Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative

Abstract

Hck, a Src family nonreceptor tyrosine kinase (SFK), has recently been established as an attractive pharmacological target to improve pulmonary function in COVID-19 patients. Hck inhibitors are also well known for their regulatory role in various malignancies and autoimmune diseases. Curcumin has been previously identified as an excellent DYRK-2 inhibitor, but curcumin's fate is tainted by its instability in the cellular environment. Besides, small molecules targeting the inactive states of a kinase are desirable to reduce promiscuity. Here, we show that functionalization of the 4-arylidene position of the fluorescent curcumin scaffold with an aryl nitrogen mustard provides a stable Hck inhibitor (K_d = 50 ± 10 nM). The mustard curcumin derivative preferentially interacts with the inactive conformation of Hck, similar to type-II kinase inhibitors that are less promiscuous. Moreover, the lead compound showed no inhibitory effect on three other kinases (DYRK2, Src, and Abl). We demonstrate that the cytotoxicity may be mediated via inhibition of the SFK signaling pathway in triple-negative breast cancer and murine macrophage cells. Our data suggest that curcumin is a modifiable fluorescent scaffold to develop selective kinase inhibitors by remodeling its target affinity and cellular stability.

Keywords: Hck inhibitor; Src family kinase; cell signaling; curcumin derivative; enzyme kinetics; kinase inhibition.

Figure 1

Design of curcumin derivatives, solution, and cellular stability.A, structural alignment of inactive (PDB: 2OIQ and 2SRC) and active (PDB: 4MXO) conformation of Src drug-binding pocket. The drug-binding pocket is shown in space filled model. B, phylogenetic tree constructed based on the kinase domain sequence of DYRK2, SFK, and Abl. C, chemical structure of compounds 1 to 5. Below the absorbance scan of the respective compound recorded for 1 h. D, confocal microscope images of live MDA-MB-231 cells treated with 4 at indicated time points. Scale bar 10 μm. E, a plot of normalized absorption versus time to demonstrate stability of 1 to 5 in pH 7.4 PBS containing 5% DMSO (see Figs. S1 and S2). PBS, phospahte buffered saline.

Figure 2

Compound 4 competitively binds with the inactive conformation of Hck kinase domainA, the plot of inhibitory constant (K_i) of 1 to 5 and imatinib for DYRK2 (100 nM), Abl (35 nM), Src (75 nM), and Hck (35 nM). B, % specific activity of Hck determined in the presence of 5 μM of 4 for the unphosphorylated (Y) and phosphorylated (pY) states. C, the plot of time-dependent decrease in tryptophan fluorescence upon binding of 4 to unphosphorylated and phosphorylated Hck. Each transient was fitted to single exponential kinetics. D, representative immunoblot showing the effect of 4 (15 μM) on Hck (250 nM) autophosphorylation at indicated time points (upper panel). The bottom panel is the plot of the relative rate of phosphorylation obtained from the densitometric analysis of the blot. E, Michaelis–Menten plot of Hck enzyme kinetics in the presence of 4 or DMSO as a control. F, tabulation of enzyme kinetic parameters of Hck at indicated concentration of 4. (n = 3 experiments; mean ± SD). All the kinetics data were recorded for 10 min (See Figs. S3–S5).

Figure 3

Binding kinetics showing 4 specifically interacts with Hck.A, the plot of time-dependent decrease in tryptophan fluorescence of kinases domains upon binding to 4. Each transient was fit to a single exponential equation to obtain observed rate constants. B, the pseudo-first-order rate constants were plotted as a function of concentration of 4 to determine the k_on and k_off from the slope and intercept of linear fitting. C, drug-binding pocket of Src and Abl kinase domain in DFG-in/c-helix out inactive state. D, a plot of % activity of wildtype and Y253F mutant of Abl determined in the presence of 10 μM of 4 and imatinib. E, table of the rate constants (k_on and k_off) measured for binding of 4. F, the kinetic transient of imatinib or 4 binding to Abl or Y253F mutant. G, a plot of the dissociation constant (K_d) for the indicated kinase domain for 4 (n = 3 experiments; mean ± SD). (See Figs. S5 and S6)

Figure 4

Compound 4 inhibits Src kinase activation and ERK1/2 translocation in MDA-MB-231 cells.A, the table contains IC₅₀ values of 1 and 4 in different cell lines. B, schematic representation of SFK signaling. Signaling modules probed are color-coded (KEGG pathway database). C, plot of the rate of wound healing measured for curcumin and 4-treated cells. D, western blot showing total Src kinase and the autophosphorylation level after treating cells with 4 followed by immunoprecipitation. Quantification of Src phosphorylation plotted as a bar diagram. E, representative images of MDA-MB-231 cells showing ERK1/2 localization after treating with 4 followed by activation with TNF-α, as indicated in each panel. F-actin and nucleus are stained with phalloidin (red) and DAPI (blue), respectively. Scale bar, 10 μm. F, quantification of nucleocytoplasmic distribution of ERK1/2 (n = 30 cells, from two independent experiments). G, representative western blot strips showing the cytoplasmic level of Bcl-2, Cytochrome C, caspase 3, and Bid in cells treated with 4. GAPDH was used as a loading control. At right panel, bar plot represents the quantification of expression level of each signaling module at indicated concentration of 4, (n = 3 experiments; mean ± SD). (See Figs. S7 and S8). Bcl-2, B-cell lymphoma 2; Bid, BH3 interacting domain death agonist; ERKI1/2, extracellular signal-regulated kinase 1/2; TNF-α, tumor necrosis factor α.

Figure 5

Compound 4 abolishes SFK signaling and STAT-3 nuclear translocation in macrophages: A, schematic of SFK signaling pathway. Signaling modules probed are color-coded (KEGG pathway database). B, Ca²⁺ flux as a function of time in RAW 264.7 cells measured after treating cells with indicated concentration of 4 followed by activation with LPS. C, representative images of RAW 264.7 cells showing STAT3 localization after treating with 4 following activation with LPS, as indicated in each panel. F-actin and the nucleus are stained with phalloidin (red) and DAPI (blue), respectively. Scale bar, 10 μm. D, quantification of nucleocytoplasmic distribution of STAT3 (n = 30 cells, from two independent experiments). E and F, western blot showing total protein and the autophosphorylation level of Src, Erk1/2, and STAT3 after treating cells with 4 following activation with LPS and immunoprecipitation. At the bottom of each panel, quantification of phosphorylation level is plotted as a function of 4 concentration. (n = 2 experiments; mean ± SD.). (See Fig. S9). ERKI1/2, extracellular signal-regulated kinase 1/2; STAT3, signal transducer and activator of transcription 3.