U.S. patent application number 09/770849 was filed with the patent office on 2001-12-13 for novel ligands and methods for preparing same.
Invention is credited to Lee, Chee Wee.
Application Number | 20010051348 09/770849 |
Document ID | / |
Family ID | 26874622 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051348 |
Kind Code |
A1 |
Lee, Chee Wee |
December 13, 2001 |
Novel ligands and methods for preparing same
Abstract
A process is disclosed for modifying a parent ligand by
attaching to the parent ligand a conjugation agent that is reactive
with a moiety of a target receptor to which the parent ligand binds
such that a covalent bond is formable between the conjugation agent
and the receptor moiety. Also disclosed are compositions, probes
and methods of detecting and/or quantifying receptors using the
modified ligands of the invention.
Inventors: |
Lee, Chee Wee; (Singapore,
SG) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Family ID: |
26874622 |
Appl. No.: |
09/770849 |
Filed: |
January 26, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60178756 |
Jan 28, 2000 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
530/408 |
Current CPC
Class: |
C07D 403/06 20130101;
G01N 33/566 20130101; C07D 403/04 20130101; C07D 405/14 20130101;
C07D 403/12 20130101; C07D 473/34 20130101; G01N 33/6803 20130101;
A61K 47/54 20170801; A61K 47/549 20170801; C07H 19/06 20130101;
G01N 33/6842 20130101; G01N 33/531 20130101; C07H 19/16
20130101 |
Class at
Publication: |
435/7.1 ;
530/408 |
International
Class: |
G01N 033/53; C07K
001/13 |
Claims
1. A process for modifying a parent ligand, comprising attaching to
said parent ligand a conjugation agent that is reactive with a
moiety of a target receptor to which said parent ligand binds,
wherein when said parent ligand binds to the receptor a covalent
bond is formed between said conjugation agent and said moiety, and
wherein the parent ligand binds specifically with a nucleoside
transporter.
2. The process of claim 1, wherein the conjugation agent is
attached to the parent ligand through a spacer.
3. The process of claim 2, wherein the spacer comprises a group
selected from the group consisting of alkyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, oxoalkyl, heterooxoalkyl, alkenyl,
hetero alkenyl, aralkyl, hetero aralkyl, aryl and heteroaryl
radicals.
4. The process of claim 1, wherein the conjugation agent is
selected from the group consisting of a sulfhydryl group specific
conjugation agent, an amino group specific conjugation agent, a
carboxyl group specific conjugation agent, a tyrosine specific
conjugation agent, an arginine specific conjugation agent, a
histidine specific conjugation agent, a methionine specific
conjugation agent, a tryptophan specific conjugation agent, and a
serine specific conjugation agent.
5. The process of claim 1, wherein the conjugation agent is a
sulfhydryl group specific conjugation agent selected from the group
consisting of N-maleimide and N-maleimide derivatives.
6. The process of claim 5, wherein the N-maleimide derivatives are
selected from the group consisting of disulfide reagents including
5'-dithiobis-(2-nitrobenzoic acid), 4,4'-dithiodipyridine,
methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl
disulfide.
7. The process of claim 1, wherein the conjugation agent is
selected from the group consisting of alkylating agents and
acylating agents.
8. A process for modifying a parent ligand, comprising attaching to
said parent ligand a sulfhydryl group specific conjugation agent
that is reactive with a sulfhydryl group of a target receptor to
which said parent ligand binds, wherein when said parent ligand
binds to the receptor a covalent bond is formed between said
conjugation agent and said sulfhydryl group, and wherein the parent
ligand binds specifically with a serotonin receptor.
9. A modified ligand produced by the process of claim 1.
10. A modified ligand produced by the process of claim 8.
11. A modified ligand having the general formula: L--R.sub.1--A (I)
wherein L is a parent ligand that binds specifically with a target
receptor comprising a nucleoside transporter; wherein A is a
conjugation agent that is reactive with a moiety of the target
receptor to which the parent ligand binds, such that when said
parent ligand binds to the receptor a covalent bond is formed
between said conjugation agent and said moiety; and R.sub.1 is an
optional spacer.
12. The modified ligand of claim 11, wherein the spacer comprises a
non-hydrolysable radical selected from the group consisting of
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl,
heterooxoalkyl, alkenyl, hetero alkenyl, aralkyl, hetero aralkyl,
aryl and heteroaryl radicals.
13. The modified ligand of claim 11, wherein the conjugation agent
is a sulfhydryl group specific conjugation agent.
14. The modified ligand of claim 12, wherein the sulfhydryl group
specific conjugation agent is selected from the group consisting of
N-maleimide and N-maleimide derivatives.
15. The modified ligand of claim 11, wherein the parent ligand
binds specifically with an es nucleoside transporter.
16. The modified ligand of claim 11, which is interactive with es
nucleoside transporter and/or
nucleoside/nucleotide/nucleobase-sensitive proteins, wherein said
modified ligand has a general formula selected from the group
consisting of: 6wherein A is N-maleimide, 2-pyridyldithio, or
halogen; X is NH, S, or O; Y is H, halogen, NH.sub.2, or O; Z is H,
halogen, or CH.sub.3; R.sub.1 is spacer arm; and R.sub.2 is H,
.beta.-D-ribose, .beta.-D-2-deoxyribose, or their 5'-mono-, 5' di-,
and 5' tri-phosphate.
17. The modified ligand of claim 11, which has a general formula
selected from the group consisting of: 7wherein R.sub.4 is
4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],
4-[p-phenyl]butyrate, N-[.gamma.-butyrate], N-[.alpha.-acetate], or
N-[.epsilon.-caproylate]; 8wherein R.sub.4 is
4-[N-methyl]cyclohexane carboxylate, N-[m-benzoate],
4-[p-phenyl]butyrate, N-[.gamma.-butyrate], N-[.alpha.-acetate], or
N-[.epsilon.-caproylate]; 9wherein R.sub.5 is
4-carbonyl-.alpha.-methyl-.alpha.-toluene,
6-[.alpha.-methyl-.alpha.-tulo- amido]-hexanoate, N-[3-propionate],
or 6-[3'-propioamido]hexanoate; and 10wherein R.sub.5 is
4-carbonyl-.alpha.-methyl-.alpha.-toluene,
6-[.alpha.-methyl-.alpha.-tuloamido]-hexanoate, N-[3-propionate],
or 6-[3 '-propioamido]hexanoate.
18. A modified ligand having the general formula: L--R.sub.1--A (I)
wherein L is a parent ligand that binds specifically with a target
serotonin receptor; wherein A is a conjugation agent that is
reactive with a sulfhydryl group of said target receptor to which
the parent ligand binds, such that when said parent ligand binds to
the receptor a covalent bond is formed between said conjugation
agent and said sulfhydryl group; and R.sub.1 is an optional
spacer.
19. The modified ligand of claim 18, wherein the parent ligand
comprises serotonin or a precursor or analog thereof.
20. The modified ligand of claim 18, wherein the modified ligand
has a structure selected from the group consisting of the
structures shown below: 11LBT3001
(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dion- e)
12LBT3002
(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxy-1H-in-
dol-3-yl)-ethyl]-butyramide) 13LBT3004
(3-(2,5-dioxo-2,5-dihydro-pyrrol-1-
-yl)-N-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-propionamide); and
14LBT3005 (4-(2,5-dioxo-2,5-dihydro-pyrrol-1-ylmethyl)-cyclohexane
carboxylic acid [2-(5-hydroxy-lH-indol-3-yl)-ethyl]-amide)
21. A composition comprising the modified ligand of claim 11 and a
pharmaceutically acceptable carrier.
22. A composition comprising the modified ligand of claim 18 and a
pharmaceutically acceptable carrier.
23. A method of detecting the presence of a target receptor in a
test sample, comprising: contacting said sample with the modified
ligand of claim 11, wherein said modified ligand binds said target
receptor if present in said test sample; and detecting the presence
of a complex comprising said modified ligand and said receptor in
said contacted sample.
24. A method of quantifying the presence of a target receptor in a
test sample, comprising: contacting said sample with the modified
ligand of claim 11, wherein said modified ligand binds said target
receptor if present in said test sample; measuring the
concentration of a complex comprising said modified ligand and said
receptor in said contacted sample; and relating said measured
complex concentration to the concentration of said receptor in said
sample.
25. A method of detecting the presence of a target receptor in a
test sample, comprising: contacting said sample with the modified
ligand of claim 18, wherein said modified ligand binds said target
receptor if present in said test sample; and detecting the presence
of a complex comprising said modified ligand and said receptor in
said contacted sample.
26. A method of quantifying the presence of a target receptor in a
test sample, comprising: contacting said sample with the modified
ligand of claim 18, wherein said modified ligand binds said target
receptor if present in said test sample; measuring the
concentration of a complex comprising said modified ligand and said
receptor in said contacted sample; and relating said measured
complex concentration to the concentration of said receptor in said
sample.
27. A probe that covalently binds to a target receptor, said probe
comprising the modified ligand of claim 11 having a reporter
molecule associated therewith.
28. Use of the modified ligand of claim 11 or of the probe of claim
27 in the study of target receptor function.
29. A probe that covalently binds to a target receptor, said probe
comprising the modified ligand of claim 18 having a reporter
molecule associated therewith.
30. Use of the modified ligand of claim 18 or of the probe of claim
29 in the study of target receptor function.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/178,756, filed Jan. 28, 2000, the
disclosure of which is incorporated herein by reference in its
entirety.
TECHNICAL FIELD
[0002] This invention relates generally to ligand-receptor
interactions. In particular, the present invention relates to
modified ligands that bind irreversibly to their cognate receptors,
and to methods for preparing such ligands. The novel ligands of the
invention have utility inter alia for investigating protein
function and as drugs (in healthcare, agricultural and
environmental applications) for more effectively inhibiting or
stimulating cognate receptor function.
BACKGROUND ART
[0003] Pharmacological receptors are intracellular or
membrane-bound proteins which produce a pharmacological effect
after binding with a specific ligand. In this regard, a
pharmacological receptor has a dual function to (a) detect a ligand
signal by forming a ligand-receptor complex and to (b) conduct and
translate the signal leading to the pharmacological effect.
[0004] Drugs can replace endogenous physiological ligands to
interact with receptors. A prerequisite for such a drug-receptor
interaction is the formation of a drug-receptor complex, just as in
the case of ligand-receptor interaction. In contrast to
physiological ligands that stimulate an effect after binding to a
receptor (receptor-mediated effects), drugs can be classified as
(a) agonists or drugs which stimulate an effect after binding to
the receptor, and (b) antagonists or drugs which do not stimulate
an effect after receptor binding.
[0005] Several types of molecular interactions are possible for
drug-receptor binding including ionic bonds, hydrogen bonds, and
hydrophobic bonds by van der Waals forces. The vast majority of
receptor interactions involve several kinds of binding
simultaneously. Ionic bonds are important for the primary phase of
drug-receptor interaction since these bonds have the greatest or
longest range. After the initial interaction, fine-tuning takes
place involving dipole-dipole-bonds, hydrogen bonds and hydrophobic
bonds. Although all these interactions also fix the drug molecule
in the receptor's active site, the bindings are nevertheless
reversible, as the force of interaction is very weak. Hence the
pharmacological effectiveness of any drug is often affected by its
own concentration in the plasma, as a decrease in plasma drug
concentration will increase the dissociation of drug molecule from
its receptor.
[0006] Several agents are known to inhibit enzymes irreversibly or
pseudoirreversibly and the precise mechanism of inhibition gives
rise to subtle differences in the inhibition profiles and the
duration of inhibition.
[0007] Many inhibitors of acetylcholinesterase react covalently
with this enzyme to form an acyl enzyme that deacylates more slowly
than the acetyl enzyme formed with the natural substrate
acetylcholine. The acetyl enzyme forms rapidly by attack of the
active site serine on the substrate. Transfer of the acyl group to
the enzyme occurs through a tetrahedral intermediate. The acetyl
enzyme is rapidly hydrolyzed, with a half time of 10 .mu.sec. These
rapid acylation and deacylation steps give rise to a turnover rate
of 10.sup.5 substrate molecules per enzyme molecule per second.
Cholinesterase inhibitors such as physostigmine and neostiginine
form methylaminocarbamyol and dimethylaminocarbamoyl enzymes, which
have half times for deacylation of several minutes. Thus, by
providing the enzyme with an alternative substrate, catalysis of
acetylcholine is precluded during the catalytic cycle for the
carbamoylating agent. The kinetic constants for the respective
acylation steps for the acetoxy and carbamoxy ester substrates do
not greatly differ; hence the longer residence time of the
carbamoyl enzyme conjugate is an important factor in favoring
inhibition.
[0008] Several other enzymes are inhibited by covalent attachment
of the inhibitor, giving rise to irreversibility. The hydrazines
(phenelzine, isocarxazid metabolites) and the acetylenic agents
(pargyline) are oxidized to reactive intermediates by monoamine
oxidase. These intermediates attack the associated flavin cofactor
on the enzyme Such agents have been termed suicide substrates since
their activation requires catalysis by the very enzyme that they
inactivate. Hence the inactivation process is mechanism-based.
