US2022206008A1PendingUtilityA1
Screening methods for parp modulators
Est. expiryApr 30, 2038(~11.8 yrs left)· nominal 20-yr term from priority
Inventors:Timothy J.N. WigleDanielle Johnston BlackwellKevin Wayne KuntzMelissa Marie VasbinderLaurie B. SchenkelKerren Kalai Swinger
G01N 33/533G01N 33/582C07D 495/04G01N 33/573G01N 2333/91142C07D 491/22G01N 33/542C07F 5/027C07D 493/10
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Claims
Abstract
The present disclosure is related to methods of identifying Poly(ADP-ribose) polymerases (PARP) inhibitors, and the methods of using PARP probes.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising:
combining (i) a polypeptide comprising a PARP catalytic domain wherein the polypeptide is labeled with a donor fluorophore, (ii) a PARP probe, wherein the PARP probe is labeled with an acceptor fluorophore, and (iii) a test compound; exposing the donor fluorophore to excitation light; measuring a signal produced by the acceptor fluorophore; and identifying the test compound as an inhibitor for PARP based on the signal produced by the acceptor fluorophore.
2 . The method of claim 1 , wherein the PARP probe is a compound having the structure:
or a salt thereof.
3 . The method of claim 1 , further comprising identifying the test compound as an inhibitor for PARP if the signal produced by the acceptor fluorophore is decreased as compared to a reference level.
4 . The method of claim 3 , wherein the reference level is the signal produced by the acceptor fluorophore in the absence of the test compound.
5 . The method of claim 1 , wherein the PARP is PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.
6 . The method of claim 1 , wherein the PARP probe is biotinylated, and the acceptor fluorophore is attached to streptavidin.
7 . The method of claim 1 , wherein the polypeptide comprises a polyhistidine tag.
8 . The method of claim 1 , wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.
9 . A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising:
combining (i) a polypeptide comprising a PARP catalytic domain, wherein the polypeptide is labeled with an acceptor fluorophore; (ii) a PARP probe, wherein the PARP probe is labeled with a donor fluorophore, and (iii) a test compound; exposing the donor fluorophore to excitation light; measuring a signal produced by the acceptor fluorophore in the presence of a test compound; and identifying the test compound as an inhibitor for PARP based on the signal produced by the acceptor fluorophore.
10 . The method of claim 9 , wherein the PARP probe is a compound having the structure:
or a salt thereof.
11 . The method of claim 9 , further comprising identifying the test compound as an inhibitor for PARP if the signal produced by the acceptor fluorophore is decreased as compared to a reference level.
12 . The method of claim 11 , wherein the reference level is the signal produced by the acceptor fluorophore in the absence of the test compound.
13 . The method of claim 9 , wherein the PARP is PARP1, PARP2, PARP3, PARP4 PARP5a, PARP5, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.
14 . The method of claim 9 , wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.
15 . A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising:
contacting a polypeptide comprising a PARP catalytic domain with a PARP probe in the presence of a test compound, wherein the PARP probe comprises a fluorophore; exposing the probe to polarized excitation light, thereby generating fluorescence; determining a fluorescence polarization value of the fluorescence; and identifying the test compound as an inhibitor for PARP based on the fluorescence polarization value of the fluorescence.
16 . The method of claim 15 , further comprising identifying the test compound as an inhibitor for PARP if the fluorescence polarization value of the fluorescence is decreased as compared to a reference level.
17 . The method of claim 16 , wherein the reference value is the fluorescence polarization value of the fluorescence in the absence of the test compound.
18 . The method of claim 15 , wherein the PARP probe is a compound having the structure:
or
or a salt thereof.
19 . The method of claim 15 , wherein the PARP is PARP1, PARP2, PARP3, PARP4, PARP5a, PARP5b, PARP6, TIPARP, PARP8, PARP10, PARP1l, PARP12, PARP13, PARP14, PARP15, or PARP16.
20 . The method of claim 15 , wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.
21 . A method of identifying an inhibitor for Poly (ADP-ribose) polymerase (PARP), the method comprising:
contacting a fusion polypeptide with a PARP probe that comprises a fluorophore, wherein the fusion polypeptide comprises a PARP catalytic domain and a luciferase enzyme; contacting the luciferase enzyme with a substrate to produce light, wherein the light can excite the fluorophore; measuring a signal produced by the fluorophore in the presence of a test compound; and identifying the test compound as an inhibitor for PARP based on the signal produced by the fluorophore.
22 . The method of claim 21 , further comprising identifying the test compound as an inhibitor for PARP if the signal produced by the fluorophore is decreased as compared to a reference level.
23 . The method of claim 22 , wherein the reference level is the signal produced by the fluorophore in the absence of the test compound.
24 . The method of claim 21 , wherein the PARP probe is a compound having the structure:
or a salt thereof.
25 . The method of claim 21 , wherein the fusion polypeptide comprises a sequence that is at least 85%, 90% or 95% identical to SEQ ID NO: 11.
26 . The method of claim 22 , wherein the PARP is PARP1, PARP2, PARP4, PARP5a, PARP5b, PARP3, PARP6, TIPARP, PARP8, PARP9, PARP10, PARP11, PARP12, PARP13, PARP14, PARP15, or PARP16.
27 . The method of claim 22 , wherein the test compound is a small molecule, a nucleic acid, a polypeptide, or an antibody or antigen-binding fragment thereof.
28 . The method of claim 1 , wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 456 to 657 of NP_056323.2 (SEQ ID NO: 1).
29 . The method of claim 1 , wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 808 to 1025 of NP_116178.2 (SEQ ID NO: 2).
30 . The method of claim 1 , wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 489 to 684 of NP_073587.1 (SEQ ID NO: 3).
31 . The method of claim 1 , wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 1611 to 1801 of NP_060024.2 (SEQ ID NO: 4).
32 . The method of claim 1 , wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 481 to 678 of NP_689828.1 (SEQ ID NO: 5).
33 . The method of claim 1 , wherein the polypeptide comprising a PARP catalytic domain comprises a sequence that is at least 85%, 90% or 95% identical to amino acid residues of 5 to 279 of NP_060321.3 (SEQ ID NO: 6).
34 . A PARP probe having a structure according to Formula (I):
or a salt thereof,
wherein:
L is a linking group having 5-30 spacer atoms selected from C, N, O, and S connecting the N atom of the piperidinyl group of Formula (I) with group A;
A is a fluorophore or an affinity tag.
35 . The PARP probe of claim 34 wherein L is
a is 0, 1, or 2;
b is 1-26; and
c is 0, 1, or 2;
wherein the sum of a+b+c is 1 to 26, or
L is a chain of 5-30 atoms in length comprising —(CH 2 CH 2 O) d — wherein d is 2-10.
36 . The PARP probe of claim 34 wherein A is:
or a salt thereof.
37 . The PARP probe of claim 34 which is a compound having the structure:
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