Compositions, systems, and methods for modulating a target gene
Methods of regulating expression of a target gene in a cell are provided. Aspects of the methods contacting the cell with a compound having a first moiety covalently linked to a second moiety, wherein: (i) the first moiety exhibits specific binding to a first endogenous protein; (ii) the second moiety exhibits specific binding to a second endogenous protein distinct from the first endogenous protein; and (iii) upon contacting the cell, the first endogenous protein and the second endogenous protein are spatially complexed to each other via the compound to yield a gain-of-function in the cell and thereby activate a process not normally controlled by either of the first and second endogenous proteins. Embodiments of the methods provide one or more beneficial features, including but not limited to: utilization of less than about 50% of an amount of the second endogenous protein present in the cell; mediation of the gain-of-function with an EC50 of less than about 1 micromolar; modulation of expression of the target gene in less than or equal to about 16 hours; etc. Also provided are compositions that find use in practicing methods of the disclosure.
1 . A method of modulating at least one BCL-6 target gene in a cell, the method comprising:
contacting the cell with a chemical inducer of proximity (CIP) comprising a structure of BR-L-BC, wherein the CIP has a molecular weight of 5,000 g/mol or less,
wherein:
BR comprises a ligand that binds to a bromodomain of bromodomain-containing protein 4 (BRD4);
BC comprises a ligand that binds to a BTB domain of B-cell lymphoma 6 (BCL-6); wherein BC is of Formula IB:
where:
D is selected from a bond, alkyl, amide, ester, carbamate, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino and substituted acylamino;
E is —CH or nitrogen;
G is nitrogen or CR 23 , wherein R 23 is selected from hydrogen, C(1-4) alkyl, —O—C(1-14) alkyl, —O—C(1-4) haloalkyl, C(1-4) haloalkyl and halogen;
J is —CH or nitrogen;
M is —CH or nitrogen;
K is —CH 2 , O, S or —NH;
represents a bond to the linker; and
each of R 16 , R 17 , R 18 , R 19 , R 20 , R 21 R 22 is independently selected from hydrogen, halogen, hydroxyl, alkoxyl, cyano, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, thiol, substituted thiol, sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfoximine or substituted sulfoximine; or
BC is of formula IB7:
where:
D is selected from a bond, alkyl, amide, ester, carbamate, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino and substituted acylamino;
E is —CH or nitrogen;
G is nitrogen or CR 23 , wherein R 23 is selected from hydrogen, C(1-4) alkyl, —O-C(1-14) alkyl, —O—C(1-4) haloalkyl, C(1-4) haloalkyl and halogen;
J is —CH or nitrogen;
M is —CH or nitrogen;
K is —CH 2 , O, S or NH;
represents a bond to the linker; and
each of R 16 , R 17 , R 18 , R 19 and R 21 is independently selected from hydrogen, halogen, hydroxyl, alkoxyl, cyano, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, thiol, substituted thiol, sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfoximine or substituted sulfoximine, and
L comprises a linker,
wherein upon contacting the cell, BCL-6 and BRD4 are spatially complexed to each other via the CIP to yield a gain-of-function in the cell,
wherein the gain-of-function is characterized by increased expression of the BCL-6 target gene in a manner dependent on presence of BRD4 bound to the CIP,
thereby modulating the at least one BCL-6 target gene.
2 . The method of claim 1 , wherein the at least one BCL-6 target gene comprises a proapoptotic gene.
3 . The method of claim 2 , wherein the proapoptotic gene is selected from the group consisting of: TP53, PUMA (BBC3), BIM (BCL2L11), BID, BAX, BAK, BOK, BAD, HRK, BIK, BMF, NOXA, CASP8, and CASP10.
4 . The method of claim 2 , wherein the modulating the at least one BCL-6 target gene comprises increasing expression of the proapoptotic gene in the cell.
5 . The method of claim 4 , wherein the method results in apoptosis of the cell.
6 . The method of claim 1 , wherein both BCL-6 and BRD4 are endogenously expressed in the cell.
7 . The method of claim 1 , wherein the cell is a diseased cell.
8 . The method of claim 7 , wherein the diseased cell overexpresses both BCL-6 and BRD4 relative to a non-diseased cell.
9 . The method of claim 7 , wherein the diseased cell is a cancer cell.
10 . The method of claim 1 , wherein the contacting comprises administering the CIP to a subject comprising the cell.
11 . The method of claim 10 , wherein the subject has a disease, a disorder, or a condition.
12 . The method of claim 11 , wherein the administering the CIP results in treatment of the disease, disorder, or condition, or a symptom associated therewith.
13 . The method of claim 11 , wherein the disease, disorder, or condition is a malignancy.
14 . The method of claim 13 , wherein the malignancy is small cell lung cancer (SCLC), diffuse large B-cell lymphoma (DLBCL), or a malignancy with high-level expression of BCL-6 protein.
15 . The method of claim 14 , wherein the DLBCL is refractory DLBCL.
16 . The method of claim 14 , wherein the refractory DLBCL is CHOP-resistant DLBCL.
17 . The method of claim 13 , wherein the malignancy has a mutation in TP53.
18 . The method of claim 10 , wherein the subject is a human.
19 . The method of claim 1 , wherein BC is of formula IB:
where:
D is selected from a bond, alkyl, amide, ester, carbamate, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino and substituted acylamino;
E is —CH or nitrogen;
G is nitrogen or CR 23 , wherein R 23 is selected from hydrogen, C(1-4) alkyl, —O—C(1-14) alkyl, —O—C(1-4) haloalkyl, C(1-4) haloalkyl and halogen;
J is —CH or nitrogen;
M is —CH or nitrogen;
K is —CH 2 , O, S or —NH;
represents a bond to the linker; and
each of R 16 , R 17 , R 18 , R 19 , R 20 , R 21 R 22 is independently selected from hydrogen, halogen, hydroxyl, alkoxyl, cyano, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, thiol, substituted thiol, sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfoximine or substituted sulfoximine.
20 . The method of claim 1 , wherein BC is of formula IB7:
where:
D is selected from a bond, alkyl, amide, ester, carbamate, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino and substituted acylamino;
E is —CH or nitrogen;
G is nitrogen or CR 23 , wherein R 23 is selected from hydrogen, C(1-4) alkyl, —O—C(1-14) alkyl, —O—C(1-4) haloalkyl, C(1-4) haloalkyl and halogen;
J is —CH or nitrogen;
M is —CH or nitrogen;
K is —CH 2 , O, S or NH;
represents a bond to the linker; and
each of R 16 , R 17 , R 18 , R 19 and R 21 is independently selected from hydrogen, halogen, hydroxyl, alkoxyl, cyano, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, acyl, substituted acyl, carboxyl, alkoxycarbonyl, substituted alkoxycarbonyl, aminoacyl, substituted aminoacyl, amino, substituted amino, acylamino, substituted acylamino, thiol, substituted thiol, sulfoxide, substituted sulfoxide, sulfone, substituted sulfone, sulfoximine or substituted sulfoximine.
21 . The method of claim 1 , wherein BC is of formula IB1:
22 . The method of claim 1 , wherein the CIP has a molecular weight of 2500 Daltons or less.
23 . The method of claim 1 , wherein L has a structure selected from the group consisting of:
wherein m, n and p are independently selected from 0 or an integer of from 1-12.