US2008096201A1PendingUtilityA1
Assay for identifying biological targets of polynucleotide-binding compounds
Individually held — no corporate assignee on recordPriority: Jun 3, 2003Filed: Feb 27, 2007Published: Apr 24, 2008
Est. expiryJun 3, 2023(expired)· nominal 20-yr term from priority
C12Q 1/32G01N 2500/00
56
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Claims
Abstract
The present invention provides methods and systems for the identification of a biological target of a chemical compound, such as saframycin A, known to bind a polynucleotide. The invention also provides methods of screening chemical compounds for those which act in a similar method and may be more potent than known compounds. The inventive methods are particularly useful in the high-throughput screening of chemical compounds that target GAPDH. The invention also provides kits useful in the practice of the inventive method. Compounds identified by inventive methods are also included in the invention.
Claims
exact text as granted — not AI-modified1 . An isolated composition comprising
a polynucleotide; saframycin A or analogue thereof; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
2 . The composition of claim 1 further comprising a solid support.
3 . The composition of claim 2 , wherein the solid support is attached to the polynucleotide.
4 . The composition of claim 3 , wherein the solid support is attached to the polynucleotide through a covalent linkage.
5 . The composition of claim 2 , wherein the solid support is attached to the saframycin A or analogues thereof.
6 . The composition of claim 5 , wherein the solid support is attached to the saframycin A or analogues thereof through a covalent linkage.
7 . The composition of claim 1 , wherein the polynucleotide and saframycin A or analogues thereof are covalently linked.
8 . The composition of claim 1 , wherein the polynucleotide is double-stranded DNA.
9 . The composition of claim 1 , wherein the polynucleotide include a sequence selected from the group consisting of 5′-GGG-3′, and 5′-GGPy-3′.
10 . The composition of claim 1 , wherein the GAPDH is bound to the polynucleotide and saframycin A or analogues thereof through a non-covalent interaction.
11 . The composition of claim 1 , wherein saframycin A or analogue thereof has the structure (I):
wherein R 1 is NR A R B , —OR A , —SR A , —C(═O)R A , —C(═S)R A , —S(O) 2 R A , or an aliphatic, heteroaliphatic, aryl, heteroaryl, (aliphatic)aryl, (aliphatic)heteroaryl, (heteroaliphatic)aryl, or (heteroaliphatic)heteroaryl moiety, wherein each occurrence of R A and R B is independently hydrogen, —(C═O)R C , —NHR C , —(SO 2 )R C , —OR C , or an aliphatic, heteroaliphatic, aryl, or heteroaryl moiety, or R A and R B , when taken together form an aryl, heteroaryl, cycloaliphatic, or cycloheteroaliphatic moiety, wherein each occurrence of R C is independently hydrogen, —OR D , —SR D , —NHR D , —(C═O)R D , or an aliphatic, heteroaliphatic, aryl, or heteroaryl moiety, wherein each occurrence of R D is independently hydrogen, a protecting group, or an aliphatic, heteroaliphatic, aryl, heteroaryl, acyl, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, or heteroarylthio moiety;
wherein R 2 is hydrogen, —OR E , ═O, —C(═O)R E , —CO 2 R E , —CN, —SCN, halogen, —SR E , —SOR E , —SO 2 R E , —NO 2 , —N(R E ) 2 , —NHC(O)R E , or an aliphatic, heteroaliphatic, aryl, or heteroaryl moiety, wherein each occurrence of R E is independently hydrogen, a protecting group, or an aliphatic, heteroaliphatic, aryl, heteroaryl, acyl, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, or heteroarylthio moiety;
wherein R 3 is hydrogen, a nitrogen protecting group, —COOR F , —COR F , —CN, or an aliphatic, heteroaliphatic, aryl, or heteroaryl moiety, wherein each occurrence of R F is independently hydrogen, a protecting group, or an aliphatic, heteroaliphatic, aryl, heteroaryl, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, or heteroarylthio moiety;
