U.S. patent application number 13/465803 was filed with the patent office on 2012-09-27 for high-throughput assay for sugar-mediated drug transport.
This patent application is currently assigned to WISCONSIN ALUMNI RESEARCH FOUNDATION. Invention is credited to John R. Fitzgibbon, Jon S. Thorson.
Application Number | 20120245054 13/465803 |
Document ID | / |
Family ID | 41431640 |
Filed Date | 2012-09-27 |
United States Patent
Application |
20120245054 |
Kind Code |
A1 |
Thorson; Jon S. ; et
al. |
September 27, 2012 |
High-Throughput Assay for Sugar-Mediated Drug Transport
Abstract
The invention provides a rapid, quantitative assay to directly
assess the impact of a diverse range of sugars upon the
sugar-mediated uptake of corresponding sugar-conjugates into
various cell types.
Inventors: |
Thorson; Jon S.; (Middleton,
WI) ; Fitzgibbon; John R.; (Madison, WI) |
Assignee: |
WISCONSIN ALUMNI RESEARCH
FOUNDATION
Madison
WI
|
Family ID: |
41431640 |
Appl. No.: |
13/465803 |
Filed: |
May 7, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12322388 |
Feb 2, 2009 |
8211654 |
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13465803 |
|
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61025024 |
Jan 31, 2008 |
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Current U.S.
Class: |
506/10 ;
506/19 |
Current CPC
Class: |
G01N 33/574 20130101;
G01N 2500/00 20130101; G01N 33/5008 20130101; G01N 2400/00
20130101 |
Class at
Publication: |
506/10 ;
506/19 |
International
Class: |
C40B 30/06 20060101
C40B030/06; C40B 40/12 20060101 C40B040/12 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
CA113297, CA084374, GM070637, and A1052218 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of assaying tumor selectivity for sugar-mediated
transport of glycoconjugates contained in a glycorandomized
library, comprising: (a) providing a glycorandomized library
containing glycoconjugates differing by attached sugars; (b)
contacting each of said glycoconjugates with cells of a first line
under conditions to allow uptake of the glycoconjugates into the
cells; and (c) assessing uptake of said glycoconjugates into the
cells wherein sugar-mediated transport of the glycoconjugates into
the cells is quantified relative to a corresponding conjugate
lacking the attached sugars; (d) repeating steps (a)-(c) with cells
of a second, differing cell line; and (e) comparing sugar-mediated
transport of said glycoconjugates between cells of the first line
and the second cell line, wherein cell line selectivity for
sugar-mediated transport of said glycoconjugates is indicated by
elevated sugar transport activity in one of the cell lines relative
to the other cell line.
2. The method of claim 1 wherein the cell line selectivity for
sugar-mediated transport of said glycoconjugates of one cell line
is at least three times higher relative to the other cell line.
3. The method of claim 1 wherein the glycoconjugates are contacted
with cells independently and in parallel.
4. The method of claim 1 wherein the cells are cancerous.
5. The method of claim 1 wherein the glycoconjugates are
sugar-fluorophore conjugates.
6. The method of claim 5 wherein the sugar-fluorophore is selected
from the group consisting of aminofluoroscein, nitrobenzoxadiazole
or dansyl chloride.
7. A kit for assaying sugar-mediated transport of glycoconjugates
contained in a glycorandomized library, comprising: (a) a
glycorandomized library comprising glycoconjugates with different
attached sugars; (b) cells of a first line; and (c) instructions
for use.
8. A kit for assaying tumor selectivity for sugar-mediated
transport of glycoconjugates contained in a glycorandomized
library, the kit comprising (a) a glycorandomized library
containing glycoconjugates differing by attached sugars; (b) cells
of a first line; (c) cells of a second, differing line; and (d)
instructions for use.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/322,388, filed Feb. 2, 2009, which claims the benefit of
U.S. Provisional Application No. 61/025,024, filed Jan. 31, 2008,
the entirety of which are hereby incorporated by reference herein
for all purposes.
PETITION FOR COLOR DRAWINGS
[0003] The patent or application file contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the U.S. Patent and Trademark Office upon request
and payment of any necessary fee.
FIELD OF THE INVENTION
[0004] The invention relates generally to methods for generating
libraries of small molecule therapeutics. More particularly, the
present invention is directed to methods of enzymatic and chemical
glycorandomization to rapidly generate and assay libraries of
glycoconjugates.
BACKGROUND OF THE INVENTION
[0005] The treatment and cure of cancer remains one of the greatest
challenges of modern medicine. The war on cancer first began with
the use of surgical strategies and radiation therapy. However, the
general inability of these early approaches to eradicate metastatic
cancers revealed a need for alternative tactics. As a result, small
molecule- and macromolecular-based chemotherapeutic compounds have
become an essential element in today's anticancer arsenal.
[0006] Prompted by the unwanted side effects of many oncolytic
drugs, and inspired by Ehrlich's original concept of the `magic
bullet`, contemporary anticancer drug development has focused on
enhancing drug selectivity and specificity through two main
strategies--molecular target-based cancer chemotherapy (often
referred to as `targeted therapy`) and the use of molecular
targeting techniques.
[0007] Molecular target-based therapies are based on an in-depth
understanding of cancer cell biology. Such drugs are designed to
act upon molecular targets specific to a tumor. Despite some of the
early successes of targeted therapies, their clinical development
has exposed unwanted adverse effects including thrombocytopenia,
myelosuppression, hypertension, neutropenia, myalgia, edema,
cardiotoxicity and pulmonary toxicity.
