDR+ cells produced by the alkylguanidiniums. In contrast, for the
alkylpyridiniums, beginning with ( \ l'\ K. differences in growth inhibition be
tween MDR—and MDR+ cells increase as a function of increasing lipophilicity.
Each point represents the average cell count of duplicate cultures, and for each
analogue experiments were repeated at least twice.
pounds. Additional corroboration is supplied by preliminary
data indicating that drug retention of l-4-[mefA>'/-14C]octylPy+
is severalfold greater in MDR—than in MDR+ cells. However,
accumulation differences between MDR—and MDR+ cells for
the nonaromatic cationic [14C]Gu+ are minimal. These results
indicate that differences in the level of drug retention correlate
qualitatively with differential cytotoxicity.
Relationship of Partition Coefficients, Chain Lengths, 50%
Inhibitory Doses, and Resistance Ratios. The octanol:H2O
partition coefficients (expressed as log P), chain lengths, 50%
inhibitory doses, and resistance ratios (MDR+/MDR—) for
6386
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SIMPLE ORGANIC CATIONS AND MDR
Table 1 Partition coefficients (log P), 50% inhibitory doses in MDR+
and MDR- cells, and resistance ratios (MDR+/MDR-) of each of
the listed alkylguanidiniums and alkylpyridiniums with
different alkyl chain lengths
Note lack of difference between 50% inhibitor) doses in MDR+ and MDRcells (resistance ratio) for the nonaromatic alkylguanidiniums. In contrast, for
the aromatic alkylpyridiniums of chain length >4, the resistance ratios increase
in accordance with increasing chain length and log P. Fifty % inhibitory doses
represent the dose of test compound which yields 50% growth (cell number) after
72-h treatment as compared to the growth of untreated cells. These values are the
average of replicate cultures in each experiment, which was repeated at least
twice.
an aromatic moiety inhibits the ability of these compounds to
access or adsorb to the P-gp for subsequent expulsion from the
cell.
Discussion
Previously, Pressman (22) demonstrated that the uptake of
the simple nonaromatic orgainic cation, guanidine, with rela
tively low lipophilicity, which enters the mitochondrial matrix
freely without being trapped within the nonpolar environment
of membranes, could serve as an accurate measure of mitochon
MDR(M)2.0
MDR+4.0
ratio2.03.01.51.31.31.01.30.81.28.58.671.037.0236.0400.0
CompoundGUAC2-GUAC3-GUAC4-GUAC5-GUAC6-GUAC8-GUACl-PYRC4-PYRC5-PYRC6-PYRC7-PYRC8-PYRC10-PYRC12-PYRLog
P-1.00-0.18+0.25+0.58+
drial membrane potential. Furthermore, he showed a relation
10~33.0
x
IO-31.0
x
ship between an increased inhibitory effect on respiration in
10-"8.0
x
I0~45.5
x
10~52.0
x
10~52.6
x
isolated mitochondria and increased lipophilicity in the same
10~51.4
x
IO'51.1
x
series of guanidinium homologues used here (22). He reasoned
10~54.0
x
10~54.0
x
1.00+
that, since electron transport and oxidative phosphorylation
10~61.3
x
10~61.7
x
1.40+2.25-2.10-1.00ND"+0.11+0.60+
10^63.0
x
10~62.5
x
take place within the membranes of mitochondria, the more
10~33.0
x
10^32.5
x
lipophilic guanidiniums would accumulate there and thus be
10~58.5
x
10~41.0
x
10~43.0
x
10~43.5
x
more potent inhibitors of these processes.
