Novel benzimidazole inhibitors bind to a unique site in the kinesin spindle protein motor domain.
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.
Sheth
Payal R
PR
Protein Science Department, Merck Research Laboratories,320 Bent Street, Cambridge, Massachusetts 02141, USA. payal.sheth@spcorp.com
Shipps
Gerald W
GW
Seghezzi
Wolfgang
W
Smith
Catherine K
CK
Chuang
Cheng-Chi
CC
Sanden
David
D
Basso
Andrea D
AD
Vilenchik
Lev
L
Gray
Kimberly
K
Annis
D Allen
DA
Nickbarg
Elliott
E
Ma
Yao
Y
Lahue
Brian
B
Herbst
Ronald
R
Le
Hung V
HV
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
D000970:Antineoplastic Agents; D001549:Benzamides; D001562:Benzimidazoles; C470243:KIF11 protein, human; D009711:Nucleotides; D011799:Quinazolines; C508757:ispinesib; C031000:benzimidazole; D000251:Adenosine Triphosphatases; D016547:Kinesins