There are now many examples of such substrates, activation of which
by the enzyme results in covalent modification of the enzyme or of
an associated cofactor. Often this occurs by conjugation or
association of the enzyme with its substrate followed by a
neighboring group attack. Several of the targets of suicide
substrates have therapeutic significance. These include the
penicillinases and alanine racemases in antibacterial design; GABA
transaminase inhibitors for antiepileptic agents; lipoxygenase and
cyclooxygenase inhibitors to control leukotriene and prostaglandin
biosynthesis, respectively; aromatase inhibitors to block formation
of estrogenic hormones; ornithine decarboxylase inhibitors as
antiparasitic agents; and dopamine .beta.-hydroxylase inhibitors to
control catecholamine biosynthesis. Many suicide substrates serve
as antimetabolites and are potential antineoplastic agents. The
effectiveness of these inhibitors depends not only on their
relative dissociation constants or K.sub.m values compared with
those of the endogenous substrate but also on kinetic competition
between turnover of the suicide substrate and the inactivation
event.
[0009] Omeprazole (PRILOSEC) is another well-known irreversible
binding drug that has been released for clinical use. This drug
inhibits gastric acid secretion by binding to the H.sup.+,
K.sup.+-ATPase present only in the apical membrane of parietal
cells. Omeprazole is especially useful in patients with
hypergastrinemia and may be valuable in those whose peptic ulcer
disease is not well controlled by H.sub.2 antagonists. Omeprazole
contains a sulfinyl group in a bridge between substituted
benzimidazole and pyridine rings. At neutral pH, this drug is a
chemically stable, lipid-soluble, weak base that is devoid of
inhibitory activity. This neutral weak base reaches parietal cells
from the blood and diffuses into the secretory canaliculi, where
the drug becomes protonated and thereby trapped. Protonated drug
rearranges to form a sulfenic acid and a sulfenamide. The
sulfenamide interacts covalently with sulfhydryl group at critical
sites in the extracellular (luminal) domain of the
membrane-spanning H.sup.+, K.sup.+-ATPase. Omeprazole must thus be
considered as prodrug that needs to be activated to be
effective.
[0010] Despite the availability of several agents that bind their
targets irreversibly, there is a dearth of methods currently
available for rationally designing ligands to irreversibly bind a
target receptor.
DISCLOSURE OF THE INVENTION
[0011] The present invention arises, at least in part, from the
unexpected discovery that by attaching a conjugation agent to a
parent ligand that reversibly binds a target receptor, wherein the
conjugation agent is reactive with a moiety of the target receptor
such that a covalent bond is formable between the conjugation agent
and the moiety, the modified ligand thus produced is capable of
binding the target receptor irreversibly Accordingly, in one aspect
of the invention, there is provided a process for modifying a
parent ligand, comprising attaching to said parent ligand a
conjugation agent that is reactive with a moiety of a target
receptor to which said parent ligand binds such that a covalent
bond is formable between said conjugation agent and said
moiety.
[0012] Suitably, the conjugation agent is attached to the parent
ligand through a spacer.
[0013] Preferably, the spacer is covalently attached to the parent
ligand.
[0014] Preferably, the spacer is covalently attached to the
conjugation agent.
[0015] The spacer is suitably radical selected from the group
consisting of alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl,
oxoalkyl, heterooxoalkyl, alkenyl, hetero alkenyl, aralkyl, hetero
aralkyl, aryl and heteroaryl radicals or any other molecular
conformation which serves the function of being a spacer. The
length of the spacer arm is suitably selected from a range of
between about 0 .ANG. and about 20 .ANG..
[0016] Preferably, the spacer is a non-hydrolysable radical under
physiological conditions.
[0017] Preferably, the conjugation agent is selected from the group
consisting of a sulfhydryl group specific conjugation agent, an
amino group specific conjugation agent, a carboxyl group specific
conjugation agent, a tyrosine specific conjugation agent, an
arginine specific conjugation agent, a histidine specific
conjugation agent, a methionine specific conjugation agent, a
tryptophan specific conjugation agent, and a serine specific
conjugation agent.
[0018] The sulfhydryl group specific conjugation agent may be
selected from the group consisting of N-maleimide, N-maleimide
derivatives and disulfide reagents including, but not restricted
to, 5'-dithiobis-(2-nitrobenzoic acid), 4,4'-dithiodipyridine,
methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl
disulfide.
[0019] The amino group specific conjugation agent may be selected
from the group consisting of alkylating agents including, but not
restricted to, .alpha.-haloacetyl compounds, aryl halides,
aldehydes and ketones, and acylating agents including, but not
restricted to, isocyanate, isothiocyanate, imidoesters,
N-hydroxylsuccinimidyl ester, .rho.-nitrophenyl ester, acyl
chloride, and sulfonyl chloride.
[0020] The carboxyl group specific conjugation agent may be
selected from the group consisting of carbodiimides and carboxyl
group esterification reagents including, but not restricted to,
diazoacetate esters and diazoacetamides.
[0021] The tyrosine specific conjugation agent may be selected from
diazonium derivatives including, but not limited to, benzidine and
bis-diazotized 3,3'-dimethylbenzidine.
[0022] The arginine specific conjugation agent may be selected from
1,2-dicarbonyl reagents including, but not restricted to, glyoxal,
phenylglyoxal, 2-3-butanedione and 1 ,2-cyclohexanedione.
[0023] The histidine specific conjugation agent is suitably
selected from the group consisting of alkylating agents including,
but not restricted to, .alpha.-haloacetyl compounds, aryl halides,
aldehydes and ketones, and acylating agents including, but not
restricted to, diethylpyrocarbonate, ethoxyformic anhydride,
isocyanate, isothiocyanate, imidoesters, N-hydroyxlsuccinimidyl
ester, .rho.-nitrophenyl ester, acyl chloride, and sulfonyl
chloride.
[0024] The methionine specific conjugation agent may be selected
from the group consisting of alkylating agents including, but not
restricted to, .alpha.-haloacetyl compounds, aryl halides,
aldehydes and ketones.
[0025] The tryptophan specific conjugation agent may be selected
from the group consisting of N-bromosuccinimide,
2-hydroxy-5-nitrobenzyl bromide and .rho.-nitrophenylsulfenyl
chloride.
[0026] The serine specific conjugation agent may be selected from
the group consisting of diisopropylfluorophosphate and
acrylsulfonyl fluorides including, but not restricted to,
phenylmethyl-sulfonylfluoride- .
[0027] The parent ligand may be any natural or non-natural ligand
but is preferably a biologically active ligand inclusive of known
drugs and naturally occurring or synthesized drug candidate
compounds.
[0028] In another aspect, the invention provides a modified ligand
produced by the process broadly described above.
[0029] In yet another aspect of the invention, there is provided a
modified ligand having the general formula:
L--R.sub.l--I--A (I)
[0030] wherein L is a parent ligand;
[0031] wherein A is a conjugation agent that is reactive with a
moiety of a target receptor to which the parent ligand binds such
that a covalent bond is formable between said conjugation agent and
said moiety; and
[0032] R.sub.1 is an optional spacer which preferably comprises a
non-hydrolysable radical selected from the group consisting of
alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, oxoalkyl,
heterooxoalkyl, alkenyl, hetero alkenyl, aralkyl, hetero aralkyl,
aryl and heteroaryl radicals.
[0033] In a preferred embodiment, the modified ligand is
interactive with es nucleoside transporter and/or
nucleoside/nucleotide/nucleobase-sensiti- ve proteins, wherein said
modified ligand has a general formula selected from the group
consisting of: 1
[0034] wherein A is N-maleimide, 2-pyridyldithio, or halogen; X is
NH, S, or O; Y is H, halogen, NH.sub.2, or O; Z is H, halogen, or
CH.sub.3; R.sub.1 is spacer arm comprising a non-hydrolysable
radical preferably under physiological conditions; R.sub.2 is H,
.beta.-D-ribose, .beta.-D-2-deoxyribose, or their 5'-mono-, 5' di-,
and 5' tri-phosphate.
[0035] Suitably, the modified ligand inhibits said es nucleoside
transporter and/or nucleoside/nucleotide/nucleobase-sensitive
proteins and has a general formula selected from the group
consisting of: 2
[0036] wherein R.sub.4 is 4-[N-methyl]cyclohexane carboxylate,
N-[m-benzoate], 4-[p-phenyl]butyrate, N-[.gamma.-butyrate],
N-[.alpha.-acetate], or N-[.epsilon.-caproylate]; 3
[0037] wherein R.sub.4 is 4-[N-methyl]cyclohexane carboxylate,
N-[m-benzoate], 4-[p-phenyl]butyrate, N-[.gamma.-butyrate],
N-[.alpha.-acetate], or N-[.epsilon.-caproylate]; 4
[0038] wherein R.sub.5 is
4-carbonyl-.alpha.-methyl-.alpha.-toluene,
6-[.alpha.-methyl-a-tuloamido]-hexanoate, N-[3-propionate], or
6-[3'-propioamido]hexanoate; and 5
[0039] wherein R.sub.5 is
4-carbonyl-.alpha.-methyl-.alpha.-toluene,
6-[.alpha.-methyl-.alpha.-tuloamido]-hexanoate, N-[3-propionate],
or 6-[3'-propioamido]hexanoate.
[0040] In another embodiment modified ligands that bind to
seratonin receptors can be used.
[0041] In another aspect, the invention resides in a composition
comprising the modified ligand as broadly described above, together
with a pharmaceutically acceptable carrier.
[0042] In a further aspect of the invention, there is provided a
method of treatment or prophylaxis of a condition associated with a
target receptor, said method comprising administering to a patient
in need of such treatment a therapeutically effective dosage of the
composition as broadly described above.
[0043] According to another aspect of the invention, there is
provided a method of detecting the presence of a target receptor in
a test sample, comprising: contacting said sample with a modified
ligand as broadly described above, wherein said modified ligand
binds said target receptor; and detecting the presence of a complex
comprising said modified ligand and said receptor in said contacted
sample.
[0044] In another aspect of the invention, there is provided a
method of quantifying the presence of a target receptor in a test
sample, comprising: contacting said sample with a modified ligand
as broadly described above, wherein said modified ligand binds said
target receptor; measuring the concentration of a complex
comprising said modified ligand and said receptor in said contacted
sample; and relating said measured complex concentration to the
concentration of said receptor in said sample.
[0045] In yet another aspect, the invention provides a method of
detecting the presence of a target receptor on a cell or cell
membrane, comprising: contacting a sample containing said cell or
cell membrane with a modified ligand as broadly described above,
wherein said modified ligand binds said target receptor; and
detecting the presence of a complex comprising said modified ligand
and said cell or cell membrane in said contacted sample.
[0046] In another aspect of the invention, there is provided a
method of quantifying the presence of a target receptor on a cell
or cell membrane, comprising: contacting a sample containing said
cell or cell membrane with a modified ligand as broadly described
above, wherein said modified ligand binds said target receptor;
measuring the concentration of a complex comprising said modified
ligand and said cell or cell membrane in said contacted sample; and
relating said measured complex concentration to the concentration
of said receptor present on said cell or cell membrane.
[0047] In another aspect, the invention extends to a probe that
covalently binds to a target receptor, said probe comprising a
modified ligand as broadly described above having a reporter
molecule associated therewith.
[0048] In one embodiment, the probe comprises the modified ligand
that is interactive with es nucleoside transporter and/or
nucleoside/nucleotide/n- ucleobase-sensitive proteins, as broadly
described above.
[0049] In this respect, the cell is preferably an animal cell, more
preferably a mammalian cell, and more preferably a human cell.
Alternatively, the cell may be a plant cell or a microbial cell.
The microbial cell includes, but is not restricted to, a cell of
bacterial, viral or fungal origin.
[0050] The invention also encompasses the use of the modified
ligand and probe as broadly described above inter alia in the
study, treatment and prevention of conditions associated with their
corresponding target receptors.
[0051] In one embodiment, there is provided process for modifying a
parent ligand, comprising attaching to said parent ligand a
conjugation agent that is reactive with a moiety of a target
receptor to which said parent ligand binds, wherein when said
parent ligand binds to the receptor a covalent bond is formed
between said conjugation agent and said moiety.
[0052] In one preferred embodiment, the conjugation agent is
positioned on the ligand at a position that promotes and/or permits
covalent bond formation with the moiety of the target receptor. In
another preferred embodiment, the receptor to which the ligand
binds is an active site associated with a biological activity. The
receptor is, for example, a cell surface receptor. The binding of
the modified ligand to the receptor in one embodiment is associated
with altered activity of the target receptor.
[0053] In another preferred embodiment, the parent ligand and/or
receptor are naturally occurring. In another embodiment, the
modified ligand is not a crosslinking agent. In another embodiment,
the modified ligand optionally does not comprise a photo-reactive
group such as a photolabel. In another embodiment, the modified
ligand does not comprise a label. In another embodiment, the
receptor to which the ligand binds is not a nucleic acid, such as
RNA or DNA. In another embodiment, the conjugation agent does not
bind to a moiety of a nucleic acid, and optionally binds to a
residue of an amino acid.
