Rational drug redesign to overcome drug resistance in cancer therapy: imatinib moving target

Abstract

Protein kinases are central targets for drug-based cancer treatment. To avoid functional impairment, the cell develops mechanisms of drug resistance, primarily based on adaptive mutations. Redesigning a drug to target a drug-resistant mutant kinase constitutes a therapeutic challenge. We approach the problem by redesigning the anticancer drug imatinib guided by local changes in interfacial de-wetting propensities of the C-Kit kinase target introduced by an imatinib-resistant mutation. The ligand is redesigned by sculpting the shifting hydration patterns of the target. The association with the modified ligand overcomes the mutation-driven destabilization of the induced fit. Consequently, the redesigned drug inhibits both mutant and wild-type kinase. The modeling effort is validated through molecular dynamics, test tube kinetic assays of downstream phosphorylation activity, high-throughput bacteriophage-display kinase screening, cellular proliferation assays, and cellular immunoblots. The inhibitor redesign reported delineates a molecular engineering paradigm to impair routes for drug resistance.

Figure 1

A, mean residence times of water molecules solvating the uncomplexed C-KIT wild-type (blue) and D816V mutant (pink) kinase domain. The mutation was introduced in silico, and the structures examined were obtained after 50 ns molecular dynamic equilibration of the uncomplexed kinases (Materials and Methods, Supplementary Material). Residue numbering follows PDB entry 1T46. The one-letter amino acid code was adopted for clarity. Columns, mean; bars, variances. Local dehydration propensities are signaled by relatively short residence times and represent weaknesses in the hydration shell of the target protein. Only residues in contact with the ligand in PDB complex 1T46 are indicated for clarity. A contact is defined by the presence of ligand atoms within a 6.2-Å sphere centered at the α-carbon. The changes in dehydration propensity introduced by the D816V mutation are most pronounced for residues F811 and A814. B, location of imatinib inhibitor with designed appended methyl group (yellow rectangle) relative to the activation loop of C-KIT kinase in the active (blue, chain B) conformation within the ligand-kinase complex PDB.1T46. The superimposed deactivating conformation (red, chain A) after full alignment of the autoinhibited form (PDB.1T45) with the active structure (PDB.1T46). Besides the activation loop (residues 808–820), the other two functional loops are also shown to give a better perspective of the inhibitor location within the ATP pocket: the P-loop (magenta, residues 594–604) and catalytic loop (light blue, residues 670–677). In addition, the side chains of the catalytic triad DFG (residues 810–812) are displayed for the active conformation. The chain conformation is indicated by virtual bonds joining α-carbons. Partially exposed F811-A814 backbone hydrogen bond involving a de-wetting hotspot in the active conformation (green). The two desolvation domains are given by 6.2-Å radius spheres (gray) centered at the α-carbons of the paired residues. By increasing the dehydration of F811 and A814, the highlighted methylation enhances the stabilization of the active loop upon ligand association to the active kinase. C, ribbon structure of C-Kit kinase target in complex with proposed imatinib modification, detailing the activation loop backbone (blue) and targeted de-wetting hotspot (green). Aligned inactive loop conformation (red).

Figure 2

The initial step in the synthesis of the imatinib derivative WBZ_7 (total synthesis in the Supplementary Material). Structure of WBZ_7, highlighting the methyl group (arrow) that substitutes the original hydrogen at position 6 in the pyperidine ring of imatinib.

Figure 3

A, downstream phosphorylation rates from spectrophotometric kinetic assay of active C-Kit kinase and active imatinib-resistant D816V mutant (Upstate, Millipore). Both kinases are inhibited by WBZ_7 (squares), whereas only the wild-type C-Kit is significantly inhibited by imatinib (triangles). Phosphorylation rate plots are displayed for wild-type kinase (blue) and for the D816V variant (red). Bars, dispersion over five runs for each kinetic assay (Materials and Methods). Each assay run consists of 11 measurements of maximum phosphorylation rate at 100-nmol/L intervals in increasing inhibitor concentration for each of the four kinase/inhibitor pairs. Notice that in contrast with imatinib, WBZ_7 is an inhibitor of the imatinib-resistant mutated as well as of the wild-type kinase. Fluctuations in initial enzyme concentrations are likely sources of experimental error particularly apparent at low inhibitor concentrations (K_m(app) ⪡ [ATP]). B to D, high-throughput screening of WBZ_7 (red) and imatinib (blue, control) over 240 human kinases displayed in a T7-bacteriophage library (Ambit Biosciences, Materials and Methods). Hit values are reported as % bound kinase.

Figure 4

A, cell proliferation assay of antitumor activity for WBZ_7 (hatched columns) and imatinib (solid columns) on cell lines GIST-ST882 (blue), HMC-1 (red), and Ba/F3 murine pro-B (green). WBZ_7 inhibits proliferation of C-Kit–positive GIST cells and of C-Kit (D816V)–positive Ba/F3 murine pro-B and HMC-1 cells. The inhibitory effect of imatinib is essentially restricted to the GIST cells that express only the wild-type kinase. Cell proliferation, expressed as % proliferating cells relative to untreated cells, was determined by the spectrophotometric assay (Materials and Methods). Assays on batteries of 24 wells for each inhibitor concentration/cell type pair were repeated four times, and the value dispersions for each battery were averaged over the four batteries (columns). SD were obtained as root mean square deviations of measured populations from the arithmetic mean. The latter was obtained for each ligand concentration/cell type pair by averaging the four mean values obtained from the four repetitions of the batteries of 24 assays. B, Western blot assay of murine Ba/F3 cells untreated (Untr.) and treated with WBZ_7 and with imatinib at different inhibitor concentrations. Top, phosphorylated D816V mutant kinase; middle, total c-Kit D816V kinase; bottom, β-actin control. C, Western blot assay of HMC-1 cells untreated and treated with WBZ_7 and with imatinib at different inhibitor concentrations. Top, phosphorylated D816V kinase; middle, total C-Kit D816V kinase; bottom, β-actin control.