10~57.0
x
IO'45.0
x
Since we had found that the positive charge for certain more
IO'62.3
x
10~48.5
x
complex compounds was involved in their recognition by
10~51.3
x
10~65.5
x
1.30ND+2.60lD5o°
10~76.0
x
10-"2.4
x
MDR+ cells (12,13,21) and others had shown this for different
x 10~5IDso
x IO'8Resistance
families of equally or more complex compounds (14-19), we
" ID50, 50% inhibitory dose; GUA. alkylguanidiniums; PYR. alkylpyridiniums;
were prompted to use these simple organic cations to probe the
ND, not determined.
relationship between lipophilicity and recognition by P-glycoprotein-mediated MDR. In addition, since the guanidiniums
each of the compounds tested are presented in Table 1. It is did not contain an aromatic moiety but the pyridiniums did, we
apparent that for alkylGu"1" and alkylPy"1", toxicity in both
utilized these two related structures to determine the impor
MDR- and MDR+ cells increases as a function of their alkyl tance, if any, that a single aromatic ring may have for recogni
tion by MDR+ cells.
chain length and their correspondingly increasing partition co
As noted above, we found that the increased inhibition of
efficients. Thus, the more lipophilic the compound, the more
growth in intact MDR+ and MDR—cells, which we detect as a
cytotoxic it is toward both MDR—and MDR+ cells. However,
for the alkylPy+ with chain lengths >4, toxicity for MDR+ cells function of increasing lipophilicities of the guanidinium com
lags significantly behind that for MDR—cells. The potencies of pounds tested here, correlates with their potency to inhibit res
longer chain alkylPy"1"for MDR— cells continue to increase
piration in isolated mitochondria (22). Whether or not inhibi
exponentially as a function of their increasing alkyl chain
tion of mitochondrial respiration is the primary mechanism by
which these compounds inhibit growth in whole cells remains
lengths. Thus, a simple correlation exists between lipophilicity
and cytotoxicity for the alkylGu"1"in both MDR—and MDR+
to be determined. More importantly, however, we find that the
cells. This relationship also applies to MDR- cells for the pyridiniums also inhibit growth as a function of their increasing
alkylPy"1",but in MDR+ cells, cytotoxicity due to these aro
lipophilicity but only in MDR—cells. In MDR+ cells, we find
that the growth-inhibitory potency of pyridinium homologues
matic cations increases less sharply when the partition coeffi
cients of the alkylPy* rise above a critical value, i.e., log P > with log P > —¿
1 does not correlate nearly as well with increas
—¿
1.00. Accordingly, the lipophilicity and aromaticity of the
ing lipophilicities, since MDR recognizes these aromatic com
pounds, which results in lowered drug accumulation. Our data
simple cationic compounds examined facilitate either their pre
sentation, or binding, to the MDR P-gp. Moreover, the results,
with reversal of resistance to the pyridinium analogues to which
with the nonaromatic cationic alkylGu"1"suggest that absence of MDR+ cells show cross-resistance, by the standard MDR
C7-PYRIDINIUM
C10-PYRIDINIUM
100 •¿
Fig. 3. Seventy-two-h growth inhibition assays
of pyridiniums with alkyl chain lengths of 7, 10,
and 12 incubated with (below) or without (above)
cotreatment of 10 »jg/mlof verapamil. Note that
differences in sensitivity to these compounds be
tween MDR- and MDR+ cells continue to in
crease as a function of increasing lipophilicity.
These differences are markedly reduced or elimi
nated when both cell types are cotreated with ver
apamil. Each point represents the average cell
count of replicate cultures, and for each analogue
experiments were repeated at least twice.
C12-PYRIDINIUM
100.
zH5OK"OJU)U^oK
A
A
50
50z0O
A$A«UA°100zIIIg
'
HA
+VP_HDA.4*
-
'
JL^_
•¿.„••4â€
•¿
'
100
150-•
50
50UJO.n'flA'
•¿A
10
10"
li"
If
10"
DRUG
10'
1»' l«'5 IO'4 IO'5 IO'2 10
CONCENTRATION
10 7 10 * 10 ! 10 ' If3
(M)
6387
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SIMPLE ORGANIC CATIONS AND MDR
modulator, verapamil, further support this interpretation. Ad
ditionally, our preliminary uptake data with radioactive octylpyridinium and radioactive guanidinium also implicate the
MDR mechanism for the pyridiniums to which MDR+ cells are
cross-resistant.