[0054] In one embodiment, there is provided process for modifying a
parent ligand, comprising attaching to said parent ligand a
conjugation agent that is reactive with a moiety of a target
receptor to which said parent ligand binds, wherein when said
parent ligand binds to the receptor a covalent bond is formed
between said conjugation agent and said moiety, and wherein the
parent ligand binds specifically with a nucleoside transporter.
[0055] In another embodiment, there is provided a process for
modifying a parent ligand, comprising attaching to said parent
ligand a sulfhydryl group specific conjugation agent that is
reactive with a sulfhydryl group of a target receptor to which said
parent ligand binds, wherein when said parent ligand binds to the
receptor a covalent bond is formed between said conjugation agent
and said sulfhydryl group, and wherein the parent ligand binds
specifically with a serotonin receptor.
[0056] In one embodiment, there is provided a modified ligand
having the general formula:
L--R.sub.1--I--A (I)
[0057] wherein L is a parent ligand that binds specifically with a
target receptor comprising a nucleoside transporter;
[0058] wherein A is a conjugation agent that is reactive with a
moiety of the target receptor to which the parent ligand binds,
such that when said parent ligand binds to the receptor a covalent
bond is formed between said conjugation agent and said moiety; and
R.sub.1 is an optional spacer.
[0059] In another embodiment, there is provided a modified ligand
having the general formula:
L--R.sub.1--A (I)
[0060] wherein L is a parent ligand that binds specifically with a
target serotonin receptor;
[0061] wherein A is a conjugation agent that is reactive with a
sulfhydryl group of said target receptor to which the parent ligand
binds, such that when said parent ligand binds to the receptor a
covalent bond is formed between said conjugation agent and said
sulfhydryl group of said receptor; and R.sub.1 is an optional
spacer.
[0062] The disclosure of all patents, patent applications,
publications and published patent applications referred to herein
are incorporated herein by reference in their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0063] The invention, as exemplified by preferred embodiments, is
described with reference to the following drawings in which:
[0064] FIG. 1 shows the NEM inhibition of equilibrative
.sup.3H-uridine transport in murine myeloma SP2/0-Ag14 cells.
[0065] FIG. 2 shows the effects of NEM on the kinetics of
.sup.3H-NBMPR equilibrium binding in murine myeloma SP2/0-Ag14
cells.
[0066] FIG. 3 shows the general reaction scheme for synthesis of
CrMCC.
[0067] FIG. 4 shows the rate of CrMCC formation.
[0068] FIG. 5 shows the light absorbance profile of CrMCC and
NBMPR.
[0069] FIG. 6 shows the inhibition of .sup.3H-NBMPR binding by
CrMCC, cytidine and SMCC in human HL-60 promyelocytic leukemia
plasma membranes.
[0070] FIG. 7 shows the effect of CrMCC on the kinetics of
.sup.3H-NBMPR binding to human HL-60 promyelocytic leukemia plasma
membranes.
[0071] FIG. 8 shows the effects of CrMCC, cytidine and SMCC on
growth of HL-60 cells.
[0072] FIG. 9 shows the time course of .sup.3H-CrMCC binding to
human HL-60 promyelocytic leukemia plasma membranes.
[0073] FIG. 10 shows the dissociation of .sup.3H-CrMCC and
.sup.3H-cytidine from the binding sites of human HL-60
promyelocytic leukemia plasma membranes.
[0074] FIG. 11 shows the concentration dependence of .sup.3H-CrMCC
binding to human HL-60 promyelocytic leukemia plasma membranes.
[0075] FIG. 12 shows the effect of pH on the dissociation of
.sup.3H-CrMCC from its binding site in human HL-60 promyelocytic
leukemia plasma membranes. FIG. 13 shows the inhibition of
.sup.3H-CrMCC binding to human HL-60 promyelocytic leukemia plasma
membranes.
[0076] FIG. 14 shows the covalent binding of sulfhydryl reactive
.sup.3H-cytidine analogs to human HL-60 promyelocytic leukemia
plasma membranes.
[0077] FIG. 15 shows the UV absorbance profile of reversed-phase
chromatography of human HL-60 promyelocytic leukemia plasma
membrane proteins.
[0078] FIG. 16 shows the radioactivity profile of reversed-phase
chromatography of human HL-60 promyelocytic leukemia plasma
membrane proteins.
[0079] FIG. 17 shows the covalent binding of sulfhydryl group
reactive .sup.3H-adenosine analogs to human HL-60 promyelocytic
leukemia plasma membranes.
[0080] FIG. 18 shows the inhibition of .sup.3H-5-HT binding to
murine brain membranes by various 5-HT analogs.
[0081] FIG. 19a shows the chemical structure of LBT3001
(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione)).
[0082] FIG. 19b shows the reaction scheme for synthesis of
LBT3001.
[0083] FIG. 20a shows the chemical structure of LBT3002
(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxyl-1
H-indol-3-yl)-ethyl]-butyramide).
[0084] FIG. 20b shows the reaction scheme for synthesis of
LBT3002.
[0085] FIG. 21a shows chemical structure of LBT3004
(3-(2,5-dioxo-2,5-dihydro-pyrrol -1-yl)-N-[2-(5-hydroxyl-1
H-indol-3-yl)-ethyl]-propionamide).
[0086] FIG. 21b shows the reaction scheme for synthesis of
LBT3004.
[0087] FIG. 22a shows the chemical structure of LBT3005
(4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexane
carboxylic acid [2-(5-hydroxy-1 H-indol-3-yl)-ethyl]-amide).
[0088] FIG. 22b shows the reaction scheme for synthesis of
LBT3005.
[0089] FIG. 23 shows the structures of exemplary ligands that can
be modified to include a conjugation agent.
DETAILED DESCRIPTION
[0090] 1. Definitions
[0091] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which the 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, preferred methods and materials are described.
For the purposes of the present invention, the following terms are
defined below.
[0092] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0093] By "attached" is meant direct or indirect attachment of a
conjugation agent to a ligand in such a manner as to resist
separation of the conjugation agent from the ligand under normal
physiological conditions. Accordingly, the term "attached" as used
herein includes within its scope one more ionic bonds, hydrogen
bonds, van der Waals forces, covalent bonds or combinations thereof
that form between the conjugation agent and the ligand or between
an intervening spacer and the conjugation agent and the ligand,
respectively, such that separation of the conjugation agent from
the ligand is resisted under normal physiological conditions.
[0094] Throughout this specification, unless the context requires
otherwise, the words "comprise", "comprises" and "comprising" will
be understood to imply the inclusion of a stated step or element or
group of steps or elements but not the exclusion of any other step
or element or group of steps or elements.
[0095] By "conjugation agent" is meant a moiety of a modified
ligand that is reactive with a moiety of a receptor that binds a
parent ligand from which the modified ligand was derived wherein a
covalent bond is formable between the conjugation agent and the
receptor moiety. In this connection, the conjugation agent may be
sufficient either on its own or in the presence of an ancillary
conjugation agent to facilitate covalent coupling with the receptor
moiety. The ancillary coupling agent may be an enzyme that
catalyzes, or an activating agent that causes, formation of a
covalent bond between the conjugation agent and said receptor
moiety.
[0096] By "isolated" is meant material that is substantially or
essentially free from components that normally accompany it in its
native state.
[0097] As used herein, the term "ligand" refers to an agent that
binds, interacts or otherwise associates with, a target receptor.
The agent may bind the target receptor when the target receptor is
in its native conformation, or when it is partially or totally
unfolded or denatured. According to the present invention, a ligand
is not limited to an agent that binds a recognized functional
region of the target receptor e.g. the hormone-binding site of a
receptor, and the like. In practicing the present invention, a
ligand can also be an agent that binds any surface or internal
sequences or conformational domains of the target receptor.
Preferably, the ligand is a molecule affecting physiological
function including a drug. The ligand can also be an endogenous
ligand.
[0098] By "obtainedfrom" is meant that a sample such as, for
example, an extract comprising a receptor is isolated from, or
derived from, a particular source such as a suitable cell or tissue
source inclusive of human, animal, plant or microbial origin.
[0099] The term "patient" as used herein refers to any organism in
which therapy or prophylaxis of a condition associated with a
receptor is desired using the methods of the invention. However, it
will be understood that "patient" does not imply that symptoms are
present. The patient may therefore include a microbe, plant or
animal. Preferably, the patient is a human or other mammal and
includes any individual it is desired to examine or treat using the
methods of the invention. Suitable mammals that fall within the
scope of the invention include, but are not restricted to,
primates, livestock animals (e.g., sheep, cows, horses, donkeys,
pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea
pigs, hamsters), companion animals (e.g., cats, dogs) and captive
wild animals (e.g., foxes, deer, dingoes).
[0100] By "pharmaceutically acceptable carrier" is meant a solid or
liquid filler, diluent or encapsulating substance that may be
safely used in topical or systemic administration.
[0101] The term "pharmaceutically acceptable salts" as used herein
refers to non-toxic salts of the modified ligands of this
invention, which are generally prepared by reacting the free base
with a suitable organic or inorganic acid. Representative salts
include the following salts: acetate, benzenesulfonate, benzoate,
bicarbonate, bisulfate, bitartrate, borate, bromide, calcium
edetate, camsylate, carbonate, chloride, clavulanate, citrate,
dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,
gluceptate, gluconate, glutamate, glycollylarsanilate,
hexylresorcinate, hydrabamine, hydrobromide, hydrochloride,
hydroxynapthoate, iodide, isothionate, lactate, lactobionate,
laurate, malate, maleate, mandelate, mesylate, methylbromide,
methylnitrate, methylsulfate, mucate, napsylate, nitrate, oleate,
oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate,
polygalacturonate, salicylate, stearate, subacetate, succinate,
tannate, tartrate, teoclate, tosylate, triethiodide, valerate.
[0102] The term "receptor" as used herein refers to a structure
including a molecule or a cluster of molecules that is specific for
one or more ligands wherein binding, interaction or otherwise
association of the ligand(s) with the receptor effects, changes or
nullifies a function of the receptor. The receptor is preferably,
but not exclusively, a protein. Representative receptors include,
but are not limited to, an insulin receptor, epidermal growth
factor receptors, .gamma.-aminobutyric acid receptors, nicotinic
acetylcholine receptors, serotonin receptors, .alpha.- and
.beta.-adrenoceptors, dop amine receptors, histamine receptors,
prostanoid receptors, adenosine receptors, cyclic nucleotide
receptors, glutamate receptors, cytokine receptors, atrial
naturetic peptide receptors, and the prostaglandin receptors. The
receptor can include transporters such as glucose, amino acids
transporters, sodium-proton exchangers, chloride-bicarbonate
exchangers, sodium pumps, calcium pumps, proton pumps; channels
such as sodium channels, potassium channels, calcium channels and
chloride channels; enzymes such as oxidoreductases, transferases,
hydrolases, lyases, isomerases, and ligases. However, it will be
understood that the receptor need not be a protein and may include,
for example, a nucleic acid in which the amino group found on
adenine, guanine and cytosine may be targeted by a conjugation
agent according to the invention. Alternatively, the receptor may
be a carbohydrate (e.g., amino-containing carbohydrates such as
aminophenyl glycosides) or a lipid (e.g., present on the phosphate
head groups of some phospholipids) having one or more carboxyl
groups, and/or one or more amino groups that may be targeted by a
conjugation agent.
[0103] By "reporter molecule" as used in the present specification
is meant a molecule that, by its chemical nature, provides an
analytically identifiable signal that allows the detection of a
complex comprising a ligand and its cognate receptor. The term
"reporter molecule" also extends to use of cell agglutination or
inhibition of agglutination such as red blood cells on latex beads,
and the like.
[0104] The term "sample" as used herein refers to any suitable
sample that may contain a target receptor according to the
invention. The sample may be extracted, untreated, treated, diluted
or concentrated from any suitable source and may contain one or
more cells and/or cell membranes. The sample may comprise whole
cells, denatured cells, cellular membranes or parts thereof.
Alternatively, the sample may contain an isolated receptor.
Suitably, the sample may comprise cells obtained from a tissue
biopsy. Alternatively, the sample may comprise cells or cell lines,
which have been cultured in vitro.
[0105] The term "spacer" as used herein refers to a chemical
linker, polymer, peptide and the like that spatially separates the
conjugation agent from the ligand. Preferably, the spacer is
selected such that it does not interfere with the binding of the
modified ligand to the receptor.
[0106] By "therapeutically effective amount", in the context of the
treatment of a condition associated with a receptor, is meant the
administration of that amount to a patient in need of such
treatment, either in a single dose or as part of a series, that is
effective for treatment of that condition. The effective amount
will vary depending upon the health and physical condition of the
individual to be treated, the taxonomic group of individual to be
treated, the formulation of the composition, the assessment of the
medical situation, and other relevant factors. It is expected that
the amount will fall in a relatively broad range that can be
determined through routine trials.