[0008] Molecular targeting techniques generally provide specificity
via tumor-selective binding or uptake of drug conjugates as
mediated by tumor-specific surface epitopes or transporters.
Anticancer molecular targeting strategies employ an extensive array
of small molecule--(e.g., folates, carbohydrates, peptides) or
macromolecule--(e.g., monoclonal antibodies) conjugates and can
provide specificity on an intracellular compartmental-, specific
cell-type--and/or even at the whole organ-level. Examples include
the oligopeptide transporter-mediated uptake of bestatin, the
folate receptor-based enhancement of tumor specificity of
folate-anticancer drugs, and the ability of organic anion
transporters to boost hepatic uptake of anticancer-bile acid drug
conjugates.
[0009] While molecular-targeted drugs have displayed both successes
and limitations, a notable advantage of identifying a successful
molecular targeting modality is its potential general applicability
to a wide range of therapeutic agents (i.e., a successful
tumor-directing small molecule can be conjugated to a variety of
anticancer agents to enhance their tumor-specificity). Thus,
molecular-targeting techniques offer a greater likelihood to
develop broadly applicable platforms for modulating the
selectivity, specificity, absorption, distribution, metabolism and
excretion of a wide range of anticancer agents (including both
conventional cytotoxics/cytostatics as well as newer targeted
therapies).
[0010] Chemoenzymatic glycorandomization employs the inherent or
engineered substrate promiscuity of sugar-activating enzymes,
coupled with inherently promiscuous natural product
glycosyltransferases (GTs), to provide a robust chemoenzymatic
means to glycodiversify natural product-based scaffolds.
Chemoenzymatic glycorandomization has been successfully applied
toward antibiotic scaffolds (novobiocin, erythromycin/megalomicin,
vancomycin), anticancer models (rebeccamycin/staurosporine/AT2433
and calicheamicin) and antihelmenthics (avermectin/ivermectin).
[0011] In contrast, neoglycorandomization relies upon the
installation of a uniquely reactive methoxyamine `handle` followed
by a direct reaction with free sugars. Neoglycorandomization is a
groundbreaking advance over classical chemical glycosylation
strategies (which require extensive steps for sugar/aglycon
protection/deprotection and sugar activation) and allows for
extensive neoglycorandomized natural product libraries in
essentially a single step.
[0012] While enzymatic glycorandomization is restricted to the
inherent specificity of the natural GTs employed,
neoglycorandomization can be accomplished essentially anywhere the
methoxyamine handle can be installed. This allows one to expand
beyond natural positions of glycosylation to explore the potential
benefits of glycosylating even non-glycosylated natural products
and therapeutics.
[0013] Therefore, a need exists for compositions and methods for
rapidly identifying and assessing compounds which can enhance the
tumor-specific uptake of drug-conjugates.
SUMMARY OF THE INVENTION
[0014] Herein, the inventors provide a systematic platform for
rapidly assessing the specific role of a diverse range of sugars to
enhance tumor-specific uptake of sugar-conjugates and, in parallel,
their impact upon the selectivity of a representative set of
cytotoxic drugs.
[0015] The present invention provides a method for assaying
sugar-mediated transport of glycoconjugates contained in a
glycorandomized library. The method comprises providing a
glycorandomized library comprising glycoconjugates with different
attached sugars; contacting each of said glycoconjugates with cells
under conditions to allow uptake of the glycoconjugates into the
cells; and assessing uptake of said glycoconjugates into the cells,
wherein sugar-mediated transport of the glycoconjugates into the
cells is quantified relative to a corresponding conjugate lacking
the attached sugars. The glycoconjugates are contacted with cells
independently and in parallel. In a preferred embodiment the cells
are cancerous animal cells, although the method may also be used
with bacteria, viruses, parasites and non-cancerous animal cells.
The glycoconjugates are preferably sugar-fluorophore conjugates,
wherein the sugar-fluorophore is preferably selected from the group
consisting of aminofluoroscein, NBD or dansyl chloride.
[0016] The invention also provides a method of assaying tumor
selectivity for sugar-mediated transport of glycoconjugates
contained in a glycorandomized library. The method comprises
providing a glycorandomized library containing glycoconjugates
differing by attached sugars; contacting each of said
glycoconjugates with cells of a first line under conditions to
allow uptake of the glycoconjugates into the cells; assessing
uptake of said glycoconjugates into the cells wherein
sugar-mediated transport of the glycoconjugates into the cells is
quantified relative to a corresponding conjugate lacking the
attached sugars; repeating steps (a)-(c) with cells of a second,
differing cell line; and comparing sugar-mediated transport of said
glycoconjugates between cells of the first line and the second cell
line, wherein cell line selectivity for sugar-mediated transport of
said glycoconjugates is indicated by elevated sugar transport
activity in one of the cell lines relative to the other cell
line.
[0017] The invention also provides a kit for assaying
sugar-mediated transport of glycoconjugates contained in a
glycorandomized library, comprising a glycorandomized library
comprising glycoconjugates with different attached sugars; cells of
a first line; and instructions for use. In an alternative
embodiment, the invention provides a kit for assaying tumor
selectivity for sugar-mediated transport of glycoconjugates
contained in a glycorandomized library, the kit comprising a
glycorandomized library containing glycoconjugates differing by
attached sugars; cells of a first line; cells of a second,
differing line; and instructions for use.
[0018] A fundamental advantage of the present invention is to
provide a rapid, quantitative assay to directly assess the impact
of a diverse range of sugars upon the uptake of sugar-drug
conjugates by GLUT transporters.
