Dual kinase-bromodomain inhibitors for rationally designed polypharmacology

Abstract

Concomitant inhibition of multiple cancer-driving kinases is an established strategy to improve the durability of clinical responses to targeted therapies. The difficulty of discovering kinase inhibitors with an appropriate multitarget profile has, however, necessitated the application of combination therapies, which can pose major clinical development challenges. Epigenetic reader domains of the bromodomain family have recently emerged as new targets for cancer therapy. Here we report that several clinical kinase inhibitors also inhibit bromodomains with therapeutically relevant potencies and are best classified as dual kinase-bromodomain inhibitors. Nanomolar activity on BRD4 by BI-2536 and TG-101348, which are clinical PLK1 and JAK2-FLT3 kinase inhibitors, respectively, is particularly noteworthy as these combinations of activities on independent oncogenic pathways exemplify a new strategy for rational single-agent polypharmacological targeting. Furthermore, structure-activity relationships and co-crystal structures identify design features that enable a general platform for the rational design of dual kinase-bromodomain inhibitors.

Figure 1. Discovery of kinase inhibitors that potently cross-react with bromodomains

a, AlphaScreen data measured at 50 μM compound concentration against BRD4(1). Shown is a correlation of observed inhibition (% inhibition) with counter screen inhibition (using a His₆ peptide lacking the bromodomain). Hits (less than 50% inhibition in counter screen and more than 60% inhibition) are highlighted in the boxed area and are labeled. b, Inhibitor structures. c, Dendrograms of the human bromodomain family temperature shift data collected on BI-2536 (BI), TG-101348 (TG), PP-242, and SB-203580 using a panel of 46 bromodomains. Bromodomains have been grouped in 8 sub-families as described. A fully labeled dendrogram is shown in Supplementary Figure 3. Temperature shifts are represented by spot size and color as indicated.

Figure 2. BI-2536 and TG-101348 displace BRD4 from chromatin and suppress c-Myc expression

a, Fluorescent Recovery After Photo-bleaching (FRAP) experiments using full-length GFP-BRD4. Time dependence of fluorescence recovery after targeted nuclear photobleaching of U2OS cells transfected with GFP-BRD4 that were untreated or exposed to the pan-BET inhibitor JQ1, BI-2536 (BI) or TG-101348 (TG) at 500 nM. Averaged recovery half times are shown in the insert and images of fluorescent nuclei are shown in Supplementary Figure 4a. b, Western blots showing down regulation of the c-Myc by JQ1 and kinase inhibitors. Top left panel: BI-2536 (BI) and TG-101348 (TG) show strong c-Myc down regulation in MM.1S cells. In contrast the PLK and JAK inhibitors GSK-461364A (GSK), ruxolitinib (Ruxo), and tofacitinib (Tofa), which are inactive against BETs, did not affect c-Myc levels. All inhibitor concentrations were 5 μM, except for JQ1 (1 μM). The other three panels show dose responses for c-Myc downregulation in MM.1s cells for JQ1, BI-2536 (BI) and TG-101348 (TG). Inhibitor concentrations are indicated. GAPDH measured on replicate blots served as the normalization control. Full blot images are shown in Supplementary Figure 4b. c, TG-101348, but not JAK inhibitors that lack BET activity, inhibits proliferation of MM.1S multiple myeloma cells. MM.1S cells were cultured for 72 h in the presence of DMSO, JQ1 (positive control at 0.5 μM), TG-101348 (TG), and the potent JAK inhibitors tofacitinib (Tofa) and ruxolitinib (Ruxo) (all at 3 μM). Error bars are the standard deviations for duplicate measurements.

Figure 3. BRD4 Binding modes of BI-2536 and TG-101348

a, Ribbon diagram showing the structure of the BRD4(1) bromodomain in complex with BI-2536. The main secondary structure elements and the N- and C-termini are labeled. b, Experimental electron density map contoured at 2σ around the inhibitor BI-2536. c, Experimental electron density map defining the binding mode of TG-101348 contoured as described in b. d, Co-crystal structure of BI-2536 with BRD4(1). Main residues interacting with the inhibitor are shown and labeled. Conserved binding site waters are shown as red spheres. e, Co-crystal structure of TG-101348 with BRD4(1). See Supplementary Table 2 for data collection and refinement statistics.