[0107] 2. Modified Ligands
[0108] The present invention resides, at least in part, in the
surprising discovery that a conjugation agent can be attached to a
parent ligand to form a modified ligand that binds irreversibly to
a target receptor to which the parent ligand binds reversibly. The
irreversible binding of the modified ligand to the receptor is
effected by formation of a covalent bond between the conjugation
agent and a moiety present on the receptor, which is preferably one
or more functional amino acid side chain groups (sometimes referred
to herein as "functional groups"). The covalent bond is formed by
association of the modified ligand with the receptor, followed by
neighboring reactive functional group attack by the conjugation
agent. This irreversible interaction of the modified ligand with
the receptor results in either permanent inhibition (for an
antagonist) or stimulation (for an agonist) of the receptor
functions. Normal function or activity of the receptor resumes only
after new receptors are synthesized.
[0109] Parent ligands that can be modified include ligands that are
interactive with es nucleoside transporter and/or
nucleoside/nucleotide/n- ucleobase-sensitive proteins. Two forms of
nucleoside transporters are classified based on their sensitivity
to inhibition by NBMPR (nitrobenzylthioinosine). See Griffith et
al., Biochim. Biophys. Acta, vol. 1286, pp. 153-181 (1996). The
group of transporters sensitive to NBMPR is designated es
(equilibrium sensitive), and the group insensitive is designated ei
(equilibrium insensitive). In one embodiment, compounds are
provided that have differential binding to es vs. ei receptors.
Compounds are provided that bind selectively and irreversibly to es
receptors. The expression of es receptors is positively correlated
with the carcinogenic state of cells.
[0110] Among the numerous reactive functional amino acid side
chains on the es nucleoside transporter proteins that can be used
for covalent attachment, the sulfhydryl group of cysteine residue
is probably the most pharmacological and biologically important.
Early studies on the effect of sulfhydryl reagents on nucleoside
transport in mammalian cells had shown to cause a marked inhibition
of uridine uptake in a variety of cultured cells (Plagemann &
Richey (1974), Biochim. Biophs. Acta, vol. 344, pp. 263-305;
Plagemann et al. (1978), J Cell. Physiol., vol. 97, pp. 49-72;
Plagemann & Wohlhueter (1980), Curr. Top. Membr. Transp., vol.
14, pp. 225-230; Belt (1983), Biochem. Biophys. Res. Commun., vol.
110, pp. 417-423; Belt (1983), Mol. Pharmacol., vol. 24, pp.
479-484). However most of the earlier studies were directed toward
the total nucleoside uptake and little attempt were made to
distinguish the differential in sensitivity towards sulfhydryl
reagents by the es and ei transport systems. Furthermore, the
sulfhydryl reagents used were mainly of organomercurial compounds,
which were demonstrated to perturb plasma structure at
concentration as low as 200 .mu.M (Belt & Noel (1985), Biochem.
J., vol. 232, pp.681-688).
[0111] Ligands also may be modified which are ligands that bind to
serotonin (5-hydroxytryptamine, or 5-HT) receptors. 5-HT receptors
include a diversity of receptor subtypes (Peroutka, S. J., CNS
Drugs, (1995), vol. 4 (Suppl 1), pp. 18-28). With the exception of
the 5-HT.sub.3 receptor, which is an ion channel (Derkash, V. et.
al., Nature, (1989), vol. 339, pp. 706-709), other 5-HT receptors
belong to the extensive family of seven transmembrane G
protein-coupled receptors. The clinical significance of the effects
of 5-HT is manifest for example, in neurological, CNS, psychiatric
and mood disorders, including migraine, anxiety, depression,
schizophrenia, obsessive compulsive disorder, psychosis,
aggression, hostility, eating disorders, gastrointestinal
disorders, hypertension, the maintenance of the circadian rhythms
in the sleep-wakefulness cycle, sexual activity, compulsive
behavior, temperature, emesis, and cardiovascular and motor
function. Drugs that target the 5-HT receptors thus have wide
clinical applications. Ligands that bind to 5-HT receptors can have
effects by binding, for example, to the cardiovascular system,
platelets, gastrointestinal tract, and the brain (Erspamer, V., Ed.
"5-Hydroxytryptamine and Related Indolealklylamines", Handbuch der
Expermentellen Pharmakologie, Vol. 19. Springer-Verlag, Berlin,
(1996), pp. 132-181).
[0112] Thus, for example, the ligand 5-HT may be modified as
disclosed herein. Other ligands that can be modified include 5-HT
precursors and 5-HT receptor agonists and antagonists known in the
art. Examples include the precursor 5-hydroxytryptophan, which has
been used as an antidepressant drug; 5-HT.sub.1A-agonists used as
tranquilizers and antihypertensives; sumatriptan, a selective
5-HT.sub.1 receptor agonist, used for migraine;
5-HT.sub.2-antagonists such as methysergide, used for migraine
prophylaxis and in carcinoid tumour syndrome;
5-HT.sub.2-antagonists such as ketaserin, used to lower the blood
pressure in hypertensive patients; and selective
5-HT.sub.3-antagonists used to treat cytostatic- and
radiation-induced emesis. (Beasley, CM., et. al.,
Psychopharmacology, (1992), vol. 107, pp. 1-10; Bolden-Watson, C.,
and Richelson, E., Life Sciences, (1993), vol. 52, pp. 1023-1029.;
Koe, B. K., J Clin. Psychiatry, (1990), vol. 51, pp. 13-17).
[0113] Serotonin binding ligands which can be modifed include those
developed for CNS disorders, such as anxiety, such as
benzodiazepines, or 5-HT.sub.1A agonists, such as buspirone,
gepirone, ipsapirone, and flesinoxan. As anxiolytic agents, they
possess advantages over benzodiazepines because they lack the
sedation and drug dependence liabilities of bezodiazepines
(Barrett, J. E., and Vanover, K. E., Psychopharmacology, (1993),
vol. 112, pp. 1-12.). Other serotonin binding ligands include those
developed for treatment of depression, such as SSRIs, for example
fluoxetine. SSRIs have also been shown to have efficacy in the
treatment of bulimia and obsessive-compulsive disorders and may be
also useful in treating obesity, panic disorders, premenstrual
syndrome, diabetic neuropathy, chronic pain, and certain cognitive
aspects of Alzheimer's disease. Other useful antidepressant drugs
which are relatively potent antagonists at 5-HT.sub.2 receptors,
include tricyclic antidepressants, such as trazadone and nefazodone
(Cowen, P. J., and Anderson. I. M, "5-Hydroxytryptamine in
Psychiatry" (Sandler, M., Coppen, A., & Harnett, S. Eds.),
Oxford University Press, New York, (1991)). Ligands include
compounds developed for the treatment of schizoprenia, including
the antipsychotic agents clozapine, risperidone, and olanzepine,
(Weil-Malherbe, H., Serotonin and schizophrenia. In "The Central
Nervous System. Vol. 3, Serotonin in Health and Disease", Essman,
W. B., Ed, Spectrum Publications, Inc., New York, (1978), pp.
231-291). Ligands associated with the treatment of eating disorders
can be used, such as the SSRIs and fenfluramine. Fenfluramine may
be useful in the treatment of other diseases, such as autism,
premenstrual syndrome, seasonal affective disorder, and attention
deficit disorder. Ligands associated with alleviation of
gastrointestinal disorders can be used, such as ondansetron and
granisetron, which are used in the treatment of radiation induced
emesis associated with cancer chemotherapy, and for ameliorating
the nausea and vomiting occurring during recovery from general
anesthesia. Other ligands include metoclopramide and cisapride.
Ligands used in control of the sleep-wakefulness cycle, such as
L-tryptophan or nonselective 5-HT agonists or 5-HT antagonists such
as ritanserin may be used (for a review, see Wauquier, A., and
Dugovic, C., Ann N.Y. Acad. Sci., (1990), vol. 600, pp. 447-459).
Other ligands include etanserin, a 5-HT.sub.2 receptor antagonist
used as an antihypertensive agent.
[0114] 2.1. Considerations Relating to Protein Structure
Reactivity
[0115] Peptides and proteins are composed of amino acids
polymerized together through the formation of peptide (amide)
bonds. The peptide-bonded polymer forms the backbone of polypeptide
structure. There are 20 common amino acids found throughout nature,
each containing an identifying side chain of particular chemical
structure, charge, hydrogen bonding capability, hydrophilicity (or
hydrophobicity), and reactivity. The side chains do not participate
in peptide formation and are thus free to interact and react with
their environment.
[0116] Amino acids may be grouped by type depending on the
characteristics of their side chains. The most significant amino
acids for covalent conjugation purposes are the ones containing
accessible ionizable side chains such as aspartic acid, glutamic
acid, lysine, arginine, histidine, cysteine, and tyrosine.
Methionine and tryptophan also containing ionizable side chains,
however, they are not easily accessible as they are usually buried
deep inside the molecular structure of receptors due to their
hydrophobic nature.
[0117] Both aspartic and glutamic acids contain carboxylate groups
that have ionization properties. Carboxylate groups in proteins may
be derivatized through the use of amide bond forming agents or
through active ester or reactive carbonyl intermediates.
[0118] Lysine, arginine, and histidine have ionizable amine
containing side chains. These amine containing side chains
typically are exposed on the surface of proteins and can be
derivatized with ease. The most important reactions that can occur
with these residues are alkylation and acylation.
[0119] Cysteine is the only amino acid containing a sulfhydryl
group. The most important reaction of cysteine groups in proteins
is the formation of disulfide cross-links with another cysteine
molecule. Cysteine sulfhydryls and cystine disulfides (called
cystine residues) may undergo a variety of reactions, including
alkylation to form stable thioether derivatives, acylation to form
relatively unstable thioesters, and a number of oxidation and
reduction processes. Cysteine and cystine groups are relatively
hydrophobic and usually found within the core of a protein. It is
often difficult to access the sulfhydryl groups of large proteins
without the presence of a deforming agent or a "driver".
Nevertheless, such steric hindrance does give the sulfhydryl groups
a leading edge in selectivity. Thus, to access the functional
reactive sulfhydryl group situated deep into the ligand-binding
site, without using deforming agents, is to utilize its
physiological ligands or drugs to direct the conjugation agent as
herein described into the inner structure of receptors. It is
possible that irreversible binding drugs that are targeted at the
sulfhydryl groups is likely to have a lower level of non-specific
binding, in clinical term: "fewer side effects", than any other
functional groups.
[0120] Tyrosine contains a phenolic side chain. Although the amino
acid is only sparingly soluble in water, the ionizable nature of
the phenolic group sometime makes it appear in hydrophilic regions
of a protein--usually at or near the surface. Thus unlike cysteine
residue, tyrosine derivatization proceeds without much need for
deforming agents to further open the protein structure. Tyrosine
may be targeted specifically for modification through its phenolate
anion by acylation.
[0121] In summary, protein molecules may contain up to nine amino
acids that are readily derivatizable at their side chains. These
nine residues contain eight principal functional groups with
sufficient reactivity for modification reactions. They are the
guanidinyl group of arginine, the .gamma.- and .beta.-carboxyl
groups of glutamic and aspartic acids, respectively, the sulfhydryl
group of cysteine, the imidazolyl group of histidine, the
.epsilon.-amino group of lysine, the thioether moiety of
methionine, the indolyl group of typtophan and the phenolic
hydroxyl group of tyrosine. Since methionine and tryptophan are
generally buried in the interior of proteins and are thereby
protected from conjugates dissolved in the solvent, they show only
some selected reactivity in intact proteins. The other ionizable
groups are normally either exposed on the surface of proteins or
can be accessed with the help of deforming agents or "drivers".
They are therefore the easy targets for conjugation. However, among
the numerous reactive functional amino acid side chains on the
proteins that can be used for labeling, the sulfhydryl group of
cysteine residue is most probably both pharmacologically and
biologically important. It has been reported that in most
macromolecules, there is at least one copy of reactive sulfhydryl
group situated at or closed to the ligand binding sites of target
macromolecules. Disruption of this reactive sulfhydryl functional
group by sulfhydryl reducing agents has been shown to affect the
functionalities of many macromolecules.
[0122] 2.2. Moieties of Receptors Permitting Conjugation
[0123] The sulfhydryl moiety, with the thiolate ion as the active
species, is the most reactive functional group in a protein. With a
pK.sub.a of about 8.6, the reactivity of the thiol is expected to
increase with increasing pH, toward and above its pK.sub.a.
[0124] In the process of modifying a parent ligand, it is
advantageous to capitalize the presence of this highly reactive
sulfhydryl group, which is typically situated near or at the
ligand-binding site of the receptor Drugs or physiological ligands
can be chemically modified to include a conjugation agent that
reacts faster with the thiol group than any other reactive
functional groups. Upon association of this modified drug with its
receptor, this sulfhydryl group directed conjugate would attack any
sulfhydryl group that is situated within its reachable proximity;
this causes covalent binding of the drug to its receptor.
[0125] In addition to sulfhydryl group, there are also other highly
reactive functional groups present on the amino acid side chains,
which can be chemically modified. Conjugates that are reactive to
these functional groups will be discussed below.