[0019] Another fundamental advantage of the present invention is
the development of a unique probe set and assay that enables the
discovery of atypical sugars which impart significantly enhanced
tumor uptake and specificity of a sugar-drug conjugate, as compared
to the standard sugar-drug conjugates of the prior art, such as
glucose, 2-deoxyglucose or fluorodeoxyglucose (FDG).
[0020] Other objects, features and advantages of the present
invention will become apparent after review of the specification,
claims and drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1. Illustration of the Warburg effect.
[0022] FIG. 2. (a) Structures of representative carbohydrates
employed in GLUT substrate-specificity studies. The sugars
represented include: 3-O-methylglucose, 1; glucosamine, 2;
2-deoxyglucose, 3; glucose, 4; maltose, 5; 2-fluoroglucose, 6;
galactose, 7; mannose, 8; fructose, 9; and xylose, 10. (b) Models
for the orientation of facilitative glucose transporter proteins in
the cell membrane. Transmembrane domains (TM) are labeled 1 to 12.
The sites of glycosylation (N) are shown. Conserved domains and
amino acids are also included. For class I transporters, the TMs
proposed to form the glucose transport channel are highlighted by
gradient shading, and regions which interact with hexose substrates
and the GLUT inhibitor cytochalasin B are indicated.
[0023] FIG. 3. An overview of the complementary enzymatic (bottom)
and chemical (top) glycorandomization strategies. E.sub.1, an
evolved sugar kinase; E.sub.2, an engineered promiscuous
nucleotidylyltransferase; GlyT, a natural product
glycosyltransferase; CSL, chemoselective ligation reactions (e.g.
Huisgen 1,3-dipolar cycloaddition). The focus of this proposal is
to utilize neoglycorandomization (top) to rapidly generate
differentially-glycosylated lipoglucopeptides (from vancomycin) and
screen them for various anti-infective activities.
[0024] FIG. 4. (a) Synthesis of sugar transporter `Probe Set A` (i)
1.5 eq DIEA , 1.5 eq chloroacyl chloride, dry THF, 24 h (75%); (ii)
3 eq NaI, MeOH, 2 h (95%); (iii) 5 eq NH.sub.2OMe, sealed vial,
45.degree. C., 24 h (85%); (iv) 2 eq reducing sugar, 3:1 DMF:AcOH,
45 C, 48 h (25-60%, 40 analogs total). (b) Synthesis of sugar
transporter `Probe Set B`.
[0025] FIG. 5. Representative sugar transporter assays. Cell lines
used for sugar transporter assays include MCF10A (immortalized
breast), MCF7 (breast cancer), A549 (non-small cell lung cancer),
Du145 (prostrate cancer), RWPE (immortalized prostrate); (a)
D-glucose-conjugate; (b) L-glucose-conjugate; (c)
N-acetyl-D-mannose-conjugate; (d) D-glucuronic acid-conjugate; (e)
L-allose-conjugate; (f) L-xylose-conjugate. For each cell line, the
dose range was 250 .mu.M, 125 .mu.M, 62.5 .mu.M, 31.3 .mu.M, 15.6
.mu.M, 7.8 .mu.M, 3.9 .mu.M and 0 .mu.M. The average error (40
compounds, 5 cell lines) was 8.3%. No uptake was observed with
parent 11 or aglycon 13 (data not shown).
[0026] FIG. 6. Fluorescence microscopy of sugar uptake in non-small
cell lung cancer cells (A549). (a) no sugar transporter probe
(negative control); (b) D-altrose conjugate; (c) D-allose
conjugate. For all three panels, general cell-staining was
accomplished with 1 .mu.M Calcein Red and imaged on an Olympus IX81
inverted microscope at 20.times. magnification.
DETAILED DESCRIPTION OF THE INVENTION
I. IN GENERAL
[0027] Before the present materials and methods are described, it
is understood that this invention is not limited to the particular
methodology, protocols, materials, and reagents described, as these
may vary. It is also to be understood that the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by any later-filed
nonprovisional applications.
[0028] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. It is also to be noted that the terms
"comprising", "including", and "having" can be used
interchangeably.
[0029] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
All publications and patents specifically mentioned herein are
incorporated by reference for all purposes including describing and
disclosing the chemicals, instruments, statistical analysis and
methodologies which are reported in the publications which might be
used in connection with the invention. All references cited in this
specification are to be taken as indicative of the level of skill
in the art. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
II. THE INVENTION
[0030] Herein, the inventors provide a versatile method for
producing large quantities of tumor-specific small molecule
therapeutics which differ solely by their attached sugars. The
method also allows the identification of tumor-specific
therapeutics which exhibit increased rates of uptake by the tumor.
The chemoenzymatic- and neo-glycorandomization of molecules for the
rapid generation of libraries of small molecule therapeutics which
differ solely by their attached sugars is the foundation of this
invention.
[0031] Cancer cells typically exhibit increased levels of glucose
uptake and glycolysis, despite reduced levels of oxygen consumption
(FIG. 1). In most cells, facilitative glucose uptake is mediated by
GLUT proteins. Intracellular glucose is accumulated in two distinct
pools--the metabolic pool used for glycolysis and the structural
pool used for the synthesis of glycoproteins and extracellular
matrix macromolecules. Under aerobic conditions, oxidative
phosphorylation ensures sufficient ATP production while under
anaerobic conditions glycolysis predominates. The Warburg effect
leads to the enhanced conversion of glucose to lactate by tumor
cells (box), even in the presence of adequate oxygen (normally used
for oxidative phosphorylation). Relevant to the present invention,
the activation of the AKT oncogene results in increased glucose
transportation and stimulation of hexokinase HK2 activity, which
enhances glycolytic rates. In a tumor cell, HIF-1
(hypoxia-inducible factor), increased by RAS and loss of VHL (von
Hippel Lindau protein--which normally mediates HIF degradation) and
stabilized by hypoxia, also transactivates glycolytic genes,
including the expression of sugar transporters.
