U.S. patent application number 13/428429 was filed with the patent office on 2012-10-25 for diagnostic and treatment methods using a ligand library.
Invention is credited to Muralidhar Reddy Moola, Jessica Schilke.
Application Number | 20120269799 13/428429 |
Document ID | / |
Family ID | 46880042 |
Filed Date | 2012-10-25 |
United States Patent
Application |
20120269799 |
Kind Code |
A1 |
Moola; Muralidhar Reddy ; et
al. |
October 25, 2012 |
DIAGNOSTIC AND TREATMENT METHODS USING A LIGAND LIBRARY
Abstract
The invention relates to diagnostic and treatment methods using
a ligand library. Specifically, the invention relates to using a
ligand library to diagnose or detect a drug induced response,
including drug adverse reaction, side effects, drug resistance, and
therapeutic efficacy. The invention further relates to identifying
biomarkers associated with a drug induced response and providing a
personalized medical treatment.
Inventors: |
Moola; Muralidhar Reddy;
(Jupiter, FL) ; Schilke; Jessica; (Jupiter,
FL) |
Family ID: |
46880042 |
Appl. No.: |
13/428429 |
Filed: |
March 23, 2012 |
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Application
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Patent Number |
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61467256 |
Mar 24, 2011 |
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61491717 |
May 31, 2011 |
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61583881 |
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Current U.S.
Class: |
424/130.1 ;
435/7.92; 506/18; 506/9; 530/389.1 |
Current CPC
Class: |
A61P 13/12 20180101;
A61P 25/28 20180101; C40B 40/04 20130101; G01N 33/566 20130101;
A61P 25/20 20180101; A61P 37/00 20180101; C07K 7/06 20130101; A61P
25/00 20180101; A61P 31/00 20180101; G01N 2800/104 20130101; A61P
19/02 20180101; A61P 29/00 20180101; A61P 21/04 20180101; C40B
60/12 20130101; A61P 31/10 20180101; G01N 2800/2821 20130101; A61P
35/02 20180101; C40B 40/10 20130101; A61P 37/02 20180101; C07K
1/047 20130101; A61P 9/00 20180101; A61P 31/04 20180101; G01N
33/57438 20130101; A61P 35/04 20180101; A61P 31/12 20180101; A61P
37/06 20180101; A61P 35/00 20180101; G01N 33/6845 20130101; A61P
17/02 20180101 |
Class at
Publication: |
424/130.1 ;
506/9; 435/7.92; 506/18; 530/389.1 |
International
Class: |
C40B 30/04 20060101
C40B030/04; C40B 40/10 20060101 C40B040/10; C07K 16/00 20060101
C07K016/00; A61K 39/395 20060101 A61K039/395; A61P 35/00 20060101
A61P035/00; A61P 25/28 20060101 A61P025/28; A61P 37/02 20060101
A61P037/02; A61P 31/12 20060101 A61P031/12; A61P 31/04 20060101
A61P031/04; A61P 31/10 20060101 A61P031/10; A61P 29/00 20060101
A61P029/00; A61P 9/00 20060101 A61P009/00; G01N 33/53 20060101
G01N033/53; A61P 35/04 20060101 A61P035/04 |
Claims
1. A method for diagnosing a drug induced response in a subject,
the method comprising: obtaining a biological sample from said
subject; screening a ligand library against said sample;
identifying binding characteristics of one or more markers in said
sample with one or more ligands in the library; and determining
whether said one or more markers are associated with said drug
induced response, thereby diagnosing said drug induced response in
said subject.
2. The method of claim 1, wherein said response is a side effect of
said drug, an adverse reaction to said drug, a resistance to said
drug, or a therapeutic dosage efficacy of said drug.
3. The method of claim 1, wherein said drug is at least one of the
drugs listed in Table 1.
4. The method of claim 1, further comprising the step of
determining whether said subject is responsive or non-responsive to
a treatment or therapy by said drug.
5. The method of claim 1, wherein said drug induced response is
associated with a disease or a stage of said disease.
6. The method of claim 1, wherein said disease is a cancer, an
autoimmune disease, an inflammatory disease, an infectious disease,
a neurodegenerative disease, or a cardiovascular disease.
7. The method of claim 6, wherein the cancer disease is breast
cancer, lung cancer, prostate cancer, cervical cancer, head and
neck cancer, testicular cancer, ovarian cancer, skin cancer, brain
cancer, pancreatic cancer, liver cancer, stomach cancer, colon
cancer, rectal cancer, esophageal cancer, lymphoma, or leukemia,
such as ligands that recognize markers unique to these disease
states.
8. The method of claim 6, wherein the autoimmune disease is lupus,
myestenia gravis, multiple sclerosis, narcolepsy, rheumatoid
arthritis, nephritis, Chagas disease, scleroderma, or Sjogren's
disease.
9. The method of claim 6, wherein the infectious disease is a
result of infection with viruses, bacteria or fungi.
10. The method of claim 6, wherein the neurodegenerative disease is
Alzheimer's disease, dementia, or Creutzfeld-Jacob disease.
11. The method of claim 1, wherein said ligand library comprises a
plurality of ligands identified in an earlier initial screening or
preselected based on known reactivity to said ligand binding
markers.
12. The method of claim 1, wherein said ligand library is a peptoid
library.
13. The method of claim 1, wherein the library comprises a compound
of formula I, ##STR00004## wherein R.sub.1 is selected from an
electron rich amino acid side chain Y; R.sub.2 is selected from H;
and R.sub.3-R.sub.6 are independently selected from the groups
consisting of H, --C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkylSCH.sub.3,
--C.sub.0-C.sub.6alkylC.sub.2-C.sub.6alkenyl,
--C.sub.0-C.sub.6alkyl C.sub.2-C.sub.6alkynyl, --C.sub.1-C.sub.6
COOH, --C.sub.1-C.sub.6alkylOH, --C.sub.1-C.sub.6alkylN(R).sub.2,
--C.sub.3-C.sub.8cycloalkyl, --C.sub.1-C.sub.6alkylaryl,
--C.sub.1-C.sub.6alkylheteroaryl,
--C.sub.1-C.sub.6alkylNC(O)C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkylcycloamide wherein any of the aryl or
heteroaryl groups may be independently substituted with --OH, Cl,
F, Br, --OCH.sub.3, --SO.sub.2NH.sub.2 or --O--CH.sub.2--O--.
14. The method of claim 1, wherein the library comprise a compound
of formula I, ##STR00005## wherein the compounds are produced by a
process which comprises use of a reactant selected from the group
consisting of (A) furfurylamine; 3,4-dimethoxyethanolamine;
benzylamine; N-(2-aminoethyl)acetamide;
N-(3-aminopropyl)-2-pyrrolidinone; ethanolamine; glycine;
diaminobutane; allylamine; piperonylamine; methylbenzylamine;
isobutylamine; 4-(2-aminoethyl)benzenesulfonamide or
cyclohexylamine; or (B) methoxyethylamine; piperonylamine;
cyclohexylamine; diaminobutane; methylbenzylamine; isobutylamine;
furfurylamine or 4-(2-aminoethyl)benzenesulfonamide; or (C)
furfurylamine, ethanolamine; glycine; diaminobutane; allylamine;
piperonylamine; methylbenzylamine; isobutylamine or
4-(2-aminoethyl)benzenesulfonamide; or (D) furfurylamine,
N-(2-aminoethyl)acetamide; N-(3-aminoethyl)-2-pyrrolidinone;
ethanolamine; glycine; diaminobutane; allylamine; piperonylamine;
methylbenzylamine; isobutylamine;
4-(2-aminoethyl)benzenesulfonamide; or (E) cysteine, glycine,
allylamine, ethanolamine, isobutylamine, methylbenzylamine,
piperonylamine, methionine, cyclohexylamine,
3,4-dimethoxyphenethylamine, benzylamine,
N-(2-aminoethyl)acetamide, N-(3-aminopropyl)-2-pyrrolidone,
4-(2-aminoethyl)benzenesulfonamide and furfurylamine; and wherein,
R.sub.1 is selected from the group consisting
--(C.sub.1-C.sub.6)SCH.sub.3; R.sub.2 is selected from H; R.sub.3
and R.sub.5 are independently selected from the groups consisting
of H, --C.sub.1-C.sub.6alkyl, --C.sub.1-C.sub.6alkylSCH.sub.3,
--C.sub.0-C.sub.6alkylC.sub.2-C.sub.6alkenyl,
--C.sub.0-C.sub.6alkyl C.sub.2-C.sub.6alkynyl, --C.sub.1-C.sub.6
COOH, --C.sub.1-C.sub.6alkylOH, --C.sub.1-C.sub.6alkylN(R).sub.2,
--C.sub.3-C.sub.8cycloalkyl, --C.sub.1-C.sub.6alkylaryl,
--C.sub.1-C.sub.6alkylheteroaryl,
--C.sub.1-C.sub.6alkylNC(O)C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkylcycloamide wherein any of the aryl or
heteroaryl groups may be independently substituted with --OH, Cl,
F, Br, --OCH.sub.3, --SO.sub.2NH.sub.2 or --O--CH.sub.2--O--;
R.sub.4 is selected from the group consisting of furfuryl or
--(C.sub.1-C.sub.6alkyl)NR.sub.7R.sub.8, R.sub.6 is selected from
the group consisting of H, 1-yl-allyl, 1-yl-2-hydroxyethyl,
isobutyl, 1-yl-n-butylamine, methylbenzyl, piperonyl, cyclohexyl,
1-yl-2-(3,4-dimethoxyphenyl)ethyl, benzyl, 1-yl-2-(acetamide)ethyl,
1-yl-3-2-pyrrolidinone, 1-yl-2-(4-benzenesulfonamide)ethyl or
furfuryl and n is 3-11.
15. The method of claim 1, wherein the library comprises a compound
having a formula Ia ##STR00006## wherein the compound is selected
from the group consisting of a compound of formula Ia wherein, (a)
R.sub.9 is n-butylamine; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
piperonyl; R.sub.12 is methylbenzyl; R.sub.13 is piperonyl;
R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-methoxyethyl; (b) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is cyclohexyl; R.sub.13 is 1-yl-2-methoxyethyl; R.sub.14
is 1-yl-2,2-dimethylethyl (isobutyl); R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(c) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (d) R.sub.9 is piperonyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is isobutyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is methylbenzyl;
R.sub.14 is cyclohexyl; R.sub.15 is isobutyl and R.sub.16 is
1-yl-n-butylamine; (e) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is cyclohexyl; R.sub.14 is
cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is piperonyl; (f)
R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isopropyl; R.sub.12 is isopropyl; R.sub.13 is 1-yl-2-methoxyethyl;
R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (g) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
piperonyl; R.sub.12 is methylbenzyl; R.sub.13 is piperonyl;
R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl; (h)
R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is methylbenzyl; R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13
is cyclohexyl; R.sub.14 is cyclohexyl; R.sub.15 is methylbenzyl and
R.sub.16 is piperonyl; (i) R.sub.9 is 1-yl-n-butylamine; R.sub.10
is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is 1-yl-n-butylamine; R.sup.14 is
cyclohexyl; R.sup.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sup.16 is cyclohexyl; (j) R.sup.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is methylbenzyl;
R.sub.11 is methylbenzyl; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-n-butylamine; R.sub.14 is methylbenzyl; R.sub.15 is isobutyl
and R.sub.16 is 1-yl-n-butylamine; (k) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is isobutyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is piperonyl; R.sub.13
is 1-yl-n-butylamine; R.sub.14 is cyclohexyl; R.sub.15 is
methylbenzyl and R.sub.16 is 1-yl-2-methoxyethyl; (l) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is isobutyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(m) R9 is 1-yl-2-methoxyethyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R12 is 1-yl-2-methoxyethyl; R13 is
methylbenzyl; R14 is 1-yl-n-butylamine; R.sub.15 is furfuryl and
R16 is furfuryl; (n) R9 is cyclohexyl; R10 is cyclohexyl; R11 is
1-yl-n-butylamine; R12 is furfuryl; R13 is 1-yl-2-methoxyethyl; R14
is 1-yl-2-methoxyethyl; R15 is 1-yl-2-(4(benzenesulfonamide)ethyl
and R16 is furfuryl; (o) R9 is 1-yl-n-butylamine; R10 is piperonyl;
R11 is 1-yl-2-(4(benzenesulfonamide)ethyl; R12 is
1-yl-2-methoxyethyl; R13 is methylbenzyl; R14 is 1-yl-n-butylamine;
R15 is 1-yl-2-methoxyethyl and R16 is methylbenzyl; (p) R9 is
cyclohexyl; R10 is cyclohexyl; R11 is piperonyl; R12 is
1-yl-n-butylamine; R13 is 1-yl-n-butylamine; R14 is
1-yl-n-butylamine; R15 is 1-yl-n-butylamine and R16 is isobutyl;
(q) R9 is piperonyl; R10 is 1-yl-n-butylamine; R11 is
1-yl-2-methoxyethyl; R12 is 1-yl-2-(4(benzenesulfonamide)ethyl; R13
is piperonyl; R14 is 1-yl-n-butylamine; R15 is methylbenzyl and
R.sub.16 is methylbenzyl; (r) R9 is methylbenzyl; R10 is
methylbenzyl; R11 is methylbenzyl; R12 is 1-yl-n-butylamine; R13 is
piperonyl; R14 is 1-yl-n-butylamine; R15 is piperonyl and R.sub.16
is 1-yl-n-butylamine; (s) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is
methylbenzyl; R.sub.15 is 1-yl-2-methoxyethyl and R.sub.16 is
piperonyl; (t) R.sub.9 is methylbenzyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
isobutyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is methylbenzyl
and R.sub.16 is 1-yl-2-methoxyethyl; (u) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isobutyl; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl
and R.sub.16 is cyclohexyl; (v) R.sub.9 is isobutyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is piperonyl and R.sub.16 is piperonyl;
(w) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
isobutyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is isobutyl; R.sub.15
is 1-yl-2-methoxyethyl and R.sub.16 is cyclohexyl; (x) R.sub.9 is
furfuryl; R.sub.10 is furfuryl; R.sub.11 is piperonyl; R.sub.12 is
cyclohexyl; R.sub.13 is piperonyl; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
cyclohexyl; (y) R.sub.9 is piperonyl; R.sub.10 is piperonyl;
R.sub.11 is 1-yl-2-methoxyethyl; R.sub.12 is 1-yl-2-methoxyethyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(z) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is isobutyl; R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.13 is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
isobutyl and R.sub.16 is 1-yl-n-butylamine; (aa) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is methylbenzyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (bb) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-methoxyethyl; R.sub.11 is
1-yl-n-butylamine; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl;
R.sub.14 is 1-yl-n-butylamine; R.sub.15 is 1-yl-n-butylamine and
R.sub.16 is piperonyl; (cc) R.sub.9 is cyclohexyl; R.sub.10 is
methylbenzyl; R.sub.11 is cyclohexyl; R.sub.12 is piperonyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is 1-yl-n-butylamine
and R.sub.16 is 1-yl-2-methoxyethyl; (dd) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is
1-yl-2-methoxyethyl; R.sub.15 is isobutyl and R.sub.16 is
cyclohexyl; (ee) R.sub.9 is 1-yl-2-methoxyethyl; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is piperonyl; R.sub.14 is isobutyl;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-n-butylamine; (ff) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl; (gg)
R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-2-methoxyethyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is 1-yl-2-methoxyethyl; R.sub.15 is
1-yl-n-butylamine and R.sub.16 is methylbenzyl; (hh) R.sub.9 is
cyclohexyl; R.sub.10 is cyclohexyl; R.sub.11 is methylbenzyl;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (ii) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
furfuryl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is furfuryl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is cyclohexyl;
(jj) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is methylbenzyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-methoxyethyl and R.sub.16 is isobutyl; (kk) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is methylbenzyl; R.sub.13
is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(ll) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (mm) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
cyclohexyl; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sub.16 is methylbenzyl; (nn) R.sub.9 is 1-yl-n-butylamine;
R.sub.10 is 1-yl-n-butylamine; R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (oo) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.10 is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is piperonyl;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(pp) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is 1-yl-n-butylamine; R.sub.12 is methylamine; R.sub.13 is
piperonyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is piperonyl and
R.sub.16 is 1-yl-2-methoxyethyl; (qq) R.sub.9 is cyclohexyl;
R.sub.10 is cyclohexyl; R.sub.11 is furfuryl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is isobutyl; R.sub.14 is cyclohexyl;
R.sub.15 is methylbenzyl and R.sub.16 is methylbenzyl; (rr) R.sub.9
is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is isobutyl;
R.sub.12 is isobutyl; R.sub.13 is 1-yl-2-methoxyethyl; R.sub.14 is
cyclohexyl; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sub.16 is 1-yl-2-(4(benzenesulfonamide)ethyl; (ss) R.sub.9 is
cyclohexyl; R.sub.10 is cyclohexyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is methylbenzyl; R.sub.15 is cyclohexyl and R.sub.16 is piperonyl;
and pharmaceutically acceptable salts thereof.
16. The method of claim 1, wherein the library comprises a compound
of the formula: ##STR00007## wherein the compound is selected from
the group consisting of a compound of formula II wherein, (a)
R.sub.9 is n-butylamine; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
piperonyl; R.sub.12 is methylbenzyl; R.sub.13 is piperonyl;
R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-methoxyethyl; (b) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is cyclohexyl; R.sub.13 is 1-yl-2-methoxyethyl; R.sub.14
is 1-yl-2,2-dimethylethyl (isobutyl); R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(c) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (d) R.sub.9 is piperonyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is isobutyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is methylbenzyl;
R.sub.14 is cyclohexyl; R.sub.15 is isobutyl and R.sub.16 is
1-yl-n-butylamine; (e) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is cyclohexyl; R.sub.14 is
cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is piperonyl; (f)
R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isopropyl; R.sub.12 is isopropyl; R.sub.13 is 1-yl-2-methoxyethyl;
R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (g) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
piperonyl; R.sub.12 is methylbenzyl; R.sub.13 is piperonyl;
R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl; (h)
R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is methylbenzyl; R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13
is cyclohexyl; R.sub.14 is cyclohexyl; R.sub.15 is methylbenzyl and
R.sub.16 is piperonyl; (i) R.sub.9 is 1-yl-n-butylamine; R.sub.10
is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is 1-yl-n-butylamine; R.sup.14 is
cyclohexyl; R.sup.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sup.16 is cyclohexyl; (j) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is methylbenzyl;
R.sub.11 is methylbenzyl; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-n-butylamine; R.sub.14 is methylbenzyl; R.sub.15 is isobutyl
and R.sub.16 is 1-yl-n-butylamine; (k) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is isobutyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is piperonyl; R.sub.13
is 1-yl-n-butylamine; R.sub.14 is cyclohexyl; R.sub.15 is
methylbenzyl and R.sub.16 is 1-yl-2-methoxyethyl; (l) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is isobutyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(m) R.sub.9 is 1-yl-2-methoxyethyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is 1-yl-2-methoxyethyl;
R.sub.13 is methylbenzyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15
is furfuryl and R.sub.16 is furfuryl; (n) R.sub.9 is cyclohexyl;
R.sub.10 is cyclohexyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
furfuryl; R.sub.13 is 1-yl-2-methoxyethyl; R.sub.14 is
1-yl-2-methoxyethyl; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl
and R.sub.16 is furfuryl; (o) R.sub.9 is 1-yl-n-butylamine;
R.sub.10 is piperonyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is methylbenzyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is 1-yl-2-methoxyethyl and R.sub.16 is
methylbenzyl; (p) R.sub.9 is cyclohexyl; R.sub.10 is cyclohexyl;
R.sub.11 is piperonyl; R.sub.12 is 1-yl-n-butylamine; R.sub.13 is
1-yl-n-butylamine; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is
1-yl-n-butylamine and R.sub.16 is isobutyl; (q) R.sub.9 is
piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
1-yl-2-methoxyethyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is piperonyl; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is methylbenzyl and R.sub.16 is
methylbenzyl; (r) R.sub.9 is methylbenzyl; R.sub.10 is
methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is piperonyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is piperonyl and R.sub.16 is
1-yl-n-butylamine; (s) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is
methylbenzyl; R.sub.15 is 1-yl-2-methoxyethyl and R.sub.16 is
piperonyl; (t) R.sub.9 is methylbenzyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
isobutyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is methylbenzyl
and R.sub.16 is 1-yl-2-methoxyethyl; (u) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isobutyl; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl
and R.sub.16 is cyclohexyl; (v) R.sub.9 is isobutyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is piperonyl and R.sub.16 is piperonyl;
(w) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
isobutyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is isobutyl; R.sub.15
is 1-yl-2-methoxyethyl and R.sub.16 is cyclohexyl; (x) R.sub.9 is
furfuryl; R.sub.10 is furfuryl; R.sub.11 is piperonyl; R.sub.12 is
cyclohexyl; R.sub.13 is piperonyl; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
cyclohexyl; (y) R.sub.9 is piperonyl; R.sub.10 is piperonyl;
R.sub.11 is 1-yl-2-methoxyethyl; R.sub.12 is 1-yl-2-methoxyethyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(z) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is isobutyl; R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.13 is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
isobutyl and R.sub.16 is 1-yl-n-butylamine; (aa) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is methylbenzyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (bb) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-methoxyethyl; R.sub.11 is
1-yl-n-butylamine; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl;
R.sub.14 is 1-yl-n-butylamine; R.sub.15 is 1-yl-n-butylamine and
R.sub.16 is piperonyl; (cc) R.sub.9 is cyclohexyl; R.sub.10 is
methylbenzyl; R.sub.11 is cyclohexyl; R.sub.12 is piperonyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is 1-yl-n-butylamine
and R.sub.16 is 1-yl-2-methoxyethyl; (dd) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is
1-yl-2-methoxyethyl; R.sub.15 is isobutyl and R.sub.16 is
cyclohexyl; (ee) R.sub.9 is 1-yl-2-methoxyethyl; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is piperonyl; R.sub.14 is isobutyl;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-n-butylamine; (ff) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl; (gg)
R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-2-methoxyethyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is 1-yl-2-methoxyethyl; R.sub.15 is
1-yl-n-butylamine and R.sub.16 is methylbenzyl; (hh) R.sub.9 is
cyclohexyl; R.sub.10 is cyclohexyl; R.sub.11 is methylbenzyl;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (ii) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
furfuryl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is furfuryl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is cyclohexyl;
(jj) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is methylbenzyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-methoxyethyl and R.sub.16 is isobutyl; (kk) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is methylbenzyl; R.sub.13
is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(ll) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (mm) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
cyclohexyl; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sub.16 is methylbenzyl; (nn) R.sub.9 is 1-yl-n-butylamine;
R.sub.10 is 1-yl-n-butylamine; R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (oo) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.10 is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is piperonyl;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(pp) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is 1-yl-n-butylamine; R.sub.12 is methylamine; R.sub.13 is
piperonyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is piperonyl and
R.sub.16 is 1-yl-2-methoxyethyl; (qq) R.sub.9 is cyclohexyl;
R.sub.10 is cyclohexyl; R.sub.11 is furfuryl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is isobutyl; R.sub.14 is cyclohexyl;
R.sub.15 is methylbenzyl and R.sub.16 is methylbenzyl; (rr) R.sub.9
is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is isobutyl;
R.sub.12 is isobutyl; R.sub.13 is 1-yl-2-methoxyethyl; R.sub.14 is
cyclohexyl; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sub.16 is 1-yl-2-(4(benzenesulfonamide)ethyl; (ss) R.sub.9 is
cyclohexyl; R.sub.10 is cyclohexyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is methylbenzyl; R.sub.15 is cyclohexyl and R.sub.16 is piperonyl;
and pharmaceutically acceptable salts thereof.