[0126] 2.3. Conjugation Agents
[0127] 2.3.1. Sulfhydryl Group Specific Conjugation Agents
[0128] N-Maleimide Derivatives.
[0129] Maleimides are considered fairly specific to the sulflhydryl
group, especially at pHs below 7 where other nucleophiles are
protonated. In acidic and near neutral solutions, the reaction rate
with simple thiols is about 100-fold faster than with the
corresponding simple amines. Although the rate increases with pH,
the reaction with the amino group also becomes significant at high
pHs. The other major competing reaction is the hydrolysis of
maleimides to maleamic acids. However, at pH 7, the apparent rate
of hydrolysis is only 3.2.times.10.sup.-4 min.sup.-1 in 0.1 M
sodium phosphate buffer at 20.degree. C., which is too slow to
interfere with the reaction with sulfhydryl groups. The rate of
decomposition becomes significant only at pH above neutrality.
Thiol undergo Michael reaction with maleimides to yield exclusively
the adduct to the double bond. The resulting thioether bond is very
stable and cannot be cleaved under physiological conditions.
[0130] Disulfide Reagents.
[0131] Disulfide interchange occurs when sulfhydryl groups react
with disulfides. Some of the most commonly used disulfide reagents
are 5,5'-dithiobis-(2-nitrobenzoic acid), 4,4'-dithiodipyridine,
methyl-3-nitro-2-pyridyl disulfide, and methyl-2-pyridyl disulfide.
The protein disulfides formed are readily reverse in the presence
of free mercaptan such as 2-mercaptoethanol or dithiothreitol. The
reduction of protein disulfide into its original sulfhydryl group
allows the protein to regain its functions. Thus, irreversible
binding drugs of this category provide additional safety mechanism
to counter various therapeutic complications such as over-dosing,
hyper reaction, etc.
[0132] 2.3.2. Amino Group Specific Conjugation Agents
[0133] The amino group is another strong nucleophile in the
protein. However, because of its abundance and omnipresence in
proteins, and the relatively high pK.sub.a of the ammonium ion,
most of the reagents that react with the amino group can also react
with other functionalities. Thus, it may not be an ideal target for
modified ligands according to the invention, unless the critical
cysteine residues are absent from the drug-binding site.
Nevertheless, many stable acylated products are still and only
formed with the amino groups. The most common reactions of amines
are alkylation and acylation reactions. Some of the important
alkylating agents that can be incorporated into ligand structures
are: .alpha.-haloacetyl compounds (e.g., haloacetate,
haloacetamides), N-maleimide derivatives, aryl halides (e.g.,
dinitrofluorobenzene, trinitrobenzenesulfonate), aldehydes (e.g.,
glutaraldehyde, formaldehyde) and ketones (e.g., pyridoxal
phosphate). Acylating agents include, but are not restricted to,
isocyanate, isothiocyanate, imidoesters, N-hydroyxlsuccinimidyl
ester, .rho.-nitrophenyl ester, acyl chloride, and sulfonyl
chloride.
[0134] 2.3.3. Carboxyl Group Specific Conjugation Agents
[0135] The most important chemical modification reactions of
carboxyl groups utilize the carbodiimide-mediated process. With
proteins, the optimum pH of the reaction is about 5, which is
difficult to achieve in most physiological conditions. Other
reagents such as diazoacetate esters and diazoacetamides can also
be used to esterify carboxyl groups. Similar to carbodiimides,
these reagents react with high specificity with carboxyl groups of
proteins under mild acid conditions.
[0136] 2.3.4. Tyrosine Specific Conjugation Agents
[0137] Tyrosine, histidine, and other aromatic residues of proteins
are rich in electrons. These residues undergo electrophilic
substitution reactions at the aromatic ring. Useful electrophiles
for reaction with tyrosine and histidine in proteins are diazonium
compounds. Other protein components such as lysine, tryptophan,
cysteine, and arginine residues react very slowly, such that
diazonium reagents can be regarded as tyrosine selective. Diazonium
ions are generally unstable even at neutral pH and maximum reaction
with the proteins is typically achieved at alkaline pH. The
phenolate ion of tyrosine also reacts similar to amino groups
toward acylating agents. However, the tyrosyl group is generally
perceived as having a lower reactivity This is not because the
phenolate ion has lower nucleophilicity, but because tyrosine
residues are usually buried in a protein and are, therefore,
generally inaccessible for reactions due to their
hydrophobicity.
[0138] 2.3.5. Arginine Specific Conjugation Agents
[0139] A predominant reaction of the guanidinyl moiety of arginine
residues is with 1,2-dicarbonyl reagents. Commonly used vicinal
diketones include glyoxal, phenylglyoxal, 2-3-butanedione and 1
,2-cyclohexanedione.
[0140] 2.3.6. Histidine Specific Conjugation Agents
[0141] While a number of alkylating agents react with the
imidazolyl moiety of histidines have been referred to earlier, the
rate of these reactions is generally inferior to other
nucleophiles. Even with .alpha.-haloacetate, N-carboxymethylation
is generally slow in comparison with sulfhydryl groups. However,
when such reactive .alpha.-halocarbonyl group is incorporated into
affinity labels (e.g., p-toluenesulfonylphenyl- anine
chloromethylketone, p-toluenesulfonyllysinechloromethylketone),
specific reaction may be achieved. Beside .alpha.-haloacetyl
groups, other alkylating agents are not as reactive towards
histidine. With acylating reagents, histidine forms acylated
products that are generally unstable and may undergo spontaneous
hydrolysis. The most important acylating agent that has been
commonly used for the modification of histidines is
diethylpyrocarbonate or ethoxyformic anhydride. The acylated
imidazole is reversed at alkaline pH, resulting in the recovery of
histidine. Deacylation can be achieved at neutral pH very rapidly
with hydroxylamine.
[0142] 2.3.7. Methionine Specific Conjugation Agents
[0143] The major chemical modification reaction of methionine is
alkylation. Because methionine is often situated in the hydrophobic
interior of proteins, it tends to provide high degree of
selectivity. Only alkylating reagents that are coupled to the
ligands are accessible to these buried methionine residues.
[0144] 2.3.8. Tryptophan Specific Conjugation Agents
[0145] Due to its hydrophobicity, tryptophan residues are generally
buried in the interior of proteins. Tryptophan residues can be
modified with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl
bromide. A distinct reagent, .rho.-nitrophenylsulfenyl chloride,
has been used for modification of the indolyl moiety. The reaction
is selective for tryptophan and cysteine residues.
[0146] 2.3.9. Serine Specific Conjugation Agents
[0147] Many reactive reagents such as diisopropylfluorophosphate,
phenylmethyl-sulfonylfluoride and other acrylsulfonyl fluorides
have been found to react with the active-site serine. Care should
be exercised in use of such reagents because of the strong
competitive reaction of hydrolysis.
[0148] 2.4. Design of Modified Ligands of the Invention
[0149] The covalent bond formation between the modified ligand and
the receptor can be catalyzed by enzymes, caused by activating
agents, or facilitated by the conjugation agent on its own. The
covalent bond is preferably formed with a functional group situated
at or near the ligand-binding site of the receptor. This strategy
is advantageous as it ensures a high degree of specificity.
[0150] The design of an irreversible binding ligand depends on the
chemical, biological and molecular properties of both ligand and
receptor. In each instance, the conjugation agent to be introduced
onto the chemical structure of the ligand may be different and may
require a certain configuration. In general, the ligand preferably
includes a functional group which permits attachment of a
conjugation agent, or which is capable of modification to contain
such a group, without affecting the activity of the ligand to bind
its receptor and to elicit a biological activity. The modified
ligand in this regard need not have the same biological activity as
the parent ligand (e.g., it may not require to be activated in vivo
by some metabolic or catabolic step).
[0151] Some of the conditions and requirements to be considered for
selection and configuration of the conjugation agent are as
follows:
[0152] 1. Determine reaction specificity towards a particular
functional group of the receptor that is required for selection of
the conjugation agent, e.g., amino, sulfhydryl, carboxyl,
guanidinyl, imidazolyl, and other amino acid side chains. Selection
will be dependent on the availability of any functional group on
the receptor to which the drug molecule will be linked The
irreversible binding conjugation agent of the modified ligand must
be specific to that functional group.
[0153] 2. Hydrophobicity and hydrophilicity of the conjugation
agent. A receptor in a hydrophobic environment may require a
hydrophobic ligand to reach the receptor. For example, a modified
ligand having a hydrophilic conjugation agent may not be able to
access a corresponding functional group situated deep inside the
hydrophobic core of the receptor.
[0154] 3. Cleavability of the conjugation agent. It may be
desirable in some cases to separate a modified ligand bound to a
receptor. For example, if a toxic drug (corresponding to a parent
ligand) is to be modified, a safety mechanism must be installed to
preempt situations like over-dosing. In this case, the use of
cleavable conjugation agents will enable the conjugation to be
reversed if complication arises. A number of cleavable bonds may be
employed for this purpose. These include disulfide bonds, amidine,
mercurial group, vicinal glycol, azo, sulfone, ester and thioester
linkages. In this regard, the conjugation agent itself may be
cleavable or, if the conjugation agent is attached to the modified
ligand through a spacer, the spacer may be cleavable. The spacer in
this regard can be selected from the group consisting of
(N-succinimidyl 3-(2-pyridyldithio)propionate,
succinimidyloxycarbonyl-.alpha.-methyl-.al-
pha.-(2-pyridyldithio)toluene, 3-(2-pyridyldithio)propionyl
hydrazide, disuccinimidyl tartarate,
N-[4-(p-azidophenylazo)-benzyol]-3-aminohexyl-N- '-oxysuccinimide
ester, 4-4'-difluoro-3,3'-dinitrophenyl-sulfone,
3-(4-azido-2-nitrobenzoylseleno)propionic acid, 2-methylmaleic
anhydride.
[0155] 4. Size and geometry of the conjugation agent. Presence of
the conjugation agent on a modified ligand must permit binding of
that ligand to the receptor. Upon binding of the modified ligand to
the receptor, the conjugation agent must be in close proximity to
the receptor moiety so that conjugation is effected. A spacer as
herein defined may be required to bring the conjugation agent in
close proximity to the receptor moiety. In such a case, the length
of the spacer will be dependent on the distance required for the
conjugation agent to reach a conjugation-effective location
adjacent to the receptor moiety. Structural analysis of receptor
using X-ray crystallography, nuclear magnetic resonance and the
like, combined with molecular modeling, can be of assistance in
identifying the receptor moiety to be conjugated and in selecting
and configuring the conjugation agent on the ligand chemical
structure. The receptor moiety may be present in the ligand-binding
site of the receptor. Preferably, the receptor moiety is present
outside the ligand-binding site of the receptor. While not being
limited to any theory, it is possible that this latter approach may
be advantageous since it would be more likely to prevent disruption
of receptor function upon binding with the modified ligand. On this
basis, a parent agonist may be modified such that it crosslinks
with its cognate receptor to thereby cause continuous stimulation
of receptor function ("receptor turn-on"). Alternatively, a parent
antagonist may be modified such that it crosslinks with its cognate
receptor to thereby cause continuous inhibition of the receptor
function ("receptor turn-off")
[0156] 3. Compositions
[0157] The invention also encompasses a composition comprising the
modified ligand as described herein, together with a
pharmaceutically acceptable carrier.
[0158] The invention also features a method of treatment or
prophylaxis of a condition associated with a target receptor,
comprising administering to a patient in need of such treatment a
therapeutically effective dosage of the composition as broadly
described above.
[0159] Depending upon the particular route of administration, a
variety of pharmaceutically acceptable carriers, well known in the
art may be used. These carriers may be selected from sugars,
starches, cellulose and its derivatives, malt, gelatine, talc,
calcium sulphate, vegetable oils, synthetic oils, polyols, alginic
acid, phosphate buffered solutions, emulsifiers, isotonic saline,
and pyrogen-free water.
[0160] Any suitable route of administration may be employed for
providing a patient with a composition of the invention. For
example, oral, rectal, parenteral, sublingual, buccal, intravenous,
intra-articular, intra-muscular, intra-dermal, subcutaneous,
inhalational, intraocular, intraperitoneal,
intracerebroventricular, transdermal and the like may be
employed.
[0161] Dosage forms include tablets, dispersions, suspensions,
injections, solutions, syrups, troches, capsules, suppositories,
aerosols, transdermal patches and the like. These dosage forms may
also include injecting or implanting controlled releasing devices
designed specifically for this purpose or other forms of implants
modified to act additionally in this fashion. Controlled release of
a modified ligand may be effected by coating the same, for example,
with hydrophobic polymers including acrylic resins, waxes, higher
aliphatic alcohols, polylactic and polyglycolic acids and certain
cellulose derivatives such as hydroxypropylmethyl cellulose. In
addition, controlled release may be effected by using other polymer
matrices, liposomes and/or microspheres.
[0162] Compositions suitable for oral or parenteral administration
may be presented as discrete units such as capsules, sachets or
tablets each containing a pre-determined amount of one or more
immunogenic agents of the invention, as a powder or granules or as
a solution or a suspension in an aqueous liquid, a non-aqueous
liquid, an oil-in-water emulsion or a water-in-oil liquid emulsion.