[0032] Unlike normal cells, tumors display a vast increase in
glucose uptake and consumption, a reliance on the glycolytic
pathway for ATP production and, as a result, an increased
production of lactate. This altered metabolic profile in tumors
and, more specifically, the corresponding enhanced sugar uptake by
said tumors, provides an immediate pathway for the targeted
tumor-specific molecules of the present invention.
[0033] The present invention provides a method for assaying
sugar-mediated transport of glycoconjugates contained in a
glycorandomized library. The method comprises providing a
glycorandomized library comprising glycoconjugates with different
attached sugars; contacting each of said glycoconjugates with cells
under conditions to allow uptake of the glycoconjugates into the
cells; and assessing uptake of said glycoconjugates into the cells,
wherein sugar-mediated transport of the glycoconjugates into the
cells is quantified relative to a corresponding conjugate lacking
the attached sugars.
[0034] By "glycoconjugate," we mean any compound or molecule that
is covalently bound to a glycosidic residue. The compounds may
include compositions of matter having biological, therapeutic
and/or diagnostic activity in animals which include, but are not
limited to: antiviral agents such as acyclovir, zidovudine and the
interferons; antibacterial agents such as aminoglycosides,
cephalosporins and tetracyclines; antifungal agents such as polyene
antibiotics, imidazoles and triazoles; antimetabolic agents such as
folic acid analogues, folic acid antagonists including
methotrexate, and purine and pyrimidine analogs; antineoplastic
agents such as the anthracycline antibiotics and plant alkaloids;
sterols such as cholesterol; carbohydrates, e.g., sugars and
starches; amino acids, peptides, proteins such as cell receptor
proteins, immunoglobulins, enzymes, hormones, neurotransmitters and
glycoproteins; dyes; radiolabels such as radioisotopes and
radioisotope-labeled compounds; radiopaque compounds; fluorescent
compounds; mydriatic compounds; bronchodilators; local anesthetics;
nucleic acid sequences such as messenger RNA, CDNA, genomic DNA and
plasmids; bioactive lipids such as ether lipids and ceramides; and
the like.
[0035] By "library," we mean a mixture of glycoconjugates of
varying sequences which can be subjected to a screening procedure
to identify compounds or molecules that exhibit enhanced
sugar-mediated transport.
[0036] The glycoconjugates are contacted with cells independently
and in parallel. In a preferred embodiment the cells are cancerous
animal cells, although the method may also be used with bacteria,
viruses, parasites and non-cancerous animal cells.
[0037] The glycoconjugates are preferably sugar-fluorophore
conjugates, wherein the sugar-fluorophore is selected from the
group consisting of aminofluoroscein, NBD or dansyl chloride. By
"fluorophore" we mean a compound that can absorb electromagnetic
energy and is capable of at least partially remitting some fraction
of that energy as electromagnetic radiation over some time period.
Suitable fluorophores include, but are not limited to, coumarins
and related dyes, xanthene dyes such as fluoresceins, rhodols, and
rhodamines, resorufins, cyanine dyes, bimanes, acridines,
isoindoles, dansyl dyes, aminophthalic hydrazides such as luminol,
and isoluminol derivatives, aminophthalimides, aminonaphthalimides,
aminobenzofurans, aminoquinolines, dicyanohydroquinones,
semiconductor fluorescent nanocrystals, fluorescent proteins and
fluorescent europium and terbium complexes and related compounds.
Fluorophores may also be environmental sensitive or be indicators
of various ions such as calcium, magnesium or pH, as is known in
the art.
[0038] Preferred fluorophores typically exhibit good quantum
yields, lifetimes, and extinction coefficients, are resistant to
collisional quenching and bleaching, and should preferably be
easily conjugated to the sugar. Particularly desirable are
fluorophores that show absorbance and emission in the red and near
infrared range, which are useful in whole animal studies, because
of reduced scattering background fluorescence, and greater
transmission through tissues. Preferred examples include
nitrobenzoxadiazole (NBD) or
5-(dimethylamino)naphthalene-1-sulfonyl chloride.
[0039] By "enhanced," we mean an increased rate of transport into
the cell line as compared to the rate of transport into the cell
line via an unmodified sugar-drug conjugate.
[0040] The invention also provides a method of assaying tumor
selectivity for sugar-mediated transport of glycoconjugates
contained in a glycorandomized library. The method comprises
providing a glycorandomized library containing glycoconjugates
differing by attached sugars; contacting each of said
glycoconjugates with cells of a first line under conditions to
allow uptake of the glycoconjugates into the cells; assessing
uptake of said glycoconjugates into the cells wherein
sugar-mediated transport of the glycoconjugates into the cells is
quantified relative to a corresponding conjugate lacking the
attached sugars; repeating steps (a)-(c) with cells of a second,
differing cell line; and comparing sugar-mediated transport of said
glycoconjugates between cells of the first line and the second cell
line, wherein cell line selectivity for sugar-mediated transport of
said glycoconjugates is indicated by elevated sugar transport
activity in one of the cell lines relative to the other cell
line.
