Thermodynamics of nucleotide and inhibitor binding to wild-type and ispinesib-resistant forms of human kinesin spindle protein.
Current antimitotic cancer chemotherapy based on vinca alkaloids and taxanes target tubulin, a protein required not only for mitotic spindle formation but also for the overall structural integrity of terminally differentiated cells. Among many innovations targeting specific mitotic events, inhibition of motor enzymes including KSP (or Eg5) has been validated as a highly productive approach. Many reported KSP inhibitors bind to an induced allosteric site near the site of ATP hydrolysis, and some have been tested in clinical trials with varying degrees of success. This allosteric site was defined in detail by X-ray crystallography of inhibitor complexes, yet complementary information on binding thermodynamics is still lacking. Using two model ATP-uncompetitive inhibitors, monastrol and ispinesib, we report here the results of thermal denaturation and isothermal titration calorimetric studies. These binding studies were conducted with the wild-type KSP motor domain as well as two ispinesib mutants (D130V and A133D) identified to confer resistance to ispinesib treatment. The thermodynamic parameters obtained were placed in the context of the available structural information and corresponding models of the two ispinesib-resistant mutants. The resulting overall information formed a strong basis for future structure-based design of inhibitors of KSP and related motor enzymes.
Sheth
Payal R
PR
Protein Science Department, Schering-Plough Research Institute, 320 Bent Street, Cambridge, Massachusetts 02141, USA. payal.sheth@spcorp.com
Basso
Andrea
A
Duca
José S
JS
Lesburg
Charles A
CA
Ogas
Polina
P
Gray
Kimberly
K
Nale
Lissette
L
Mannarino
Anthony F
AF
Prongay
Andrew J
AJ
Le
Hung V
HV
D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D019943:Amino Acid Substitution; D001549:Benzamides / Q000378:metabolism / Q000494:pharmacology*; D055162:Biocatalysis; D002151:Calorimetry; D002942:Circular Dichroism; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D016547:Kinesins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D011743:Pyrimidines / Q000737:chemistry / Q000378:metabolism; D011799:Quinazolines / Q000378:metabolism / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D013871:Thiones / Q000737:chemistry / Q000378:metabolism; D044366:Transition Temperature
D001549:Benzamides; D004791:Enzyme Inhibitors; C470243:KIF11 protein, human; D009711:Nucleotides; D011743:Pyrimidines; D011799:Quinazolines; D011993:Recombinant Fusion Proteins; D013871:Thiones; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; C400223:monastrol; D000255:Adenosine Triphosphate; C508757:ispinesib; D000251:Adenosine Triphosphatases; D016547:Kinesins; D008274:Magnesium