Multidrug Resistance Essay

Terms & Definitions and General Perspectives

Drug resistance: The ability of a cell or microorganism to withstand the
effects of a drug that is lethal to most members of their equivalents.

Malignant neoplasms vary in their response to cytotoxic drugs: some are

sensitive, others resistant. Understanding of the biochemical basis of this resistance might lead
to the development of markers that would correlate with the clinical response to the drugs - or
even lead to ways of overcoming the resistance.

Multidrug resistance:
1 The resistance of Tumor cells to more than one
chemotherapeutic agent. Resistance may be
attributed to a P-glycoprotein transmembrane pump
that lowers the concentration of drugs in the cell.

2 The resistance of bacteria, especially

Mycobacterium tuberculosis, against more than two of
the antibiotics that were once effective.

Multidrug resistance " multiple drug resistance in some

malignant cell lines"= Resistance to many structurally
unrelated chemotherapy agents in cells that were discovered
to have developed natural resistance to a single cytotoxic
compound.
 Discovery
Multidrug resistance was first described in 1970 after
selection of Chinese hamster ovarian cancer cells
exposed to increasing concentrations of actinomycin
D.1 Though the cells had been selected by a single
agent, they proved to be resistant to a range of
clinically important anticancer drugs, including the
Anthracyclines (doxorubicin and daunomycin), the
Vinca alkaloids (vincristine, vinblastine, and
vindesine), Etoposide, and Colchicine.

Criteria of multidrug resistant cells

Riordan and Ling went on to show that the multidrug resistant cells had

 lower concentrations of the drug –

 a drug accumulation deficit - and
 that a membrane glycoprotein of 170 kDa was responsible. 2

At first the deficit was thought to be due to a fault in Permeation and therefore the
glycoprotein was named P glycoprotein (P-gp). 3

Genetics

The gene for this, mdr-1, has been cloned and

sequenced, and the amino acid has been
sequenced and the amino acid sequence
derived.4 P glycoprotein has 1280 amino acids,
and its structure suggests that it arose from the
fusion of two closely related genes; it uses ATP
as a source of energy.
The precise molecular mechanism whereby P
glycoprotein can transport such a wide range of
structurally diverse drugs is uncertain, and
several models have been proposed.

P glycoprotein& multidrug
resistance associated protein
(MRP).
P glycoprotein belongs to a superfamily of ATP binding
cassette transporters, whose members include the cystic
fibrosis transmembrane regulator, the chloroquine
transporter of Plasmodium falciparum, and a yeast
transporter.8

Recently a new member of this family was cloned from a

human small cell lung cancer cell line, H69AR.

Though it was resistant to doxorubicin and vincristine, it did

not express P glycoprotein. 9 This new protein has 1522 amino
acids and has been termed multidrug resistance associated
protein (MRP).

Cells with this type of resistance may not have accumulation

deficits of doxorubicin. One possible explanation is that
multidrug resistance associated protein facilitates the
sequestration of cytotoxic drugs into intracellular organelles.9

Whether this protein binds to the drugs against which it

confers resistance is not known, but the emergence of a
potential new target for modulation, if it is widely expressed in
human tumours, is clearly an exciting development.

Several probes have been developed that can detect the P glycoprotein
gene, its mRNA, and its protein product.10,11 Studies have shown that the
gene expressed in normal gastrointestinal mucosal cells, renal tubular
cells, biliary canalicular cells, and adrenocortical cells .
11
Tumours that are initially sensitive to chemotherapy but resistant on relapse commonly show
increases in expression of P glycoprotein.

Leukaemias with high concentrations of P glycoprotein tend to be resistant to inductive

treatment with chemotherapy based on anthracycline.12

Expression of P glycoprotein is an adverse factor in multivariate prognostic models for

childhood sarcoma and neuroblastoma.13,14

BEFORE LONG IT MAY BE PRACTICABLE TO “TISSUE TYPE” TUMOURS ACCORDING TO

EXPRESSION OF P GLYCOPROTEIN AND, PERHAPS, TO AVOID THE TOXICITY AND REDUCED
QUALITY OF LIFE ASSOCIATED WITH TREATMENT WITH INEFFECTIVE ANTICANCER DRUGS.