17. The method of claim 1, wherein said ligand library is a bead
based ligand library comprising a plurality of beads having ligands
bound to the beads.
18. The method of claim 1, wherein said screening is performed by
an array.
19. The method of claim 18, wherein said array is a microarray, a
microscope slide, a plate, a chip, or a population of beads.
20. The method of claim 18, wherein said array comprising a solid
substrate having a surface; a plurality of ligands bound to said
substrate, each of said ligand comprising an anchor segment stably
bound to the substrate surface, a peptoid segment, and a linker
segment connecting and separating the anchor and peptoid
segments.
21. The method of claim 18, wherein said array comprises between
about 1000 and 100,000 distinct ligands.
22. The method of claim 18, wherein said array comprises between
about 2000 and 50,000 distinct ligands.
23. The method of claim 18, wherein said array comprises between
about 4000 and 25,000 distinct ligands.
24. The method of claim 18, wherein said array comprises between
about 6000 and 15,000 distinct ligands.
25. The method of claim 18, wherein said array comprises between
about 7000 and 12,500 distinct ligands.
26. The method of claim 18, wherein said array comprises between
about 8000 and 10,000 distinct ligands.
27. The method of claim 18, wherein said array comprises a ligand
binding moiety cross-linked to said array.
28. The method of claim 1, wherein the step of determining
comprises detecting the binding molecules fluorescently.
29. The method of claim 18, wherein said array comprises
fluorescently labeled ligands.
30. The method of claim 1, wherein said screening comprises an
initial screening against a first ligand library and a subsequent
screening against a second ligand library.
31. The method of claim 30, wherein said initial screening is
performed against a first sample collected prior to administering
said drug to said subject and said subsequent screening is
performed against a second sample collected after administering
said drug to said subject.
32. The method of claim 30, said first ligand library is screened
for a first response and said second ligand library is screened for
a second response.
33. The method of claim 32, wherein said first response is
associated with said disease and said second response is associated
with said drug induced response.
34. The method of claim 32, wherein said first response is
associated with said disease and said second response is associated
with a stage of said disease.
35. The method of claim 30, wherein said first ligand library
comprises a first set of ligands and said second ligand library
comprises a second set of ligands.
36. The method of claim 35, wherein said first set of ligands are
different from said second set of ligands.
37. The method of claim 35, wherein one or more of said first set
of ligands are same as one or more of said second set of
ligands.
38. The method of claim 1, wherein said biological sample is
tissue, cell, urine, serum, whole blood, cerebrospinal fluid,
sputum, saliva, or semen.
39. The method of claim 1, wherein said subject is a human or
animal subject.
40. The method of claim 1, wherein said diagnostic steps are
performed in accordance with instructions in a package insert.
41. The method of claim 40, wherein said package insert comprises
instructions for performing said diagnostic steps, instructions for
determining a drug administration, and instructions for
administering said drug based on the determination.
42. The method of claim 40, wherein said package insert is an
insert in a kit, said kit comprising said library and components
for performing said diagnostic steps.
43. A method for treating a disease in a subject, the method
comprising: obtaining a biological sample from said subject;
screening a ligand library against said sample; identifying binding
characteristics of one or more markers in said sample with one or
more ligands in the library; determining whether said one or more
markers are associated with a response to a drug for treating said
disease; administering said drug to said subject, based on the
determination of association between said one or more markers to
said response, thereby treating said disease in said subject.
44. A method for treating a disease in a patient, the method
comprising: obtaining a biological sample from said patient;
screening said sample with a library comprising at least one ligand
to determine a ligand binding profile; administering a drug to said
patient, based on the results of said ligand binding profile in
said patient, wherein said at least one ligand has an affinity to
one or more antibodies associated with said disease.
45. A method for monitoring a treatment by a drug in a subject, the
method comprising: obtaining a biological sample from said subject;
screening a ligand library against said subject; identifying
binding characteristics of one or more markers in said sample with
one or more ligands in the library; determining whether said one or
more markers are associated with a response to a drug for treating
said disease; administering said drug to said subject, based on the
determination of association between said one or more markers to
said response, thereby monitoring said treatment by said drug in
said subject.
46. A method for detecting a risk of adverse reaction to a drug in
a subject, the method comprising: obtaining a biological sample
from said subject; screening a ligand library against said sample;
identifying binding characteristics of one or more markers in said
sample with one or more ligands in the library; and determining
whether said one or more markers are associated with said risk,
thereby detecting said risk of adverse reaction to said drug in
said subject.
47. A method for profiling one or more subjects for a response to a
drug to treat a disease, the method comprising: obtaining a
biological sample from said subject; screening a ligand library
against said subject; identifying binding characteristics of one or
more markers in said sample with one or more ligands in the
library; and determining whether said one or more markers are
associated with said response for said drug, thereby profiling said
one or more subjects for said response to said drug to treat said
disease.
48. A method for diagnosing a drug induced response in a subject,
the method comprising: obtaining a biological sample from said
subject; screening a peptoid library against said sample;
identifying binding characteristics of one or more markers in said
sample with one or more ligands in the library, wherein at least
one of said one or more markers is an autoantibody capable of
binding to a peptoid ligand; and determining whether said one or
more markers are associated with said drug induced response,
thereby diagnosing said drug induced response in said subject.
49. A method for providing a prognosis of a drug induced response
for treating a disease in a subject, the method comprising:
obtaining a biological sample from said subject; screening a ligand
library against said sample; identifying binding characteristics of
one or more markers in said sample with one or more ligands in the
library; and determining whether said one or more markers are
associated with said drug induced response, thereby providing said
prognosis of said drug induced response for treating said disease
in said subject.
50. A kit for diagnosis of a disease in accordance with claim 1
comprising: a ligand library, detection reagents for screening said
ligand library against a biological sample, adjuvants for said
screening, and a package insert, wherein said package insert
comprises instructions for performing said diagnostic steps,
instructions for determining a drug administration, and
instructions for administering said drug based on the
determination.
51. A kit for treating a disease in accordance with claim 43
comprising: a ligand library, detection reagents for screening said
ligand library against a biological sample, adjuvants for said
screening, and a package insert, wherein said package insert
comprises instructions for performing said diagnostic steps,
instructions for determining a drug administration, and
instructions for administering said drug based on the
determination.
52. A method for identifying a marker associated with a drug
induced response for treating a disease, the method comprising:
obtaining a biological sample from a subject; screening a ligand
library against said biological sample; determining the binding
characteristics of a marker in said sample to a ligand in the
library; and determining whether said marker is associated with
said drug induced response for treating said disease, thereby
identifying said marker associated with said drug induced response
for treating said disease.
53. The method of claim 52, wherein said library comprises about
350,000 to about 150 MM ligands.
54. The method of claim 52, wherein said ligand library is a random
ligand library.
55. The method of claim 52, wherein said ligand library is a
non-random ligand library.
56. The method of claim 52, wherein said ligand library is a
peptoid library.
57. The method of claim 52, wherein at least one ligand in said
ligand library has an affinity to an autoantibody to antigen that
is associated with said drug induced response for treating said
disease.
58. The method of claim 52, wherein said marker is an autoantibody
capable of binding to a peptoid ligand.
59. The method of claim 52, wherein the ligand library is a bead
based ligand library comprising a plurality of beads having ligands
bound to the beads.
60. The method of claim 52, wherein said screening is performed by
an array.
61. The method of claim 60, wherein said array is a microarray, a
microscope slide, a plate, a chip, or a population of beads.
62. The method of claim 60, wherein said array comprising a solid
substrate having a surface; a plurality of ligands bound to said
substrate, each of said ligand comprising an anchor segment stably
bound to the substrate surface, a peptoid segment, and a linker
segment connecting and separating the anchor and peptoid
segments.
63. The method of claim 52, wherein the library comprises a
compound of formula I, ##STR00008## wherein R.sub.1 is selected
from an electron rich amino acid side chain Y; R.sub.2 is selected
from H; and R.sub.3-R.sub.6 are independently selected from the
groups consisting of H, --C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkylSCH.sub.3,
--C.sub.0-C.sub.6alkylC.sub.2-C.sub.6alkenyl,
--C.sub.0-C.sub.6alkyl C.sub.2-C.sub.6alkynyl, --C.sub.1-C.sub.6
COOH, --C.sub.1-C.sub.6alkylOH, --C.sub.1-C.sub.6alkylN(R).sub.2,
--C.sub.3-C.sub.8cycloalkyl, --C.sub.1-C.sub.6alkylaryl,
--C.sub.1-C.sub.6alkylheteroaryl,
--C.sub.1-C.sub.6alkylNC(O)C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkylcycloamide wherein any of the aryl or
heteroaryl groups may be independently substituted with --OH, Cl,
F, Br, --OCH.sub.3, --SO.sub.2NH.sub.2 or --O--CH.sub.2--O--.
64. The method of claim 52, wherein the library comprise a compound
of formula I, ##STR00009## wherein the compounds are produced by a
process which comprises use of a reactant selected from the group
consisting of (A) furfurylamine; 3,4-dimethoxyethanolamine;
benzylamine; N-(2-aminoethyl)acetamide;
N-(3-aminopropyl)-2-pyrrolidinone; ethanolamine; glycine;
diaminobutane; allylamine; piperonylamine; methylbenzylamine;
isobutylamine; 4-(2-aminoethyl)benzenesulfonamide or
cyclohexylamine; or (B) methoxyethylamine; piperonylamine;
cyclohexylamine; diaminobutane; methylbenzylamine; isobutylamine;
furfurylamine or 4-(2-aminoethyl)benzenesulfonamide; or (C)
furfurylamine, ethanolamine; glycine; diaminobutane; allylamine;
piperonylamine; methylbenzylamine; isobutylamine or
4-(2-aminoethyl)benzenesulfonamide; or (D) furfurylamine,
N-(2-aminoethyl)acetamide; N-(3-aminoethyl)-2-pyrrolidinone;
ethanolamine; glycine; diaminobutane; allylamine; piperonylamine;
methylbenzylamine; isobutylamine;
4-(2-aminoethyl)benzenesulfonamide; or (E) cysteine, glycine,
allylamine, ethanolamine, isobutylamine, methylbenzylamine,
piperonylamine, methionine, cyclohexylamine,
3,4-dimethoxyphenethylamine, benzylamine,
N-(2-aminoethyl)acetamide, N-(3-aminopropyl)-2-pyrrolidone,
4-(2-aminoethyl)benzenesulfonamide and furfurylamine; and wherein,
R.sub.1 is selected from the group consisting
--(C.sub.1-C.sub.6)SCH.sub.3; R.sub.2 is selected from H; R.sub.3
and R.sub.5 are independently selected from the groups consisting
of H, --C.sub.1-C.sub.6alkyl, --C.sub.1-C.sub.6alkylSCH.sub.3,
--C.sub.0-C.sub.6alkylC.sub.2-C.sub.6alkenyl,
--C.sub.0-C.sub.6alkyl C.sub.2-C.sub.6alkynyl, --C.sub.1-C.sub.6
COOH, --C.sub.1-C.sub.6alkylOH, --C.sub.1-C.sub.6alkylN(R).sub.2,
--C.sub.3-C.sub.8cycloalkyl, --C.sub.1-C.sub.6alkylaryl,
--C.sub.1-C.sub.6alkylheteroaryl,
--C.sub.1-C.sub.6alkylNC(O)C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkylcycloamide wherein any of the aryl or
heteroaryl groups may be independently substituted with --OH, Cl,
F, Br, --OCH.sub.3, --SO.sub.2NH.sub.2 or --O--CH.sub.2--O--;
R.sub.4 is selected from the group consisting of furfuryl or
--(C.sub.1-C.sub.6alkyl)NR.sub.7R.sub.8, R.sub.6 is selected from
the group consisting of H, 1-yl-allyl, 1-yl-2-hydroxyethyl,
isobutyl, 1-yl-n-butylamine, methylbenzyl, piperonyl, cyclohexyl,
1-yl-2-(3,4-dimethoxyphenyl)ethyl, benzyl, 1-yl-2-(acetamide)ethyl,
1-yl-3-2-pyrrolidinone, 1-yl-2-(4-benzenesulfonamide)ethyl or
furfuryl and n is 3-11.
65. The method of claim 52, wherein the library comprises a
compound having a formula Ia ##STR00010## wherein the compound is
selected from the group consisting of a compound of formula Ia
wherein, (tt) R.sub.9 is n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is piperonyl; R.sub.12 is methylbenzyl;
R.sub.13 is piperonyl; R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-methoxyethyl; (uu) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is cyclohexyl; R.sub.13 is 1-yl-2-methoxyethyl; R.sub.14
is 1-yl-2,2-dimethylethyl (isobutyl); R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(vv) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (ww) R.sub.9 is piperonyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is isobutyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is methylbenzyl;
R.sub.14 is cyclohexyl; R.sub.15 is isobutyl and R.sub.16 is
1-yl-n-butylamine; (xx) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is cyclohexyl; R.sub.14 is
cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is piperonyl;
(yy) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is isopropyl; R.sub.12 is isopropyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (zz) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
piperonyl; R.sub.12 is methylbenzyl; R.sub.13 is piperonyl;
R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl;
(aaa) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is methylbenzyl; R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13
is cyclohexyl; R.sub.14 is cyclohexyl; R.sub.15 is methylbenzyl and
R.sub.16 is piperonyl; (bbb) R.sub.9 is 1-yl-n-butylamine; R.sub.10
is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is 1-yl-n-butylamine; R.sup.14 is
cyclohexyl; R.sup.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sup.16 is cyclohexyl; (ccc) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is methylbenzyl;
R.sub.11 is methylbenzyl; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-n-butylamine; R.sub.14 is methylbenzyl; R.sub.15 is isobutyl
and R.sub.16 is 1-yl-n-butylamine; (ddd) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is isobutyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is piperonyl; R.sub.13
is 1-yl-n-butylamine; R.sub.14 is cyclohexyl; R.sub.15 is
methylbenzyl and R.sub.16 is 1-yl-2-methoxyethyl; (eee) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is isobutyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(fff) R9 is 1-yl-2-methoxyethyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R12 is 1-yl-2-methoxyethyl; R13 is
methylbenzyl; R14 is 1-yl-n-butylamine; R.sub.15 is furfuryl and
R16 is furfuryl; (ggg) R9 is cyclohexyl; R10 is cyclohexyl; R11 is
1-yl-n-butylamine; R12 is furfuryl; R13 is 1-yl-2-methoxyethyl; R14
is 1-yl-2-methoxyethyl; R15 is 1-yl-2-(4(benzenesulfonamide)ethyl
and R16 is furfuryl; (hhh) R9 is 1-yl-n-butylamine; R10 is
piperonyl; R11 is 1-yl-2-(4(benzenesulfonamide)ethyl; R12 is
1-yl-2-methoxyethyl; R13 is methylbenzyl; R14 is 1-yl-n-butylamine;
R15 is 1-yl-2-methoxyethyl and R16 is methylbenzyl; (iii) R9 is
cyclohexyl; R10 is cyclohexyl; R11 is piperonyl; R12 is
1-yl-n-butylamine; R13 is 1-yl-n-butylamine; R14 is
1-yl-n-butylamine; R15 is 1-yl-n-butylamine and R16 is isobutyl;
(jjj) R9 is piperonyl; R10 is 1-yl-n-butylamine; R11 is
1-yl-2-methoxyethyl; R12 is 1-yl-2-(4(benzenesulfonamide)ethyl; R13
is piperonyl; R14 is 1-yl-n-butylamine; R15 is methylbenzyl and
R.sub.16 is methylbenzyl; (kkk) R9 is methylbenzyl; R10 is
methylbenzyl; R11 is methylbenzyl; R12 is 1-yl-n-butylamine; R13 is
piperonyl; R14 is 1-yl-n-butylamine; R15 is piperonyl and R.sub.16
is 1-yl-n-butylamine; (lll) R.sub.9 is 1-yl-n-butylamine; R.sub.10
is 1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is
methylbenzyl; R.sub.15 is 1-yl-2-methoxyethyl and R.sub.16 is
piperonyl; (mmm) R.sub.9 is methylbenzyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
isobutyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is methylbenzyl
and R.sub.16 is 1-yl-2-methoxyethyl; (nnn) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isobutyl; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl
and R.sub.16 is cyclohexyl; (ooo) R.sub.9 is isobutyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is piperonyl and R.sub.16 is piperonyl;
(ppp) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
isobutyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is isobutyl; R.sub.15
is 1-yl-2-methoxyethyl and R.sub.16 is cyclohexyl; (qqq) R.sub.9 is
furfuryl; R.sub.10 is furfuryl; R.sub.11 is piperonyl; R.sub.12 is
cyclohexyl; R.sub.13 is piperonyl; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
cyclohexyl; (rrr) R.sub.9 is piperonyl; R.sub.10 is piperonyl;
R.sub.11 is 1-yl-2-methoxyethyl; R.sub.12 is 1-yl-2-methoxyethyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(sss) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is isobutyl; R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.13 is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
isobutyl and R.sub.16 is 1-yl-n-butylamine; (ttt) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is methylbenzyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (uuu) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-methoxyethyl; R.sub.11 is
1-yl-n-butylamine; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl;
R.sub.14 is 1-yl-n-butylamine; R.sub.15 is 1-yl-n-butylamine and
R.sub.16 is piperonyl; (vvv) R.sub.9 is cyclohexyl; R.sub.10 is
methylbenzyl; R.sub.11 is cyclohexyl; R.sub.12 is piperonyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is 1-yl-n-butylamine
and R.sub.16 is 1-yl-2-methoxyethyl; (www) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is
1-yl-2-methoxyethyl; R.sub.15 is isobutyl and R.sub.16 is
cyclohexyl; (xxx) R.sub.9 is 1-yl-2-methoxyethyl; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is piperonyl; R.sub.14 is isobutyl;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-n-butylamine; (yyy) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl;
(zzz) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-methoxyethyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is 1-yl-2-methoxyethyl; R.sub.15 is
1-yl-n-butylamine and R.sub.16 is methylbenzyl; (aaaa) R.sub.9 is
cyclohexyl; R.sub.10 is cyclohexyl; R.sub.11 is methylbenzyl;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (bbbb) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
furfuryl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is furfuryl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is cyclohexyl;
(cccc) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is methylbenzyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-methoxyethyl and R.sub.16 is isobutyl; (dddd) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is methylbenzyl; R.sub.13
is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(eeee) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (ffff) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
cyclohexyl; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sub.16 is methylbenzyl; (gggg) R.sub.9 is 1-yl-n-butylamine;
R.sub.10 is 1-yl-n-butylamine; R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (hhhh) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.10 is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is piperonyl;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(iiii) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is methylamine; R.sub.13 is
piperonyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is piperonyl and
R.sub.16 is 1-yl-2-methoxyethyl; (jjjj) R.sub.9 is cyclohexyl;
R.sub.10 is cyclohexyl; R.sub.11 is furfuryl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is isobutyl; R.sub.14 is cyclohexyl;
R.sub.15 is methylbenzyl and R.sub.16 is methylbenzyl; (kkkk)
R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isobutyl; R.sub.12 is isobutyl; R.sub.13 is 1-yl-2-methoxyethyl;
R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (llll) R.sub.9 is cyclohexyl;
R.sub.10 is cyclohexyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
methylbenzyl; R.sub.15 is cyclohexyl and R.sub.16 is piperonyl; and
pharmaceutically acceptable salts thereof.