Thus, in comparison to other studies with substantially more
complex organic cations in which it was shown that two planar
aromatic moieties were required for modulatory activity in
MDR+ cells (18), our results with simple organic cations show
that only a single aromatic moiety is necessary for MDR-recognition. Moreover, we define a critical chain length or lipophilicity which must be attained before these compounds can be
recognized by the P-gp-mediated MDR mechanism.
Although it has been shown that MDR P-gp has homology to
a number of eukaryotic and prokaryotic glycoproteins known to
function as pumps or transporters of specific proteins and
inorganic ions (26-30), the MDR P-gp does not display a
typically selective lock-and-key fit, characteristic of enzymesubstrate interactions. The wide variety of compounds which
are processed by the MDR P-gp-effluxing system argues rather
in favor of a less selective process, such as adsorption. In this
regard, Sehested et al. (5) showed that MDR+ cells undergo
accelerated endocytosis and proposed a nonspecific adsorptive,
lysosomal ion-trapping mechanism to account for expulsion of
cationic drugs from MDR-t- cells. However, compounds with
hard cationic charges such as rhodamine 123 (and the alkylPy*
used here) which are processed by the MDR mechanism are not
candidates for ion trapping (31) by acidic lysosomes. Therefore,
nonselective ion-trapping alone cannot entirely account for the
handling of all MDR drugs. More recently, Warren et al. (6)
have demonstrated in an MDR-t- cell line in which P-gp is
overexpressed that the content of lysosomal enzymes is reduced
and that the relative rates of execretion of these enzymes are
significantly greater as compared to those of its MDR—coun
terpart. Their data suggest a linkage between accelerated lyso
somal enzyme elimination and enhanced drug efflox, since both
processes are inhibited by verapamil.
Higgins and Gottesman (7) suggested that the MDR P-gp
may function as a flippase, which would also invoke an adsorp
tive process to explain the wide variety of lipophilic organic
compounds effluxed from MDR+ cells. While the invocation of
a flippase to explain MDR remains speculative, it is of interest
that this mechanism also involves a relatively unselective ad
sorptive process. Our data, showing that a critical lipophilicity
for the aromatic alkylPy"1"must be reached before MDR+ cells
show cross-resistance to them and that cross-resistance in
creases as a function of increasing lipophilicity, are more con
sistent with an adsorptive process than with a substrate selective
pump mechanism.
Regardless of the mechanism(s) involved, the data presented
here clearly demonstrate that for simple cytotoxic organic cat
ions, an aromatic moiety and a critical degree of lipophilicity
are required for MDR recognition. The question of whether the
same physical chemical properties which we have identified for
differential toxicity by MDR+ and MDR-cells will also prevail
for MDR modulation toward other chemotherapeutic agents
such as Adriamycin, with the pyridinium and guanidinium an
alogues used here, remains to be determined.
The data presented could serve to provide a rational basis
for better understanding the structural requirements of more
complex molecules that interact with the MDR mechanism. If
the principles derived from this study apply to the chemother
apeutic drugs which are currently used in the clinic, but are
compromised by MDR, then it may be possible to design ana
logues with the appropriate chemical structures which would
protect them from attenuation by the P-gp-mediated MDR
mechanism.
Acknowledgments
We thank You-Fang Shi for performing the uptake studies and Dr.
K. Carraway and Dr. G. E. Conner for reviewing the manuscript.
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SIMPLE ORGANIC CATIONS AND MDR
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Structural Requirements of Simple Organic Cations for
Recognition by Multidrug-resistant Cells
Marc Dellinger, Berton C. Pressman, Cesar Calderon-Higginson, et al.
Cancer Res 1992;52:6385-6389.
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