After an initial observation by Tsuruo et al,15 several groups

have shown that transport defect mediated by P glycoprotein
can be blocked by many non-cytotoxic drugs, including
nifedipine, verapamil, quinine, chloroquine, progestogens,
tamoxifen, cyclosporin A and its analogues, reserpine and
tricyclic anti-depressants.16 Clinical trials have assessed the
combination of conventional chemotherapy with modulators
of P glycoprotein; in one study of relapsed non-Hodgkin's
lymphoma 13 of 18 patients responded to infusional
chemotherapy with high doses of verapamil.17 Unfortunately,
the amounts of verapamil needed to reverse drug resistance
produced congestive cardiac failure and heart block. Research
groups are currently looking for potent, non-toxic modulators
of P glycoprotein to combine with conventional
chemotherapy. Cyclosporin A has been reported to reverse
clinical multidrug resistance in myeloma. 18 The combination
of quinidine with the antineoplastic agent epirubicin was
recently assessed in a prospective, placebo controlled
randomised study in patients with advanced breast cancer. 19
Tumour response rates and survival were similar in the two
arms of the trial, a finding that probably reflects the low
potency of quinidine to bind P glycoprotein. 7

Another possible application of the genetics of P glycoprotein is in gene therapy. Transgenic

mice that express human P glycoprotein in their bone marrow are resistant to chemotherapy, 20
so in theory if human bone marrow could be transfected with the gene that might protect it
against myelosuppression from anticancer chemotherapy.8

Novel, improved modulators of P glycoprotein seem likely to be developed in parallel with mechanistic
research on the function of the gene, and thus should lead to the rational design of inhibitor. Future
phase II clinical trials with new modulators of P glycoprotein must include pharmacokinetic studies since
Etoposide's activity is increased by
interactions can occur with cytotoxic drugs - for example,

80% when it is given in combination with cyclosporin A. These trials should focus on
21

those cancers that are known to express high concentrations of P glycoprotein (including

renal and colorectal cancers and sarcomas).

Since P glycoprotein is expressed in normal tissues there is

some concern that its modulators might alter the cellular
transport of physiological metabolites such as bilirubin and
thereby delay the clearance of cytotoxic drugs that undergo
hepatobiliary excretion. These clinical trials should therefore
be performed in units used to handling the toxicity associated
with intensive chemotherapy.

Oxford Journals

 Volume95, Issue4

 Pp. 255-257.

Multidrug Resistance: Can New Drugs Help

Chemotherapy Score Against Cancer?
1. Bruce Goldman
For the vast stretch of evolutionary time preceding refrigeration, running water, and
airtight packaging, life was short and food was dirty. In today’s industrialized world,
things are different. But the mechanisms that evolved in living cells to protect them from
dangerous substances introduced through ingestion, inhalation, and infection remain
intact.

Prominent among these mechanisms is a family of cell-membrane-anchored proteins

that act as “goalies” for the cell, denying entry to foreign toxins that would otherwise
diffuse through the cell membrane. The first such pump to be identified, P-glycoprotein
or simply P-gp, is common in the gut, liver, and kidney—where toxins abound—as well
as the testes and blood-brain barrier.

This goalie is ultra-versatile: A variety of structurally diverse chemicals are substrates for
P-gp, which makes good evolutionary sense, because there are not enough genes to code
for all the pumps that would be required if each were narrowly specific. Unfortunately,
many widely used chemotherapy drugs are expelled by P-gp before they can do their job
of damaging the cell beyond all hope of replication. In a tumor, clones of cells can
spontaneously arise that produce large amounts of P-gp and thereby acquire
chemoresistance. Other tumors, derived from tissues expressing P-gp, are resistant to
begin with.

Given these observations, it is reasonable to ask: Can blocking P-gp— “tripping the
goalie”—safely and effectively reverse a tumor’s chemotherapy resistance? After more
than two decades of focused attention, there has been no definitive answer to this
question, largely because of the absence of ideal P-gp-inhibiting drugs that would
provide unambiguous results.

Redundant Mechanisms
“Virtually all of the roughly half-million annual cancer deaths in the United States can be
said to have occurred because chemotherapy failed,” said Tito Fojo, M.D., Ph.D., a senior
investigator at the National Cancer Institute’s Center for Cancer Research. Such failures
happen because patients’ tumors either were resistant at the outset or eventually
developed resistance after exposure to the drugs.