66. The method of claim 52, wherein the library comprises a
compound of the formula: ##STR00011## wherein the compound is
selected from the group consisting of a compound of formula II
wherein, (tt) R.sub.9 is n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is piperonyl; R.sub.12 is methylbenzyl;
R.sub.13 is piperonyl; R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-methoxyethyl; (uu) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is cyclohexyl; R.sub.13 is 1-yl-2-methoxyethyl; R.sub.14
is 1-yl-2,2-dimethylethyl (isobutyl); R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(vv) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (ww) R.sub.9 is piperonyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is isobutyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is methylbenzyl;
R.sub.14 is cyclohexyl; R.sub.15 is isobutyl and R.sub.16 is
1-yl-n-butylamine; (xx) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is cyclohexyl; R.sub.14 is
cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is piperonyl;
(yy) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is isopropyl; R.sub.12 is isopropyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (zz) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
piperonyl; R.sub.12 is methylbenzyl; R.sub.13 is piperonyl;
R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl;
(aaa) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is methylbenzyl; R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13
is cyclohexyl; R.sub.14 is cyclohexyl; R.sub.15 is methylbenzyl and
R.sub.16 is piperonyl; (bbb) R.sub.9 is 1-yl-n-butylamine; R.sub.10
is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is 1-yl-n-butylamine; R.sup.14 is
cyclohexyl; R.sup.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sup.16 is cyclohexyl; (ccc) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is methylbenzyl;
R.sub.11 is methylbenzyl; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-n-butylamine; R.sub.14 is methylbenzyl; R.sub.15 is isobutyl
and R.sub.16 is 1-yl-n-butylamine; (ddd) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is isobutyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is piperonyl; R.sub.13
is 1-yl-n-butylamine; R.sub.14 is cyclohexyl; R.sub.15 is
methylbenzyl and R.sub.16 is 1-yl-2-methoxyethyl; (eee) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is cyclohexyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is isobutyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(fff) R.sub.9 is 1-yl-2-methoxyethyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is methylbenzyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is furfuryl and R.sub.16 is furfuryl;
(ggg) R.sub.9 is cyclohexyl; R.sub.10 is cyclohexyl; R.sub.11 is
1-yl-n-butylamine; R.sub.12 is furfuryl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is 1-yl-2-methoxyethyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is furfuryl; (hhh)
R.sub.9 is 1-yl-n-butylamine; R.sub.10 is piperonyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is methylbenzyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is 1-yl-2-methoxyethyl and R.sub.16 is
methylbenzyl; (iii) R.sub.9 is cyclohexyl; R.sub.10 is cyclohexyl;
R.sub.11 is piperonyl; R.sub.12 is 1-yl-n-butylamine; R.sub.13 is
1-yl-n-butylamine; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is
1-yl-n-butylamine and R.sub.16 is isobutyl; (jjj) R.sub.9 is
piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
1-yl-2-methoxyethyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is piperonyl; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is methylbenzyl and R.sub.16 is
methylbenzyl; (kkk) R.sub.9 is methylbenzyl; R.sub.10 is
methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is piperonyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is piperonyl and R.sub.16 is
1-yl-n-butylamine; (lll) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is
methylbenzyl; R.sub.15 is 1-yl-2-methoxyethyl and R.sub.16 is
piperonyl; (mmm) R.sub.9 is methylbenzyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
isobutyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is methylbenzyl
and R.sub.16 is 1-yl-2-methoxyethyl; (nnn) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isobutyl; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl
and R.sub.16 is cyclohexyl; (ooo) R.sub.9 is isobutyl; R.sub.10 is
1-yl-n-butylamine; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-n-butylamine; R.sub.15 is piperonyl and R.sub.16 is piperonyl;
(ppp) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
isobutyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is isobutyl; R.sub.15
is 1-yl-2-methoxyethyl and R.sub.16 is cyclohexyl; (qqq) R.sub.9 is
furfuryl; R.sub.10 is furfuryl; R.sub.11 is piperonyl; R.sub.12 is
cyclohexyl; R.sub.13 is piperonyl; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
cyclohexyl; (rrr) R.sub.9 is piperonyl; R.sub.10 is piperonyl;
R.sub.11 is 1-yl-2-methoxyethyl; R.sub.12 is 1-yl-2-methoxyethyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is 1-yl-n-butylamine;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(sss) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11
is isobutyl; R.sub.12 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.13 is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
isobutyl and R.sub.16 is 1-yl-n-butylamine; (ttt) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is methylbenzyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (uuu) R.sub.9 is
1-yl-n-butylamine; R.sub.10 is 1-yl-2-methoxyethyl; R.sub.11 is
1-yl-n-butylamine; R.sub.12 is isobutyl; R.sub.13 is cyclohexyl;
R.sub.14 is 1-yl-n-butylamine; R.sub.15 is 1-yl-n-butylamine and
R.sub.16 is piperonyl; (vvv) R.sub.9 is cyclohexyl; R.sub.10 is
methylbenzyl; R.sub.11 is cyclohexyl; R.sub.12 is piperonyl;
R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is 1-yl-n-butylamine
and R.sub.16 is 1-yl-2-methoxyethyl; (www) R.sub.9 is
1-yl-2-methoxyethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-2-methoxyethyl; R.sub.13 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.14 is
1-yl-2-methoxyethyl; R.sub.15 is isobutyl and R.sub.16 is
cyclohexyl; (xxx) R.sub.9 is 1-yl-2-methoxyethyl; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is piperonyl; R.sub.14 is isobutyl;
R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-n-butylamine; (yyy) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
methylbenzyl; R.sub.11 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.12 is methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14
is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is cyclohexyl;
(zzz) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-methoxyethyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is 1-yl-2-methoxyethyl; R.sub.15 is
1-yl-n-butylamine and R.sub.16 is methylbenzyl; (aaaa) R.sub.9 is
cyclohexyl; R.sub.10 is cyclohexyl; R.sub.11 is methylbenzyl;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (bbbb) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
furfuryl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is furfuryl; R.sub.14
is cyclohexyl; R.sub.15 is methylbenzyl and R.sub.16 is cyclohexyl;
(cccc) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is methylbenzyl;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is
1-yl-2-methoxyethyl; R.sub.14 is methylbenzyl; R.sub.15 is
1-yl-2-methoxyethyl and R.sub.16 is isobutyl; (dddd) R.sub.9 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is methylbenzyl; R.sub.13
is methylbenzyl; R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is methylbenzyl;
(eeee) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is 1-yl-n-butylamine;
R.sub.12 is 1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14
is 1-yl-n-butylamine; R.sub.15 is methylbenzyl and R.sub.16 is
1-yl-n-butylamine; (ffff) R.sub.9 is 1-yl-n-butylamine; R.sub.10 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.11 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
cyclohexyl; R.sub.15 is 1-yl-2-(4(benzenesulfonamide)ethyl and
R.sub.16 is methylbenzyl; (gggg) R.sub.9 is 1-yl-n-butylamine;
R.sub.10 is 1-yl-n-butylamine; R.sub.11 is piperonyl; R.sub.12 is
1-yl-2-(4(benzenesulfonamide)ethyl; R.sub.13 is 1-yl-n-butylamine;
R.sub.14 is methylbenzyl; R.sub.15 is methylbenzyl and R.sub.16 is
cyclohexyl; (hhhh) R.sub.9 is 1-yl-2-(4(benzenesulfonamide)ethyl;
R.sub.10 is methylbenzyl; R.sub.11 is methylbenzyl; R.sub.12 is
1-yl-n-butylamine; R.sub.13 is methylbenzyl; R.sub.14 is piperonyl;
R.sub.15 is 1-yl-n-butylamine and R.sub.16 is 1-yl-2-methoxyethyl;
(iiii) R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine;
R.sub.11 is 1-yl-n-butylamine; R.sub.12 is methylamine; R.sub.13 is
piperonyl; R.sub.14 is 1-yl-n-butylamine; R.sub.15 is piperonyl and
R.sub.16 is 1-yl-2-methoxyethyl; (jjjj) R.sub.9 is cyclohexyl;
R.sub.10 is cyclohexyl; R.sub.11 is furfuryl; R.sub.12 is
1-yl-2-methoxyethyl; R.sub.13 is isobutyl; R.sub.14 is cyclohexyl;
R.sub.15 is methylbenzyl and R.sub.16 is methylbenzyl; (kkkk)
R.sub.9 is piperonyl; R.sub.10 is 1-yl-n-butylamine; R.sub.11 is
isobutyl; R.sub.12 is isobutyl; R.sub.13 is 1-yl-2-methoxyethyl;
R.sub.14 is cyclohexyl; R.sub.15 is
1-yl-2-(4(benzenesulfonamide)ethyl and R.sub.16 is
1-yl-2-(4(benzenesulfonamide)ethyl; (llll) R.sub.9 is cyclohexyl;
R.sub.10 is cyclohexyl; R.sub.11 is 1-yl-n-butylamine; R.sub.12 is
methylbenzyl; R.sub.13 is 1-yl-n-butylamine; R.sub.14 is
methylbenzyl; R.sub.15 is cyclohexyl and R.sub.16 is piperonyl; and
pharmaceutically acceptable salts thereof.
67. The method of claim 59, wherein said bead based library
comprises a ligand binding moiety cross-linked to beads.
68. The method of claim 52, wherein the step of determining
comprises detecting the binding molecules fluorescently.
69. The method of claim 52, wherein said library comprises
fluorescently labeled ligands.
70. The method of claim 52, wherein said biological sample is
obtained at early stage of said disease.
71. The method of claim 52, wherein said biological sample is
obtained at late stage of said disease.
72. The method claim 52, wherein the method further comprises
identifying a marker associated with a stage of said disease.
73. The method of claim 52, wherein said disease is a cancer, an
autoimmune disease, an inflammatory disease, an infectious disease,
a neurodegenerative disease, or a cardiovascular disease.
74. The method of claim 73, wherein the cancer disease is breast
cancer, lung cancer, prostate cancer, cervical cancer, head and
neck cancer, testicular cancer, ovarian cancer, skin cancer, brain
cancer, pancreatic cancer, liver cancer, stomach cancer, colon
cancer, rectal cancer, esophageal cancer, lymphoma, or
leukemia.
75. The method of claim 73, wherein the autoimmune disease is
lupus, myestenia gravis, multiple sclerosis, narcolepsy, rheumatoid
arthritis, nephritis, Chagas disease, scleroderma, or Sjogren's
disease.
76. The method of claim 73, wherein the infectious disease is a
result of infection with viruses, bacteria or fungi.
77. The method of claim 73, wherein the neurodegenerative disease
is Alzheimer's disease, dementia, or Creutzfeld-Jacob disease.
78. The method of claim 52, wherein said response is a side effect
of said drug, an adverse reaction to said drug, a resistance to
said drug, or a therapeutic dosage efficacy of said drug.
79. The method claim 52, wherein the method further comprises
identifying a marker associated with a population or subpopulation
of patients.
80. The method of claim 52, wherein at least one ligand has an
affinity to an autoantibody to antigen that is associated with a
stage of said disease in a patient population or subpopulation.
81. The method of claim 52, wherein said drug is at least one of
the drugs listed in Table 1.
82. The method of claim 52, wherein the differences in binding
differentiates between a subject who is and is not responsive to a
treatment or therapy by said drug.
83. The method of claim 52, wherein said screening comprises an
initial screening against a first ligand library and a subsequent
screening against a second ligand library.
84. The method of claim 83, wherein said first ligand library is a
random ligand library and said second ligand library is a random or
a non-random ligand library.
85. The method of claim 83, wherein said second ligand library
comprises a plurality of ligands identified in said initial
screening or preselected based on known reactivity to said ligand
binding markers.
86. The method of claim 83, wherein said first ligand library
comprises a first set of ligands and said second ligand library
comprises a second set of ligands.
87. The method of claim 86, wherein said first set of ligands are
different from said second set of ligands.
88. The method of claim 86, wherein one or more of said first set
of ligands are same as one or more of said second set of
ligands.
89. The method of claim 83, wherein said initial screening is
performed against a first sample collected prior to administering
said drug to said subject, and said subsequent screening is
performed against a second sample collected after administering
said drug to said subject.
90. The method of claim 83, said first ligand library is screened
to identify one or more biomarkers associated with a first response
and said second ligand library is screened to identify one or more
biomarkers associated with a second response.
91. The method of claim 83, said first ligand library is screened
to identify one or more biomarkers associated with a response and
said second ligand library is screened to validate the association
of said one or more biomarkers with said response.
92. The method of claim 83, said first ligand library is screened
to identify one or more biomarkers associated with said disease and
said second ligand library is screened to identify one or more
biomarkers associated with a stage of said disease.
93. The method of claim 83, said first ligand library is screened
to identify one or more biomarkers associated with said disease and
said second ligand library is screened to identify one or more
biomarkers associated with said drug induced response.
94. The method of claim 83, said first ligand library is screened
to identify one or more biomarkers associated with said disease
and/or said drug induced response and said second ligand library is
screened to validate the association of said one or more biomarkers
with said disease and/or said drug induced response.
95. The method of claim 52, wherein said biological sample is
tissue, cell, urine, serum, whole blood, cerebrospinal fluid,
sputum, saliva, or semen.
96. The method of claim 52, wherein said subject is a human or
animal subject.
97. A marker associated with a response to a drug for treating a
disease, wherein said marker is identified by the method of claim
52.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Applications 61/467,256, 61/491,717, and 61/583,881 filed Mar. 24,
2011, May 31, 2011, and Jan. 6, 2012, respectively, all of which
are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to diagnostic and treatment methods
using a ligand library and active compounds derived therefrom.
Specifically, the invention relates to using a ligand library to
find ligands that are used to determine disease biomarker profiles
and to diagnose or detect a drug induced response, including drug
adverse reaction, side effects, drug resistance, and therapeutic
efficacy. The invention further relates to identifying biomarkers
associated with a drug induced response and providing a
personalized medical treatment.
BACKGROUND OF THE INVENTION
[0003] Small-molecule microarrays are becoming increasingly
important tools in combinatorial chemistry. The arrays are
generally produced by first synthesizing a combinatorial library on
a suitable bead resin, separating the beads into the wells of
microtiter plates, and then releasing the compounds from the beads.
The resulting solutions then are spotted robotically onto a
chemically modified glass slide such that the library-derived
molecule is attached covalently to the surface. Alternatively,
methods exist for the synthesis of certain classes of compounds in
situ on the array surface. By far the most common application of
small-molecule microarrays has been as a versatile platform for
library screening, usually with the goal of identifying
small-molecule ligands for a given protein of interest.
[0004] U.S. patent publication 2007/0003954 discloses protein and
antibody profiling using small molecule microarrays. The
application discloses ligands, which bind to ligand binding
moieties wherein the ligands are arranged in arrays of synthetic
molecules, which are used to screen for biomarkers and molecular
fingerprints. The specific arrays described therein include, for
example, a peptoid microarray having 7680 different compounds bound
to the array. In that disclosure, bead based libraries were
utilized as the initial means to make peptoids which were then
transferred to microarrays with addressable locations on the
microarray to screen biological fluids. The screening results in a
unique pattern or molecular fingerprint on the array for
any-particular protein in a complex biological mixture. U.S. patent
application 2010/0303805, hereby incorporated by reference,
discloses certain peptoids and diagnostic arrays useful in
screening biological fluids for biomarkers associated with central
nervous system disorders. The specific monomers disclosed therein
utilized to form the arrays therein may also be utilized in the new
screening methodology of the present invention provided the
libraries are enlarged to a much greater number of beads/peptoids
or beads/ligands, e.g., between greater than I00K to 150 MM.
[0005] The inventors of the instant application have found that
significantly larger bead based libraries (relative to microarray
based screens for antibody biomarkers or bead based screens for
cells) can, under the right conditions, be used to directly screen
complex biological samples to find drug response associated
biomarkers as well as a significantly larger pool of ligands which
bind to such ligand-binding moieties. This significantly larger
pool includes a significantly improved number of high affinity
ligands that serve as diagnostic tools as well as potential
therapeutics.
SUMMARY OF THE INVENTION
[0006] In one embodiment, the invention provides a ligand library.
In an exemplary embodiment, the invention provides a large bead
based random ligand library including a random peptoid ligand
library.
[0007] In another embodiment, the invention provides a method for
diagnosing a drug induced response in a subject, the method
comprising: obtaining a biological sample from said subject;
screening a ligand library against said sample; identifying binding
characteristics of one or more markers in said sample with one or
more ligands in the library; and determining whether said one or
more markers are associated with said drug induced response,
thereby diagnosing said drug induced response in said subject. In
one exemplary embodiment, said drug induced response is an adverse
reaction. In another exemplary embodiment, said drug induced
response is a side effect. In another exemplary embodiment, said
drug induced response is a resistance to said drug. In another
exemplary embodiment, said drug induced response is its therapeutic
efficacy including dosage efficacy.
[0008] In another embodiment, the invention provides a method for
treating a disease in a subject, the method comprising: obtaining a
biological sample from said subject; screening a ligand library
against said sample; identifying binding characteristics of one or
more markers in said sample with one or more ligands in the
library; determining whether said one or more markers are
associated with a response to a drug for treating said disease;
administering said drug to said subject, based on the determination
of association between said one or more markers to said response,
thereby treating said disease in said subject.
[0009] In another embodiment, the invention provides a method for
detecting a risk of adverse reaction to a drug in a subject, the
method comprising: obtaining a biological sample from said subject;
screening a ligand library against said sample; identifying binding
characteristics of one or more markers in said sample with one or
more ligands in the library; and determining whether said one or
more markers are associated with said risk, thereby detecting said
risk of adverse reaction to said drug in said subject.
[0010] In another embodiment, the invention provides a method for
profiling one or more subjects for a response to a drug to treat a
disease, the method comprising: obtaining a biological sample from
said subject; screening a ligand library against said sample;
identifying binding characteristics of one or more markers in said
sample with one or more ligands in the library; and determining
whether said one or more markers are associated with said response
for said drug, thereby profiling said one or more subjects for said
response to said drug to treat said disease.
[0011] In another embodiment, the invention provides a method for
identifying a marker associated with a drug induced response for
treating a disease, the method comprising: obtaining a biological
sample from a subject; screening a ligand library against said
biological sample; determining the binding characteristics of a
marker in said sample to a ligand in the library; and determining
whether said marker is associated with said drug induced response
for treating said disease, thereby identifying said marker
associated with said drug induced response for treating said
disease.
