key: cord-0962435-wc49hilr
authors: Chakraborty, Manas Pratim; Bhattacharyya, Sudipta; Roy, Souryadip; Bhattacharya, Indira; Das, Rahul; Mukherjee, Arindam
title: Selective targeting of the inactive state of hematopoietic cell kinase (Hck) with a stable curcumin derivative
date: 2021-02-20
journal: J Biol Chem
DOI: 10.1016/j.jbc.2021.100449
sha: 6c4dda9379f8e51243eb2270d5dc919c875acaba
doc_id: 962435
cord_uid: wc49hilr

Hck, a Src family non-receptor 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 signalling 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 remodelling its target affinity and cellular stability.

Curcumin, an active molecule against inflammation, microbial infections, cancer, and neurodegenerative diseases, loose its drug-like potential due to its instability in the cellular environment. (1) Besides, the planar symmetry makes curcumin less druggable towards asymmetric binding pockets like kinases. (2) Albeit, it is undeniable that pure curcumin shows excellent selectivity towards the DFG-Asp-in, active conformation of dual-specificity tyrosine regulated kinase 2 (DYRK2). ( 3) The drugability of curcumin may be improved by nanoformulation and modification at relevant positions to enhance stability and specificity. A recent report suggests that curcumin self-assembles in the presence of Zn(II) and F-moc histidine to adopt nanoformulations enhancing stability. (4) The functionalization of the methylenic position in the diketone motif of curcumin provides stable derivatives and introduces non-planarity in the conformation. (5) (6) (7) Analysis of the recent DYRK2 kinase domain structure in complex with curcumin suggests that the modification at the methylenic position in the diketone motif may prevent the docking of the curcumin derivatives to the ATP binding pocket (Figure 1a) . We asked if the functionalization of the methylenic position will remodel the target preference of the curcumin compounds towards other kinases viz. Src, Hck, and Abl ( