Supplemental Table 1. KIT, BRAF, and NRAS mutations assayed by mass-spectroscopy genotyping.

KIT: A829P, D816H, D816V, K642E, L576P, N566D, R634W, V559A, V559D, V560D, V825A, Y553N.

BRAF: A598V, D594E, D594G, D594V, E586K, F595S, G455R, G464E, G464R, G464V, G466A, G466E, G466R, G469E, G469R, G469S, G469V, G606E, G615R, G616P, H608R, I582M, I592M, I592V, K439Q, K475M, K601E, K601N, L588R, L597Q, L597R, L597S, L597V, L618S, R444W, S605C, S605N, S607P, S614P, S616P, T599I, V600A, V600D, V600E, V600E, V600G, V600K, V600M, V600R, W604G, W619R.

NRAS: A18T, E49K, G12A G12C, G12D, G12R, G12S, G12V, G13A, G13C, G13D, G13R, G13V, Q61E, Q61H, Q61K, Q61L, Q61P, Q61Q, Q61R, S65C, Y64N

Supplemental Methods:

All molecular dynamics (MD) simulations were carried out by using the sander and the pmemd module within the AMBER 9 suite of programs (1), and the parm99 all-atom force field by Cornell et al (2). working in parallel on 64 processors of the IBM/BCX calculation cluster of the CINECA calculation centre of Bologna. The crystallographic coordinates of the active KIT structure in complex with the imatinib (1T46.pdb,)(3) was obtained from the protein data bank was employed as starting geometries for both the wild-type and the mutant protein simulations. For having the same reference conditions, the crystallographic structure of the complex was modified substituting imatinib with dasatinib, using a superposition technique between the binding site of 1T46.pdb and the one of 2GQG.pdb (KIT-dasatinib complex). The mutation was introduced into the corresponding wild-type KIT structure using the Biopolymer module of Insight II (v. 2001, Accelrys Inc., San Diego, CA, USA) by swapping the mutant residue into the specific site and choosing the appropriate residue rotamer according to a validated procedure (4). All MD simulations were run with explicit water, using periodic boundary conditions, and the particle mesh Ewald approach to introduce long-range electrostatic effects. An opportune number of counterions was introduced using the leap module of AMBER 9, replacing the water molecules, when superposition occurred, to reproduce the correct salt concentration present in an organic solution. All energetic analysis were done for only a single MD trajectory of each KIT/ligand complex considered, with unbound protein and substrate snapshots taken from the snapshots of that trajectory. According to the so-called molecular mechanics/Poisson-Boltzmann surface area method (MM_PBSA)(5), the binding free energy Gbind between imatinib (or dasatinib) and KIT can be calculated as:

Gbind = EMM + Gsolv - TS	[1]

where:

Gsolv = GPB + GNP	[2]

The term EMM on eq. [1] denotes the sum of molecular mechanics (MM) energies of the molecules, and can be further split into contributions from electrostatic (EEL) and van der Waals (EvdW) energies:

EMM = EEL +EvdW	[3]

Gsolv in eqs. [1] and [2] represents the solvation free energy. The polar solvation process (i.e., GPB in eq. [2]) is equivalent to the transfer of a protein from one medium with dielectric constant equal to that of the interior of the protein to another medium with dielectric constant equal to that of the exterior of the protein. This term yields a free energy because it corresponds to the work done to reversibly charge the solute, and it is a polarization free energy because the work goes to the polarization of the solvent. The non polar solvation contribution (i.e., GNP in eq. [2]) includes cavity creation in water and van der Waals interactions between the modeled nonpolar protein and water molecules. This term can be imagined as transferring a nonpolar molecule with the shape of the protein from vacuum to water. The polar component of Gsolv was evaluated with the Poisson-Boltzmann (PB) approach (6), while the non-polar contribution to the solvation energy was calculated with the molsurf program present in AMBER 9 (7). Finally, the normal-mode analysis approach was followed, using the nmode program present in MM_PBSA, to estimate the entopic contribution –TS.

Supplemental References

1. Case DA, Darden TA, Cheatham TE III., Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Pearlman DA, Crowley M, Walker RC, Zhang W, Wang B, Hayik S, Roitberg A, Seabra G, Wong KF, Paesani F, Wu X, Brozell S, Tsui V, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Beroza P, Mathews DH, Schafmeister C, Ross WS, Kollman PA. AMBER 9, University of California, San Francisco, CA USA, 2006.

2. Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA. A second generation force field for the simulation of proteins and nucleic acids. J Am Chem Soc 1995;117:5179-97.

Mol CD, Lim KB, Sridhar V, Zou H, Chien EY, Sang BC, Nowakowski J, Kassel DB, Cronin CN, McRee DE. Structure of a c-Kit product complex reveals the basis for kinase transactivation. J Biol Chem 2003;278:31461-4.

Pricl S, Fermeglia M, Ferrone M, Tamborini E. T315I-mutated Bcr-Abl in chronic myeloid leukemia and imatinib: insights from a computational study. Mol Cancer Ther 2005;4:1167-74.

Kollman PA, Massova I, Reyes C, Kuhn B, Huo S, Chong L, Lee M, Lee T, Duan Y, Wang W, Donini O, Cieplak P, Srinivasan J, Case DA, Cheatham TE. Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acct Chem Res 2000;33:889-97.

Sitkoff D, Sharp KA, Honig B. Accurate calculation of hydration free energies using macroscopic solvent models. J Phys Chem 1994;98:1978-1988.

Connoly ML. Analytical molecular surface calculation. J Appl Cryst 1983;16:548-58.