[0012] In another embodiment, the invention provides a method for
identifying a marker associated with a response to a drug for
treating a disease, the method comprising: obtaining a first set of
biological samples from one or more subjects exhibiting said
response to said drug for treating said disease; obtaining a second
set of biological samples from one or more subjects not exhibiting
said response to said drug for treating said disease; screening a
random ligand library against said first and second biological
samples; determining the differences in binding of one or more
markers to one or more ligands in the library between said first
and second biological samples; and identifying a marker associated
with said response to said drug for treating said disease. In an
exemplary embodiment, the marker is an autoantibody capable of
binding to a peptoid ligand.
[0013] Other features and advantages of the present invention will
become apparent from the following detailed description examples
and figures. It should be understood, however, that the detailed
description and the specific examples while indicating preferred
embodiments of the invention are given by way of illustration only,
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows a basic chemical schematic of the preparation
of a library of Tentagel beads (KN1B) used to screen Alzheimer's
serum samples. FIG. 1A shows starting from a polystyrene bead
having an amino group as the reactant (a PEG or equivalent or
alternative linker may be formed between the bead and the terminal
amino group). FIG. 1B shows the starting amino acid on the bead as
methionine and which is then reacted to form, the compound shown in
B. FIG. 1C shows the submonomers (monomeric amines and haloacetic
acids) utilized to form the oligomeric library of compounds.
[0015] FIG. 2 shows a basic chemical schematic of the preparation
of a library of Tentagel beads (JC3B) also used to screen
Alzheimer's serum samples. FIG. 1A shows starting from a
polystyrene bead having an amino group as the reactant (a PEG or
equivalent or alternative linker may be formed between the bead and
the terminal amino group). FIG. 1B shows the starting amino acid on
the bead as methionine and which is then reacted to form the
compound shown in B, FIG. 1C shows the submonomers (monomeric
amines and haloacetic acids) utilized to form the oligomeric
library of compounds. JC3B was also used to screen pancreatic
cancer serum.
[0016] FIG. 3 shows a basic chemical schematic of the preparation
of a library of Tentagel beads (JC4B) used to screen Alzheimer's
serum samples. FIG. 1A shows starting from a polystyrene bead
having an amino group as the reactant (a PEG or equivalent or
alternative linker may be formed between the bead and the terminal
amino group). FIG. 1B shows the starting amino acid on the bead as
methonine and which is then reacted to form the compound shown in
B, FIG. 1C shows the submonomers (monomenc amines and haloacetic
acids) utilized to form the oligomeric library of compounds.
[0017] FIG. 4 shows a basic chemical schematic of the preparation
of a library of Tentagel beads (JC5B) used to screen Alzheimer's
serum samples. FIG. 1A shows starting from a polystyrene bead
having an amino group as the reactant (a PEG or equivalent or
alternative linker may be formed between die bead and the terminal
amino group). FIG. 1B shows the starting amino acid on the bead as
methionine and which is then reacted to form the compound shown in
B. FIG. 1C shows the submonomers (monomeric amines and haloacetic
acids) utilized to form the oligomeric library of compounds. JC5B
monomers included Isobutylamine, 2-Methoxyethylamine,
Diaminobutane, Furfurylamine, Cyclohexylamine, R-Methylbenzylamine,
Piperonylamine and 4-(Aminoethyl)Benzenesulfonamide.
[0018] FIG. 5 shows a basic chemical schematic of the preparation
of a library of Tentagel beads (JC7B) used to screen serum samples.
FIG. 3A shows starting from a polystyrene bead having an amino
group as the reactant (a PEG or equivalent or alternative linker
may be formed between the bead and the terminal amino group). FIG.
1B shows the starting amino acid on the bead as methionine and
which is then reacted to form the compound shown in B. FIG. 1C
shows the submonomers (monomeric amines and haloacetic acids)
utilized to form the oligomeric library of compounds.
[0019] FIG. 6 shows a schematic of the process of the invention to
screen a complex biological sample using head based libraries of
peptoid ligands.
[0020] FIG. 7 shows normal control (NC) Dynabead hits after QDot
addition in a peptoid library (JOB) prepared to screen against an
Alzheimer's normal control serum sample and Alzheimer's diseased
serum sample. The hits were picked out and the remaining ligand
bound beads were used in the disease based screen.
[0021] FIG. 8 shows the Tentagel bead screening of diseased serum
from Alzheimer's patient blood samples after the NC hits were
removed. The hits are shown in red, which is the Qdot secondary
antibody bound to the disease associated biomarker (antibody) in
the serum which is bound to a peptoid linked through a PEG linker
to the bead.
[0022] FIG. 9 shows a reproducibility test which uses a normal
control sample (NC 030093) after SDS wash and QDOT addition. The
arrow shows which NC peptoid hits were picked to sequence.
[0023] FIG. 10 shows a reproducibility test which uses a normal
control sample (NC 050047) after SDS wash and QDOT addition.
[0024] FIG. 11 shows a reproducibility test which uses a diseased
sample after SDS wash and QDOT addition.
[0025] FIG. 12 shows the peptoid sequences of the putative hits
selected from the Alzheimer's screen from the JC3B library. The
C-terminus is on the right side of the sheet and the N-terminus is
on the left side.
[0026] FIG. 13 shows the chemical structures of the preferred high
affinity hits from the Alzheimer's screen from the JC3B library, in
this example, the structures shown have a cysteine residue and were
resynthesized after determining the structure of the initial hit in
the preliminary screen. The JC3B library contained an analogous
peptoid but which had a methionine residue on the C-terminus and
not a cysteine residue.
[0027] FIG. 14 shows a competition experiment between a high
affinity ligand (ADTGI) in solution versus ADTG-1-ADTG-42 on a
microarray support. The competition experiment shows that ADTGI in
solution bonded to the same antibody that would have bound to
peptoids ADTG-1, ADTG14, ADTG24, ADTG25, ADTG3I, ADTG35 and ADTG40
on the microarray. Similar experiments were conducted on each of
the peptoids to find four sets of peptoids, which bound to four
distinct Alzheimer's autoantibodies.
[0028] FIG. 15 shows the four groups of distinct peptoids, which
bind to different autoantibodies in the Alzheimer's screen. Each
group on the figure has the higher affinity binder at the top.
[0029] FIG. 16A shows AD test data (blinded) for a pool of patients
using Plaag1 (JC3B-1) peptoid and FIG. 16B shows test data
(blinded) for the same pool of AD patients using Plaag2 (JC3B-21).
Each peptoid is presented on a microarray.
[0030] FIG. 17A shows AD test data (blinded) for a pool of patients
using Plaag3 (JC3B-7) peptoid and FIG. 17B shows test data
(blinded) for the same pool of AD patients using Plaag4 (3C3B-5).
Each peptoid is presented on a microarray.
[0031] FIG. 18A shows AD test data (blinded) for a pool of patients
using Plaag5 (JC3B-R8) peptoid and FIG. 18B shows test data
(blinded) for the same pool of AD patients using Plaag6 (JC3B-R
12). Each peptoid is presented on a microarray.
[0032] FIG. 19A shows microarray data for ADP2 in the same pool of
patients for the tests conducted using Plaag1-6. FIG. 19B shows
comparative data using Plaag4 with the same set of patients. The
data shows a clear correlation between the results achieved with a
previously identified ADP2 and the newly identified Plaag4 in the
same patient pool.
[0033] FIG. 20A shows microarray data for ADP3 in the same pool of
patients for the tests conducted using Plaag1-6, FIG. 20B shows
comparative data using Plaag2 with the same set of patients. The
data shows a clear correlation between the results achieved with a
previously identified ADP3 and the newly identified Plaag2 in the
same patient pool.
[0034] FIG. 21 shows a validation of Plaag5 (putative hit 5 or
JC3B-R8) on TentaGel beads in a comparison of diseased AD serum
versus healthy control (pooled) at 40 ug/mL.
[0035] FIG. 22A shows the peptoid hits in the pancreatic cancer
screen using QDot 655 and using the JC5B library. FIGS. 22B and C
show confirmation of hits using QDot 655 (arrows point to
hits).
[0036] FIG. 23 shows pancreatic peptoid hit validation and compares
disease serum addition and detection with QDot 655 versus normal
serum addition.
[0037] FIG. 24 shows hit validation by mixing AD markers and PC
markers. The data shows that, the PC marker was detected while
there was no detectable antibody on the AD peptoid bead in the
pancreatic cancer serum (Serum 1).
[0038] FIG. 25 shows the pancreatic cancer screen hit sequences
from the JC3B library.
[0039] FIG. 26 shows the pancreatic cancer screen hit sequences
from the JC5B library.
[0040] FIGS. 27 A, B and C show the results of an SLE (Lupus)
screen. A is normal control and B and C are SLE serum from two
different groups 1 and 2. The arrows point to the hits.
[0041] FIG. 28 shows the SLE hits from the KN1B library. The
C-terminus is on the right side of the sheet.
[0042] FIG. 29 shows a hit validation for peptoid KN1B-20. Group 1
is pooled diseased serum at a concentration of about 0.374 mg/mL
(left picture) (the hits are shown with a red tinge on the bead).
Non-diseased pooled serum (center picture) is provided at a
concentration of about 0.378 mg/mL and the far right picture shows
a no serum control.
[0043] FIG. 30 shows the binding/detection of one of the SLE
(lupus) peptoids to ELISA plates using two different binding
methods at different concentrations of peptoid using a fluorescein
tag.
[0044] FIG. 31 shows a competition assay between plate bound
KN1B-20-biotin-fluorescein versus free KN1B-20-biotin in solution
at various concentrations. Signal dampening occurs as the
concentration of free KN1B-20-biotin increases from equimolar
concentrations of bound versus free.
[0045] FIG. 32 shows an ELISA plate having peptoid at various
concentrations and clearly shows a difference between diseased
serum (AD) (P column 1) and normal control serum (column 3) [1:200
doubling each well to 1:400, 1:800, I; 1,600, 1:3, 200, 1:6,400,
1:12,800]. The arrow points to the 1:800 dilution in 1XTBST buffer.
The peptoid concentration in the wells is 10 mM. FIG. 32 also shows
validation of the TentaGel bead platform to distinguish between
diseased and control sera.
[0046] FIG. 33 shows an ELISA plate with 10 mM ADP3 and at various
dilutions of AD sera versus control sera. The arrow points to the
1:800 dilution.
[0047] FIG. 34 shows an ELISA plate with 10 mM SLE-KNJ B-20 and at
various dilutions of AD sera versus control sera. The arrow points
to the 1:800 dilution.
[0048] FIG. 35 shows an AD serum ELISA graph using 10 mM ADP3
prepared in binding buffer at various serum dilutions. Separation
between normal and diseased serum occurred, over the dilution range
of 1:200 through approximately 1:10,000. The starting dilutions
were 0.1:200 (Group 1 AD serum 0.394 mg/mL and non-diseased serum
at 0.386 mg/mL).
[0049] FIG. 36 shows an SLE serum ELISA graph using 10 mM KN1B-20
prepared in binding buffer at various serum dilutions. Separation
between normal and diseased serum occurred over the dilution range
of 1:200 through approximately 1:10,000. The starting dilutions
were 1:200 (Group 1 SLE serum 0.375 mg/mL and non-diseased serum at
0.396 mg/mL).
[0050] FIG. 37 shows an SLE serum ELISA graph using 1.0 mM KN1B-20
prepared in DMSO at various serum dilutions. Separation between
normal and diseased serum occurred over the dilution range of 1:200
through approximately 1:10,000. The starting dilutions were 1:200
(Group 1 SLE serum 0.367 mg/mL and non-diseased serum at 0.322
mg/mL).
[0051] FIG. 38 shows a FAGS platform for Tentagel beads hits
validation.
[0052] FIG. 39 shows the degree of separation between beads having
an acetyl group and beads having a 2,5-dintrophenyl group (DNP) at
various concentrations of sera (100 ug/mL to 1,000 ug/mL) and in
response to treatment with an anti-DNP labeled secondary antibody.
The Mean fluorescence intensity (MFI) separation was greatest at
the higher dilution of 1,000 ug/mL sera.
[0053] FIG. 40 shows that there is a direct competition between
free ethanolamine-DNP and the binding of DNP (on a plate) to
anti-DNP antibody at 1,000 ug/mL sera concentration.
[0054] FIG. 41 shows ADP3 bound anti-antibody from pooled normal
control sera and pooled AD sera. The data shows good separation at
sera concentration ranges of 20 and 140 ug/mL using two different
secondary antibodies (goat anti-human Dylight 649 and goat
anti-human Alexa 647).
[0055] FIG. 42 shows ADP3 bound auto-antibody from normal control
and AD sera after background subtraction at various sera
concentration ranges. There is a significant degree of separation
at most sera concentration ranges from less than 20 ug/mL to 1.20
ug/mL or greater.
[0056] FIGS. 43 and 44 show the structures of the SLE (lupus)
resynthesized peptoid ligand hits.
[0057] FIG. 45 shows the preparation of ADP3 on 10 um Tentagel
beads and the subsequent cleavage using CNBr along with a mass
specttoraetry reading of the lactone shown.
[0058] FIG. 46 shows ADP3 bound autoantibody from normal control
and Alzheimer's disease sera at different concentrations. The beads
were preblocked for 3 hours with 1X TBST and then detected using
Goat anti-human Alexa 647 secondary antibody.
[0059] FIG. 47 shows the ADP3 bound autoantibody from normal
control and Alzheimer's disease sera at different sera
concentrations and also shows DNP values.
[0060] FIGS. 48 and 49 show ADP3 bound autoantibody from normal
controls versus Alzheimer's disease sera using pre-blocking
conditions such as E. coli lysate and lysine.
[0061] FIG. 50 shows a simple schematic of the preparation of and
distinction between peptoids that are used in microarrays versus
those peptoids that are placed on ELISA plates. Schematic for how
peptoid microarrays are made: individual beads are segregated into
the wells of microtiter plates and the peptoids are cleaved from
the beads to make a concentrated stock solution. Note that each
well will now contain a single kind of peptoid. Several thousand
peptoids are then spotted onto chemically-modified glass microscope
slides in such a way that they bind covalently to the surface.
Several thousand slides can be produced highly reproducibly from a
single synthetic library. The ELISA production is similar except
that there is no PEG chain on the surface but the density of
peptoids on the ELISA plate may be different than it is on the
microarrays.
[0062] FIG. 51 shows ELISA experiments with a clear distinction
between normal control and diseased serum at a serum dilution of
1:800 using horseradish peroxidase linked to a secondary antibody
that detects the disease associated antibody-peptoid complex. The
colorless substrate is added and changes color (blue) upon reaction
with the bound HRP enzyme.
[0063] FIG. 52 shows titration data that compares various AD
peptoids in an ELISA test at various serum dilutions of diseased
serum (A) versus normal serum (B). There is no intensity of the
signals in the normal serum but clear distinction and intensity of
all of the AD peptoids as the concentration increases from 1:12,800
to 1:200.
[0064] FIG. 53 provides a diagram that validates the correlation
between the clinical diagnosis of the unblinded sample set of AD
patients at various stages of Alzheimer's disease (or not) versus
the data obtained from the same patient serum samples (blinded) and
which were screened against ADP3 peptoid to detect disease
associated antibodies. The results shown are from a blinded study
of plasma samples from Mayo Clinic Jacksonville. UND=Undecided. The
plot was derived from taking a single serum concentration (1:800)
dilution. A reading of >1. was considered positive, a reading
between 1 and 0.7 was considered undecided and a reading below 0.7
was considered negative.
[0065] FIG. 54 provides a diagram that validates the correlation
between the clinical diagnosis of the unblinded sample set of AD
patients at various stages of Alzheimer's disease (or not) versus
the data obtained from the same patient serum samples (blinded) and
which were screened against the various AD peptoids (plot is
average value of results of 9 peptoids) of the invention to detect
disease associated antibodies. The results shown are from a blinded
study of plasma samples from Mayo Clinic Jacksonville.
UND=Undecided. The plot was derived from taking a single serum
concentration (1:800) dilution, A reading of >3 was considered
positive, a reading between 1 and 0.7 was considered undecided and
a reading below 0.7 was considered negative.
[0066] FIG. 55 provides a diagram that validates the correlation
between the clinical diagnosis of the unblinded sample set of AD
patients at various stages of Alzheimer's disease (or not) versus
the data obtained from the same patient serum samples (blinded) and
which were screened against the various AD peptoids of the
invention to detect disease associated antibodies. The results
shown are from a blinded study of plasma samples from Mayo Clinic
Jacksonville. UND=Undecided. The plot was derived from taking a
single serum concentration (1:800) dilution. A reading of >1 was
considered positive, a reading between 1 and 0.7 was considered
undecided and a reading below 0.7 was considered negative. The data
also shows performance on other dementias where MC1/depression
samples are labeled and Lewis Body Dementia samples are marked as
well. The data shows that at least three MCI patients have serum
samples with detectable amounts above 1 of the antibodies captured
by the AD selective peptoids of the invention.
[0067] FIGS. 56A-D provide data on that subset of samples from
patients that have disagreements between the Opko Health peptoid
diagnostic assay using multiple AD peptoids versus the clinical
diagnosis after this information was provided when unblinded. FIG.
56A shows the data for peptoids ADP3 and others as shown for a
patient that was diseased clinically but for which the Opko peptoid
Plaag4 was below 1.0 (UND at a single point; Titration AD
positive). All other Opko peptoids were positive for AD (i.e.,
above 1.0). FIG. 56B shows that all Opko peptoids were positive for
disease associated antibodies in a patient that was currently
diagnosed as normal (non-demented) suggesting pre-AD. FIG. 56C
shows that none of the Opko AD peptoids showed an intensity above 1
at any dilution point in a patient that was clinically diagnosed
with AD suggesting that this patient had some other form of
dementia. FIG. 56D shows that in a clinically positive AD patient,
multiple Opko AD peptoids were not positive for disease associated
antibodies but two peptoids (Plaag6 and Plaag4) were positive, thus
UND at a single point and UND even after titration.
[0068] FIG. 57 shows the cluster diagram generated from previous AD
samples using a microarray spotted with ADP3. There is a clear
correlation between diseased versus control in the microarray data
and data generated using the ELISA platform. FIG. 57 also shows
that the ADP3 peptoid is selected for disease associated antibodies
associated with Alzheimer's disease and not Parkinsons or Lupus
(SLE).
[0069] FIG. 58 provides a summary of ELISA analysis using a total
of 106 serum samples tested.
[0070] FIG. 59 provides the chemical structures of Plaag7-9.
DETAILED DESCRIPTION OF THE INVENTION
[0071] In the following detailed description, numerous specific
details are set forth in order to provide a thorough understanding
of the invention. However, it will be understood by those skilled
in the art that the present invention may be practiced without
these specific details. In other instances, well-known methods,
procedures, and components have not been described in detail so as
not to obscure the invention.
[0072] The invention encompasses a ligand library and uses thereof.
Specifically, the invention encompasses screening of molecules,
pharmacoproteomics, diagnosis, treatment, and other uses of a
random library.
[0073] In one embodiment, the invention provides a ligand library
for a personalized medicine. The terms "personalized medicine," as
used herein, may refer to the use of a test (or diagnostic) to
target a drug (or therapy) at patients that are most likely to
benefit therefrom, or to identify patients who may be at risk of
harm from said therapy. Before a drug or diagnostic product can be
marketed in the United States and most other countries, it is
subjected to strict regulatory review of its safety and efficacy.
In the case of a diagnostic for personalized medicine this will
likely require the testing of tissue or bodily fluids from patients
that received the drug to ascertain whether there is a link between
their response to therapy and the presence of a particular marker.
Accordingly, in one embodiment, provided herein is a method for
diagnosing a response for a drug to treat a disease in a subject,
the method comprising: obtaining a biological sample from said
subject; screening a ligand library against said sample;
identifying binding characteristics of one or more markers in said
sample with one or more ligands in the library; and determining
whether said one or more markers are associated with said response
for said drug, thereby diagnosing said response for said drug to
treat said disease in said subject. Examples of a response include,
but are not limited to, an adverse reaction, a side effect, a drug
resistance, and a therapeutic efficacy including dosage
efficacy.
