Targeted polypharmacology: discovery of dual inhibitors of tyrosine and phosphoinositide kinases

Abstract

The clinical success of multitargeted kinase inhibitors has stimulated efforts to identify promiscuous drugs with optimal selectivity profiles. It remains unclear to what extent such drugs can be rationally designed, particularly for combinations of targets that are structurally divergent. Here we report the systematic discovery of molecules that potently inhibit both tyrosine kinases and phosphatidylinositol-3-OH kinases, two protein families that are among the most intensely pursued cancer drug targets. Through iterative chemical synthesis, X-ray crystallography and kinome-level biochemical profiling, we identified compounds that inhibit a spectrum of new target combinations in these two families. Crystal structures revealed that the dual selectivity of these molecules is controlled by a hydrophobic pocket conserved in both enzyme classes and accessible through a rotatable bond in the drug skeleton. We show that one compound, PP121, blocks the proliferation of tumor cells by direct inhibition of oncogenic tyrosine kinases and phosphatidylinositol-3-OH kinases. These molecules demonstrate the feasibility of accessing a chemical space that intersects two families of oncogenes.

Figure 1. Structural and sequence comparison of tyrosine kinases and PI3-Ks

(a) Backbone traces of crystal structures of the kinase domain of c-Src aligned to the kinase domain of the Src-family tyrosine kinase Hck (left), the receptor tyrosine kinase VEGFR2 (center) and the PI3-K p110γ (right). Statistics for the pairwise sequence identity and backbone r.m.s.d. are shown below. The number of residues used for each alignment is shown in parentheses. (b) Sequence alignment of the kinase domains of the tyrosine kinases c-Src, Hck, and VEGFR2 and the PI3-K p110γ. Conserved residues relative to c-Src are colored red. The p110γ sequence was manually aligned to c-Src using x-ray structures of the two proteins that superimpose key secondary structural elements. The VEGFR2 insert comprising residues 944–1001 is omitted.

Figure 2. Biochemical target selectivity of pyrazolopyrimidine inhibitors

(a) Experimental strategy for the discovery of dual inhibitors, and IC₅₀ values (µM) for eight molecules tested against 14 tyrosine and phosphoinositide kinases (10 µM ATP). IC₅₀ values less than 0.1 µM are shaded red. Pyrazolopyrimidine N4 and N5, which make hydrogen bonds to the kinase, are labelled. (b) Percent inhibition of 84 tyrosine kinases (right) and 135 serine/threonine kinases (left) by 7 inhibitors from this study (right columns) and 5 reference compounds (left columns). PP inhibitors were tested at 1 µM drug and, typically, 10 µM ATP. Data from the Invitrogen SelectScreen Assay. (c) Principal component analysis of the target selectivity of 172 pyrazolopyrimidine inhibitors and 8 reference compounds. Key compounds are labelled.

Figure 3. Crystal structures of S1 and S2 bound to human p110γ

(a) Binding mode of S1 to p110γ, viewed from the entrance to the ATP binding pocket (left) and above the ATP binding pocket (right). Dashed lines indicate hydrogen bonds. (b) Binding mode of S2 to p110γ.

Figure 4. Structural comparison of pyrazolopyrimidine binding to tyrosine kinases and PI3-Ks

(a) Correlation between IC₅₀ values for inhibitors against Src (x-axis) and either Hck or the gatekeeper mutant Src T338I (y-axis). (b) Binding orientation of S1 relative to ATP in c-Src (top) and p110γ (bottom). (c) Overlay of co-crystal structures of inhibitors bound to c-Src (protein colored red, drugs orange: S1, PP102, PP121, and PP494) and p110γ (protein blue, compounds gray: S1 and S2). The gatekeeper residues Thr338 (c-Src) and Ile879 (p110γ) are highlighted. (d) (top) The catalytic lysine (Lys295) makes a hydrogen bond to Glu310 in active c-Src. (center) Helix C and Glu310 are disordered in c-Src structures containing PP102. (bottom) PP121 makes a hydrogen bond to Glu310 and orders helix C when bound to c-Src.

Figure 5. PP121 directly inhibits p110α/mTOR

(a) Schematic of signaling downstream of tyrosine kinases. Not all arrows represent direct physical interactions. Drugs used in this study and their key targets are highlighted. (b) LN229 and (c) U87 glioblastoma cells in serum (10%) were treated with PP102 or PP121 (0.040 to 10 µM). Cells were lysed and phosphorylation of signalling proteins was probed by western blotting. pS6 (Ser235/236), pErk (Thr202/Tyr204). (d) Proliferation of tumor cells was measured following 72 h treatment with PP102, PP121, PI-103, PIK-90, or sorafenib (0.040 to 10 µM). Each cell line was tested at three serum concentrations (0.5%, 2%, and 10%). (e) Cell cycle analysis by flow cytometry following treatment with PP121 or PI-103 (2.5 µM) or vehicle (0.1% DMSO) for 24 h.

Figure 6. PP121 directly inhibits Src

(a) NIH3T3 cells transformed with v-Src(Thr338) were treated with the indicated concentration of each inhibitor (2 h), lysed, and blotted for indicated proteins. Molecular weights are indicated adjactent to phosphotyrosine (pTyr) blots. (b) v-Src(Thr338) transformed NIH3T3 cells were treated with the indicated inhibitors (2.5 µM, 24 h) and then stained with FITC-phalloidin (actin) and DAPI (DNA). The percentage of cells acquiring actin stress fibers was quantitated by counting while blinded to sample identity.

Figure 7. PP121 directly inhibits Ret

(a) TT thyroid carcinoma cells were treated with the indicated concentration of each inhibitor (2 h), lysed, and blotted for indicated proteins. pRet (Tyr905). (b) TT cells were treated with a dose response of each inhibitor (0.040 to 10 µM) and cell number was quantitated after 13 days. Drug was replenished every three days.

Figure 8. PP121 reduntantly targets Bcr-Abl and PI3-K/mTOR in CML cells

(a) BaF3 cells expressing Bcr-Abl (left column) or Bcr-Abl T315I (right column) were treated with PP121, PI-103, or Imatinib (0.080 to 20 µM) for 120 min. Cells were lysed and phosphorylation of signaling proteins was probed by western blotting. (b) Proliferation of BaF3 Bcr-Abl and BaF3 Bcr-Abl T315I cells in response to selected drugs (2.5 µM). (c) Percentage of cells undergoing apoptosis in response to drug treatment. BaF3 Bcr-Abl cells (2.5 µM, 36 h), BaF3 Bcr-Abl T315I and K562 cells (5 µM, 72 h). (d) Cell cycle analysis of live cells remaining following treatment in panel c.