Such compositions may be prepared by any of the methods of pharmacy
but all methods include the step of bringing into association one
or more modified ligands as described above with the carrier which
constitutes one or more necessary ingredients. In general, the
compositions are prepared by uniformly and intimately admixing the
modified ligands of the invention with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product into the desired presentation.
[0163] The modified ligands may be in the form of a
pharmaceutically acceptable salt as is known in the art.
[0164] The above compositions may be administered in a manner
compatible with the dosage formulation, and in such amount as is
therapeutically effective. In this regard, the dose of modified
ligand administered to a patient should be sufficient to effect a
beneficial response in the patient over time such as an
amelioration of the condition to be treated. The quantity of the
modified ligand(s) to be administered may depend on the subject to
be treated inclusive of the age, sex, weight and general health
condition thereof. In this regard, precise amounts of the modified
ligand(s) for administration will depend on the judgement of the
practitioner. In determining the effective amount of the modified
ligand to be administered in the treatment or prophylaxis of the
condition associated with the target receptor, the physician may
evaluate progression of the condition.
[0165] In any event, those of skill in the art may readily
determine suitable dosages of the modified ligands of the
invention. Such dosages may be in the order of nanograms to
milligrams of the modified ligands of the invention.
[0166] 4. Detection of Target Receptors and Cells or Cell Membranes
Containing Same
[0167] The invention also features a method of detecting the
presence of a target receptor in a test sample. The method
comprises contacting the sample with a modified ligand as described
in Section 2, wherein said modified ligand binds the target
receptor, and detecting the presence of a complex comprising said
modified ligand and said receptor in the contacted sample.
[0168] The invention also encompasses a method of quantifying the
presence of a target receptor in a test sample. The method
comprises contacting the sample with a modified ligand as broadly
described above, wherein the modified ligand binds said target
receptor, measuring the concentration of a complex comprising the
modified ligand and the receptor in the contacted sample, and
relating the measured complex concentration to the concentration of
the receptor in the sample.
[0169] The invention also provides a method of detecting the
presence of a target receptor on a cell or cell membrane. The
method comprises contacting a sample containing the cell or cell
membrane with a modified ligand as broadly described above, wherein
the modified ligand binds the target receptor, and detecting the
presence of a complex comprising the modified ligand and the cell
or cell membrane in the contacted sample.
[0170] Also encompassed by the invention is a method of quantifying
the presence of a target receptor on a cell or cell membrane. The
method comprises contacting a sample containing the cell or cell
membrane with a modified ligand as broadly described above, wherein
the modified ligand binds said target receptor. The concentration
of a complex comprising the modified ligand and the cell or cell
membrane is then measured in the contacted sample, and the measured
complex concentration is related to the concentration of the
receptor present on the cell or cell membrane.
[0171] The modified ligands can be used as a tool to identify the
actual drug pocket or drug binding area on the receptor molecules.
Since the binding of these modified ligands to their targets is
irreversible, the actual site that the drugs interact with can be
identified on the receptors using various techniques such as
peptide finger printing using Mass Spec. This information can be
used to identify molecules that bind to that particular drug
pocket.
[0172] Any suitable technique for determining formation of the
complex may be used. For example, a modified ligand according to
the invention, having a reporter molecule associated therewith
(sometimes referred to herein as a "probe") may be utilised in any
suitable assay known in the art for detecting and/or quantifying
ligand-receptor interactions. For example, scintillation counting,
autoradiography, fluorography, flow cytometry, UV spectroscopy,
fluorescence spectroscopy, chemiluminescence imaging, fluorescence
microscopy, confocal microscopy, electron microscopy, etc may be
used in this regard.
[0173] The reporter molecule may be associated with the any
suitable part of the modified ligand including the conjugation
agent and the spacer, if included. Preferably association of the
reporter molecule with the modified ligand is selected such that
the reporter molecule does not interfere with binding of the
modified ligand to the receptor.
[0174] It will be appreciated that the reporter molecule associated
with the antigen-binding molecule may include the following:
[0175] direct attachment of the reporter molecule to the modified
ligand;
[0176] indirect attachment of the reporter molecule to the modified
ligand; i.e., attachment of the reporter molecule to another assay
reagent which subsequently binds to the modified ligand; and
[0177] attachment to a subsequent reaction product of the modified
ligand.
[0178] The reporter molecule may be selected from a group including
a chromogen, a chromophore, a catalyst, an enzyme, a fluorochrome,
a chemiluminescent molecule, a lanthanide ion such as Europium
(Eu.sup.34), a radioisotope, a spin label, and a direct visual
label.
[0179] In the case of a direct visual label, use may be made of a
colloidal metallic or non-metallic particle, a dye particle, an
enzyme or a substrate, an organic polymer, a latex particle, a
liposome, or other vesicle containing a signal producing substance
and the like.
[0180] A large number of enzymes suitable for use as reporter
molecules is disclosed in U.S. Pat. No. 4,366,241, U.S. 4,843,000,
and U.S. 4,849,338. Suitable enzymes useful in the present
invention include alkaline phosphatase, horseradish peroxidase,
luciferase, .beta.-galactosidase, glucose oxidase, lysozyme, malate
dehydrogenase and the like. The enzymes may be used alone or in
combination with a second enzyme that is in solution.
[0181] Suitable fluorochromes include, but are not limited to,
fluorescein isothiocyanate (FITC), tetramethylrhodamine
isothiocyanate (TRITC), R-Phycoerythrin (RPE), and Texas Red. Other
exemplary fluorochromes include those discussed by Dower et al.
(International Publication WO 93/06121). Reference also may be made
to the fluorochromes described in U.S. Pat. No. 5,573,909 (Singer
et al), U.S. Pat. No. 5,326,692 (Brinkley et al). Alternatively,
reference may be made to the fluorochromes described in U.S. Pat.
Nos. 5,227,487, 5,274,113, 5,405,975, 5,433,896, 5,442,045,
5,451,663, 5,453,517, 5,459,276, 5,516,864, 5,648,270 and
5,723,218.
[0182] Preferably, the reporter molecule is a radioisotope such as
.sup.3H, 125I, .sup.14C, .sup.32P, .sup.33P, and .sup.35S.
[0183] 5. Applications
[0184] Therapeutic.
[0185] The modified ligands of the invention, which preferably
represent irreversible binding drugs, have substantial advantages
over conventional reversible binding drugs. Firstly, the
dose-related inhibition or stimulation can be persisted long after
the drugs disappear from the plasma. In other words, the drug
effect is likely to last longer than would be predicted from its
plasma elimination half-life. Secondly, due to longer lasting drug
effects, these irreversible binding drugs can be administered less
frequently and at a lower dosage. This minimizes adverse side
effect and prevent cumulative toxicity induce by the drug
themselves or by their metabolites. Thirdly, since the binding of
the irreversible antagonist to its receptor is permanent, the
blockade of receptor response is not longer a competitive
inhibition mechanism. This irreversible antagonism prevents the
agonist, at any concentration, from producing a maximum effect on a
given receptor. Furthermore, if the modified ligand is rendered
highly radioactive, it may be used as a therapeutic for
specifically killing cells bearing the cognate receptor or may be
used for imaging.
[0186] Diagnostic.
[0187] Another application of this ligand modification technology
is to further elucidate various receptor subpopulations that can be
targeted to relieve dysfunctions in the various complex
physiological processes More importantly, the modified drugs allow
us to develop/identify animal models that are "deficient" in
certain receptors without undergoing lengthy, tedious and
complicated manipulation of the genetic materials Hence the complex
physiological mechanisms and functions of various receptors in
"real-life" situations can be studied and analyzed. Furthermore,
one can also study how the various different receptors are
interlinked and influenced by each other's functions. The
availability of such animal models will also enable investigators
to predict and reveal therapeutic outcomes of various drugs by
simultaneously blocking multiple receptor populations of interest.
Thus, the present invention can be used to profile different
receptors present on a cell as well as in a tissue, organ or
system. Such receptor profiling can be used advantageously to
discover novel drug targets, to predict the possible side-effects
of drugs and to determine how various cells communicate with each
other, their state of health, and whether they respond to certain
external stimuli (e.g., to drugs).
[0188] The invention will now be described with reference to the
following non-limiting examples.
EXAMPLES
Example 1:
[0189] Effects of N-ethylmaleimide on the Equilibrative Nucleoside
Transporter in Murine Myeloma SP2/0-Ag14 Cells.
[0190] Exemplary N-maleimide derivatives were used as a sulfhydryl
reagent that is advantageously specific to the sulfhydryl group,
reacting only with certain accessible sulfhydryl groups on the
proteins, making it possible for specific inhibitions, and good
penetration into cells due to the uncharged nature of the compound.
N-Ethylmaleimide (NEM) is the smallest maleimide reagent capable of
forming stable thio esters with the reactive sulfhydryl groups of
proteins. The sensitivity of es and ei nucleoside transport systems
towards NEM was demonstrated. Various different cell lines were
used to show that the sensitivity of nucleoside transport system(s)
toward NEM is a general phenomenon and not restricted to certain
cell or tissues types.
[0191] Previous studies of the es nucleoside transporter system
(Paterson et al. (1977), Mol. Pharmacol., vol. 13, pp. 114; Gati et
al. (1983), Mol. Pharmacol., vol. 23, pp. 146-1520) suggested that
the sugar component of nucleosides was important for binding of a
nucleoside to the es nucleoside transporter site. Thus in the
present invention, that linker moiety was attached to the
pyrimidine/purine ring of the nucleosides to avoid destruction of
effective inhibition for the es nucleoside transporter to provide
novel probes for that transporter regulatory site.
[0192] The present inventor has found that a novel group of
nucleosides, cytidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylic
acid (CrMCC) and derivatives and analogues thereof, have the
ability to irreversible inhibiting the es nucleoside transporter
proteins of human cells. A direct, rapid synthetic route used to
synthesize CrMCC or
1-[[4-[(4-amino-1-.beta.-D-ribofuranosyl-2(1H)-pyrimidione)carbonyl]
cyclohexyl]methyl]-1H-pyrrole-2,5-dione, is set out in FIG. 3.
[0193] To demonstrate the sensitivity of es and ei nucleoside
transport systems to NEM, various mammalian cell lines were treated
with varying concentrations of NEM from 0 to 30 mM for 1 min prior
transport assay. FIG. 1 shows the equilibrative transport (measured
at 5 s uptake interval) of .sup.3H-uridine (final concentration 50
.mu.M) in murine myeloma SP2/0-Ag14 cells was inhibited by NEM in
an apparent biphasic manner. About 60-70% of the transport activity
was inhibited by NEM with IC.sub.50 value of 0.15 mM. The remaining
30-40% of transport activity was gradually abolished by NEM at
concentrations above 3 mM. Similar biphasic inhibition by NEM was
also observed in human HL-60 and human MCF-7 cells, which possess
both the es and ei transport systems (Lee et aL (1995), Biochim.
Biophys. Acta, vol. 1268, pp. 200-208). For cells that possess only
es (murine EL-4 cells) or only ei (rat Morris 7777) transport
system, monophasic .sup.3H-uridine transport curves in NEM
dose-response experiments were observed. The IC.sub.50 values were
approximately 0.1 and 1.5 mM for EL-4 and Morris 7777 cells,
respectively. These results suggested that the biphasic curve of
total .sup.3H-uridine transport observed in NEM dose-response
experiments is a reflection of the presence of two distinct
equilibrative nucleoside transport systems in those cells. The
NEM-sensitive component is the es transport system and the
NEM-insensitive component is the ei transport system. The ei
transporter, although less sensitive to NEM inhibition, can be
inhibited at higher concentrations of NEM or by prolonged exposure
to NEM. This observation is in agreement with the general notion
that sulfhydryl groups of enzymes display a considerable variation
in their reactivity, ranging from unreactive, through several
stages of sluggishness, to free and being immediately reactive.
[0194] To further demonstrate that the reduction in uridine influx
by es transport system is due to NEM-induced chemical modification
on the carrier protein which subsequently affected the transport
affinity, and to confirm the notion that the changes in es
nucleoside transport activity can be demonstrated by changes in
.sup.3H-NBMPR binding ability, murine myeloma SP2/0-Ag14 cells
pretreated with or without 0.3 mM NEM for 1 min were assayed for
the availability of high-affinity .sup.3H-NBMPR binding sites on
the cell membranes. FIG. 2 shows that the K.sub.d value (corrected
for non-specific binding determined in the presence of 20 .mu.M of
NBTGR) for .sup.3H-NBMPR binding was changed significantly in
response to NEM treatment. Shortly after 1 min of NEM exposure, the
apparent K.sub.d value was 1.9.+-.0.4 nM, as compared to
0.16.+-.0.02 nM for untreated cells. However, the Bmax values of
40,000.+-.2,600 and 41,000.+-.1,000 sites/cell for NEM treated and
untreated cells, respectively, were not significantly different.
