Methods for modulating checkpoint activation through TopBP1

Abstract

ATR kinase is a key regulator of checkpoint responses to incompletely replicated and damaged DNA. Without this checkpoint, cells will enter mitosis prematurely, likely resulting in cell death. The invention provides methods and reagents to either block or activate the activation of the ATR kinase checkpoint, through, for example, either blocking or activating the expression of an ATR activator TopBP1. The invention also provides screening methods to identify additional ToBP1 inhibitors or activators that may be used to modulate the activity of the ATR checkpoint.

1 . A method for modulating ATR activation by TopBP1, comprising administering one or more modulator of TopBP1 activity. 
     
     
         2 . The method of  claim 1 , wherein the modulator affects the transcription and/or expression of TopBP1, affects the binding of TopBP1 to the ATR-ATRIP complex, and/or affects the activation of the ATR kinase activity by TopBP1. 
     
     
         3 . The method of  claim 1 , wherein the modulator is an inhibitor of TopBP1 activity. 
     
     
         4 . The method of  claim 1 , wherein the inhibitor is: an siRNA, a microRNA, a shRNA, an antisense oligonucleotide, a ribozyme, a DNA enzyme, a Morpholino antisense, a small molecule inhibitor, an antibody or functional fragment thereof, a peptide, a dominant negative mutant TopBP1 or fragment thereof, or a peptidomimetic. 
     
     
         5 . The method of  claim 1 , wherein the modulator is an activator of TopBP1 activity. 
     
     
         6 . The method of  claim 5 , wherein the activator is: a TopBP1 activator, a TopBP1 transcriptional activator, a TopBP1 stabilizer, or an ATR activation domain of TopBP1. 
     
     
         7 . The method of  claim 1 , further comprising contacting ATR with one or more modulators of ATR activity. 
     
     
         8 . The method of  claim 1 , wherein ATR and TopBP1 are inside a cell. 
     
     
         9 . The method of  claim 8 , wherein the cell is a vertebrate cell. 
     
     
         10 . The method of  claim 1 , wherein ATR is at least about 90% identical to human or  Xenopus  ATR. 
     
     
         11 . The method of  claim 1 , wherein TopBP1 is at least about 90% identical to human or  Xenopus  TopBP1. 
     
     
         12 . A method for treating cancer, comprising administering to a patient in need thereof an effective amount of a therapeutic composition comprising an inhibitor of ATR activation by TopBP1. 
     
     
         13 . The method of  claim 12 , wherein the inhibitor is an inhibitor of TopBP1 activity. 
     
     
         14 . The method of  claim 13 , further comprising administering an inhibitor of ATR activity. 
     
     
         15 . The method of  claim 12 , further comprising administering a treatment and/or an agent that damages DNA and/or inhibits DNA replication. 
     
     
         16 . The method of  claim 15 , wherein the treatment is ionizing radiation. 
     
     
         17 . The method of  claim 15 , wherein the agent is a chemotherapeutic agent. 
     
     
         18 . A method to screen for a modulator of ATR activation by TopBP1, the method comprising:
 (1) providing a mixture comprising TopBP1 and ATR;   (2) contacting the mixture with a candidate compound;   (3) determining the binding of TopBP1 to ATR, and/or the activation of the kinase activity of ATR;   wherein a statistically significant change either in the binding of TopBP1 to ATR or the activation of the kinase activity of ATR or both in the presence of the test compound compared to those in the absence of the test compound is indicative that the test compound is a modulator of TopBP1 activation of ATR.   
     
     
         19 . The method of  claim 18 , further comprising determining the extent of ATR activity change by the test compound in the absence of TopBP1 in the mixture. 
     
     
         20 . The method of  claim 18 , wherein TopBP1 and ATR forms a complex in the mixture. 
     
     
         21 . The method of  claim 20 , wherein the complex further comprises ATRIP. 
     
     
         22 . The method of  claim 18 , wherein TopBP1 is a full length protein, or a functional fragment comprising amino acid sequences between the sixth and seventh BRCT domains of TopBP1. 
     
     
         23 . The method of  claim 18 , wherein TopBP1 or ATR is from human or  Xenopus.    
     
     
         24 . The method of  claim 18 , which is an in vivo assay. 
     
     
         25 . An ATR activator comprising a polypeptide at least about 90% identical to the ATR activation domain of TopBP1, said ATR activator activates the kinase activity of ATR. 
     
     
         26 . The ATR activator of  claim 25 , wherein said ATR activation domain of TopBP1 comprises residues 1050-1192 of human TopBP1.