title: Targeting the replication checkpoint using SCH 900776, a potent and functionally selective CHK1 inhibitor identified via high content screening.
abstract: Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds.
authors.affiliation: Merck Research Laboratory, Cambridge, Masachusetts, USA.
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Guzi
authors.forename: Kamil
authors.initials: K
authors.lastname: Paruch
authors.forename: Michael P
authors.initials: MP
authors.lastname: Dwyer
authors.forename: Marc
authors.initials: M
authors.lastname: Labroli
authors.forename: Frances
authors.initials: F
authors.lastname: Shanahan
authors.forename: Nicole
authors.initials: N
authors.lastname: Davis
authors.forename: Lorena
authors.initials: L
authors.lastname: Taricani
authors.forename: Derek
authors.initials: D
authors.lastname: Wiswell
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Seghezzi
authors.forename: Ervin
authors.initials: E
authors.lastname: Penaflor
authors.forename: Bhagyashree
authors.initials: B
authors.lastname: Bhagwat
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Danling
authors.initials: D
authors.lastname: Gu
authors.forename: Yunsheng
authors.initials: Y
authors.lastname: Hsieh
authors.forename: Suining
authors.initials: S
authors.lastname: Lee
authors.forename: Ming
authors.initials: M
authors.lastname: Liu
authors.forename: David
authors.initials: D
authors.lastname: Parry
mesh_terms: D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000008:administration & dosage / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D017209:Apoptosis / Q000187:drug effects; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000008:administration & dosage / Q000494:pharmacology; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003497:Cyclic N-Oxides; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004261:DNA Replication / Q000187:drug effects*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D006657:Histones / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007212:Indolizines; D051379:Mice; D008819:Mice, Nude; D015394:Molecular Structure; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011720:Pyrazoles / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D011726:Pyridinium Compounds / Q000008:administration & dosage / Q000494:pharmacology; D011743:Pyrimidines / Q000008:administration & dosage / Q000737:chemistry / Q000494:pharmacology*; D034622:RNA Interference; D047368:Tumor Burden / Q000187:drug effects; D023041:Xenograft Model Antitumor Assays; D000093542:Gemcitabine
chemical_list: D000964:Antimetabolites, Antineoplastic; D019086:Bridged Bicyclo Compounds, Heterocyclic; D003497:Cyclic N-Oxides; C468783:H2AX protein, human; D006657:Histones; D007212:Indolizines; C559815:MK-8776; D047428:Protein Kinase Inhibitors; D011720:Pyrazoles; D011726:Pyridinium Compounds; D011743:Pyrimidines; D003841:Deoxycytidine; C553669:dinaciclib; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; C578633:Chek2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D018844:Cyclin-Dependent Kinases; D000093542:Gemcitabine