[0041] The invention also provides a kit for assaying
sugar-mediated transport of glycoconjugates contained in a
glycorandomized library, comprising a glycorandomized library
comprising glycoconjugates with different attached sugars; cells of
a first line; and instructions for use. In an alternative
embodiment, the invention provides a kit for assaying tumor
selectivity for sugar-mediated transport of glycoconjugates
contained in a glycorandomized library, the kit comprising a
glycorandomized library containing glycoconjugates differing by
attached sugars; cells of a first line; cells of a second,
differing line; and instructions for use.
[0042] The facilitative and sodium-dependent glucose transport
processes of cells are mediated by two distinct families of
structurally-related glucose transporters (Table 1).
[0043] The passive, facilitative glucose transporters (GLUTs) allow
the energy independent transport of glucose across the hydrophobic
cell membrane and down its concentration gradient. Thirteen members
of this family have been identified, GLUT1-12 and HMIT
(H.sup.+/myo-inositol transporter) and sequence comparison suggests
structural conservation exists throughout the entire family (FIG.
2).
[0044] Based upon phylogenetic analysis, the GLUT family has been
divided into three classes. Class I (48-63% identity) includes the
well-characterized GLUT1-4. Class II (36-40% identity) contains the
fructose transporter GLUT5 and three recently described proteins
GLUT7, 9 and 11. Class III (19-41% identity) is composed of newer
members GLUT6, 8, 10, 12 and HMIT.
[0045] A model for the orientation of GLUTs in the cell membrane
has been proposed based upon the amino acid sequences of GLUTs
(FIG. 2b). Based upon this model, GLUTs transverse the cell
membrane twelve times with 12-helical transmembrane (TM) domains
and the NH.sub.2- and COOH-termini both terminate on the
cytoplasmic side of the membrane. A high degree of homology exists
between GLUT proteins across the TM domains with significant
variation limited primarily to loop and NH.sub.2/COOH-- terminal
sequences.
[0046] Two major distinctions among classifications emerge from
this analysis. First, the class I and II members of this family
have a large extracellular loop between TMs 1 and 2, the
glycosylation of which modulates the efficiency of glucose
transport.
[0047] In contrast, the class III members contain a large
extracellular loop between TMs 9 and 10 which carries putative N-
and O-glycosylation motifs. Second, class II members lack the
tryptophan residue within the GPGPIPW motif of TM10 critical for
binding the inhibitory ligands cytochalasin B and forskolin.
[0048] Based upon expression profiling and/or Western blotting,
many GLUT transporters have unique tissue distribution. These
distinctions in tissue distribution, along with the differences in
substrate specificities provide the basis for functional assignment
(Table 1). Among transporters, GLUT1 is the most ubiquitously
distributed isoform and is involved in basal glucose uptake. GLUT2
expression occurs mainly in kidney and intestinal epithelial cells,
where it participates in the release of absorbed glucose, and it
also provides glucose-sensing functions for insulin secretion in
pancreatic .gamma.-cells. GLUT3 is considered to be the
neuron-specific transporter while GLUT4 is predominately expressed
in insulin sensitive tissues.
[0049] Under insulin stimulation, GLUT4 undergoes a rapid
intracellular-to-cell surface translocation, resulting in a
dramatic increase in cellular glucose transport activity. GLUT5 is
considered to be responsible for most of the fructose uptake in the
small intestine and GLUT7, most closely related to GLUT5, is
distributed predominately in the small intestine and colon. GLUT8
is unique as it more closely resembles hexose transporters found in
plants and bacteria. GLUT8, which may also undergo an
intracellular-to-cell surface translocation, has been postulated to
play a major role in providing glucose to mature spermatozoa. GLUT9
is primarily detected in the kidney and the liver and at least
three isoforms are known to be upregulated in diabetes. GLUT10 is
expressed at highest levels in the liver and pancreas but basal
GLUT10 expression has also been detected in a variety of other
tissues.
[0050] GLUT10 has received special interest because of its link to
the human chromosomal region 20q12-13.1 (a susceptibility locus for
Type 2 diabetes). Isoforms of GLUT11, another transporter with
significant similarity to GLUT5, have been found in a variety of
tissues. GLUT12 was initially cloned from breast cancer tissue and
its expression appears to be restricted to insulin-sensitive
tissues. Little is known regarding the tissue distribution of
GLUT6.
[0051] Substrate specificities (FIG. 2a) for many of the GLUT
transporters have been established via expression of single
isolated GLUTs in `GLUT-free` systems. Transfected Xenopus oocytes
have served as the predominate model systems in this regard but
COS-7, CHO and yeast variants have also been employed. Notably,
substrate specificity studies to date have been primarily
restricted to the availability of radio-labeled carbohydrates
(e.g., [U-.sup.14C]fructose, 2-deoxy-D-[2,6-.sup.3H]glucose,
D-[2,6-.sup.3H]glucose) where alternative `substrates` are often
identified through their ability to inhibit the transport of one of
these standard radio-labeled agents (e.g. in these cases, the
`affinity` is actually a measurement of K.sub.i, not a direct
measure of transport).
[0052] From a combination of such general methods and models, GLUT1
specificity studies revealed low affinity (greater than 15 mM) for
3-O-methylglucose, galactose, mannose and glucosamine and high
affinity (approximately 3-5 mM) for 2-deoxyglucose and glucose.
GLUT2 specificity studies revealed low affinity (greater than 15
mM) for glucose, galactose, mannose and fructose but high affinity
(approximately 1 mM) for glucosamine. GLUT3 transports glucose and
2-deoxyglucose with high affinity (approximately 1.5 mM) and a
variety of carbohydrates (including galactose, mannose, maltose,
and xylose) with moderate to low affinity (greater than 8.5 mM).