[0074] Adverse drug reactions are a principal cause of the low
success rate of drug development programs (less than one in four
compounds that enters human clinical testing is ultimately approved
for use by the U.S. Food and Drug Administration (FDA)).
Drug-induced disease or toxicity presents a unique series of
challenges to drug developers, as these reactions are often not
predictable from preclinical studies and may not be detected in
early clinical trials involving small numbers of subjects. When
such effects are detected in later stages of clinical development
they often result in termination of a drug development program.
When a drug is approved despite some toxicity, its clinical use is
frequently severely constrained by the possible occurrence of
adverse reactions in even a small group of patients. The likelihood
of such a compound becoming first line therapy is small (unless
there are no competing products). Clinical trials that use this
invention may allow for improved predictions of possible toxic
reactions in studies involving a small number of subjects. The
methods of this invention offer a quickly derived prediction of
likely future toxic effects of an intervention. Accordingly, in
another embodiment, provided herein is a method for detecting a
risk of adverse reaction to a drug in a subject, the method
comprising: obtaining a biological sample from said subject;
screening a ligand library against said subject; identifying
binding characteristics of one or more markers in said sample with
one or more ligands in the library; and determining whether said
one or more markers are associated with said risk, thereby
detecting said risk of adverse reaction to said drug in said
subject.
[0075] The present invention is based, in part, on the surprising
discovery that a combination of peptoid binding biomarkers can be
used to individualize therapy in patients. Given the high
inter-patient variability in response to a drug, the assay methods
of the present invention are particularly advantageous because they
utilize a combinatorial strategy that takes into account
differences in binding characteristics of multiple molecular
determinants (e.g., peptoid binding biomarkers) to determine
whether a disease in a patient has a high likelihood of responding
to treatment with a specific drug or combination of drugs. If the
patient is classified as a responder, a dosing regimen tailored to
that patient can then be created to achieve therapeutic efficacy
without inducing toxic side-effects. Consequently, patients
classified as responders can receive the full benefits of drug
induced therapy without experiencing the side-effects associated
with such therapy. Similarly, patients already undergoing treatment
with a drug can experience a reduction in toxic side-effects
without compromising therapeutic efficacy by adjusting the
subsequent dose of the drug. Likewise, patients already undergoing
treatment with a drug can be monitored to assess whether resistance
to the drug has developed and an alternative therapy should be
administered.
[0076] As a result, the methods of the present invention enable
treating a disease in a subject, the method comprising: obtaining a
biological sample from said subject; screening a ligand library
against said subject; identifying binding characteristics of one or
more markers in said sample with one or more ligands in the
library; determining whether said one or more markers are
associated with a response to a drug for treating said disease;
administering said drug to said subject, based on the determination
of association between said one or more markers to said response,
thereby treating said disease in said subject. In another
embodiment, the invention provides monitoring a treatment by a drug
in a subject, the method comprising: obtaining a biological sample
from said subject; screening a ligand library against said subject;
identifying binding characteristics of one or more markers in said
sample with one or more ligands in the library; determining whether
said one or more markers are associated with a response to a drug
for treating said disease; administering said drug to said subject,
based on the determination of association between said one or more
markers to said response, thereby monitoring said treatment by said
drug in said subject.
[0077] In another embodiment, the invention provides a ligand
library for pharmacoproteomics and identifying a marker associated
with a response to drug. Accordingly, in one embodiment, provided
herein is a method for identifying a marker associated with a drug
induced response for treating a disease, the method comprising:
obtaining a biological sample from a subject; screening a ligand
library against said biological sample; determining the binding
characteristics of a marker in said sample to a ligand in the
library; and determining whether said marker is associated with
said drug induced response for treating said disease, thereby
identifying said marker associated with said drug induced response
for treating said disease. In an exemplary embodiment, the response
to drug is a patient's response to medication, its dosage, or
adverse reaction.
[0078] In one embodiment, one or more putative hits or leads of
biomarkers are identified using a ligand library of the invention.
A biological sample may be obtained from a subject or a plurality
of subjects (e.g., patient population or sub-population). In some
embodiments, a first set of biological samples may be obtained from
a plurality of subjects exhibiting a response (e.g., drug induced
response or response to a disease) and a second set of biological
samples may be obtained from a plurality of subjects not exhibiting
a response. The ligand library may be screed against the first and
second set biological samples. The differences in binding
characteristics of one or more markers to one or more ligands in
the library between the first and second set biological samples may
be determined. Based on the differences in binding characteristics,
the putative hits or leads of biomarkers can be identified. In some
embodiments, the putative hits or leads are biomarkers that
recognize antoantibodies to antigens associated with a response,
for example, a patient's response to a drug, response to a disease,
and response to a particular stage of a disease.
[0079] In one embodiment, for identifying putative hits or leads of
biomarkers, a bead-based large random peptoid ligand library having
peptoid ligands ranging from about 350K to about 250 MM may be
used. The putative hits or leads of biomarkers identified in the
initial random library screening may then be used to screen against
the samples in a subsequent screening for diagnostics or companion
diagnostics discussed herein.
[0080] In some embodiments, the putative hits or leads are
validated in a subsequent screening. In one embodiment, the same
ligand library that was used in the initial screening may be used
in the validation subsequent screening. In another embodiment, a
different ligand library (different from the one that was used in
the initial screening) may be used in the validation subsequent
screening.
[0081] In one embodiment, the putative hits or leads associated
with a first trait (e.g., response to a disease) are identified in
the initial screening and the identified putative hits or leads are
used to screen against the samples to determine their association
with a second trait (e.g., response to a drug). In one embodiment,
the putative hits or leads associated with a disease are identified
in the initial screening and the identified putative hits or leads
are used to screen against the samples to both validate and
determine an association with a specific stage of said disease.
[0082] In some embodiments, the initial screening for identifying
putative hits may be performed on a bead-based device, using a
large random ligand library, and the subsequent screening for
diagnostics or companion diagnostics may be performed using any
platform, for example, microarray, using a non-random or random
ligand library.
[0083] In one embodiment, in a subsequent screening for diagnostics
or companion diagnostics, a first screening may be performed
against a first ligand library and a subsequent screening against a
second ligand library, wherein the first ligand library comprises a
first set of ligands and the second ligand library comprises a
second set of ligands. In one example, the first ligand library is
screened to identify markers associated with a disease, and a
second ligand library is screened in a subsequent screening to
identify markers associated with a drug induced response.
[0084] In one example, the putative hits or leads are used in a
first screening in patient samples, collected prior to drug
treatment, to determine their association with a disease. After
drug treatment, the samples may be collected from patients, and in
one embodiment, the same putative hits used in the pre-treatment
group may be used to identify those patients having certain disease
stages or responsive changes to drug treatment. In another
embodiment, different putative hits may be used to monitor drug
related side effects or treatment effects that are due to drug
administration and are not necessarily correlated or related to the
pretreatment profile. In some embodiments, another random library
may be used to find additional biomarkers that might be associated
with the drug treatment.
[0085] The term "drug," as used herein, may refer to any drug,
including, but not limited to, a synthetic inorganic or organic
compound, a protein, a peptide, a polysaccharides and other sugars,
a lipid, DNA and RNA nucleic acid sequences, an antisense
oligonucleotide, an antibody; a receptor ligand, an enzyme, an
adhesion peptide, an antigen, a hormone, a growth factor, a
ribozyme, and a retroviral vector.
[0086] The invention encompasses any suitable drug known one of
skilled in the art. These drugs are listed in The Merck Index;
Physicians' Desk Reference, PDR Network, 2011 Edition edition (Dec.
1, 2010); U.S. Pat. No. 7,932,294, and U.S. Patent Publications
20060046967, 20110274695, 20110269722, 20110269709, and
20060205674, all of which are incorporated by reference herein in
their entirety.
[0087] In one embodiment, the drug is selected from one or more of
the following categories/groups: Antibiotics, Sedatives, Hypnotics,
Antidepressants, Antipsychotics, Antimanics, Analgesics,
Antipyretics, Antimigraine agents, Anticonvulsants, Drugs used in
parkinsonism and movement disorders, Drugs for dementia,
Antiemtics, drugs for Vertigo, CNS Stimulants activators,
Antiinfective eye preparations, Antiinflammatory, antiallergic
preparations, antiglucoma drugs, preparations to cure eye diseases,
aural preparations, nasal preparations, oropharyngeal preparations,
Antiarrhythemic drugs, Antihypertensives, alfa/beta-blockers,
channel blockers, ACE inhibitors, Angiotensin II receptor
antagonists, diuretics, Antianginals, nitrates, calcium channel
blockers. Drugs for cardiac failure and shock, Vasodilators,
Coagulants, Anticoagulants, Thrombolytics, antiplatelet drugs,
Respiratory stimulants, Antitissives, Expectorants, Mucolytics,
Decongestants, Antihistamine agents, antiasthmatics; Antiulcer,
Antisecretory drugs, H.sub.2 receptor antagonists, Proton Pump
Inhibitors, Prostaglandin analogues, Antacids, Antispasmodics,
drugs modifying intestinal motility, Antidiarrhoeals, antimotility
drugs, antimicrobial drugs, drugs acting on gall bladder, Urinary
antiinfectives, Diuretics, Urinary analgesics, Antispasmodics,
Antiinfective drugs acting on urethra and vagina, drugs acting on
uterus, Drugs for prostatic hypertrophy, alfa blockers,
antiandrogens, Drugs for erectile dysfunction, Spermicidals,
nonhormonal contraceptives, Emollients, keratolytics, topical
antiinfectives, topical antifungals, topical parasiticidals,
topical steroids, topical drugs for acne vulgaris, drugs for
psoriasis, pigmentation disorders, and Antiseborrhoeics, Non
Steroidal Anti Inflammatory Drugs (NSAIDs), COX-2 inhibitors,
Antiarthritic agents, Immunosuppressants, Topical analgesics,
Muscle relaxants, Neuromuscular Drugs, Penicillin antibiotics,
Cephalosporin antibiotics, Quinolone, Fluoroquinolone antibiotics,
Macrolide antibiotics, Chloramphenicol, Tetracyline antibiotics,
Sulfonamides, Antianaerobics, Metronidazole, Antitubercular drugs,
Antileprosy drugs, Antifungals, Antiprotozoals, Anthelminthics,
Antiinfestive Drugs, Antimalarials, Antivirals, Anabolics,
androgenic steroids, Corticosteroids, Oestrogens, Progestogens and
Hormonal contraceptives, Fertility Agents, Trophic hormones and
related drugs, Thyroid and antithyroid drugs, Antidiabetics and
hyperglycaemics, Vitamins, Amino acids, Anti-obesity drugs,
Hypolipidaemic drugs, fibric acid derivatives, statins, HMG CoA
reductase inhibitors, nicotinic acid group, drugs used for Gout,
drugs affecting bone metabolism, bisphosphonates, Anticancer drugs,
alkylating agents, cytotoxic antibiotics, antimetabolites,
cytarbine, Fludarbine, 5-Fluorouracil, Mercaptopurine, Thioguanine,
Vinca alkaloids, Etoposide, Taxanes, Topoisomerase 1 inhibitors,
Cytotoxic immunosuppressants, Immunostmulants, Cytoprotectives,
Amifostine, Oestrogens, Progestogens, hormon antagonists,
antineoplastic drugs, Antiallurgics, non-sedative antihistamins,
Cetirizine, Desloratadine, Terfenadine, Fexofenadine, sedative
histamines, histamine receptor blockers, Local anaesthetics,
intravenous anaesthetics, inhalation anaesthetics, and muscle
relaxants.
[0088] In another embodiment, the drug is selected from one or more
of the drugs listed in Table 1 below.
TABLE-US-00001 TABLE 1 Selected Examples of Branded Drugs in Market
Generic Name Brand Names Indications Atorvastatin Lipitor .RTM.
Cholesterol Clopidrogel Plavix .RTM. Atherosclerosis Etanercept
Enbrel .RTM. RA, JRA, Ps, PsA, AS Fluticasone Salmetrol Advair
.RTM. Asthma Infliximab Remicade .RTM. RA, UC, CD, Ps, PsA, AS
Valsartan Diovan .RTM. Hypertension Rituximab Rituxan .RTM. NHL, RA
Esomaprazole Nexium .RTM. Ulcers Bevacizumab Avastin .RTM. Colon
cancer Aripiprazole Abilify .RTM. Schizophrenia Trastuzumab
Herceptin .RTM. Breast Cancer Olanzapine Zyprexa .RTM.
Schizophrenia Quetiapine Seroquel .RTM. Schizophrenia Adalimumab
Humira .RTM. RA, Ps, JIA, PsA, AS, CD Ontelukast Singulair .RTM.
Asthma Enoxaparin Lovenox .RTM. Anticoagulant DVT Venlafaxine
Effexor .RTM. Depression Pioglitazone Actos .RTM. Diabetes
Candersartan Atacand .RTM., Hypertension Blopress .RTM.
Escitalopram Lexapro .RTM., Depression Cipralex .RTM. Glatiramer
Copaxone .RTM. Multiple Sclerosis NHL Non Hodgkin's Lymphoma, RA
Rheumatoid Arthritis, JRA Juvenile Rheumatoid Arthritis, JIA
Juvenile Idiopathic Arthritis, Ps Psoriasis, PsA Psoriatic
arthritis, CD Crohn's Disease; UC Ulcerative Colitis, AS Ankylosing
Spondylitis.
[0089] In another embodiment, the drug is selected from one or more
of the following: Abacavir, Aripiprazole, Arsenic Trioxide,
Atomoxetine, Atorvastatin, Azathioprine, Boceprevir, Brentuximab
Vedotin, Busulfan, Capecitabine, Carbamazepine, Carisoprodol,
Carvedilol, Celecoxib, Cetuximab (1), Cetuximab (2), Cevimeline,
Chlordiazepoxide and Amitriptyline, Chloroquine, Citalopram (1),
Citalopram (2), Clobazam, Clomiphene, Clomipramine, Clopidogrel,
Clozapine, Codeine, Crizotinib, Dapsone, Dasatinib, Desipramine,
Desloratadine and Pseudoephedrine, Dexlansoprazole (1),
Dexlansoprazole (2), Dextromethorphan and Quinidine, Diazepam,
Doxepin, Drospirenone and Ethinyl, Estradiol, Erlotinib,
Esomeprazole, Fluorouracil, Fluoxetine, Fluoxetine and Olanzapine,
Flurbiprofen, Fluvoxamine (1), Fluvoxamine (2), Fluvoxamine (3),
Fulvestrant, Galantamine, Gefitinib (1), Gefitinib (2),
Iloperidone, Imatinib (1), Imatinib (2), Imatinib (3), Imatinib
(4), Imipramine, Indacaterol, Irinotecan, Isosorbide and
Hydralazine, Lapatinib, Lenalidomide, Maraviroc, Mercaptopurine,
Metoprolol, Mivacurium, Modafinil (1), Modafinil (2), Nefazodone,
Nelfinavir, Nilotinib (1), Nilotinib (2), Nortriptyline,
Omeprazole, Panitumumab (1), Panitumumab (2), Pantoprazole,
Paroxetine, Peginterferon alfa-2b, Perphenazine, Phenyloin,
Pimozide, Prasugrel, Pravastatin, Propafenone, Propranolol,
Protriptyline, Quinidine, Rabeprazole, Rasburicase, Rifampin,
Isoniazid, Pyrazinamide, Risperidone, Sodium Phenylacetate and
Sodium Benzoate, Sodium Phenylbutyrate, Tamoxifen, Telaprevir,
Terbinafine, Tetrabenazine, Thioguanine, Thioridazine, Ticagrelor,
Timolol, Tiotropium, Tolterodine, Tositumomab, Tramadol and
Acetaminophen, Trastuzumab, Tretinoin, Trimipramine, Valproic Acid,
Vemurafenib, Venlafaxine, Voriconazole, Warfarin (1), and Warfarin
(2).
[0090] In another embodiment, the invention comprises a companion
diagnostic to a drug that targets a known drug target. In another
embodiment, the invention comprises a companion diagnostic to a
drug target that is undergoing a clinical trial. Examples of a drug
target that is undergoing clinical trial include, but are not
limited to, Bapineuzumab, Solanezumab, Intravenous immunoglobulin
(IVIg), Latrepirdine (Dimebon), Scyllo-inositol/ELND 005,
Methylthioninium chloride (Rember), CERE-110, PBT2,
Davenutide/AL-108, BMS-708163, PF-04494700/TTP488, Tideglusib/NP-12
(Nypta), Belimumab, Atacicept, Mapatumumab, Apomab, Dulanermin,
Odanacatib, AMG-785, DG-041, OC-000459, PLX-4032, LX-1031, and
LX-1032.
[0091] A binding profile of one or more sample components (e.g.,
biomarkers) can be used to predict, diagnose, assess, or treat, any
disease, known to one of skilled in the art. The terms "disease" or
"condition" are commonly recognized in the art and designate the
presence of signs and/or symptoms in an individual or patient that
are generally recognized as abnormal. Diseases or conditions may be
diagnosed and categorized based on pathological changes. Signs may
include any objective evidence of a disease such as changes that
are evident by physical examination of a patient or the results of
diagnostic tests. Symptoms are subjective evidence of disease or a
patient's condition, i.e. the patient's perception of an abnormal
condition that differs from normal function, sensation, or
appearance, which may include, without limitations, physical
disabilities, morbidity, pain, and other changes from the normal
condition experienced by an individual. Various diseases or
conditions include, but are not limited to; those categorized in
standard textbooks of medicine including, without limitation,
textbooks of nutrition, allopathic, homeopathic, and osteopathic
medicine. In certain aspects of this invention, the disease or
condition is selected from the group consisting of the types of
diseases listed in standard texts such as Harrison's Principles of
Internal Medicine, 14.sup.th Edition (Fauci et al, Eds., McGraw
Hill, 1997), or Robbins Pathologic Basis of Disease, 6.sup.th
Edition (Cotran et al, Ed. W B Saunders Co., 1998), or the
Diagnostic and Statistical Manual of Mental Disorders: DSM-IV,
4.sup.th Edition, (American Psychiatric Press, 1994), or other text
books, which are incorporated herein in their entirety. The
diseases are also listed in U.S. Patent Publications 2007/0003954
and 2011/0092384, which are incorporated herein in their
entirety.
[0092] Examples of a disease or condition include, but are not
limited to cancer, autoimmune disease, inflammatory disease,
infectious disease, neurodegenerative disease, cardiovascular
disease, bacterial infection, viral infection, fungus infection,
prion infection, physiologic state, contamination state, or health
in general.
[0093] The random ligand library screening methods of the invention
can use the binding characteristics to differentiate between
different forms of a disease or its state, including pre-disease
states or the severity of a disease state. For example, the methods
may be used to determine the metastatic state of a cancer or the
susceptibility to an agent or disease state. In some embodiments,
the invention includes methods and compositions for assessing
ligand binding moieties present in or associated with a cancer, for
example, but not limited to, breast cancer, lung cancer, prostate
cancer, cervical cancer, head & neck cancer, testicular cancer,
ovarian cancer, skin cancer, brain cancer, pancreatic cancer, liver
cancer, stomach cancer, colon cancer, rectal cancer, esophageal
cancer, lymphoma, and leukemia.
[0094] In some embodiments, the invention includes methods and
compositions for assessing ligand binding moieties present in
autoimmune diseases, for example, but not limited to, myasthenia
gravis, chronic active hepatitis, primary biliary cirrhosis,
dilated cardiomyopathy, myocarditis, autoimmune polyendocrine
syndrome type I (APS-I), autoimmune hepatitis, cystic fibrosis
vasculitides, acquired hypoparathyroidism, goodpasture syndrome,
Crohn disease, coronary artery disease, pemphigus foliaceus,
pemphigus vulgaris, Guillain-Barre syndrome, Type 1 diabetes, stiff
man syndrome, Rasmussen encephalitis, autoimmune gastritis, Addison
disease, insulin hypoglycemic syndrome (Hirata disease), Type B
insulin resistance, acanthosis, systemic lupus erythematosus (SLE),
pernicious anemia, treatment-resistant Lyme arthritis,
polyneuropathy, multiple sclerosis, demyelinating diseases,
Rheumatic fever, atopic dermatitis, primary biliary cirrhosis,
Graves disease, autoimmune hypothyroidism, vitiligo, thyroid
associated ophthalmopathy, autoimmune thyroiditis, autoimmune
Hashimoto thyroiditis, coeliac disease, ACTH deficiency, myositis,
dermatomyositis, polymyositis, dermatomyositis, Sjogren syndrome,
systemic sclerosis, progressive systemic sclerosis, systemic
sclerosis, scleroderma, morphea, primary antiphospholipid syndrome,
bullous pemphigoid, herpes gestationis, cicatricial pemphigoid,
chronic idiopathic urticaria, connective tissue syndromes,
necrotizing and crescentic glomerulonephritis (NCGN), systemic
vasculitis, Wegener granulomatosis, Churg-Strauss syndrome,
polymyositis, Raynaud syndrome, chronic liver disease, visceral
leishmaniasis, autoimmune C1 deficiency, membrane proliferative
glomerulonephritis (MPGN), prolonged coagulation time, autoimmune
thrombocytopenia purpura, immunodeficiency, atherosclerosis,
neuronopathy, paraneoplastic pemphigus, paraneoplastic stiff man
syndrome, paraneoplastic encephalomyelitis, subacute autonomic
neuropathy, cancer-associated retinopathy, paraneoplastic
opsoclonus myoclonus ataxia, lower motor neuron syndrome,
Lambert-Eaton myasthenic syndrome, and paraneoplastic cerebellar
degeneration.