wherein R 4 and R 6 are each independently hydrogen, or an aliphatic, heteroaliphatic, aryl, heteroaryl, acyl, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, or heteroarylthio moiety;
wherein R 5 and R 7 are each independently hydrogen, —OR G , —C(═O)R G , —CO 2 R G , —CN, —SCN, halogen, —SR G , —SOR G , —SO 2 R G , —NO 2 , —N(R G ) 2 , —NHC(O)R G , or an aliphatic, heteroaliphatic, aryl or heteroaryl moiety, wherein each occurrence of R G is independently hydrogen, a protecting group, or an aliphatic, heteroaliphatic, aryl, heteroaryl, acyl, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, or heteroarylthio moiety;
wherein R 8 is hydrogen, alkyl, —OH, protected hydroxyl, ═O, —CN, —SCN, halogen, —SH, protected thio, alkoxy, thioalkyl, amino, protected amino, or alkylamino;
wherein m is 0-5;
wherein X 1 , X 2 , X 3 and X 4 are each independently hydrogen, —OR H , ═O, —C(═O)R H , —CO 2 R H , —CN, —SCN, halogen, —SR H , —SOR H , —SO 2 R H , —NO 2 , —N(R H ) 2 , —NHC(O)R H , or an aliphatic, heteroaliphatic, aryl, or heteroaryl moiety, wherein each occurrence of R H is independently hydrogen, a protecting group, or an aliphatic, heteroaliphatic, aryl, heteroaryl, acyl, alkoxy, aryloxy, alkylthio, arylthio, heteroaryloxy, or heteroarylthio moiety;
whereby if at least either X 1 and X 2 or X 3 and X 4 are doubly bonded to the 6-membered ring, then the dotted bonds in either or both of the 6-membered rings represent two single bonds and one double bond, and a quinone moiety is generated, or if at least either X 1 and X 2 or X 3 and X 4 are singly bonded to the 6-membered ring, then the dotted bonds in either or both of the 6-membered rings represent two double bonds and one single bond, and a hydroquinone moiety is generated;
whereby each of the foregoing aliphatic, heteroaliphatic and alkyl moieties may independently be substituted or unsubstituted, branched or unbranched, or cyclic or acyclic, and each of the foregoing aryl or heteroaryl moieties may independently be substituted or unsubstituted.
12 . A method of identifying a cellular target, the method comprising steps of:
providing a solid support; providing a polynucleotide; providing a chemical compound; attaching the polynucleotide to the solid support; contacting the chemical compound with the polynucleotide attached to the solid support under suitable conditions to form a binary complex between the chemical compound and the polynucleotide; contacting the binary complex attached to the solid support with a cell lysate under suitable conditions for a biological target of the binary complex to bind; and identifying biological target bound to binary complex.
13 . The method of claim 12 , wherein the solid support is a resin.
14 . The method of claim 12 , wherein the solid support is a plastic or glass plate.
15 . The method of claim 12 , wherein the polynucleotide is DNA.
16 . The method of claim 12 , wherein the polynucleotide is a modified DNA.
17 . The method of claim 12 , wherein the polynucleotide is RNA.
18 . The method of claim 12 , wherein the polynucleotide is a modified RNA.
19 . The method of claim 12 , wherein the polynucleotide contains a sequence of bases recognized by the chemical compound.
20 . The method of claim 12 , wherein the polynucleotide comprises up to 100 bases.
21 . The method of claim 12 , wherein the polynucleotide comprises up to 50 bases.
22 . The method of claim 12 , wherein the polynucleotide comprises up to 30 bases.
23 . The method of claim 12 , wherein the polynucleotide is double-stranded.
24 . The method of claim 12 , wherein the polynucleotide is single-stranded.
25 . The method of claim 12 , wherein the chemical compound is a natural product.
26 . The method of claim 12 , wherein the chemical compound is a small molecule.
27 . The method of claim 12 , wherein the chemical compound is a saframycin.
28 . The method of claim 12 , wherein the chemical compound is a saframycin analogue.