Is P-gp the major determinant of chemotherapy resistance? Victor Ling, Ph.D., now vice
president of research at the British Columbia Cancer Agency in Vancouver, B.C.,
discovered P-gp in the late 1970s when, after he challenged cultured tumor cells with the
cytotoxic drug colchicine, some cells became resistant not only to colchicine but also to
chemically unrelated drugs. However, several molecular pumps similar to P-gp (not to
mention several unrelated mechanisms) have now been implicated in drug resistance.
One such pump is MRP1 (for multidrug resistance protein), discovered in 1992 by Susan
Cole, Ph.D., and Roger Deeley, Ph.D. “MRP1 is distributed fairly ubiquitously in almost all
normal tissues, usually at low levels,” said Cole, professor of pathology, oncology, and
toxicology at Queens University in Kingston, Ontario.

There are redundant toxin-expelling systems in the digestive tract. “In colorectal and
renal cancer, the large majority of tumors express P-gp,” said Branimir Sikic, M.D., an
oncologist, professor of medicine, and program director of Stanford University Medical
Center’s Clinical Research Center. “And we know that, uniformly, P-gp-related cancer
drugs aren’t active in those tumors. Yet, even at doses of compounds that inhibit P-gp
substantially in patients, we haven’t been able to convert inactive drugs into active
agents. I think the reason is co-expression of multiple defense mechanisms by the cell.”

Still, Sikic and others think that P-gp is the most prevalent and single most important
cause of multidrug resistance. In the laboratory, P-gp is the molecular pump whose
expression most commonly gets upregulated by cancer cell lines after exposure to
chemotherapy drugs. And the pump is overexpressed in acute myelogenous leukemia
(AML) and breast, lung, and other tumors whose healthy tissue counterparts typically
express only low levels, said Susan Bates, M.D., head of the Molecular Therapeutics
Branch at the NCI’s Center for Cancer Research.

Old Drugs, New Tricks?

With Ling’s discovery of P-gp, the search was on for P-gp-inhibiting agents that, when
co-administered with chemotherapy, could prolong, restore, or enhance the effectiveness
of chemotherapeutic drugs. The first batch turned up on pharmacy shelves, already
approved for other uses: both the calcium-channel blocker verapamil and the
immunosuppressant cyclosporine, for example, inhibited P-gp. But their strong
pharmacological effects in their intended indications made them undesirable as P-gp
inhibitors. They blocked P-gp in the laboratory, but in humans the doses needed to get
pump-blocking quantities into tumors proved toxic to healthy tissues.

These first-generation P-gp blockers were succeeded by a second generation of

compounds that were tweaked to reduce side effects and increase potency. Two
prominent examples were valspodar (Novartis Pharmaceuticals, East Hanover, N.J.) and
biricodar (Vertex Pharmaceuticals, Cambridge, Mass.). Valspodar, in combination with
standard chemotherapy, advanced all the way to phase III trials in AML and ovarian
cancer, but neither trial yielded statistically significant results.

Neither drug was potent or long-acting, and both drugs had the confounding
characteristic of inhibiting MRP1 as well as P-gp. “In tumors where both pumps are
playing a role, using two distinct blockers in combination might be very useful. But often
it’s just a single pump that’s active,” said NCI’s Fojo. And MRP1’s prevalence throughout
the body portends potential side effects—just what oncologists administering maximal
doses of cytotoxic drugs do not want.

Novartis and Vertex have curtailed active development of these drugs. The second-
generation P-gp blockers were defeated above all by a factor that became clear to
investigators only late in the drugs’ development: pharmacokinetic interactions. The
agents interfered with the metabolism of cytotoxic compounds, slowing their clearance
and increasing their toxicity to healthy tissues. To compensate, oncologists reduced
dosages of the cytotoxics administered in late-phase trials. But each patient’s
metabolism is different, therefore some patients were effectively receiving too much, and
others too little, of their chemotherapy drugs.

New Drugs, New Trials

The early P-gp blockers’ low potency, poor specificity, short duration of action, and
pharmacokinetic interactions bedeviled efforts to make the inhibitors a feasible addition
to the clinic. But a new generation of P-gp blockers—whose attributes should allow
rigorous testing of the hypothesis that P-gp reversal can restore, enhance, or prolong
chemotherapeutic drug sensitivity—is moving to center stage.

Two third-generation candidates, tariquidar (Xenova, Slough, U.K.) and zosuquidar (Eli
Lilly, Indianapolis), are extremely potent and P-gp-specific. Neither drug blocks MRP1 or
exhibits direct toxicity. Tariquidar is particularly long-acting: In one assay employing
circulating “natural killer” cells that express P-gp in high amounts, “tariquidar completely
blocked P-gp for at least 48 hours after a half-hour infusion,” said the NCI’s Bates.
Chemotherapeutic drugs administered anytime during that blockade cannot be expelled
by P-gp.