GLUT4 displays high affinity (less than or equal to 5 mM) for both
glucose and glucosamine. Conversely, GLUT5 has little or no
affinity for glucose, but instead has a high affinity for fructose
(approximately 1 mM). GLUT7, while most closely related to GLUT5,
has high affinity (greater than 0.5 mM) for both glucose and
fructose but does not transport galactose, 2-deoxy-D-glucose or
xylose. GLUT8 also displays high affinity (approximately 2 mM) for
glucose while GLUT9 exhibits glucose and 2-deoxy-glucose
transport.
[0053] GLUT10 displays high affinity (greater than 0.5 mM) for both
glucose and galactose while GLUT11 provides for transport of
fructose and glucose, but not galactose. GLUT12 displays
preferential affinity for glucose over other hexoses. Little is
known regarding the specificity of GLUT6.
[0054] In addition to the limitations of GLUT substrate specificity
assays, such methods cannot be used to directly assess the ability
of GLUT transporters to facilitate the uptake of sugar-drug
conjugates. Therefore, a fundamental advantage of the present
invention is to provide a rapid, quantitative assay to directly
assess the impact of a diverse range of sugars upon the uptake of
sugar-drug conjugates by GLUT transporters.
[0055] Nearly all tumors over-express GLUT family members (Table 1)
normally expressed in the respective tissues of origin, most
notably GLUT1. In some cases, tumors can also induce expression of
GLUTs not normally expressed in the respective tissue of origin.
Overexpression of GLUT1 has been widely observed in tumors
(bladder, brain, breast, cervical, colorectal, cutaneous squamous
cell, embryonic, esophageal, gastric, head and neck,
leiomyosarcomas, lung, liver, ovarian, pancreatic, penile, thyroid,
uterine, and vascular hemangiomas) and, in most cases reported, the
level of GLUT1 overexpression correlates with metastasis and/or
poor prognosis. GLUT5 has been found to be expressed in malignant
breast cells but not in normal breast cells and blocking GLUT5
expression has been shown to reduce the growth of malignant
cancerous cells. GLUT4 and GLUT12 overexpression has also been
observed in breast tumors and, in the case of GLUT4, also in
gastric and lung cancers.
TABLE-US-00001 TABLE 1 Facilitative GLUT Transporters Proposed
Overexpression in Protein Class Isoform.sup.a K.sub.m.sup.b
Distribution Function human cancer.sup.c GLUT1 I 492 3-7
Ubitquitous Basal glucose Bladder (A), brain (A), distribution
uptake cutaneous squamous cell (NR), embryonic (NR), esophageal
(NA), gastric (A), head and neck (A), leiomyosarcomas (A), lung
(A), ovarian (A), pancreatic (NR), penile (NR), thyroid (A), uterus
(NR), vascular- hemangioma (NR) GLUT2 I 524 17 Liver, islets,
kidney, High capacity, Gastric (A) small intestine low affinity
transport GLUT3 I 496 1.4 Brain and nerve cells Neuronal Brain (A),
breast (NR), transport gastric (NR), head and neck (NA), lung (A),
meningiomas (NR), ovarian (NR) GLUT4 I 509 6.6 Muscle, fat, heart
Insulin- Breast (NR), gastric regulated (NR), lung (NR) transport
GLUT5 II 501 --.sup.d Intestine, kidney, testis Fructose Lung (A)
transport GLUT7 II 524 .3 Small intestine, colon, Fructose testis
transport GLUT9 II 511/540 N.D..sup.e Liver, kidney N.D..sup.e
GLUT11 II 496 N.D..sup.e Heart, muscle Fructose transport GLUT6 III
507 N.D..sup.e Spleen, leukocytes, N.D..sup.e brain GLUT8 III 477 2
Testis, blastocyst, Fuel supply for brain, muscle, spermatozoa,
adipocytes insulin- responsive transport in blastocysts GLUT10 III
541 .3 Liver, pancreas N.D..sup.e GLUT12 III 617 N.D..sup.e Heart,
prostrate, H.sup.+/myo-inositol Breast (NR) mammary gland
co-transport HMIT III 618/629 N.D..sup.e brian .sup.anumber of
amino acids; .sup.bmillimolar (mM); .sup.c`A` indicates an
association between overexpression and metastatis/poor prognosis,
`NA` indicates no association between overexpression and
metastatis/poor prognosis has not been reported; .sup.dnot a
substrate; .sup.enot determined.
[0056] Such observations present the opportunity to exploit
GLUT-dependent strategies in cancer treatment and diagnostics. One
such early study was to attempt to use 2-deoxyglucose or
5-thioglucose as antimetabolites to interfere with the glycolytic
dependency of tumors. More recent work has focused upon the
generation of glucose-conjugates. Examples of this approach include
2-G1uSNAP, a glucose-conjugated nitric oxide donor which has shown
promise in ovarian carcinoma.
[0057] In a similar manner, glucose-conjugated
O.sup.6-methylguanine-DNA methyltransferase inhibitors (a major
determinant of resistance to alkylating antitumor agents) presented
selective activity enhancements. Improvements upon oxaliplatin via
glucose conjugation have also been recently reported. Perhaps the
most notable example is the glucose-conjugate of oxazaphosphorine
ifosfamide (glufosfamide) which has demonstrated lower
myelotoxicity and a higher antitumor activity than the parent
ifosfamide. In glufosfamide, the glucose moiety stabilizes the drug
and provides for preferential uptake via sugar transporters.
