Novel poly(adp-ribose) polymerase genes

Patent metadata

Field	Value
Publication number	US-2016010067-A1
Application number	US-201514732603-A
Country	US
Kind code	A1
Filing date	Jun 5, 2015
Priority date	Jun 5, 1998
Publication date	Jan 14, 2016
Grant date	—

Abstract

The invention relates to poly(ADP-ribose)polymerase (PARP) homologs which have an amino acid sequence which has
a) a functional NAD + binding domain
and

b) no zinc finger sequence motif of the general formula

CX 2 CX m HX 2 C

in which
m is an integral value from 28 or 30, and the X radicals are, independently of one another, any amino acid;

and the functional equivalents thereof; nucleic acids coding therefor; antibodies with specificity for the novel protein; pharmaceutical and gene therapy compositions which comprise products according to the invention; methods for the analytical determination of the proteins and nucleic acids according to the invention; methods for identifying effectors or binding partners of the proteins according to the invention; novel PARP effectors; and methods for determining the activity of such effectors.

First claim

1 . A poly(ADP-ribose) polymerase (PARP) homolog derived from a human or non-human mammal which has an amino acid sequence which has
a) a functional NAD + binding domain and b) no zinc finger sequence motif of the general formula

CX 2 CX m HX 2 C

in which
m is an integral value from 28 or 30, and the X radicals are, independently of one another, any amino acid.

2 . A PARP homolog as claimed in claim 1 , wherein the functional NAD + binding domain comprises one of the following general sequence motifs:

PX n (S/T)GX 3 GKGIYFA,

(S/T)XGLR(I/V)XPX n (S/T)GX 3 GKGIYFA

or

LLWHG(S/T)X 7 IL(S/T)XGLR(I/V)XPX n (S/T)GX 3 GKGIYFAX 3 SKS

AXY

in which
n is an integral value from 1 to 5, and the X radicals are, independently of one another, any amino acid.

3 . A PARP homolog as claimed in claim 1 , comprising at least another one of the following part-sequence motifs:

LX 9 NX 2 YX 2 QLLX (D/E)X 10/11 WGRVG,

AX 3 FXKX 4 KTXNXWX 5 FX 3 PXK,

QXL(I/L)X 2 IX 9 MX 10 PLGKLX 3 QIX 6 L,

FYTXIPHXFGX 3 PP;

and

KX 3 LX 2 LXDIEXAX 2 L,

in which the X radicals are, independently of one another, any amino acid.

4 . A PARP homolog as claimed in claim 1 , selected from human PARP homologs, which has the amino acid sequence shown in SEQ ID NO: 2 (human PARP2) or SEQ ID NO: 4 or 6 (human PARP3 type 1 or 2); or murine PARP homologs which have the amino acid sequence shown in SEQ ID NO:8 (mouse PARP long form) or SEQ ID No:10 (mouse PARP short form).

5 . A binding partner for having specificity for PARP homologs as claimed in claim 1 , selected from
a) antibodies and fragments thereof, b) protein-like compounds which interact with a part sequence of the protein, and c) low molecular weight effectors which modulate the catalytic PARP activity or another biological function of a PARP molecule.

6 . A nucleic acid comprising
a) a nucleotide sequence coding for at least one PARP homolog as claimed in claim 1 , or the complementary nucleotide sequence thereof; b) a nucleotide sequence which hybridizes with a sequence as specified in a) under stringent conditions; or c) nucleotide sequences which are derived from the nucleotide sequences defined in a) and b) through the degeneracy of the genetic code.

7 . A nucleic acid as claimed in claim 6 , comprising
a) nucleotides +3 to +1715 shown in SEQ ID NO:1; b) nucleotides +242 to +1843 shown in SEQ ID NO:3; c) nucleotides +221 to +1843 shown in SEQ ID NO:5; d) nucleotides +112 to +1710 shown in SEQ ID NO:7; or e) nucleotides +1 to +1584 shown in SEQ ID NO:9.

8 . An expression cassette comprising, under the genetic control of at least one regulatory nucleotide sequence, at least one nucleotide sequence as claimed in claim 6 .

9 . A recombinant vector comprising at least one expression cassette as claimed in claim 8 .

10 . A recombinant microorganism comprising at least one recombinant vector as claimed in claim 9 .

11 . A transgenic mammal comprising a vector as claimed in claim 9 .

12 . A PARP-deficient mammal or PARP-deficient eukaryotic cell, in which functional expression of at least one gene which codes for a PARP homolog as claimed in claim 1 is inhibited.

13 . An in vitro detection method for PARP inhibitors, which comprises
a) incubating an unsupported or supported polyADP
ribosylatable target with a reaction mixture comprising
a1) a PARP homolog as claimed in claim 1 ,
a2) a PARP activator; and
a3) a PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected;
b) carrying out the polyADP ribosylation reaction; and c) determining the polyADP ribosylation of the target qualitatively or quantitatively.

14 . The method as claimed in claim 13 , wherein the PARP homolog is preincubated with the PARP activator and the PARP inhibitor or an analyte in which at least one PARP inhibitor is suspected, before the polyADP ribosylation reaction is carried out.

15 . The method as claimed in claim 13 , wherein the polyADP-ribosylatable target is a histone protein.

16 . The method as claimed in claim 13 , wherein the PARP activator is activated DNA.

17 . The method as claimed in claim 13 , wherein the polyADP ribosylation reaction is started by adding NAD + .

18 . The method as claimed in claim 13 , wherein the polyADP ribosylation of the supported target is determined using anti-poly(ADP-ribose) antibodies.

19 . The method as claimed in claim 13 , wherein the unsupported target is labeled with an acceptor fluorophore.

20 . The method as claimed in claim 19 , wherein the polyADP ribosylation of the unsupported target is determined using anti-poly(ADP-ribose) antibody which is labeled with a donor fluorophore which is able to transfer energy to the acceptor fluorophore.

21 . The method as claimed in claim 19 , wherein the target is biotinylated histone, and the acceptor fluorophore is coupled thereto via avidin or streptavidin.

22 . The method as claimed in claim 20 , wherein the anti-poly(ADP-ribose) antibody carries a europium cryptate as donor fluorophore.

23 . An in vitro screening method for binding partners for a PARP molecule, which comprises
a1) immobilizing at least one PARP homolog as claimed in claim 1 on a support; b1) contacting the immobilized PARP homolog with an analyte in which at least one binding partner is suspected; and c1) determining, where appropriate after an incubation period, analyte constituents bound to the immobilized PARP homolog; or a2) immobilizing on a support an analyte which comprises at least one possible binding partner for a PARP molecule; b2) contacting the immobilized analyte with at least one PARP homolog as claimed in claim 1 for which a binding partner is sought; and c

Assignees

• Abbvie Deutschland

Inventors

• Kock Michael
• Höger Thomas
• Kröger Burkhard
• Otterbach Bernd
• Lubisch Wilfried
• Lemaire Hans-Georg

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61K49/00

  Preparations for testing in vivo · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N9/1077 Primary

  Pentosyltransferases (2.4.2) · CPC title