title: Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.
abstract: Affinity selection-mass spectrometry (AS-MS) screening of kinesin spindle protein (KSP) followed by enzyme inhibition studies and temperature-dependent circular dichroism (TdCD) characterization was utilized to identify a series of benzimidazole compounds. This series also binds in the presence of Ispinesib, a known anticancer KSP inhibitor in phase I/II clinical trials for breast cancer. TdCD and AS-MS analyses support simultaneous binding implying existence of a novel non-Ispinesib binding pocket within KSP. Additional TdCD analyses demonstrate direct binding of these compounds to Ispinesib-resistant mutants (D130V, A133D, and A133D + D130V double mutant), further strengthening the hypothesis that the compounds bind to a distinct binding pocket. Also importantly, binding to this pocket causes uncompetitive inhibition of KSP ATPase activity. The uncompetitive inhibition with respect to ATP is also confirmed by the requirement of nucleotide for binding of the compounds. After preliminary affinity optimization, the benzimidazole series exhibited distinctive antimitotic activity as evidenced by blockade of bipolar spindle formation and appearance of monoasters. Cancer cell growth inhibition was also demonstrated either as a single agent or in combination with Ispinesib. The combination was additive as predicted by the binding studies using TdCD and AS-MS analyses. The available data support the existence of a KSP inhibitory site hitherto unknown in the literature. The data also suggest that targeting this novel site could be a productive strategy for eluding Ispinesib-resistant tumors. Finally, AS-MS and TdCD techniques are general in scope and may enable screening other targets in the presence of known drugs, clinical candidates, or tool compounds that bind to the protein of interest in an effort to identify potency-enhancing small molecules that increase efficacy and impede resistance in combination therapy.
authors: Protein Science Department, Merck Research Laboratories,320 Bent Street, Cambridge, Massachusetts 02141, USA. payal.sheth@spcorp.com
authors: Payal R
authors: PR
authors: Sheth
authors: Gerald W
authors: GW
authors: Shipps
authors: Wolfgang
authors: W
authors: Seghezzi
authors: Catherine K
authors: CK
authors: Smith
authors: Cheng-Chi
authors: CC
authors: Chuang
authors: David
authors: D
authors: Sanden
authors: Andrea D
authors: AD
authors: Basso
authors: Lev
authors: L
authors: Vilenchik
authors: Kimberly
authors: K
authors: Gray
authors: D Allen
authors: DA
authors: Annis
authors: Elliott
authors: E
authors: Nickbarg
authors: Yao
authors: Y
authors: Ma
authors: Brian
authors: B
authors: Lahue
authors: Ronald
authors: R
authors: Herbst
authors: Hung V
authors: HV
authors: Le
mesh_terms: D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000970:Antineoplastic Agents / Q000037:antagonists & inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D001549:Benzamides / Q000378:metabolism; D001562:Benzimidazoles / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D001665:Binding Sites; D002942:Circular Dichroism; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry; D009711:Nucleotides / Q000037:antagonists & inhibitors / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011799:Quinazolines / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D001562:Benzimidazoles; C470243:KIF11 protein, human; D009711:Nucleotides; D011799:Quinazolines; C508757:ispinesib; C031000:benzimidazole; D000251:Adenosine Triphosphatases; D016547:Kinesins