These agents do not appear to interfere with the metabolism of the chemotherapy drugs
themselves. Tariquidar has been tested in clinical trials with paclitaxel, doxorubicin, and
vinorelbine, best-selling representatives of three distinct categories of chemotherapeutic
drugs (taxanes, anthracyclines, and vinca alkaloids) and shown not to necessitate any
changes in chemotherapeutic dosing schedules.

Zosuquidar and tariquidar are in phase III trials in, respectively, AML and non-small-cell
lung cancer (NSCLC), two diseases with very short median survival times (and the
prospect of relatively fast results). Importantly, both trials employ P-gp blockers as first-
line therapy in combination with chemotherapy, with the intention of destroying tumors
before they evolve resistance mechanisms. Because the emergence of resistant clones
may be prevented by the destruction of just a few P-gp-expressing cells, time to relapse
and overall survival—not tumor shrinkage—will be key endpoints.

The zosuquidar trial, led by Larry Cripe, M.D., director of clinical affairs at Indiana
University Cancer Center, will focus on older patients, whose transformed leukocytes are
generally known to be more likely to express high amounts of P-gp than those of
younger patients. About 450 patients with AML will be randomly assigned to receive 6-
hour infusions of either a placebo or zosuquidar plus the chemotherapy drugs
daunorubicin (a P-gp substrate) and cytarabine. Results are expected in about 5 years,
Cripe said. Numerous studies of AML patients published since 1995 have shown an
association between P-gp expression on chemotherapy recipients’ tumors and clinical
outcomes. However, daunorubicin is a substrate for both P-gp and MRP1. This could
complicate attempts to reverse resistance by blocking P-gp, because MRP1 is fairly
common on the leukocytes in which AML arises.

In the clinical trial of tariquidar, patients with NSCLC will receive a tariquidar infusion for
about one-half hour preceding chemotherapy with paclitaxel and carboplatin in one 500-
patient trial, and with vinorelbine in another, equal-sized study for poorer-performing
patients. As with AML, MRP1 abounds in NCSLC tumors. But paclitaxel—a strong P-gp
substrate—has little affinity for MRP1. (MRP1’s appetite for vinorelbine is not yet clear.)
Final data should be available in 2005, said Graeme Boniface, Ph.D., director of research
for oncology at QLT, Vancouver, B.C., the company to which Xenova licensed tariquidar
for cancer indications.

There is much debate as to whether P-gp appears on NSCLC tumors frequently enough to
provide statistically meaningful trial results. Arguing that the benefits of P-gp inhibitors
are limited to tumors expressing P-gp, some authorities propose studies that enroll only
patients whose tumors are already P-gp-positive. Fojo and others counter that current
detection methods lack reliability, tend to underestimate the incidence of P-gp
expression in NCSLC, and fail to distinguish between tumors with zero P-gp expression
and those with low expression. But the latter tumors may still be prone to develop
resistance and, therefore, may be responsive to co-administration of a blocking agent
along with chemotherapy.

At the NCI, Fojo and Bates are developing better surrogate assays using radioactive dyes
to diagnose P-gp expression and monitor P-gp inhibition in cells and tissues. An
ultimate goal: to catalog each tumor cell for all mechanisms of resistance and tailor
treatment accordingly with highly targeted agents.

Meanwhile, with the advent of highly specific P-gp inhibitors, the stage is now set to see
if “tripping the goalie” will work. Given chemotherapy’s present failure rate, even a small
but significant reduction in drug resistance may turn into thousands of lives saved.

 Oxford University Press

Nanoparticles may play a role in inhibiting the multidrug resistance in

chemotherapy

(Nanowerk Spotlight) Multidrug resistance, the principal mechanism by which many

cancers develop resistance to chemotherapy drugs, is a major factor in the failure of
many forms of chemotherapy. New research by Chinese scientists suggests that
nanoparticle surface chemistry and size as well as the unique properties of the magnetic
nanoparticles themselves may contribute to a synergistic enhanced effect of drug
uptake of targeted cancer cells. These findings could result in promising biomedical
applications for cancer therapy.

Professor Xuemei Wang from the the State Key Laboratory of Bioelectronics (Chien-
Shiung Wu Laboratory) in Nanjing, PR China, together with several of her colleagues
from Southeast University, recently published a paper titled "Synergistic enhancement
effect of magnetic nanoparticles on anticancer drug accumulation in cancer cells" in the
June 26, 2006 online issue of Nanotechnology.