[0095] In one embodiment, the invention includes methods and
compositions for assessing ligand binding moieties present in
infectious diseases, for example, but are not limited to, Acquired
immunodeficiency syndrome (AIDS), Anthrax, Botulism, Brucellosis,
Chancroid, Chlamydial infection, Cholera, Coccidioidomycosis,
Cryptosporidiosis, Cyclosporiasis, Diphtheria, Ehrlichiosis,
Arboviral Encephalitis, Enterohemorrhagic Escherichia coli (E.
coli), Giardiasis, Gonorrhea, Haemophilus influenzae, Hansen's
disease (leprosy), Hantavirus pulmonary syndrome, Hemolytic uremic
syndrome, Hepatitis A, Hepatitis B, Hepatitis C, Human
immunodeficiency virus (HIV), Legionellosis, Listeriosis, Lyme
disease, Malaria, Measles, Meningococcal disease, Mumps, Pertussis
(whooping cough), Plague, Paralytic Poliomyelitis (polio),
Psittacosis (parrot fever), Q Fever, Rabies, Rocky Mountain spotted
fever, Rubella, Congenital rubella syndrome, Salmonellosis, Severe
acute respiratory syndrome (SARS), Shigellosis, Smallpox,
Streptococcal disease (invasive Group A), Streptococcal toxic shock
syndrome (STSS), Streptococcus pneumoniae, Syphilis, Tetanus, Toxic
shock syndrome, Trichinosis, Tuberculosis, Tularemia, Typhoid
fever, Vancomycin-Intermediate/Resistant Staphylococcus aureus,
Varicella, Yellow fever, variant Creutzfeldt-Jakob disease (vCJD),
Dengue fever, Ebola hemorrhagic fever, Echinococcosis (Alveolar
Hydatid disease), Hendra virus infection, Human monkeypox,
Influenza A H5N1 (avian influenza), Lassa fever, Marburg
hemorrhagic fever, Nipah virus, O'nyong-nyong fever, Rift Valley
fever, Venezuelan equine encephalitis, and West Nile virus.
[0096] The ligand library of the invention may be used to screen
for any stage of a disease, for example, an early stage of a
disease or an advanced late stage of a disease.
[0097] In yet another embodiment, the invention includes methods
and compositions for assessing ligand binding moieties present in
neurodegenerative diseases, for example, but are not limited to,
stroke, hypovolemic shock, traumatic shock, reperfusion injury,
multiple sclerosis, AIDS, associated dementia; neuron toxicity,
Alzheimer's disease, head trauma, adult respiratory disease (ARDS),
acute spinal cord injury, Huntington's disease, and Parkinson's
Disease.
[0098] The invention comprises compositions which comprises
particle based libraries of compounds selected from peptoids,
peptides, oligomers, small molecules and any molecule naturally
derived or synthetically made and which can be placed cm a support
system such as a bead or small particle.
[0099] In accordance with the present invention, there is provided
compositions comprising peptoid(s) that bind antibodies indicative
of a response to a drug and methods of detecting antibodies in an
antibody-containing sample comprising contacting an
antibody-containing sample with a support having affixed thereto a
peptoid. Ligand libraries can include compounds of formula I
wherein the R groups on either the amine side chain or die alpha
carbon are independently selected from the group consisting of
hydrogen; alkyl; allyl; methyl: ethyl; n-propyl; isopropyl;
n-butyl; isobutyl; n-butylamine; sec-butyl; tert-butyl; pentyl;
hexyl; isopentyl; aryl; heteroaryl; furanyl; indolyl; thiophenyl;
thiazolyl; imidazolyl; isoxazoyl; oxazoyl; piperonyl; pyrazoyl;
pyrrolyl; pyrazinyl; pyridyl; pyrimidyl; pyrimidinyl; purinyl;
cinnolinyl; benzofuranyl; benzothienyl; benzotriazolyl;
benzoxazolyl; quinoline; isoxazolyl; isoquinoline cycloalkyl;
alkenyl; cycloalkenyl; phenyl; pyridyl; methoxyeihyl;
(R)-methylbenzyl; C.sub.0-6 alkylaryl; C.sub.0-6 alkylheteroaryl;
C.sub.0-6 alkyl substituted with a group selected from OH, SH, a
halogen, OR.sup.15, COOR.sup.15, NR.sup.15 (wherein R.sup.15 is
selected from the group consisting of H or C.sub.1-6, alkyl or
C.sub.1-6 alkynl) or R.sup.16 (wherein R.sup.16 is selected from
the group consisting of H or C.sub.1-6 alkynl); OC.sub.1-6 alkyl;
C.sub.2-6 alkenyl; C.sub.2-6 alkynyl; C.sub.2-6 alkenyl; and
C.sub.2-6 alkynyl--including one or more chemical group described
in Tables 1 and 2 in U.S. Provisional Patent Application
61/467,256, which is incorporated by reference herein in its
entirety.
[0100] In one embodiment, the ligand libraries of the invention may
comprise a compound of formula 1 on a support,
##STR00001##
wherein R.sub.1 is selected from an electron rich amino acid side
chain Y; and R.sub.2-R.sub.6 are independently selected from the
groups consisting of H, --C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkylSCH.sub.3;
--C.sub.0-C.sub.6alkylC.sub.2-C.sub.6alkenyl,
--C.sub.0-C.sub.6alkyl C.sub.2-C.sub.6alkynyl, --C.sub.1-C.sub.6
COOH, --C.sub.1-C.sub.6alkylOH, --C.sub.1-C.sub.6alkylN(R).sub.2,
--C.sub.3-C.sub.8cycloalkyl, C.sub.1-C.sub.6alkylaryl,
--C.sub.1-C.sub.6alkylheteroaryl,
--C.sub.1-C.sub.6alkylNC(O)C.sub.1-C.sub.6alkyl,
--C.sub.1-C.sub.6alkykycloamide wherein any of the aryl or
heteroaryl groups may be independently substituted with --OH, Cl,
F, Br, --OCH.sub.3, --SO.sub.2NH.sub.2 or --O--CH.sub.2--O--.
[0101] Compounds of the random ligand library (e.g. the ligand
libraries) for screening a complex biological fluid are fully
described in U.S. Provisional Patent Application 61/467,256 and
61/491,717 which are incorporated by reference herein in their
entirety.
[0102] The large ligand libraries of the invention can be used
directly in biological fluid, under the appropriate experimental
conditions, to screen for biomarkers and without the need to use
fewer support members (e.g. about 100,000 or less) or requirement
to transfer such peptoids or ligands to a microarray before
screening the biological fluid. In addition, the ligand libraries
may also be used to screen for cell based receptors that
specifically relate to a particular cell surface marker. The
present invention, unlike prior methods, permits the inclusion of
greater numbers of beads/resins and thus larger libraries in either
the ligand binding agent screen or the cell receptor screen to
directly screen the complex biological samples.
[0103] As previously described with respect to microarray systems,
virtually any molecule or compound may be used to build a random
bead or resin based library. These "molecules" or "compounds" may
include natural products or man-made compounds or synthetically
derived molecules. The source of such molecules can be from
biological systems as well as non-biologically derived sources. The
preferred ligands for purposes of the initial screen using large
bead libraries under the conditions claimed in the present
invention are made, in part, from submonomers, which are selected
from any known monomeric amine and from any known acetic acid
halide or substituted acetic acid halide. For example, Table 1 in
U.S. Provisional Patent Application 61/467,256, which is
incorporated by reference herein in its entirety, provides a range
of R groups on a monosubstituted amine.
[0104] In some embodiments, the monomers and/or submonomers are
selected from, the group consisting of cysteine, glycine,
methionine, allylamine, ethanol amine, isobutylamine,
diaminobutane, methylbenzylamine (racemic or enantiomeric),
piperonylamine, cyclohexylamine, 3,4 dimethoxyphenethylamine,
benzylamine, N-(2-aminoethyl)acetamide,
N-(3-aminopropyl)-2-pyrrolidinone,
4-(2-aminoethyl)benzenesulfonamide or furfurylamine.
[0105] Acetic acid halides and/or R substituted acetic acid,
halides wherein R is selected from any amino acid side chain or
from any other group including those groups or variables on the
monosubstituted amines are also utilized as submonomers.
Alternatively; the combination of any amine and any acetic acid
halide may be reacted to form a monomer, which is then reacted,
with another reactive monomer on a growing peptoid chain to form an
oligomer of the invention.
[0106] Combinatorial libraries of peptoids may be prepared as
follows: Peptoids having a cysteine or methionine monomeric amino
acid attached to a support, or a linker on a support or resin or
bead may be prepared by first adding the protected amino acid to a
support or linker on a support. Following addition of said amino
acid (or any amino acid desired which can serve a functional or
other purpose in the oligomer or a diagnostic having said
oligomer), remaining monomers can be added using standard peptide
chemistry or using submonomers of bromoacetic acid (or
.alpha.-substituted bromoacetic acid or similar reactant) and a
monosubstituted amine wherein the amine is substituted with an R
group. The R group may be selected from any known peptoid
substituted including those described in, for example, U.S. Pat.
Publication Nos. 2010/0303805 or 2010/0303835 and/or those
described in Zuckermann and various Kodadek publications, which are
incorporated by reference herein in their entirety. The preferred
amines are those selected from the libraries recited herein wherein
particular monomeric amines are added to each library to build
1-250 MM bead or resin libraries. The preferred library size ranges
from 1 MM to 50 MM beads or resins having said diversity of
compounds thereon.
[0107] The process to make each peptoid generally involves (1)
preparation of an amino acid reactant on a support (including an
optional linker on a support); (2) reaction of the amino acid
moiety on said support with an acyl halide such as bromoacetic acid
or chloroacetic acid to form a halogenated derivative (3) reaction
of the halogenated derivative with a monosubstituted amine to form
an amide and (4) repeat of steps (2) and (3) to form a peptoid.
Methionine containing peptoids are generally made in the large
libraries. Cysteine containing peptoids are typically made when
larger scale quantities of high affinity peptoids are desired and
following the initial screening of the large bead or resin
libraries. In the large bead based libraries used to initially
screen complex biological fluid such as serum, there is no need for
or requirement for a long PEG linker which is typically necessary
for microarray screens. A PEG linker may be on the bead or resin
provided it is a short linker of less than about 10 monomeric
units. In the diagnostic kits comprising heads or tentagel beads of
less than about 50 microns (e.g. 10 microns), it is useful to use
both short PEG linkers (e.g. between 2-10 PEG monomers) or longer
PEG oligamers may be utilized.
[0108] The conditions used to perform each step in the oligomer
building process utilize solvents such as DMF or acetonitrile or
dichloromethane. Trifluoroacetic acid is utilized for cleavage
purposes and piperidine or other suitable base is used as a base in
the reaction between a bromo derivative and an amine. Various
protecting groups are utilized in the preparation of the amino acid
reactant. In a preferred embodiment, diaminobutane is utilized as
the first amine submonomer in the chain adjacent to the cysteine
residue at the C-terminus of the peptoid. In the first step of the
process, the selected beads or resins (in grain or milligram
quantities) are swollen in a suitable solvent such as DMF, If the
beads are "protected" with a protecting group on the reactive amine
on said bead, a base solution such as piperidine is repeatedly
added with subsequent washing with DMF to deprotect the head. Once
the bead is deprotected or if a bead such as a tentagel bead is
initially utilized, it may be reacted with a suitable amino acid
such as cysteine or methionine (protected with Fmoc or other
suitable protecting group on the nitrogen and protected with Trt
(triphenylmethyl) on the sulfur and in sufficient molar quantities
to react with each bead) in a suitable solvent such as DMF. HBTU
(tetramethyluronium hexafluorophosphate (coupling reagent) and
4-methylmorpholine (base) along with the protected amino acid are
added to the bead solution in a beaker (or tube or flask) and
shaken at room temperature to form the Fmoc/Trt protected amino
acid on the resin (or on a linker on the resin). The beads are then
washed multiple times in a solvent such as DMF. The Fmoc group is
then deprotected using a suitable reagent which permits reaction of
the amine on the amino acid with another reactant such as another
protected amino acid or a submonomer such as bromoacetic acid and
an activating agent e.g. DIG (3-isopropylcarbodiimide) in a
suitable solvent under heat (microwave with stirring). The
resultant beads are then washed multiple times and then treated
with a desired monomeric amine (in slight mole excess) in a
suitable solvent under heat. The resultant beads are washed
multiple times and then treated repeatedly with bromoacetic acid
and the amine of choice to build the oligomer and oligomeric
library. The peptoids may be cleaved from the beads using
trifluoroacetic acid. Art alternative or other suitable process for
peptoids comprising a preferred embodiment--e.g., those peptoids
having cysteine adjacent to a monomer having a 1-yl-n-butylamine
includes building a peptoid having two amino acids on the
C-terminus followed by a process that further includes adding any
of the monomers built in a submonomer process wherein the second
amino acid is lysine. This further includes the selection of any
monomer or submonomer to make .alpha.-substituted bromoacetic acid
submonomers wherein the carbon substituents may be selected from
typical amino acid side chains to form, after reaction of the
reactants, .alpha.-substituted peptoids wherein an R group is found
on either or both of the carbon on the peptoid chain or the
nitrogen on the peptoid chain. The substituents on either the
.alpha.-carbon or nitrogen may be virtually any substituent as
recited herein.
[0109] Combinatorial libraries of small molecules may be obtained
commercially or prepared using methods known in the art. See for
example, Bichler et al. 1995; Cho et al, 1999; LePiae et al., 2002;
Ostergaard and Holm, 1997; Yang et al, 1999). In addition, U.S.
Pat. No. 6,344,334 and publications Gallop et al, (1994), Gordon et
al, (1994); Thompson and Eilman (1996) are also sources of such
molecules and libraries.
[0110] Combinatorial libraries of peptides may be obtained
commercially or prepared using methods known in the art. See, for
example, Stewart and Young (1984); Tam et al. (1983); MerrifteM
(1986); and Barmy and Merritleld (1979), each of which are hereby
incorporated by reference.
[0111] Combinatorial libraries of, nucleic acids including RNA or
DNA may be obtained, commercially or prepared using methods known
in die art. Combinatorial libraries of oligosaccharides may be
obtained commercially or prepared using methods known in the
art.
[0112] In each instance, the "ligands" or random ligands may be
added to support resins or beads to form screening libraries can be
used, under the conditions described, herein, to screen for
biomarkers in complex biological fluid. The preferred ligands are
peptoid ligands.
[0113] In addition to building and/or using such libraries, it may
be necessary or desired to characterize, purify and/or synthesize
or re-synthesize any such ligand. Such methods are known in the art
and include the entire gamut of purification methods such as HPLC
via chromatographic means or purification methods via chemical
means; characterization methods such as mass spec or NMR or
combinations of any of these methods. Such methods axe--farther
described in, for example, US Pat. Publication 2007/0003954, which
is hereby incorporated by reference. In such cases, any such
purified ligand may be referred to as a compound or substantially
purified compound.
[0114] In the initial screening methodology of the invention, beads
and/or resins are utilized as the support means having an oligomer
operably coupled to said, support. In diagnostic kits or other kits
having "hits" or "putative hits" from such initial screen, the
support systems can be broadened to virtually any support system
including microarrays or any other known diagnostic platforms. In
these cases, it is necessary to ensure that such kits or other
support, systems with the putative hits also have or are adapted to
have a detector or detection methods to permit detection of ligands
having ligand binding moieties attached to such ligands. The
preferred detection methods include, for example, ELISA or other
methods which involve the use of labeled secondary antibodies.
[0115] Supports can be made of any suitable material. Materials
utilized to make such supports can include, for example, glass,
plastic, ceramic or polymeric resins or beads. Supports may also
include materials such as nickel, brass, steel or other metals or
mixtures of metals. The supports may also be conditioned to have
linkers and/or other means to bind to or connect to or react with a
ligand or active group on a ligand. Such groups are also described
in U.S. Pat. Pub. No. 2007/0003954. In the present invention, the
number of resins or beads having individual ligands bonded thereto
or to a linker and then to said support ranges from greater than
100K to about 150 million (MM). The preferred number utilized in
the initial screening methods of the invention ranges between 1 MM
and 2 MM ligands/resins.
[0116] TentaGel.RTM. resins are most preferred for the large ligand
screening methodology of the invention. These resins are grafted
copolymers consisting of a low crosslinked polystyrene matrix on
which polyethylene glycol (PEG or FOB) is grafted. TentaGel resins
are commercially available (Rapp Polymere GmbH). As PECS is a
"cameleon type" polymer with hydrophobic and hydrophilic
properties, the graft copolymer shows modified chemical properties.
According to the manufacturer, there are in principle two ways to
introduce PEG onto the modified polystyrene matrix. The simplest
immobilization procedure is to couple PEG via one of its terminal
hydroxyl groups to chloromethylated polystyrene according to the
classical ether synthesis or to use other bifunctional PEG's for
coupling onto the solid support. The manufacturer found that by
means of anionic graft copolymerization setting up the PEG step by
step directly on the matrix, PEG chains of molecular masses up to
20 kilo dalton have been immobilized on functionalized crosslinked
polystyrenes. Graft copolymers with PEG chains of about 2000-3000
dalton proved to be optimal in respect of kinetic rates, mobility,
swelling and resin capacity. As there is no procedure to get
monodisperse PEG with more than 10 ethylene oxide units by any
polymerization techniques, there is theoretically no way to
introduce monodisperse PEG chains with more than 10 ethylene oxide
units to the resin or to get monodisperse PEG by direct
polymerization onto the polystyrene backbone (monodisperse is
defined as: PEG without any molecular weight distribution). These
graft copolymers are pressure stable and can be used in batch
processes as well as under continuous flow conditions. The
copolymer contains about 50-70% PEG (w/w). The properties of these
polymers are highly dominated by the properties of PEG and versus
by the polystyrene matrix.
[0117] Setting up a chemical library or peptide library by the "one
bead one compound" approach it is essential to know the number of
beads which are available within a certain amount of resin as well
as the capacity of single beads. Table 1 summarizes some particle
sizes and correlates them to the corresponding capacity of a single
bead. The calculations are based on a typical loading of TentaGel
beads which are in the range of 0.25-0.3 mmol/g. For analytical
characterization at least 5 pmol of resin-bound peptide are needed
for sequencing on a bead. In order to estimate the optimum resin
quantity for the library, which can be handled economically one,
has to take into account the bead sizes and bead capacities. In
respect to homogeneity of diffusion process and kinetic rates as
well as for single bead analysis and single bead quantification,
all our beads show a very narrow size distribution.
TABLE-US-00002 resin size [.mu.m] beads/g capacity/bead TentaGel
NH.sub.2 750 .mu.m 4.62 .times. 10.sup.3 65 nmol TentaGel NH.sub.2
500 .mu.m 1.5 .times. 10.sup.4 19 nmol TentaGel NH.sub.2 300 .mu.m
6.4 .times. 10.sup.4 4 nmol TentaGel NH.sub.2 200 .mu.m 2.15
.times. 10.sup.5 1.3 nmol TentaGel NH.sub.2 130 .mu.m 8.87 .times.
10.sup.5 280-330 pmol TentaGel NH.sub.2 90 .mu.m 2.86 .times.
10.sup.6 80-100 pmol TentaGel M NH.sub.2 35 .mu.m 4.55 .times.
