Ku inhibitors and their use
The present disclosure relates to certain compounds having binding affinity for Ku, and uses thereof. Specifically, the present disclosure relates to the use of Ku inhibitors as described herein in site-specific genome engineering technologies, including but not limited to CRISPR/Cas9, Zinc finger nuclease (ZFN), Transcription activator-like effector nuclease (TALEN), and meganuclease. The present disclosure also relates to kits useful for site-specific genome engineering that include at least one compound as described herein.
1 . A method of gene editing comprising
a. contacting a compound with at least one cell comprising at least one programmable nuclease,
wherein the at least one programmable nuclease comprises a Cas9 endonuclease; and
wherein the compound is of formula I
wherein
X is absent
Z is O or S;
R 1 is H, —CO 2 R 6 , or —S(O) 2 NR 6 R 7 ;
R 2 is H, —CO 2 R 6 , —CN, or tetrazolyl;
R 3 is C 1 -C 6 alkyl;
R 4 is selected from the group consisting of
R 5 is H;
R 6 and R 7 are each independently H, or C 1 -C 6 alkyl;
R 10 is halogen;
“*” represents the point of attachment as indicated in Formula I; and
is either a single bond or a pi-bond; and
b. contacting the at least one cell with a plasmid encoding a guide RNA (gRNA) comprising a Clustered Regularly Interspaced Short Palindromic repeat RNA (crRNA) and a trans-activating RNA (tracrRNA).
2 . The method of claim 1 , wherein the compound is of the formula Ia
3 . The method of claim 2 , wherein the compound is of the formula Ib
4 . The method of claim 1 , wherein R 1 is H, —CO 2 H, —S(O) 2 NH 2 , or —CO 2 Et; and R 2 is H, —CO 2 H, —CO 2 Et, —CN, or tetrazolyl, provided that at least one of R 1 and R 2 is not H.
5 . The method of claim 4 , wherein R 1 is H, and R 2 is tetrazolyl.
6 . The method of claim 4 , wherein R 1 is —S(O) 2 NH 2 and R 2 is H.
7 . The method of claim 1 , wherein the Cas9 endonuclease is encoded in a plasmid.
8 . The method of claim 1 , wherein the at least one cell is a bacterial cell, a mammalian cell, a yeast cell, or a plant cell.
9 . The method of claim 1 , wherein R 3 is methyl.
10 . The method of claim 1 , wherein R 4 is selected from the group consisting of
wherein * represents the point of attachment of R 4 to the amide nitrogen.
11 . The method of claim 1 , wherein the compound is selected from the group consisting of
12 . The method of claim 1 , wherein the compound is selected from the group consisting of
13 . The method of claim 1 , wherein X is absent,
14 . The method of claim 13 , wherein X is C
15 . The method of claim 1 , wherein
X is absent,
R 1 is H, —CO 2 H, —S(O) 2 NH 2 , or —CO 2 Et;
R 2 is H, —CO 2 H, —CO 2 Et, —CN, or tetrazolyl;
R 3 is methyl;
R 4 is selected from the group consisting of
“*” represents the point of attachment as indicated in Formula I; and
is either a single bond or a pi-bond.
16 . The method of claim 1 , wherein
X is absent claim 1 , wherein
R 1 is H, —CO 2 H, —S(O) 2 NH 2 , or —CO 2 Et;
R 2 is H, —CO 2 H, —CO 2 Et, —CN, or tetrazolyl;
R 3 is methyl;
R 4 is selected from the group consisting of
“*” represents the point of attachment as indicated in Formula I; and
is either a single bond or a pi-bond.
17 . A method of gene editing comprising
a. contacting a compound with at least one cell comprising at least one programmable nuclease,
wherein the at least one programmable nuclease comprises a Cas9 endonuclease; and
wherein the compound is of formula I
wherein
X is absent,
Z is O or S;
R 1 is H, —CO 2 H, —S(O) 2 NH 2 , or —CO 2 Et;
R 2 is H, —CO 2 H, —CO 2 Et, —CN, or tetrazolyl;
R 3 is methyl;
R 4 is selected from the group consisting of
“*” represents the point of attachment as indicated in Formula I; and
is either a single bond or a pi-bond; and
b. contacting the at least one cell with a plasmid encoding a guide RNA (gRNA) comprising a Clustered Regularly Interspaced Short Palindromic repeat RNA (crRNA) and a trans-activating RNA (tracrRNA).
18 . The method of claim 17 , wherein R 4 is selected from the group consisting of
19 . The method of claim 1 , wherein Z is O.
20 . The method of claim 17 , wherein Z is O.