In it, the researchers describe their investigation of the synergistic effect of three kinds
of magnetic nanoparticles, nano Fe3O4, Ni and Fe2O3, on the drug uptake of anticancer
drug daunorubicin in leukemia K562 cells.

They show how Fe3O4 nanoparticles could remarkably enhance the uptake or diffusion
efficiency of anticancer drugs into target cancer cells (especially drug resistance cancer
cells). If Fe3O4 nanoparticles, which are biocompatible and very stable, are fixed at the
ailing area by using external magnetic field during the tumor treatment, the
chemotherapy effect could be considerably enhanced by combination of the application
of the new magnetic nanoparticles in drug delivery systems for achieving the targeting
and controlled drug release.

Microscopy images (200 µm × 200 µm) of Fe3O4 incubated leukemia cells in the
absence and presence of a magnetic field. The cells numbered in the images illustrate
the movement of the cells, while squared cells in the left image were observed to move
out of vision after the external magnetic field was applied, and the cycled cells in the
right image appeared upon application of the external magnetic field. The arrows
indicate the direction of the magnetic field and the picture was captured after the
magnetic field was applied for 5 s. (Reprinted with permission from IOP Publishing)

"Our results illustrate that the presence of magnetic nanoparticles could facilitate the
drug accumulation of daunorubicin inside leukemia cells and the enhancement effect of
nano Fe3O4 is much stronger than that of the other two magnetic nanoparticles" Wang
explains the findings to Nanowerk. "These observations are consistent with the results
of our recent biological experimental studies, which indicates that the presence of Fe3O4
nanoparticles could apparently inhibit the growth of the respective leukemia cells
(Interestingly, the Fe3O4 nanoparticle itself could also inhibit the cell growth somehow);
especially, when treated the target cells by anticancer drug daunorubicin together with
Fe3O4 nanoparticles, the growth of leukemia cells could be much more remarkably
inhibited than that with only daunorubicin or other nanoparticles. Since these three kinds
of nanoparticles were all capped with the tetraheptylammonium, our observations
suggest that both the size and the unique properties of magnetic nanoparticles
themselves may contribute to the synergistic enhanced effect of the drug uptake of
targeted cancer cells."

The magnetic targeting offers a unique opportunity to treat tumors without systemic
toxicity. It is known that the cure efficiency of cancer chemotherapy depends not only on
the anticancer drug itself but also on how it is delivered to its targets. As already
reported in some literature, it has been observed that the magnetic particles can be
targeted and concentrated in some tumor tissue at significantly high level.

"Our observations indicate that magnetic nanoparticles with different size and surface
chemistry have different ability to enter target cells and thus the relative efficiency of the
drug delivery systems by the conjugation of drugs with nanoparticles will be critically
dependent upon nanoparticle surface chemistry and size of the functionalized
nanoparticles" says Wang.

"Based on these observations, our future research with regard to cancer therapy may
focus on the relative mechanisms of new magnetic nanoparticles" Wang describes a
possible direction for her group's future research. "Magnetic nanomaterials are
especially promising for the early diagnosis of some cancers and for efficiently targeting
chemotherapy.

Multidrug resistance
Mechanism and function of multidrug transporters in pro- and eukaryotic cells

The development of resistance to multiple drugs is a major problem in the treatment of

cancer cells and infections by pathogenic microorganisms. One of the mechanisms by
which human cells and bacteria can acquire multidrug resistance involves the expression of
membrane proteins which mediate the active extrusion of drugs from the cell. Current
research focuses on the molecular properties of four distinct multidrug transport proteins.
  
Multidrug transporter LmrP
In Lactococcus lactis LmrP mediates drug resistance by extruding amphiphilic, organic
compounds from the inner leaflet of the cytoplasmic membrane. LmrP is a secondary
multidrug transporter which is typical for prokaryotes. The antibiotic specificity of LmrP is
exceptionally broad and includes tetracyclins, quinolones, aminoglucosides, lincosamides,
macrolides, streptogramins and others. To facilitate functional and structural studies,
histidine-tagged LmrP protein was overexpressed in L. lactis, solubilized with
dodecylmaltoside, purified to homogeneity by nickel chelate affinity chromatography, and
functionally reconstituted in proteoliposomes. Interestingly, LmrP is able to transport
detergents such as Triton X-100. Therefore, the choice of the detergent used for
solubilization of the protein was critical.
  