10.sup.7 5.5 pmol TentaGel M NH.sub.2 20 .mu.m 2.4 .times. 10.sup.8
1.0 pmol TentaGel M NH.sub.2 10 .mu.m 1.95 .times. 10.sup.9 0.13
pmol Correlation of particle size, number of beads per gram resin
and capacity per single bead. Calculation of single bead capacity
is based on a capacity of 0.25-0.3 mmol/g resin
[0118] There are several types of TentaGel resins available showing
tailored properties dependent on their application:
TentaGel S Resins:
[0119] The PEG spacer is attached to the polystyrene backbone via
an alkyl linkage. This linkage is not sensitive to acids or bases.
This type of resin is a standard type of resin used for peptide
synthesis, solid phase organic synthesis or combinatorial
chemistry.
TentaGel PAP Resins:
[0120] The PEG is attached to the polystyrene backbone via a benzyl
ether linkage. This benzyl ether linkage is sensitive to harsh acid
conditions like 100% TFA or mixtures of TFA/FMSBr.
[0121] These specially tailored resins are used for immunization
procedures or for synthesizing PEG modified derivatives (PEG
Attached Products), Using harsh acid conditions, the PEG spacer is
cleaved together with the synthesized compound from the solid
support to get soluble PEG modified compounds by applying solid
phase conditions (e.g. PEG modified peptides).
TentaGel N Resins:
[0122] The PEG spacer is attached to the polystyrene backbone via a
benzyl ether linkage. These tailored resins are used in
oligonucleotide chemistry for small and large scale oligormcleolide
synthesis. In comparison to CPG glass the capacity is increased by
a factor of K).
[0123] As TentaGel resins are copolymers composed from polystyrene
and polyethylene glycol, chemical and physico chemical properties
of both base polymers have to be taken in account.
[0124] PEG itself is a hygroscopic polymer. It is known from
literature that PEG esters are not very stable and easily
hydrolyzed. Dependent on the storage conditions and storage time,
PEG itself can be oxidized along the polyether chain to form
peroxides or esters. Consequently, acid treatment or treatment with
bases hydrolyzes the formed PEG-esters which result in a small
amount of "PEG-leakage". This leakage can be noticed by MS or NMR
as PEG signals and impurities in the final product. This chemical
behavior is true to all PEG's--and PEG based polymers.
TABLE-US-00003 TentaGel S: "S" means Standard resin, applicable to
a large number of applications, useful in batch and flow through
systems. TentaGel R: a special suited resin for research purpose
synthesis. The resin shows an increased swelling volume but is less
pressure resistant. Well suited for large peptides and difficult
sequences. TentaGel HL: this highloaded version of TentaGel
combines a significant higher capacity with the advantages of
TentaGel resins. TentaGel MB: TentaGel Macrobeads are highlighted
by extraordinary large particle diameters and high capacities based
on the TentaGel technology and designed for single bead synthesis
and single bead analysis. TentaGel N: this resin type is designed
for automated large scale oligonucleotide synthesis. TentaGel J:
this resin type has been developed for polymeric immunoconjugates.
TentaGel M: the microspherical shape of 10, 20, 30 .mu.m of this
TentaGel and it's monodispersity allows applications in automated
sorters, for creating huge libraries, high speed synthesis etc.
TentaGel B: describes bifunctional TentaGel resins, where the
reactive sites on the outer surface of the bead is orthogonally
protected to the reactive sites located in the internal volume of
the bead and hybrid resins for sequentional cleavage.
[0125] In addition to TentaGel beads, other resins and/or particles
may be utilized build a one ligand per bead library. For example,
lightly cross-linked polystyrene resins or polyamide resins may be
utilized. The group that joins the substrate to the resin bead can
be an essential part of solid phase synthesis. The linker is a
specialized protecting group, in that much of the time, the linker
will tie up a functional group, only for it to reappear at the end
of the synthesis. The linker must not be affected by the chemistry
used to modify or extend the attached compound. And finally the
cleavage step should proceed readily and in a good yield. The best
linker must allow attachment, and cleavage in quantitative
yield.
[0126] In certain aspects, the support may be a bead, a plate, a
dipstick, a filter, a membrane a pin, or a well. Detecting may
comprise RIA, FIA, ELISA, Western blot, flow cytometry, FRET, or
surface plasmon resonance.
Carboxylic Acid Linkers
[0127] The first linking group used for peptide synthesis bears the
name of the father of solid phase synthesis. Merrifield resin is
cross-linked polystyrene functionalised with a chloromethyl group.
The carbonyl group is attached by the nucleophilic displacement of
the chloride with a cesium carboxylate salt in DMF. Cleavage to
regenerate the carboxylic acid is usually achieved by hydrogen
fluoride.
[0128] The second class of linker used for carboxylic acid is the
Wang linker. Hits linker is generally attached to cross-linked
polystyrene, TentaGel and polyacrylamide to form Wang resin, it was
designed for the synthesis of peptide carboxylic acids using the
Fmoc-protection strategy, and due to the activated benzyl alcohol
design, the carboxylic acid product can be cleaved with TFA.A more
acid-labile form of the Wang resin has been developed. The SA.SRIN
resin has the same structure as the Wang linker but with the
addition of a methoxy group to stabilise the carbomum ion formed
during acid catalysed, cleavage.
Carboxamide Linkers
[0129] The rink linker is generally preferred for generating
primary carboxamide on solid phase, to the present invention, this
Sinker is utilized when manufacturing or re-synthesizing the hits
or putative hits from the primary screen of the invention. In such
cases, cysteine is the first monomer reacted with the rink linker
and then the process involves either subsequent monomer addition to
build the oligomer or subsequent submonomer chemistry to build the
oligomer. The greater acid sensitivity in the rink linker is a
consequence of the two additional electron donating methoxy groups.
In the generation of primary carboxamide, the starting material is
attached, to the linker as a carboxylic acid and after synthetic
modification is cleaved from the resin with TEA.
##STR00002##
[0130] The use of Rink resin to produce carboxamide following
TFA-catalysed cleavage.
Alcohol Linkers
[0131] A hydroxyl linker based on the tetrahydropyranyl (THP)
protecting group has been developed by Thompson and Ellmann. All
type of alcohols readily add to dihydropyraa and the resulting THP
protecting group is stable to strong base, but easily cleaved with
acid. This linker is attached to a Merrifield resin. The trityl
group is a good acid-labile protecting group for a lot of
heteroatoms. The trityl group has been used to anchor alcohols in
the synthesis of a library of .beta.-mercaptoketones.
Carbamates and Amines Linker
[0132] Carbamates linker has been used for the synthesis of a
combinatorial library of 576 polyamines prepared in the search of
inhibitors of trypanosomal parasitic infections. Two linkers were
investigated. One based on hydroxymethylbenzoic acid 1, and the
other one, an electron-donating group has been added 2. The last
one allowed cleavage by TFA while the first one could be cleaved
with strong acidic conditions.
##STR00003##
[0133] A very useful linker has been recently developed for the
generation of tertiary amine. (Tertiary amines are commonly used in
drag molecules.) Primary and secondary amines are introduced to the
linker by Michael addition. The amine may be alkylated to gives a
resin-bound quaternary aminonium ion. In mildly basic condition,
Hoffmann elimination occurs to give a tertiary amines of high
purity.
Traceless Linkers
[0134] In some case, the starting materials are loaded onto the
resin in one form, such as carboxylic acid, and cleaved in another
form; a carboxamide for example. This is perfectly acceptable if
the target, compound requires the released function. (Peptides
invariably contain a carboxylic acid or carboxamide.) However, the
growth in interest in combinatorial libraries of low molecular
weight non-peptides has elicited a need in new types of linker.
These linkers show non-specific function after cleavage, Traceless
linkers are so called because an examination of the final compound
reveals no trace of the point of linkage to the solid phase.
Samples
[0135] As discussed previously, the complex biological fluids
prepared for analysis in the process of the invention include or
can include a host of potential biomarkers including markers
expressed on cells (non-adherent cells, including T-cells or other
immune effector cells), microorganisms, proteins, peptides, lipids,
polysaccharides, small molecules, organic molecules, inorganic
molecules, biological molecules and including any detectable or
readable moiety in such complex milieu. In a preferred embodiment,
such markers are antibodies and, in particular, are antibodies
generated as a result of a disease or condition. In a preferred
embodiment, body fluids such as serum, plasma, saliva or other
fluids or samples derived from a patient or animal or organism are
the source of such markers. Each sample or tissue or biologically
derived, or environmentally derived or obtained sample is
conditioned, or treated or diluted, or otherwise handled in order
to expose said sample to either the initial screening or any
subsequent screening using putative hits or ligands which have
affinity for such biomarkers. The samples are diluted pursuant to
the methods recited herein to provide or permit sufficient
distinction between background levels or noise and signals
associated with the binding of a ligand to a ligand binding
moiety.
[0136] The time and/or conditions necessary to expose the
ligands/supports to such samples depend upon the particular sample
and other factors. The preferred conditions for the process of the
claimed invention are further described herein. In almost all
cases, washing or eluting steps and other conditioning means are
utilized following exposure of the biological fluid to the large
ligand library and/or ligands or kits derived from such library.
Aqueous solutions are utilized including buffered solutions such as
HEPES buffer, Iris buffer or phosphate buffered saline. Support
systems may also be treated with energy absorbing materials to
facilitate desorption or ionization of a "complex" from a support
surface. Chemical means are also utilized to decouple or remove
ligand-ligand binding moiety complexes from supports.
[0137] Detection methods for detecting ligand-ligand binding moiety
complexes on a support include photometric and non-photometric
means. Such methods include ensuring that the process includes a
method to detect and measure absorbance, fluororescence, refractive
index, polarization or light, scattering. These include direct
and/or indirect means to measure such parameters. Methods involving
fluorescence include fluorescent tagging in immunological methods
such as ELISA or sandwich assay. Methods involving refractive index
include surface plasmon resonance (SPR), grating coupled methods
(e.g. sensors uniform grating couplers, wavelength-interrogated
optical sensors (WIOS) and chirped grating couplers), resonant
mirror and interferometric techniques. Methods involving
polarization include ellipsometry. Light scattering methods may
also be used. Other means for tagging and/or separating and/or
detecting can also include magnetic means. Magnetic resonance
imaging, gas phase ion spectrometry, MRI may all be used.
[0138] Analysis of the data generated typically involves
quantification of a signal due to the detected biomarker versus a
control or reference. The data can be analyzed by any suitable
means. Computers and computer programs may be utilized to generate
and analyze the data. Beads and/or other supports may be computer
coded or coded for identification purposes. Data analysis includes
analysis of signal strength under the particular conditions of the
assay or detection method, Ligands, ligand binding moieties or
reference moieties and/or secondary detection moieties may be
labeled or radio-labeled or tagged with a detectable moiety. One of
ordinary skill in the art can assess the difference and/or
distinction between biological fluid samples that have disease
associated biomarkers versus those control or healthy patient,
samples that do not contain such markers. One of ordinary skill in
the art can also determine, pursuant to the methods described
herein, the presence of false positives or other hits that are or
may be found in control samples to account for and/or remove such
"hits" and one of ordinary skill in the art, pursuant to the
methods described herein, can continue the process of determining
or finding disease associated biomarkers in patient samples having
tray disease or condition. The "detection" of such hits, in all
cases, is accomplished by means for detecting the binding of a
ligand-binding moiety such as a disease associated biomarker or
other marker to ligands in a ligand library such as those described
herein.
[0139] Biomarkers associated with the diseases and/or conditions
recited herein will vary depending upon the particular stage of the
disease and/or condition of the particular patient or animal or
other organism assessed. The ligands, which are the putative hits
and the compounds recited herein, are expected to, in most cases,
mimic the natural antigen that initiates the immune response and/or
formation of antibodies or immune cells in the first instance. The
present invention and screening process claimed and recited herein
does not require knowledge of either the particular antigen or the
antibody generated in response to the antigen. The ligands,
however, may be useful in their own right as vaccines or drug
candidates in addition to being useful in the screens and
diagnostic methods recited herein. The present invention thus
includes compounds and pharmaceutical compositions.
Peptoid Screens:
[0140] To screen one-bead-one-compound (OBOC) combinatorial peptoid
libraries, tens of thousands to millions of peptoid bearing beads
are prepared and then mixed with a complex biological sample. The
initial complex biological sample is preferably a control sample
and a subsequent complex biological sample treated with a ligand
library that has "removed" the control hits is then treated and/or
screened against a diseased complex biological sample. The
ligands/beads that interact, with at least one disease associated
biomarker are then detected, identified and isolated and/or
characterized. In a preferred embodiment, a Tentagel screening
protocol is used which comprises (1) bead preparation, (2)
screening of complex biological fluid and (3) detection of
hits.
Peptide Screens;
[0141] To screen one-bead-one-compound (OBOC) combinatorial peptide
libraries, tens of thousands to millions of peptide bearing beads
are prepared and then mixed with a complex biological sample
following the processes described herein. The beads that interact
with disease associated biomarkers are then identified and isolated
for compound structure determination. For example, OBOC peptide
library screening using streptavidin (SA) as probe protein, labeled
with a red fluorescent dye and using the COPAS BIO-BEAD flow
sorting equipment to separate fluorescent from nonfluorescent beads
may be performed. See Manmi et al., J. Comb. Chein. 2009, 11 (1),
pp 146-150. The red dyes which may be used are ATTO 590 and Texas
Red. After incubating the library with the SA-red fluorescent dye
conjugate, positive beads caused by peptide-SA interaction are
obtained. The beads are analyzed by matrix-assisted laser
desorption ionization time-of-flight mass spectrometry (MALDI-TOF
MS). Thus, peptide libraries may be used in a manner that is
analogous to the process described herein with peptoids wherein
initial control biological fluid samples are used to remove any
ligand/bead hits from the starting compound library and wherein the
remaining members of the library are used to then screen for any
hits in a diseased complex biological fluid sample. These hits are
the putative hits which are then carried forward in any diagnostic
kits.
[0142] In a similar manner, any ligand may be screened on the beads
or supports using the processes described herein. These ligands
include, in addition to peptoids or peptides, nucleic acid
oligomers, polysaccharides, small molecules and/or any combination
thereof which can be build into libraries and, under the conditions
recited herein, used to screen complex biological fluid.
Kits and Diagnostic Tools
[0143] Any of the compounds or compositions described herein may be
further utilized in diagnostic kits either in a clinical or
laboratory setting. These kits can range from simple point of care
diagnostic assays to complex and multiplex instruments or probes.
The support systems and "packaging" surrounding the core
support/ligand system can be selected from current commercial kits
that are designed to include the putative hits and or hits that are
resynthesized and installed on such suitable platforms or they can
be used in newly designed diagnostic kits. The kits will typically
be accompanied by all suitable reagents and instructions to use the
kits to screen for and/or diagnosis the particular disease or
condition the kit is designed for. Any such kit or method will
comprise at least one putative hit or ligand that has been
identified pursuant, to the screening method recited herein. This
ligand or plurality of ligands may be selected from the same ligand
or a mixture of ligands which comprise the compounds of the
invention. The ligands may be selected based upon their affinity
for a disease associated biomarker for one particular disease state
or a group or battery of diseases or conditions. The preferred
ligands are peptoid ligands. The kits will also contain
instructions for the physicians diagnosing a particular disease or
condition and specific labeling for the particular kit and disease
state or condition. The present invention thus includes a
combination of a kit including all of its essential components such
as the putative peptoids or ligands found from an initial screening
using any one of the libraries disclosed herein and/or known
pursuant to the specific methods recited herein and labeling
instructions. It is also envisioned that the particular processes
and methods and materials disclosed herein may be utilized in a
clinical and laboratory setting under the supervision of a skilled
operator. The kits and/or instruments or equipment comprise ligands
such as peptoids that are specific for disease associated
antibodies and/or ceils. The "kit" may comprise a complete
diagnostic kit and or screening kit or the "kit" may comprise
components or sub-components containing or comprising the
diagnostic peptoids, antibodies discovered and characterized
through such peptoids or native antigens that are discovered and
purified and/or characterized as a result of interaction with and
discovery from the autoantibody. Such antibodies and purified
antigens comprise part of the present invention.
[0144] In one embodiment, provided herein is a kit for diagnosis of
a disease. In another embodiment, provided herein is a kit for
treating a disease. The kit may comprise a ligand library,
detection reagents for screening the ligand library against a
biological sample, adjuvants for the screening, and a package
insert. The package insert may include instructions for performing
the diagnostic steps, instructions for determining a drug
administration, and instructions for administering the drug based
on the determination. In some embodiments, the kit may include a
package insert that is a label approved by FDA or a drug approval
authority in other countries.
[0145] The ligand libraries of the present invention are utilized
to find and determine ligands that bind to disease associated
biomarkers. Such ligands are then utilized in the kits and/or
methods described generally above to assess, screen or diagnose
disease states or conditions. These diagnostic methods typically
involve screening for and finding disease associated biomarkers
which comprise antibodies and/or other biological markers. As
stated above, these antibodies can be further identified and
characterized using the ligands of the invention on suitable
columns to pull out or remove such antibodies from blood samples.
The antibodies can in turn be used to probe for and discover the
native antigen associated with such antibody. The present invention
thus includes both the antibodies and purified antigens associated
with such antibodies and which are discovered, isolated and
characterized using the methods of the invention.
[0146] Kits and/or other means to screen for and/or diagnose
disease states or conditions must, in the first instance, be
assessed against patient samples. These patient samples may be
derived from normal control samples or from patient samples wherein
said patient, has been identified as a patient that has or is
suspected of having that disease or condition. The patient may have
other symptoms associated with the disease beyond the "presence" of
a disease associated biomarker. The patient may be in an early
stage of the disease, may not have the disease or condition at all
or may be in a late stage of a particular disease. In any clinical
context and under appropriate guidelines and controls, patient and
clinical samples may be provided in a blinded fashion and then
assessed using the compounds of the invention. The data generated
as a result of the screening may then be analyzed after un-blinding
to find or not find statistically significant results or
correlations with known or underlying data about any particular
patient or group of patients. The present invention comprises a
method of screening for the presence of a disease or condition
comprising (1) screening a biological sample from a patient with at
least one compound of the invention: (2) screening a control
biological sample under the same conditions using said at least one
compound and (3) comparing the healthy control data versus the
patient data to determine the presence or absence of a disease
associated, biomarker. A group of patients or patient samples
having or suspected of having disease X may be screened against a
kit or diagnostic probe having at least one compound of the
invention and the data generated with respect to each patient may
be utilized on a case by case basis to confirm or validate a
disease state or condition or lack thereof. Such data generated
herein may be used in combination with the total, information known
about that particular patient, to assess the patient's condition
and to provide guidance to the medical practitioner providing
treatment options. The "information" generated as a result of any
such screen may be used in the clinical trial setting to assess
individual patients that are taking drag therapy. The present
invention thus includes a method of assessing clinical trial
progression comprising use of a screen performed, according to the
methods described herein. In a preferred embodiment, the present
invention relates to a method of screening for or diagnosing an
early disease state comprising use of a screen or compound claimed
herein to detect a disease associated biomarker. The invention is
particularly useful, in the context of early disease intervention
wherein detection of such biomarkers is expected to occur well
before aggressive progression of the disease. In another context;
early intervention in cardiovascular disease and/or metabolic
disease as well as neurological disease is expected to save lives
and prevent or be useful for preventing further development of such
diseases without early medical intervention or treatment.
[0147] The present invention also includes methods to increase the
resolution or efficiency of the difference between a control or
standard solution and the complex biological fluid containing the
disease associated biomarker. For example, methods include
preconditioning or pre-treating or pre-blocking the system/serum
with buffers and/or conditioning agents such as E. coli lysate
and/or lysine.
[0148] In yet another embodiment, there is provided a method of
treating a subject suspected of having a disease comprising (a)
contacting an antibody-containing sample from said subject with one
or more supports having affixed thereto a peptoid comprising a
peptoid of the formulas recited herein (b) detecting antibodies
bound to said peptoids; and (c) making a treatment decision based
on the result of step (b). The method may further comprise
obtaining said sample from a subject. The method may also further
comprise making a diagnosis of a disease for a subject from which
said sample was obtained if antibody binding to the peptoid is
greater than, that observed for control non-diseased patients. The
method may also further comprise making a treatment decision for
said subject. The sample may be contacted with more than one
peptoid of formulas recited herein. The sample may be contacted
with a multiplex platform for the purposes of diagnosing multiple
disease states or conditions. The support may be a bead, a plate, a
dipstick, a filter, a membrane a pin, or a well. The sample may be
blood, serum, saliva or CSF. Detecting may comprise RIA, FLA,
ELISA, Western blot, flow cytometry, FRET, or surface plasmon
resonance.