[0058] For cancer diagnostics/imaging, the most widely used tumor
positron emission tomography (PET) imaging agent is
[.sup.18F]fluorodeoxyglucose (FDG)..sup.29 FDG (FIG. 2a, 6) is
transported, phosphorylated and metabolically trapped in tumor
cells as a glucose surrogate. The overexpression of GLUT
transporters, and glycolytic enzymes such as hexokinase, in tumors
enable the tumor-selective imaging with FDG. The demonstrated
success of both glucose surrogates and glucose-conjugates in cancer
imaging and treatment provide validation for GLUT-dependent
cancer-targeting strategies.
[0059] A fundamental advantage of the present invention is the
development of a unique probe set and assay that enables the
discovery of atypical sugars which impart significantly enhanced
tumor uptake and specificity of a sugar-drug conjugate, as compared
to the standard sugar-drug conjugates of the prior art, such as
glucose, 2-deoxyglucose or FDG.
[0060] Kits. In an alternate embodiment of the invention, a kit for
assaying sugar-mediated transport of glycoconjugates contained in a
glycorandomized library according to the present invention is
provided. In one embodiment the kit includes a glycorandomized
library comprising glycoconjugates with different attached sugars;
cells of a first line; and instructions for use.
[0061] In another embodiment, the present invention provides a kit
for assaying tumor selectivity for sugar-mediated transport of
glycoconjugates contained in a glycorandomized library. In one
embodiment the kit includes a glycorandomized library comprising
glycoconjugates with different attached sugars; cells of a first
line; cells of a second, differing line; and instructions for
use.
[0062] By "instructions for use" we mean a publication, a
recording, a diagram, or any other medium of expression which is
used to communicate the usefulness of the invention for one of the
purposes set forth herein. The instructional material of the kit
can, for example, be affixed to a container which contains the
present invention or be shipped together with a container which
contains the invention. Alternatively, the instructional material
can be shipped separately from the container or provided on an
electronically accessible form on a internet website with the
intention that the instructional material and the biocompatible
hydrogel be used cooperatively by the recipient.
[0063] The following examples are, of course, offered for
illustrative purposes only, and are not intended to limit the scope
of the present invention in any way. Indeed, various modifications
of the invention in addition to those shown and described herein
will become apparent to those skilled in the art from the foregoing
description and the following examples and fall within the scope of
the appended claims.
III. EXAMPLES
Example 1
Synthesis of a Fluorescence-Based Sugar Transporter Probe
Library
[0064] To directly assess the impact of sugars upon the specificity
and efficiency of cellular uptake of glycoconjugates, the inventors
synthesized a neoglycorandomized sugar transporter probe (FIGS. 4A
& b, Probe Set A and Probe Set B) containing several distinct
members differing solely by the sugars attached. By "probe" we mean
a compound useful as marker or environmental indicator, or
modifying reagent for use with the present invention. Probes may
comprises fluorescent, spectroscopic or modifying moieties as
described herein. The different sugars used can be seen in Table
2.
TABLE-US-00002 TABLE 2 Sugars used in the sugar-drug conjugates of
the present invention. D-lyxoside D-lactoside 6-deoxy-D-glucoside
L-rhamnoside 3-deoxy-D-glucoside D-maltoside D-xyloside
N-acetyl-D-glucosaminoside D-riboside D-cellobioside
2-deoxy-D-galactoside 6-deoxy-6-chloro-D-galactoside L-lyxoside
6-deoxy-6-bromo-D-galactoside L-glucoside
6-deoxy-6-azido-D-galactoside D-fucoside
4-deoxy-4-azido-D-glucoside L-mannoside D-glucorono-6,3-lactonide
D-alloside 2-deoxy-2-amino-D-glucoside L-galactoside
3-O-methyl-D-glucoside maltotrioside
2,3,4-tri-O-acetyl-L-rhamnoside L-fucoside mycaroside
D-glucuronoside 2,3,4,6-tetra-O-benzyl-D-glucopyranoside
2-deoxy-D-glucoside 2,3,4-tri-O-benzyl-L-fucopyranoside L-xyloside
2,3,5-tri-O-benzyl-D-arabinofuranoside 3-amino-3-deoxy-L-xyloside
2,3,5-tri-O-benzyl-D-ribofuranoside 3-azido-3-deoxy-L-xyloside
6-deoxy-6-fluoro-D-glucoside 3-thio-3-deoxy-L-xyloside
4-O-(-D-galacto pyranosyl-D- D-Galacturonoside mannopyranoside)
2-deoxy-L-riboside 6-deoxy-6-acyl-D-galactoside D-arabinoside
L-taloside 2-deoxy-D-riboside 6-thio-D-mannose dimer L-riboside
6-deoxy-6-N-decanoyl-D-glucosaminoside D-melibioside
3-deoxy-3-N-decanoyl-D-glucosaminoside D-altroside
3-deoxy-3-carbamic acid allyl ester-D- L-arabinoside glucoside
D-mannoside 6-deoxy-3-carbamic acid allyl ester-D-
N-acetyl-D-galactosaminoside glucoside novioside
2,3,4,6-tetra-O-benzyl-D- mannopyranoside L-alloside
3-deoxy-3-azido-D-glucoside D-taloside D-Digitoxoside L-altroside
6-deoxy-6-amino-D-glucoside 3-fluoro-3-deoxy-D-glucoside
D-galactoside 2-fluoro-2-deoxy-D-glucoside
6-deoxy-6-thio-acyl-D-galactoside N-acetyl-D-mannosaminoside
D-idoside L-idoside D-guloside D-glucoside
[0065] The library synthesis was accomplished in four simple steps
with an overall yield of 15-37% starting from
commercially-available aminofluoroscein (FIG. 5a, 11). All members
were purified by solid phase extraction to provide a library with
an average purity greater than or equal to 93%.
