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Most gastrointestinal stromal tumors (GISTs) exhibit aberrant activation of the receptor tyrosine kinase (RTK) KIT. The efficacy of the inhibitors imatinib mesylate and sunitinib malate in GIST patients has been linked to their inhibition of these mutant KIT proteins. However, patients on imatinib can acquire secondary KIT mutations that render the protein insensitive to the inhibitor. Sunitinib has shown efficacy against certain imatinib-resistant mutants, although a subset that resides in the activation loop, including D816H/V, remains resistant. Biochemical and structural studies were undertaken to determine the molecular basis of sunitinib resistance. Our results show that sunitinib targets the autoinhibited conformation of WT KIT and that the D816H mutant undergoes a shift in conformational equilibrium toward the active state. These findings provide a structural and enzymologic explanation for the resistance profile observed with the KIT inhibitors, Prospectively, they have implications for understanding oncogenic kinase mutants and for circumventing drug resistance. kinase inhibitor | signal transduction | targeted therapy | resistance mechanism | cancer

The crystallographic and kinetic characterization of two small molecule inhibitors that bind to an allosteric site in the proximity of the checkpoint kinase 1 (CHK1) substrate site which is a key element in the DNA damage response pathway and plays a crucial role in the S-G2-phase checkpoint is presented. Cocrystal structures of the inhibitors bound to CHK1 revealed an allosteric site, unique to CHK1, located in the C-terminal domain and consisting of a shallow groove linked to a small hydrophobic pocket and could find potential application for the development of highly selective CHK1 inhibitors.

Combination of kinetic, biophysical, and structural methods were applied to perform highly purified preparation and characterize the receptor tyrosine kinase VEGFR2 catalytic domain without (VEGFR2-CD) and with (VEGFR2-CD/JM) the juxtamembrane (JM) domain. The results obtained provide insight into the thermodynamic origin of the potency differences of VEGFR2 JM domain and identified potential functions of the VEGFR2 JM domain with implications to both receptor biology and inhibitor design.

Modifications to a 7 T nano-LC micro-ESI Fr-ICR mass spectrometer, including a shorter octopole, ~100% duty cycle, improved nano-LC micro-ESI emitter tips, and reverse-phase HPLC resins that require no ion-pairing agent, combine to achieve attomole detection limit. Three peptides in a mixture totaling 500 attomoles (amol) each in water (10 [micro]L, 50 amol/[micro]L) are separated and detected, demonstrating detection from a mixture at low endogenous biological concentration. Two peptides in a mixture totaling 500 amol each in artificial cerebrospinal fluid (1 [micro]L, 500 amol/[micro]L) are separated and detected, demonstrating detection from a mixture at a biological concentration in a biological solvent. The highest sensitivity is attained with [arg.sup.8]-vasotocin, in which a total of 300 amol is detected in artificial cerebrospinal fluid (1 [micro]L, 300 amol/[micro]L) and a total of 100 amol in water (1 [micro]L, 100 amol/[micro]L). [Arg.sup.8]-vasotocin isolated from the pineal gland of rainbow trout is detected, demonstrating the ability of Fr-ICR to detect and identify a true endogenous biological analyte.