These results provide additional evidence that a critical
disulfhydryl bond is located near or close to the nucleoside
transporting/binding site of the es transport system, where
formation of disulfhydryl bond with NEM affects the transport
affinity of the carrier. The suggestion that the critical
sulfhydryl group on the es transporter protein is probably located
or close to but not on the nucleoside transporting/binding site is
derived from the observations that NBMPR, dilazep, dipyridamole at
30 .mu.M and uridine at 10 mM were incapable of protecting this
sulffiydryl group from NEM modification (Lee et al. (1995),
Biochim. Biophys. Acta, vol. 1268, pp. 200-208). Although NEM is
effective as an inhibitor of es transporter protein, it is toxic
and should be modified for therapeutic purposes.
Example 2
[0195] Synthesis and Characterization of CrMCC
[0196] The strategy to selectively irreversibly inhibit the es
transporter protein is to attach a reactive group specific covalent
binding agent (maleimide) to a driver so that it can deliver the
covalent binding agent to the desired target. The most suitable
driver will be the physiological ligand itself (nucleoside).
Cytidine, a pyrimidine nucleoside, is selected among other
physiological nucleosides due to its "function inertness", thus
minimizes "non-specific" drug binding. As for the spacer arm that
links the maleimide to the cytidine, a cyclohexane carboxylic acid
is chosen for pilot studies. This configuration is to mimic the
chemical structure of NBMPR (FIG. 5b). Furthermore, other
advantages like hydrophobicity provided by cyclohexane) and
stability (provided by carboxylic acid) are also taking into
consideration.
[0197] A direct, rapid synthetic route used to synthesize CrMCC or
1-[[4-[(4-amino-1-.beta.-D-ribofuranosyl-2(1H)-pyrimidione)carbonyl]cyclo-
hexyl]methyl]-1H-pyrrole-2,5-dione, is set out in FIG. 3.
[0198] N-Succinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) and cytidine
were dissolved in anhydrous dimethysulfoxide (DMSO) separately
prior reaction. The reaction started when these two reagents were
mixed at room temperature and shielded from light. The molar
concentration ratio of SMCC:cytidine in the mixture was 0.10 M:0.15
M with pH 7.5-8.0 in the reaction system. Within 4 hrs, CrMCC was
found in the reaction mixture and can be separated from cytidine
and SMCC by a C.sub.18 reversed-phase column (Resource RPC,
Pharmacia) operated on a HPLC (AKTA Purifier, Pharmacia) using the
absorbance wavelength of 300 nm. Cytidine was eluted by 100% water
and CrMCC and SMCC were eluted by 15% acetonitrile in water with
the flow rate 1.5 ml/min. The hydrophobicity of CrMCC is greater
than that of cytidine but is less than that of SMCC. Formation of
CrMCC reached its near maximum after 48 hrs of reaction between
SMCC and cytidine at 22-24.degree. C. (FIG. 4). The area under the
peak was calculated using a computer program UNICORN (version
2.0).
[0199] HPLC purified CrMCC was lyophilized by freeze-drying. The
activity of CrMCC was stable for at least three months if it was
stored at -20.degree. C. in a desiccator. The molecular weight of
CrMCC was determined by LC-MS (Waters). Briefly, CrMCC was eluted
out of a C.sub.18 reversed-phase column (Resource RPC, Pharmacia)
by 20% acetonitrile in water at a flow-rate of 3 ml/min. The
capillary voltage of micromass mass spectrometer was set to 3.0-3.5
V, and the cone voltage was set at 20 V. N.sub.2 gas flow was at
700 L/hr and electrospray was negative. Mass spectra were gathered
under a full-scan operation, scanning range 1-1000 m/z. The
molecular weight of CrMCC was determined by monitoring the
protonated molecular ion, and was similar to the predicted value of
462.5. The purity of CrMCC synthesized was consistently greater
than 95%.
[0200] The light absorbance spectrum of CrMCC was measured by
uv-vis spectrophotometer. FIG. 5A shows there are two
.lambda..sub.max's for the light absorbance spectrum of CrMCC. One
of them is at 254 nm (similar to the .lambda..sub.max of cytidine)
and the other one is at 300 nm (similar to the .lambda..sub.max of
SMCC). Such an absorbance profile of CrMCC with two
.lambda..sub.max's is very similar to that of NBMPR (FIG. 5B). This
indicated that CrMCC consists of both pyrimidine and cyclohexane
rings on its chemical structure. Subsequent structural studies
using NMR confirmed this finding.
Example 3
[0201] Effects of CrMCC on the Binding of .sup.3H-NBMPR in Human
HL-60 Promyelocytic Leukemia Plasma Membranes
[0202] To synthesize .sup.3H-CrMCC, radioactive cytidine
(.sup.3H-cytidine) was used. Since high concentration of substrate
increases the yield of CrMCC, nonradioactive cytidine was pre-mixed
with radioactive .sup.3H-cytidine at a concentration ratio of 100:1
prior to reaction with SMCC (see Example 2).
[0203] Purified HL-60 plasma membranes suspended in reaction buffer
(0.13 M NaCl, 0.02 M NaHCO.sub.3, pH 7.0) were pretreated with
graded concentrations of cytidine, SMCC, and CrMCC for 5 min prior
exposed to .sup.3H-NBMPR (5 .mu.M) for additional 30 min. The
reaction was terminated by membrane filtration method (Lee &
Jarvis (1988), Biochem. J., vol 249, pp. 557-564). The data shown
in FIG. 6 were corrected for non-specific binding determined in the
presence of 20 .mu.M of NBTGR. FIG. 6 shows the specific of
.sup.3H-NBMPR to HL-60 unsealed plasma membranes was inhibited by
CrMCC with IC.sub.50 value of 10 .mu.M. In contrast, the IC.sub.50
values for SMCC and cytidine in inhibiting .sup.3H-NBMPR binding
were 100 .mu.M and >1 mM, respectively. The Ki value for CrMCC
was calculated to be 1 .mu.M.
[0204] To analyse the effect of CrMCC on the binding kinetics of
.sup.3H-NBMPR, purified HL-60 plasma membranes were pretreated with
0, 10, and 50 .mu.M of CrMCC for 5 min prior incubated with graded
concentrations of .sup.3H-NBMPR (0.2 to 8 nM) for additional 30
min. A double reciprocal plot of the results is presented in FIG.
7. The lines of the plots were intersected at the abscissa
indicating a changed value for B.sub.max but an unchanged value for
K.sub.d in the presence of CrMCC. For the data shown, the apparent
K.sub.d values of .sup.3H-NBMPR binding were 0.36.+-.0.04,
0.31.+-.0.03 and 0.39.+-.0.05 nM with B.sub.max values of
1.56.+-.0.05, 0.59.+-.0.01 and 0.30.+-.0.01 pmol/mg protein for
membranes treated with 0, 10 and 50 .mu.M of CrMCC, respectively.
Data were corrected for non-specific binding determined in the
presence of 20 .mu.M of nitrobenzylthioguanosine (NBTGR), a
non-radioactive competitive ligand. These results suggested a
non-competitive inhibition of .sup.3H-NBMPR binding by CrMCC. This
is a unique feature of the irreversible antagonism.
[0205] Any clinically useful drugs must have little or no
cytotoxicity at their therapeutic dosage range. HL-60 cells in
logarithmic growth at an initial cell density of 5.times.10.sup.4
cells/ml in RPMI medium containing 10% FBS were exposed to graded
concentrations of CrMCC, cytidine, and SMCC (0 to 100 .mu.M) for 3
days. The cell density was counted using an electronic particle
analyzer (Sysmex). FIG. 8 shows both CrMCC and cytidine had little
or no effect on HL-60 cell growth at concentrations as high as 100
.mu.M after 3 days of exposure. In contrast, SMCC, one of the
parent compounds, was extremely toxic to HL-60 cells with IC.sub.50
value of less than 0.5 .mu.M on cell growth. The toxicity of SMCC
is attributed to its non-specific interaction with every accessible
sulfhydryl groups on the cells. Little or no inhibition on cell
growth by cytidine is expected as this nucleoside is rather "inert"
and does not induce nucleotide imbalance at concentration range
tested
Example 4
[0206] Binding of .sup.3H-CrMCC to the Human HL-60 Promyelocytic
Leukemia Plasma Membranes
[0207] The availability of radioactive CrMCC (.sup.3H-CrMCC) makes
it possible to study the biochemical properties of CrMCC on the es
nucleoside transporter. This experiment was conducted to
investigate the rate of .sup.3H-CrMCC binding to the unsealed
plasma membranes of HL-60 cells. FIG. 9 shows the binding of
.sup.3H-CrMCC (30 .mu.M final concentration) to purified HL-60
plasma membranes was rather slowed. A minimum of 5 min is needed to
achieve the maximum binding value of 12 nmoles per mg of HL-60
plasma membrane protein. This is unlike the binding of reversible
antagonists such as NBMPR, dipyridamole and dilazep, which the
binding was known to be rapid and mostly completed within first
minute of incubation. The binding reaction in FIG. 9 was terminated
by membrane filtration method and the data were corrected for
filter blanks.
[0208] It is important to confirm the binding of .sup.3H-CrMCC to
the unsealed HL-60 plasma membranes is indeed irreversible.
Purified unsealed HL-60 plasma membranes were incubated with 30
.mu.M of .sup.3H-CrMCC for 10 min. After which the mixtures were
diluted 20 folds and the diluted mixtures were sat at room
temperature for various time intervals to allow dissociation to
occur. FIG. 10 shows little or no dissociation of .sup.3H-CrMCC
from its binding sites occurred for at least 60 min after a 20-fold
dilution. Even 1 mM of cytidine presence in the dilution medium
failed to displace .sup.3H-CrMCC from its binding sites. In
contrast, the binding of 30 .mu.M of .sup.3H-cytidine to HL-60
plasma membranes was low and dissociated rapidly and completely
upon dilution (inset of FIG. 10). This finding together with the
non-competitive inhibition of .sup.3H-NBMPR binding by CrMCC (FIG.
7) suggested the interaction of CrMCC to its binding sites is
indeed irreversible. The dissociation reaction shown in FIG. 10 was
terminated by membrane filtration method and the data were
corrected for filter blanks. To determine the concentration
dependence of .sup.3H-CrMCC binding to its binding sites, purified
unsealed HL-60 plasma membranes were incubated with graded
concentration of .sup.3H-CrMCC (3 to 800 .mu.M) for 10 min and the
reaction was terminated by membrane filtration method (FIG. 11).
The data can be resolved into at least two components, a high
affinity component (K.sub.d=23.8.+-.2.2 .mu.M) and a low affinity
component (inset of FIG. 11). The kinetic constants for the low
affinity component cannot be estimated with the existing
concentration range of .sup.3H-CrMCC. It is also possible that this
low affinity component is a non-specific .sup.3H-CrMCC binding
site. The data shown in FIG. 11 were corrected for filter
blanks.
[0209] It is important to determine the stability of
.sup.3H-CrMCC-receptor complexes in various pH conditions. Purified
unsealed HL-60 plasma membranes were incubated with 30 .mu.M of
.sup.3H-CrMCC for 5 min. The reaction mixture was then diluted
20-fold with reaction buffer of various pH values (pH 0.85 to 7.5).
The dissociation reaction was terminated by membrane filtration
method. The data were corrected for filter blanks. FIG. 12 shows
.sup.3H-CrMCC-receptor complexes were very stable at physiological
pH. However, the ligand-receptor complexes began to break down at
pH below 3.5.
[0210] Early studies with sulfhydryl reagent NEM had suggested that
a cysteine residue is probably situated very close to but not on
the nucleoside transporting/binding site of the es nucleoside
transporter protein. Thus, if .sup.3H-CrMCC is bind to the same
site that NEM binds, then the substrates that failed to inhibit NEM
action (Lee et al. (1995), Biochim. Biophys. Acta, vol. 1268, pp.
200-208) should similarly have little or no effect on .sup.3H-CrMCC
binding. FIG. 13 shows 1 mM of physiological nucleosides (e.g.
cytidine, uridine, adenosine and inosine), and 20 .mu.M of es
nucleoside transport inhibitors (e.g. NBMPR and dipyridamole)
indeed failed to inhibit the binding of .sup.3H-CrMCC (30 .mu.M
final concentration) to the HL-60 plasma membranes. In contrast, 1
mM of NEM was as effective as 0.5 mM of CrMCC in inhibiting the
binding of .sup.3H-CrMCC. This finding suggested that NEM and CrMCC
reacted to the same sulfhydryl group on the es nucleoside
transporter protein.