29 . The method of claim 12 , wherein the chemical compound is ecteinascidin 743.
30 . The method of claim 12 , wherein the chemical compound is a protein.
31 . The method of claim 12 , wherein the chemical compound is a peptide.
32 . The method of claim 12 , wherein the chemical compound alkylates the polynucleotide.
33 . The method of claim 12 , wherein the chemical compound covalently binds to the polynucleotide.
34 . The method of claim 12 , wherein the suitable conditions are physiological conditions.
35 . The method of claim 12 , wherein the suitable conditions includes a pH between 7.2 and 7.5.
36 . The method of claim 12 , wherein the suitable conditions include a temperature ranging from 20-40° C.
37 . The method of claim 12 , wherein the suitable conditions include a temperature of approximately 37° C.
38 . The method of claim 12 , wherein the suitable conditions include a temperature of approximately 25° C.
39 . The method of claim 12 , wherein the cell lysate is a human cell lysate.
40 . The method of claim 12 , wherein the cell lysate is a human tumor cell lysate.
41 . The method of claim 12 , wherein the cell lysate is derived from a tissue or organ.
42 . The method of claim 12 , wherein the cell lysate is a mammalian cell lysate.
43 . The method of claim 12 , wherein the cell lysate is a yeast cell lysate.
44 . The method of claim 12 , wherein the cell lysate is a bacterial cell lysate.
45 . The method of claim 12 , wherein the step of identifying comprises sequencing the target.
46 . A method of identifying a cellular target, the method comprising steps of:
providing a solid support; providing a polynucleotide; providing a chemical compound; attaching the chemical compound to the solid support; contacting the polynucleotide with the chemical compound attached to the solid support under suitable conditions to form a binary complex between the chemical compound and the polynucleotide; contacting the binary complex attached to the solid support with a cell lysate under suitable conditions for a biological target of the binary complex to bind; and identifying biological target bound to the binary complex.
47 . A method of screening, the method comprising steps of:
providing at least one chemical compound; providing a polynucleotide attached to a solid support; providing a target; contacting the polynucleotide attached to a solid support with a chemical compound under suitable conditions for the chemical compound to bind the polynucleotide; contacting the target with the polynucleotide attached to a solid support after it has been contacted with the chemical compound; and quantitating the amount of target bound to the solid support for each chemical compound.
48 . The method of claim 47 , wherein the step of providing at least one chemical compound comprises providing at least 100 chemical compounds.
49 . The method of claim 47 , wherein the step of providing at least one chemical compounds comprises providing a combinatorial library of chemical compounds.
50 . The method of claim 47 , wherein the solid support is a multi-well plate.
51 . The method of claim 47 , wherein the target is GAPDH.
52 . The method of claim 47 , wherein the target is HMG 1.
53 . A kit for assaying comprising
a multi-well plate modified to bind polynucleotides; a target; and reagents for detecting bound target quantifiably.
54 . The kit of claim 53 further comprising a polynucleotide.
55 . The kit of claim 53 , wherein the target is GAPDH.
56 . The kit of claim 53 , wherein the reagents comprises antibodies.
57 . The kit of claim 53 , wherein the reagents comprises fluorescent dye-labeled antibodies.
58 . The kit of claim 53 , wherein the reagents comprises antibodies directed against GAPDH.
59 . A method of screening, the method comprising steps of:
providing at least one chemical compound; providing a polynucleotide attached to a solid support; providing a GAPDH; providing a saframycin or saframycin analogue; contacting the polynucleotide attached to a solid support with the saframycin or saframycin analogue under suitable conditions for the saframycin to bind the polynucleotide; contacting the binary complex of saframycin and polynucleotide with a chemical compound under suitable conditions that the chemical compound competes with the saframycin for binding to the polynucleotide; contacting the target with the polynucleotide attached to a solid support after it has been contacted with the chemical compound; and quantitating the amount of target bound to the solid support for each chemical compound.
60 . Compounds identified by the method of claim 47 or 59 .Join the waitlist — get patent alerts
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