[0066] In contrast to the few prior reports of sugar-fluorophore
conjugates (limited to D-glucose, D-glucosamine, 2-deoxy-D-glucose,
2-deoxyfluoroglucose, galactose and fructose), the neoglycosyl
chemistry of the present invention enables the rapid synthesis of
large probe libraries by eliminating most of the tedious
protection, deprotection and anomeric activation steps associated
with conventional glycosylation strategies.
[0067] In addition, as additional comparators to the D-glucose
`standard`, the 2-deoxy-D-glucose probe displayed similarly broad
specificity but slightly lower uptake levels, while the FDG probe
also displayed slightly lower uptake levels but a preference for
the prostrate and non-small cell lung cell lines (data not
shown).
Example 2
Sugar Transport Assay
[0068] For the sugar transporter assay of the present invention,
cells were plated at a density of 30,000 cells/ml (100 .mu.L/well)
in a 96-well clear bottom microtiter plates. Cells were allowed to
attach overnight, and then incubated in glucose-free medium for an
additional 24 hours. Compounds were added (from 96-well compound
stock plates at a 1:100 dilution) using a Biomek FX liquid handler
equipped with a 96 channel head. The plates were incubated for 1
hour and then the compound solution was removed by gentle
aspiration. The cells were subsequently washed (1.times.100 .mu.L)
with phosphate buffered saline to remove residual fluorescent
dye.
[0069] Sugar-conjugate uptake was assessed via intracellular
fluorescence (FIG. 4b-4f) as determined using a Safire2 microplate
reader .delta..sub.ex=485 nm, .delta..sub.em=535 nm) and normalized
to cell number by a subsequent Cell-Titer Glo assay. For the
Cell-Titer Glo assay, 50 .mu.L Cell-Titer Glo of was added to each
well of the 96 well plate and the plates were incubated for 10
minutes at room temperature with gentle shaking The Cell Titer Glo
assay was analyzed on a Safire2 microplate reader for luminescence
at 1 second per well. As an initial preliminary study, the
transport assay with a representative set of probes was also
analyzed using microscopy (FIG. 6).
[0070] The resultant data show that the present invention provides
the first example of a systematic approach for rapidly assessing
the specific role of a wide range of sugars to enhance the specific
uptake of sugar-conjugates and extends well beyond the standard
small set of sugars employed in typical sugar transport studies.
Most importantly, the inventors' results illustrate that slight
perturbations in sugar structure can lead to drastic consequences
in terms of in vitro cellular uptake. For instance, the
neoglycorandomized L-glucose of the present invention exhibits a
non-specific enhancement of overall uptake (FIG. 5B, L-glucose) in
comparison to D-glucose (FIG. 5A). Similarly, the
neoglycorandomized N-acetyl-D-mannosamine of the present invention
exhibits a nearly complete exclusion (FIG. 5C,
N-acetyl-D-mannosamine). Further, some neoglycorandomized sugars of
the present invention exhibit a tumor-preference--prostrate tumor
versus `normal` prostrate (FIG. 5D, D-glucuronic acid), or
non-small cell lung (FIG. 5E, L-allose) or breast tumor versus
`normal` breast (FIG. 5F, L-xylose).
[0071] In summary, the sugars identified herein impart remarkable
cell line selectivities (up to a greater than or equal to an
eight-fold increased selective uptake in tumor versus normal cell
lines in 1 hr) and are far superior (up to greater than or equal to
a 10-fold increased uptake in 1 hr versus the `standard` D-glucose
conjugate) to the conventional sugars (D-glucose, 2-deoxy-D-glucose
or FDG) employed for uptake enhancement.
[0072] While the method of the present invention is described
herein as used to identify and assess enhanced conjugates for
cancer treatment and prevention, the method and composition of the
present invention may also be used to identify and assess enhanced
conjugates for other issues as well. For instance, the invention
may be used to probe for the best sugars to enhance oral
bioavailability and/or gut uptake and thereby enhance oral
bioavailabity/uptake of drugs, such as with standard Caco-2
monlayers. In addition, the invention may be used to probe for the
best sugars for targeting specific bacterial pathogens (e.g. via
screening for uptake into different bacterial pathogens) and
thereby sugar target antibiotics. Similarly, the invention may be
used to probe for the impact of sugars upon whole body
distribution. Finally, the present invention may be use used to
provide a convenient HT assay for GLUTs or SGLTs. The inhibition of
GLUTs are known to kill cancer cells and the inhibition of SGLTs
are relevant to reduction of sugar uptake (e.g. for diabetes drugs)
or even anti-parasite/viral drug development.
[0073] It is understood that the invention is not confined to the
specific reagents, formulations, reaction conditions, etc., herein
illustrated and described, but embraces such modified forms thereof
as come within the scope of the following claims. Other embodiments
and uses of the invention will be apparent to those skilled in the
art from consideration from the specification and practice of the
invention disclosed herein, as well as from the attached pages,
which are incorporated by reference herein. All references cited
herein for any reason, including all journal citations and
U.S./foreign patents and patent applications, are specifically and
entirely incorporated herein by reference.
* * * * *