Example 5
[0211] Effects of other Sulfhydrl Reactive Covalent Arms on the
Binding of .sup.3H-cytidine Molecule to Human HL-60 Promyelocytic
Leukemia Plasma Membranes
[0212] It was successfully demonstrated that maleimide, a
sulfhydryl group reactive agent, linked to cytidine via a
cyclohexane carboxylic acid spacer arm is effective in irreversibly
inhibiting the binding of .sup.3H-NBMPR to the es transporter
protein. In this experiment, the binding capability of
.sup.3H-cytidine molecule to the HL-60 plasma membranes after being
linked to the same or different sulfhydryl group reactive agent via
different spacer arms was compared, to provide an indirect
indication on the suitability of the spacer arms used in creating
the irreversible es nucleoside transport inhibitor. Thus,
.sup.3H-cytidine was chemically modified to incorporate various
sulfhydryl reactive side arms onto the 6-amino position of the
pyrimidine ring. Chemicals used to modify .sup.3H-cytidine include
m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), N-succinimidyl
4-[p-maleimidophenyl]butyrate (SMPB),
N-[.gamma.-maleimidobutyryloxy]succ- inimide,
4-suceinimidyloxycarbonyl-.alpha.-methyl-.alpha.-[2-pyridylthio]t-
oluene (SMPT), N-succinimidyl 3-[2-pyridyldithio] propionate
(SPDP), N-succinimidyl maleimidoacetate (AMAS), and
N-[.epsilon.-maleimidocaproyl- oxy]succinimide (EMCS ), in addition
to N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC). Purified HL-60 plasma membranes were incubated with 100
.mu.M of these chemically modified .sup.3H-cytidine analogs for 5
min. The reaction was terminated by membrane filtration method.
FIG. 14 shows N-.alpha.-maleimidoacetoxyli- c acid (AMA) covalent
arm promotes highest irreversible binding of .sup.3H-cytidine to
the HL-60 plasma membranes followed by 3-[2-pyridyldithio]propionic
acid (PDP) However, N-.epsilon.-maleimidocap- roylic acid (EMC),
m-maleimidobenzoic acid (MB), N .gamma.-maleimidobutyry- lic acid
(GMB) and 4-[p-maleimidophenyl]butyrylic acid (MPB) were equally
effective as covalent arms as compare to N-maleimidomethyl
cyclohexane carboxylic acid (MCC). In contrast,
.alpha.-methyl-.alpha.-[2-pyridyldith- io]toluene carbonic acid
(MPT) was least effective. Symbol X in FIG. 14 represents
cytidine.
Example 6
[0213] Identification of .sup.3H-CrMCC Binding Proteins in Human
HL-60 Promyelocytic Leukemia Plasma Membranes
[0214] Attempts were made to identify the plasma membrane proteins
that were labelled by .sup.3H-CrMCC. .sup.3H-CrMCC (20 .mu.M)
labelled HL-60 plasma membrane proteins solubilized in SDS were
loaded onto a C.sub.18 reversed-phase column (Resource RPC,
Pharmacia) operated on a FPLC (ATKA FPLC, Pharmacia) with the
following conditions: column volume (CV, 3 ml), starting buffer A
(0.05% TFA in water), eluent B (0.065% TFA in acetonitrile), flow
rate (2 ml/min), detection (280 nm), elution (0% B in 3 CV, 0-5% B
in 1 CV, 5% B in 5 CV, 5-100% B in 15 CV, wash-out 100% B). FIG. 15
shows the general UV (.lambda.=280 nm) absorbance profile of HL-60
plasma membranes after being separated by FPLC using a C.sub.18
reversed-phase column. The arrows on the chromatogram indicate the
protein peaks that were labelled by .sup.3H-CrMCC (refer to FIG.
16). The eluents were collected at 1 mmin and the radioactivity was
determined by a liquid scintillation counter. FIG. 16 shows there
are at least three major radioactive peaks located at fraction
numbers 43-44, 49-50, and 57-59 of the chromatogram. The sharp
radioactive peak at fraction number 6 is due to degradation product
of .sup.3H-CrMCC (i.e. .sup.3H-cytidine) and the non-specific
binding of .sup.3H-CrMCC to the membrane lipids.
Example 7
[0215] Development of Irreversible Binding Drugs of Adenosine
Origin
[0216] In view of the success of creating irreversible inhibitor of
es transporter protein using cytidine as a lead compound,
reproduction of the invention can be implemented by attaching a
covalent arm to other physiological nucleosides such as adenosine
(FIG. 17). .sup.3H-Adenosine was chemically modified to incorporate
various sulfhydryl reactive covalent arms onto the 6-amino position
of the purine ring according to the general procedure set out in
FIG. 3, and using the chemicals listed in Example 5. HL-60 plasma
membranes were incubated with 100 .mu.M of these chemically
modified .sup.3H-adenosine analogs for 5 min. The reaction was
terminated by membrane filtration method. FIG. 17 shows
4-[p-maleimidophenyl]butyrylic acid (MPB) covalent arm promotes
highest irreversible binding of .sup.3H-adenosine (100 .mu.M) to
the HL-60 plasma membranes followed by N-.alpha.-maleimidoacetic
acid (AMA). However, 3-[2-pyridyldithio]-propionic acid (PDP),
N-.epsilon.-maleimidocaproylic acid (EMC), m-maleimidobenzoic acid
(MB), and N-maleimidomethyl cyclohexane carboxylic acid (MCC) were
equally lesser effective as covalent arms. In contrast,
N-.gamma.-maleimidobutyrylic acid (GMB) and
.alpha.-methyl-.alpha.-[2-pyridyldithio]toluene carbonic acid (MPT)
were least effective.
Example 8
[0217] Inhibitory Effects of Irreversible Interaction 5-HT Analogs
on the Binding of .sup.3H-5-HT in Murine Brain Membranes
[0218] A. Compound Synthesis
[0219] The compounds shown in FIGS. 19a, 20a, 21a and 22a (chemical
structures of LBT3001
(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-- dione),
LBT3002 (4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-N-[2-(5-hydroxyl-lH-
-indol-3-yl)-ethyl]-butyramide), LBT3004
(3-(2,5-dioxo-2,5-dihydro-pyrrol--
1-yl)-N-[2-(5-hydroxyl-1H-indol-3-yl)-ethyl]-propionamide), and
LBT3005 (4-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl-methyl)-cyclohexane
carboxylic acid [2-(5-hydroxy-1H-indol-3-yl)-ethyl]-amide),
respectively) demonstrated efficacy as irreversible binding
compounds, and provide examples of modified ligands which are
modified 5-HT receptor binding ligands comprising conjugation
agents.
[0220] The reaction scheme for synthesis of LBT3001 (FIG. 19b) is
as follows: 5-Hydroxytryptamine (212.7 mg, 1.0 mmol) was dissolved
in saturated sodium bicarbonate (25 ml) solution at 0.degree. C.
N-Ethoxycarbonyleimide (177.6 mg, 1.05 mmol) was added under
stirring. After 30 minutes, the ice-water bath was replaced with
warm water bath (30.degree. C. to 40.degree. C.). The reaction
solution was then stirred in the warm water bath for about one
hour. The solution was then extracted with ethyl acetate
(3.times.50 ml). The ethyl acetate layer was then washed with
deionized water until pH close to neutral (2.times.20 ml) and then
with brine (20 ml). The organic layer was then dried with anhydrous
MgSO.sub.4. The solvent was removed under reduced pressure to
obtain a crude product in yellow oil. Purification through flash
chromatography (30% ethyl acetate in hexane) afforded the product
as a yellow to orange crystal (187.2 mg, 73%).
[0221] The reaction scheme for synthesis of LBT3002 (FIG. 20b) is
as follows: 5-Hydroxytryptamine hydrochloride (42.5 mg, 0.2 mmol)
and 3-maleimidobutyric acid (40.3 mg, 0.22 mmol) were suspended in
1 ml 2-methoxyethyl ether (DME). To the solution, N-methyl
morpholine (NMM, 25 .mu.l, 0.22 mmol) and
1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22 mmol) were added.
The solution was stirred for 3 hours at the room temperature. The
product was purified by flash chromatography directly using a
gradient solvent eluent (0% to 3% methanol in methylene chloride)
to generate brownish oil (40.7 mg, 60%).
[0222] The reaction scheme for synthesis of LBT3004 (FIG. 21b) is
as follow: 5-Hydroxytryptamine hydrochloride (42.5 mg, 0.2 mmol)
and 3-maleimidopropionic acid (37.2 mg, 0.22 mmol) were suspended
in 1 ml methoxyethyl ether (DME). To the solution, N-methyl
morpholine (NMM, 25 .mu.l, 0.22 mmol) and
1,3-dicyclohexylcarbodiimide (DCC, 45.4 mg, 0.22 mmol) were added.
The solution was stirred for 3 hours at the room temperature. The
product was purified by flash chromatography directly using a
gradient solvent eluent (0% to 1.5% to 5% methanol in methylene
chloride) to generate an orange crystal (46.0 mg, 70%). The
reaction scheme for synthesis of LBT3005 (FIG. 22b) is as follow:
4-Aminomethyl-cyclohexanecarboxylic acid (2.83 g, 18.0 mmol) in
saturated NaHCO.sub.3 (80 ml) was stirred vigorously at 0.degree.
C. To the solution, N-ethoxycarbonyleimide (finely grounded, 3.05
g, 18.0 mmol) was added portion by portion. When the addition was
completed (c.a. 10 minutes), deionized water (80 ml) was added and
the mixture was stirred at room temperature for 40 minutes. The
resulting mixture was bought to pH 1-2 with 1 M of HCl and
extracted with ethyl acetate (3.times.80 ml). The organic layer was
washed with deionized water (50 ml) and then brine (20 ml). The
organic phase was dried over magnesium sulfate and evaporated under
reduced pressure to generate a crude product. Purification by flash
chromatography (hexane: ethyl acetate : acetic acid, 60: 39: 1) to
afford the product as a solid (2.69 g, 63%). 5-Hydroxytryptamine
hydrochloride (42.5 mg, 0.2 mmol) and maleimidomethyl-cyclohexane
carboxylic acid (52.2 mg, 0.22 mmol) were suspended in 1 ml
methoxyethyl ether (DME). To the solution, N-methyl morpholine
(NMM, 25 .mu.l, 0.22 mmol) and 1,3-dicyclohexylcarbodiimide (DCC,
45.4 mg, 0.22 mmol) were added. The solution was stirred for 3
hours at the room temperature. The product was purified by flash
chromatography directly using a gradient solvent eluent (ethyl
acetate: hexane (1:1) to ethyl acetate: hexane (3:1)) to generate a
light brown oil (28.3 mg, 35.8%).
[0223] Other 5-HT receptor binding compounds such as those shown in
FIG. 23 can be similarly modified for irreversible binding to their
target sites using one of the reaction schemes illustrated in FIGS.
19b to 22b.
[0224] B. Assay
[0225] FIG. 18 shows the inhibitory effects of various 5-HT analogs
on the high affinity binding of .sup.3H-5-HT to murine brain
membranes. Purified murine brain membranes suspended in reaction
buffer (0.13 M NaCl, 0.02 M NaHCO.sub.3, pH 7.0) were pretreated
with graded concentrations of LBT3001 (.box-solid.), LBT3002
(.quadrature.), LBT3004 (.smallcircle.), and LBT3005
(.circle-solid.) for 5 min prior exposure to .sup.3H-5-HT (5 nM
final concentration) for additional 30 min. The reaction was
terminated by membrane vacuum filtration method. The data shown
were corrected for non-specific binding determined in the presence
of 1 mM of non-radioactive 5-HT. The results were plotted against
control binding determined in the absence of inhibitors.
[0226] FIG. 18 shows the binding of .sup.3H-5-HT to murine brain
membranes was inhibited by all analogs of 5-HT in an apparent
biphasic manner.
[0227] LBT3001
(1-[2-(5-hydroxy-1H-indol-3-yl)-ethyl]-pyrrole-2,5-dione), an
analog containing no spacer arm between the 5-HT and the maleimide
molecules (FIG. 19a), effectively inhibited the binding of
.sup.3H-5-HT to its high and low affinity binding sites with
IC.sub.50 values of about 0.001 and 50 .mu.M, respectively.
LBT3002, an analog containing 3 carbon molecules in the spacer arm
(FIG. 20a), inhibited the binding of .sup.3H-5-HT to its high and
low affinity binding sites with IC.sub.50 values of about 0.00003
and 200 .mu.M, respectively. LBT3004, an analog containing 2 carbon
molecules in the spacer arm (FIG. 21a), inhibited the binding of
.sup.3H-5-HT to its high and low affinity binding sites with
IC.sub.50 values of about 0.2 and 50 .mu.M, respectively. LBT3005,
an analog containing cyclohexane carboxylic molecule in the spacer
arm (FIG. 22a), inhibited the binding of .sup.3H-5-HT to its high
and low affinity binding sites with IC.sub.50 values of 0.5 and 300
.mu.M, respectively. These results clearly indicate irreversible
binding drugs can be designed using the technology described.
Additionally, the technology is applicable to many small molecules
and can be applied to further improve the efficacy of existing
molecules.
[0228] The entire disclosures of all publications, patents and
patent applications referred to herein are hereby incorporated by
reference.
[0229] Throughout the specification the aim has been to describe
the preferred embodiments of the invention without limiting the
invention to any one embodiment or specific collection of features.
Those of skill in the art will appreciate that, in light of the
instant disclosure, various modifications and changes can be made
in the particular embodiments exemplified without departing from
the scope of the present invention. All such modifications and
changes are intended to be included within the scope of the
appendant claims.
* * * * *