Multidrug transporter LmrA
A second protein in L. lactis, LmrA, mediates antibiotic resistance via a similar mechanism,
and with a similar specificity as the secondary multidrug transporter LmrP. Unlike other
known bacterial multidrug resistance proteins, LmrA is an ATP-binding cassette (ABC)
transporter. LmrA is homologous to the human multidrug resistance P-glycoprotein,
encoded by the MDR1 gene, overexpression of which is one of the major causes of
resistance of human cancers to chemotherapy. In collaboration with Prof. C.F. Higgins in
Oxford, LmrA was expressed in human lung fibroblast cells to compare the
pharmacological properties of LmrA with those of P-glycoprotein. Surprisingly, LmrA was
targeted to the plasma membrane and conferred typical multidrug resistance on these
human cells. The pharmacological characteristics of LmrA and P-glycoprotein- expressing
lung fibroblasts were very similar, and the affinities of both proteins for vinblastine and
magnesium-ATP indistinguishable. Blockers of P-glycoprotein-mediated multidrug
resistance also inhibited LmrA-dependent drug resistance. Kinetic analysis of drug
dissociation from LmrA, expressed in plasma membranes of insect cells, revealed the
presence of two allosterically-linked drug binding sites indistinguishable from those of P-
glycoprotein. These findings have implications for the reversal of antibiotic resistance in
pathogenic microorganisms. Taken together, these observations demonstrate that
bacterial LmrA and human P-glycoprotein are functionally interchangeable and that this
type of multidrug resistance efflux pump is conserved from bacteria to man. LmrA has
been overexpressed in L. lactis up to a level of 35% of total membrane protein. This
protein is functional, and has been purified and reconstituted into proteoliposomes to
facilitate detailed structure-function analyses. Currently, mg quantities of reconstituted
LmrA can be obtained rather easily.
  
Multidrug transporter HorA
Lactococcal LmrA is homologous to other prokaryotic ABC transporters such as the hop-
resistance protein HorA in the beer-spoilage bacterium Lactobacillus brevis. To study HorA
in more detail, the protein was functionally expressed in L. lactis. Studies in whole cells
and membrane vesicles derived thereof demonstrate that HorA is a multidrug transporter
able to transport hop compounds (iso-a-acids), with a substrate specificity similar to that
of LmrA. Recently, the protein has been purified and reconstituted into proteoliposomes.
  
Cholate transporter
Micro-organisms able to colonize the gastrointestinal tract in mammals must tolerate high
levels of bile salts, powerful detergents that disrupt biological membranes. Physiological
evidence has been obtained for the presence of an ATP-dependent cholate transporter in L.
lactis. Interestingly, this transport activity may be related to the transport activities for
fluorescent anionic dyes BCECF and FTUG in this organism. Current work aims at the
isolation of the gene(s) encoding the cholate transporter.
  
Multidrug resistance-associated protein
Multidrug resistance in humans is caused by the overexpression of two multidrug
transporters: P-glycoprotein and the Multidrug Resistance-Associated Protein (MRP1).
Although MRP1 contains two nucleotide binding domains (NBDs), it is not known whether
both NBDs can function as an active ATPase, and whether drug-protein interactions in
these domains play a role in the drug-stimulated ATPase activity of MRP1. The NBDs of
MRP1 were expressed in fusion with glutathione S-transferase, purified by affinity
chromatography, cleaved from the fusion partner by thrombine, and purified to
homogeneity by gel filtration. Both NBDs hydrolyze ATP, with a Km of 340 mM Mg-ATP and
Vmax of 6.0 nmol Pi/mg/min for NBD1, and a Km of 910 mM Mg-ATP and Vmax of 7.5
nmol Pi/mg/min for NBD2. The properties of NBD1 were further studied. Surprisingly, the
Vmax of the NBD1 ATPase reaction was stimulated more than 3-fold by the MRP substrate
decyl glutathione but not by decyl maltoside or decanol, whereas the Km of the reaction
was not affected. The interaction between S-alkyl glutathione conjugates and NBD1 was
analyzed by using the intrinsic tryptophan fluorescence of NBD1 as a reporter. The
tryptophan fluorescence was quenched in a concentration-dependent manner by S-alkyl
glutathione conjugates, but not by alkyl maltosides or alkanols. The apparent KD of NBD1
for S-alkyl glutathione conjugates decreased with an increasing length of the alkyl chain.
Thus, NBD1 is suggested to have a high-affinity binding site for S-alkyl glutathione
conjugates.