Figure 4. Structure-activity relationships for dihydropteridinone interactions with kinases and bromodomains and structural comparison of inhibitor binding modes

a, Dendrograms of the human kinase and bromodomain families showing inhibitor structures interactions with BI-2536 and BI-D1870 (see Supplementary Fig. 6 for BI-D1870 structure and hinge/acetyl-lysine binding moieties). Selectivity against the kinase family is shown in the left panels for each of the two compounds. Temperature shifts against bromodomains are shown in the right panels. The primary, intended kinase targets are indicated by blue spheres. The radii of the spheres correspond to inhibitor potency (K_d) or temperature shift at 10 μM inhibitor concentration as indicated. b, Details of the binding mode of TG-101348 with BRD4(1) (left panel) and JAK2 kinase (right panel). The JAK2/TG-101348 structure is a model consistent with the co-crystal structure of the closely related inhibitor TG-101209 bound to JAK2 (PDB ID 4J19). c, Details of the binding mode of BI-2536 with BRD4(1) (left panel) and PLK1 kinase (PDB ID 2RKU) (right panel). Critical elements of inhibitor interaction are labeled and highlighted. Conserved water molecules are shown as transparent cpk spheres and key residues are depicted in stick representation.

Figure 5. Responses of FLT3 inhibitor-sensitive and -resistant AML cell lines to TG-101348 and BET inhibitors

a, TG-101348, but not JAK inhibitors that lack FLT3 and BET activity, potently inhibits proliferation of MV4-11 AML cells. MV4-11 cells were tested against the indicated inhibitors for 72 h. Viable cell percent of DMSO control (PoC) is plotted on the y-axis. IC₅₀ values (μM): JQ1 (0.028), TG-101348 (TG, 0.079), I-BET (0.20), ruxolitinib (Ruxo, ~10), and tofacitinib (Tofa, >10). Error bars are the standard deviations for duplicate measurements. b, Molm14 AML cells with acquired resistance to a FLT3 inhibitor retain sensitivity to BET inhibition. Molm14 (parental) and two FLT3 inhibitor-resistant derivatives harboring acquired resistance mutations in the FLT3 kinase domain (D835Y and F691L) were treated with JQ1 at multiple concentrations for 72 h and data were analyzed as described in the Online Methods. All Molm14 cell lines were inhibited by JQ1 with similar potencies (IC₅₀’s ~200 nM). c, JQ1 inhibits c-Myc expression with similar potencies in all FLT3 inhibitor-sensitive and -resistant Molm14 cell lines. c-Myc and GAPDH (internal control run on a parallel blot) levels were measured by western blotting as described in Online Methods post 6 h treatment with JQ1 at the indicated concentrations. Full blot images are shown in Supplementary Figure 7.

Figure 6. Functional activities of dual kinase/bromodomain inhibitors in primary human cell disease models

a, Test agents and kinase or BET benchmark inhibitors were tested for effects on protein biomarkers (x-axis) in 12 primary human cell-based BioMAP systems (Supplementary Table 4) at 7 doses. The TG-101348 (TG) BioMAP profile in selected systems is overlaid with profiles of inhibitors targeting JAK (tofacitinib (Tofa) and ruxolitinib, (Ruxo) or BET (PFI-1 and I-BET) (all at 3.3 μM). Biomarkers modulated by TG in a manner similar to either JAK or BET inhibitors are labeled in blue and green, respectively. See Supplementary Figure 9 for full BioMAP profile. b, BI-2536 (BI) and TG-101348 (TG) and inhibitors targeting either kinases, including JAK (tofacitinib (Tofa) and ruxolitinib (Ruxo)) or PLK (GSK-461364A (GSK)), or BET bromodomains (JQ1, I-BET, PFI-1, and I-BET 151) were profiled in the BioMAP Diversity PLUS panel at 6 doses to generate dose-specific compound signature activity profiles. Statistical measures of profile similarity were used to cluster compounds with shared phenotypic responses to graphically represent their functional similarity. Dot color and size indicate compound and dose, respectively. Compound profiles similar above the selected threshold (Pearson > 0.8) are represented by connected dots. Target-related clusters reflect a common phenotypic signature in these primary cell models. c, Dose-response plots for inhibitors of JAK (Tofa, Ruxo), BET (JQ1, PFI-1), and the dual inhibitor (TG) against a BET biomarker (TNFα). Biomarker ratio to DMSO control is plotted on the y-axis. Error bars are the SEM (n = 3).