[0149] A further embodiment is directed to an antibody composition
isolated from a biological fluid that is indicative of a disease,
in certain embodiments the antibodies are isolated by contacting a
sample having such antibodies with a peptoid composition that
specifically binds antibodies indicative or associated with a
disease. The antibodies can be removed, isolated, or purified from
other non-antibody and non-D specific components. The antibodies
can then be washed and/or disassociated from the peptoid capture
agent(s).
[0150] In certain embodiments, a peptoid array made from the
peptoids discovered in the process described herein is hybridized
with a biological sample that has been supplemented with, a
bacterial lysate, e.g., an E, coli lysate. The biological sample
includes a control sample and a sample having a marker for a
central nervous system disorder. For example, microarray slides are
covered with a hybridization chamber and equilibrated with 1X TBST
(50 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween20) for about 15
minutes. The slides are then blocked with a bacterial lysate at a
concentration at least, at most, or about 0.5, 1, 1.5, 2 mg/ml of
lysate. The lysate is removed and the slides are incubated with
about a milliliter of biological sample (having an approximate
protein concentration of 5, 10, 15, 20 or 25 Dg/ml including all
ranges and values there, between) in bacterial lysate with gentle
shaking. Microarrays are then washed with 1X TBST and hybridized
with labeled Anti-IgG antibodies (e.g., at 1:400 dilution). The
slides are then washed with an appropriate buffer. The slides are
dried using a centrifuge (e.g., 5 min spin at 1500 rpm) and scanned
on a microarray scanner, for example, using a 635-nm laser at 100%
power and a 600 or 650 photomultiplier tube gain. The present
invention thus also relates to a method of reducing background
antisera noise in a diagnostic assay comprising treating the
control plasma sample and the diseased sample with, an E. coli
lysate and contacting said samples with a peptoid or ligand array.
It is believed that this process can be used to support treatment
of any array used to detect and distinguish antibodies in sera in
the context of comparing a control sample to a diseased sample.
[0151] It is contemplated that any method or composition described
herein can be implemented with respect to any other method or
composition described herein.
[0152] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or snore," "at least one," and "one or more than one."
[0153] It is contemplated that any embodiment discussed in this
specification can be implemented with respect to any method or
composition of the invention, and vice versa. Furthermore,
compositions and kits of the invention can be used to achieve
methods of the invention.
[0154] Throughout this application, the term "about" is used to
indicate that a value includes the inherent, variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0155] It is understood that any one of the putative hits or
peptoids discovered through the process recited herein may also be
a therapeutic drug or vaccine candidate. The present invention thus
relates to a process for discovering drug candidates or vaccines
comprising use of the screen pursuant to the methods described
herein.
[0156] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way be construed, however, as limiting the broad scope of the
invention.
EXAMPLES
Example 1
Library Preparation
Protocol for Peptoid Synthesis (Cyst-Peptoid or
Methionine-Peptoid)
[0157] The following example demonstrates how peptoid libraries of
the invention were generated. The materials utilized in the example
include reaction flasks or beakers, plastic tubing, 10-15 3 ml
syringes with needles. Latex gloves, 10-15 15 ml polypropylene test
tubes and micropipettes with solvent safe tips (1000 .mu.l), glass
pipettes and Resin beads. The chemicals and/or reagents utilized
included N,N Dimethylformamide, Bromoacetic acid (BMA), Anhydrous
Dimethylformamide, Piperidine, Acetonitrile,
3-diisopropylcarbodimide (D1C), Trifluoroacetie acid,
5(6)-Carboxyfluorescein, Dichloromethane (DCM), and
4-Methylmorpholine (NMM). The various amines utilized in each
library preparation were also used as well as HBTU
(Tetramethyluronium Hexafluorophosphate) and triethylsilane.
Peptoid Preparation
[0158] The concentration of each amine used in the process is
calculated using following the formula:
V-FW/d/1000.times.2M.times.5 ml
Procedure:
Step 1
[0159] Swelling of the Resin Beads.
(a) 250 milligrams of resin beads were placed into a clean dried
reaction flask and 5 mls of hydrous DMF was added to the beads
which were allowed to swell over a period of an hour or less. The
beads were then washed with DMF multiple times (2 or 3.times.)
under vacuum.
Step 2
[0160] Steps (b), (c) and (d) were omitted when "unprotected beads"
(e.g., TeMa.about.Gel) were used.
[0161] 20% solution of Piperidine (base) using anhydrous DMF as the
solvent was used in the following process:
[0162] The following process comprising steps (b), (c) and (d) was
done 2 times when using "protected beads" [0163] (b) 2.5 ml of 20%
piperidine solution was added to the protected beads; [0164] (c)
After adding piperidine, the reaction flask was placed on a
shaker/incubator for 20 minutes, set at 200 rpm @ 25.degree. C.
[0165] (d) the reaction flask was then washed 8-10 times with
hydrous DMF using 5 mls of DMF.
[0166] The following solutions were also prepared: [0167] 468 mg
Fmoc-Cys(Trt)-OH in anhydrous DMF (2 ml volume) (solution A).
[0168] 161.6 mg of NMM, in anhydrous DMF 2 ml [0169] 303.2 mg HBTU
was added to NMM vial (solution B).
Addition of HBTU/NMM and Fmoc-Cys
[0170] 1 ml of each of solution A and solution 8 was added to the
beads--(HBTU/NMM) and Fmoc-Cys(Trt)-OH) and shake for 1 hour.
[0171] The beads were washed in DMF 5-10 times.
[0172] The remaining 1 ml solution of solution A and B were added
to the beads which were shaken for a period of 1 hour and then
washed again in DMF 5-10 times.
[0173] The following solutions were also prepared;
20% Piperidine (in anhydrous DMF) 2M Bromoacetic acid
50% D1C/A. DMF
[0174] 2M solution of each amine
[0175] The following steps (a), (b) and (c) were performed 2 times,
2.5 ml of 20% piperidine solution was added; (b) the reaction flask
was shaken at 200 rpm at 25 degrees C. and then (c) the beads were
washed with DMF 8X to 10X.
[0176] A 10 ml solution of 2M Bromoacetic acid was prepared.
[0177] A 10 ml solution of 50% 3.2M D1C/anhydrous DMF (v/v) was
also prepared.
[0178] 2M amine solutions were prepared of each amine in and for
each library.
[0179] For peptoid synthesis, 1 ml of 2M stock solution was used
each time an amine was added on the peptoid chain.
Step 3
[0180] 1 ml of Bromoacetic acid was added to the reaction vessel;
(b) 1 ml of 50% DIC/DMF solution was then added and the resultant
solution, was (c) microwaved for 15 seconds @ 10% power.
[0181] Step (c) was performed 2 times swirling the flask side to
side between sets of micro waving.
[0182] A white precipitate was formed after each micro waving step.
The beads were then washed 8-10 times with DMF.
Step 4
[0183] One ml of the first amine in the sequence was added to the
reaction flask containing the bromo intermediate from the preceding
step and the vessel was shaken to evenly distribute the amine on
the beads. The reaction was then initiated using the microwave for
15 seconds @ 10% power 2 times. The reacted beads were then washed
with hydrous DMF 8-10 times.
[0184] Steps 3 and 4 were repeated until all amines were added to
make the target peptoids.
Step 5
[0185] The heads were then washed with dichloromethane (DCM) 3
times and allowed to dry.
Step 6
[0186] The peptoids were then cleaved from the beads using a 95%
TFA solution (5 mls). The peptoids were then collected off the
beads which were washed with a solvent (CH3CN and water) to remove
residual peptoids. Argon gas was used to remove any residual TFA.
The peptoids were then lyophilized and characterized and purified
as necessary.
[0187] The reaction conditions specified above may be modified on
an as-needed basis depending upon the quantities needed for any
particular bead composition.
[0188] FIGS. 1-5 generally demonstrate how the library of the
invention was prepared for a disease, for example, AD diagnostics,
pancreatic cancer diagnostics and lupus. In general the beads
having an amine moiety were linked to an amino acid residue through
a series of steps using standard peptide chemistry which was then
reacted with an activated carbonyl moiety having a halide group
which was then reacted with a monomeric amine having an R group.
Steps 2 and 3 of the cycle were repeated as shown in the Figures to
create large peptoid libraries having 1 MM to 2MM distinct ligands.
The initial screening library prepared on Tentagel resin or beads
typically had a methionine amino acid as the first monomer in the
chain. The present inventor uses such an amino acid to facilitate
cleavage from a bead or resin that does not have a cleavable
linker. The Rink resin used to build the cysteine containing
peptoids have linkers which do not need or require the use of
methionine as the first amino acid. The cysteine containing
peptoids were typically resynthesized after the initial screen
found the putative hits. The cysteine sulfur group permits reaction
of the peptoid chain with, for example, another reactive moiety on
a diagnostic platform substrate. The peptoids which were
resynthesized also contained a 1-yl-n-butylamino moiety as the
first side chain in the chain after the amino acid amine. It is
believed this group is necessary to display the peptoid and to
solubilize the peptoid in aqueous containing solutions.
Example 2
General Screening Methodology
[0189] 160 micron Tentagei beads attached to a peptoid of choice
were swelled overnight in DMF. Beads were then washed ten times in
a reaction vessel with Millipore water and vigorous shaking. Fresh
Millipore water was added each time, and on the 10.sup.1h wash,
beads were allowed to shake overnight at 150-200 rpm. The next day,
beads were washed in the same fashion with IX TBST and allowed to
shake at 150-200 rpm for at least 3 hours.
[0190] Beads were then split evenly into 15 ml conical tubes, about
0.5 grams per tube in 1.times.TBST. TBST was removed, and 4 ml of
diluted normal human serum was added to each tube. Serum stock made
in IX TBST was Nano-dropped to get desired concentration of 20
ug/ml. Tubes containing serum and heads were then tumbled overnight
at 4 degrees Celsius in the dark. Serum was then pipetted out of
the tubes, and replaced with 4 ml 1X TBST. Tubes were then slowly
inverted to re-suspend then beads, and then allowed to settle. TBST
was removed and added twice more, for a total of three TBST
washes.
[0191] Secondary antibody solution was then prepared, by preparing
5 ul of goat anti-human IgG Qdot 655 per 1 ml 1X TBST. Once the
last TBST addition was removed from the beads, 4 ml of the Qdot
solution was added, and beads were tumbled for 2 hours at four
degrees Celsius in the dark. The beads were then allowed to settle,
and the Qdot solution was removed. The beads were then washed three
times with 4 ml of 1X TBST. Beads were then poured into a clear
Petri dish of viewed under a UV microscope containing a DAP I
filter. All beads stained red were removed.
[0192] After the first screen was completed, beads were poured back
into 15 ml conical tubes, and tumbled at four degrees Celsius for
at least four hours before the next serum sample addition. Disease
serum was then added to the beads in the same fashion as normal
serum addition, with the exception that the serum was diluted in
PBS starting block as opposed to 1X TBST. However, the original
stock was prepared in 1X TBST in order to obtain the proper
concentration with the nanodrop. The serum addition and secondary
antibody addition is the same as with the normal serum.
[0193] Once diseased "hits" were removed, they were pooled into a
1.5 ml eppendorf tube, and heated at 95 degrees Celsius for 25-30
minutes in 1% SDS. The SDS was then removed from the tube, and
replaced with Millipore water. Beads were then tumbled for 15
minutes at four degrees Celsius. The water was then replaced with
fresh water, and beads were tumbled for another 15 minutes. The
water was then removed and replaced with 50/50 Acetonitrile/water
solution and allowed to tumble another 15 minutes. Beads were then
separated into individual wells in a 96 well plate and allowed to
dry.
[0194] A solution of 20-30 mg Cyanogen Bromide, 500 ul
Acetonitrile, 400 ul Glacial Acetic Acid, and 100 ul Millipore
water was made, and 20 ul of solution was added to each well
containing a hit bead. The plate was covered and allowed to shake
for 16 hours at 100 rpm. The cover was then removed, and the
cleavage solution was evaporated from the wells. The hits were then
spotted onto a MSMS plate and sequenced using a 4800 MALDI/TOF/FOF
analyzer.
[0195] FIG. 6 provides a general schematic of the screening
methodology disclosed and claimed herein.
Example 3
Diagnosing a Response to Drug for Treating a Disease
[0196] Five hundred milligrams of 160 micron Tentagel beads (JC3B
library) is added to a fifteen milliliter conical tube. Five
milliliters of DMF is added to the tube, and the beads are allowed
to sit overnight to swell. The next day, DMT' is pipetted out of
the tube and replaced with five milliliters of 1X TBST. The tube is
inverted to mix, and beads are then allowed to settle to the bottom
and 1X TBST is removed. Five milliliters of 1X TBST is added and
removed five more times.
[0197] Normal serum samples are prepared by adding 4 milliliters of
PBS starting block to a tube, and adding 7 ul of each of four
separate drug treated samples to the same tube. The serum is added
to the washed beads and beads and serum are allowed to tumble
overnight at four degrees Celsius in the dark. The next morning,
beads are removed from the tumbler and allowed to settle before the
serum is pippeted out of the tube. Four milliliters of 1X TBST is
added to the tube, and the tube is inverted to mix. The TBST is
then pipetted out of the tube and replaced with four milliliters of
fresh 1X TBST and removed again.
[0198] DYNA-bead solution is then prepared by adding 50 ul of
well-mixed goat anti-human IgG DYNA beads to four milliliters of 1X
TBST. The mixture is then added to the washed beads. The beads are
then allowed to rumble for two hours at four degrees Celsius in the
dark.
[0199] Without washing the beads, the DYNA beads screen is
performed. The tube is placed in a magnet holder and filled to the
brim with 1X TBST. The magnet and tube are slowly agitated for two
minutes, and the beads are allowed to settle in the magnet holder.
The TBST and free beads that settled to the bottom are removed
carefully, to not touch the hit beads attached to the sides by the
magnet and replaced with fresh 1X TBST, The process is repeated two
to three times, until no beads could be seen attached to the sides
of the tube. The hit beads are then combined into one tube.
[0200] Remaining non-hit beads are divided into 15 milliliter
tubes, inverted and quickly pulsed centrifuged. The supernatant is
removed and replaced with fresh 1X TBST. This process repeated 6-8
times until no more DYNA beads are visible in the bead/TBST
solution. The hit beads are washed in the same fashion.
[0201] Beads are combined back into a 15 ml tube, and normal serum
is added to the beads in the same way as previously stated, and
allowed to tumble overnight at 4 degrees Celsius in the dark. In
addition, three Milliliters of each four normal samples is added to
1 milliliter of PBS starting block, and this solution is added to
the DYNA bead "hit" bead tube. The next day, beads are washed in
the same fashion as with the normal serum addition.
[0202] 20 ul of goat-anti human IgG Quantum Dot 655 is diluted in 4
milliliter 1X TBST (20 ul Qdot in I M1 IX TBST for the "hit" tube),
and added to the beads. The solution is tumbled for two hours at
four degrees Celsius in the dark. Both hit and non-hit tube is
washed four times with 1X TBST and screened for bright red beads
under a UV microscope. Remaining beads are tumbled in tour
milliliters of 1X TBST for one hour, and disease or drug treated
serum sample is added in the same fashion as the normal serum
sample. The magnetic screen and Qdot additions are performed in the
same manner as previously stated. The hits are then sequenced on a
MALD1 TOF/TOF mass spectrometer.
Example 4
Microarray Data with A Single Measurement
[0203] Microarrays were prepared as described in U.S. Pat.
Publication No. 2010/0303805 which is hereby incorporated by
reference; Microarray slides are covered with hybridization chamber
and equilibrated with IXTBST (50 roM Iris, pH 8.0, 150 mM NaCL 0.1%
Tween20) for 15 minutes. The slides are then blocked with 1 ml of
blocking buffer for 1 hour at 4.degree. C. The blocking buffer is
removed and the slides are incubated with 1 nil of serum (20 mg/ml)
for 16 hours at 4.degree. C. with gentle shaking. In an alternative
method, the slides are blocked with 1 nil of E. coli lysate (1.5
mg/ml) for 1 hour at 4.degree. C. The E. coli lysate is removed and
the slides are incubated with 1 ml of serum (15 mg/ml) in E. coli
lysate (1.5 mg/ml) for 18 hours at 4.degree. C. with gentle
shaking. Microarrays are then washed three times with IXTBST and
hybridized with Alexa-647 labeled Anti IgG antibody (5 mg/ml) for 2
hours on orbital shaker at 4.degree. C. The chamber cassettes were
removed from microarray slides and washed with IXTBST (3.times.15
min) followed by 0.1XTBST (1.times.10). The slides are then dried
on centrifuge (5 min at 1500 RPM) and scanned on microarray scanner
(Gene Pix Autoloader 4200) by using 635-nm laser at 100% power and
600 or 650 photomultiplier tube gain. All the scanned images were
analyzed by the Gene Fix Pro 6.0 software and Genespring
software.
Example 5
ELISA Protocol
[0204] 96 well Maleimide-activated plates were obtained from Thermo
Scientific, and washed three times with 400 uL/well wash buffer
(0.1M sodium phosphate, 0.15M sodium chloride, 0.05% Tween 20, pH
7.2), using a plate washer from Beckman Coulter. The peptoid of
interest was diluted to 10 mM in PBS binding buffer (0.1M sodium
phosphate, 0.15M sodium chloride, 10 mM EDTA, p.H 7.2), and 200 ul
of the peptoid solution was added to the appropriate wells. The
plate was then allowed to incubate in the dark for two hours at
room temperature with shaking at 500 rpm. The peptoid solution was
then aspirated from the wells using die plate washer, and again
washed three times with 400 ul/well of wash buffer. L-Cysteine HCL:
H20 (Thermo Scientific) was diluted to 10 ug/mL in binding buffer,
and 200 ul per well was added. The plate was then incubated for one
hour in the dark at room temperature with shaking at 500 rpm, and
washed three times. 200 ul StartingBloek.TM. (PBS) Blocking Buffer
(Thermo Scientific) was added to the wells and the plate was
incubated for one hour at 4.degree. Celsius. in the dark with
shaking at 500 rpm. The plate was washed three times with the plate
washer, and serum samples were prepared by serially diluting in
binding buffer from 1:200 downward. Concentrations of the 1:200
sample stocks were taken using a nano-drop (Thermo Scientific), to
make sure that they were similar. Each diluted sample was voilexed
before preparing the next dilution. 200 ul of the appropriate
dilution for serum (both disease and normal) was added to die
plate, as well as binding buffer without serum as a control. The
serum was allowed to incubate for two hours at room temperature in
the dark with 500 rpm shaking. The plate was again washed, and 200
ul of a 1;30,000 dilution of goat anti-human IgG HRP (MilHpore) in
binding buffer was added to the appropriate wells and incubated at;
room temperature for 30 minutes with 500 rpm shaking in the dark.
The plate was washed three times, and 100 ul of TMB
(3,3',5,5'.about.tetramethylbenzidine) solution was added to each
well, and color was allowed to develop for 30 minutes on the bench
in the dark. L00 ul of 2M Sulfuric acid stop solution was added to
stop the reaction, and the wells were read at an absorbance of 450
using a plate reader.
[0205] Thus, in each case and with respect to each drug treatment,
the process of the invention may be utilized to rapidly discover
biomarkers associated with a drug response (e.g., an adverse
reaction, a drug resistance, and a therapeutic dosage efficancy)
and ligands which bind to such markers. These ligands--this larger
pool of ligands--can then be used for multiple diagnostic and/or
therapeutic purposes. The diagnostic platforms include microarrays,
bead based methods and ELISA systems. The conditions utilized above
comprise an important aspect of the invention. These conditions
include dilution ranges for sera as well as the concentration of a
particular peptoid on a bead or in a well and detection methods.
The number of beads having a peptoid on a bead may vary depending
upon the particular test kit or screening kit. These numbers may
also vary depending upon whether beads/ligands are used in the
initial screening protocol and method recited herein and/or are
used in a test kit based upon the discovery of a high affinity
ligand.
[0206] It will be appreciated by those skilled in the art that
changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is
understood, therefore, that this invention is not limited to the
particular embodiments disclosed, but it is intended to cover
modifications that are within the spirit and scope of the
invention, as defined by the appended claims.
* * * * *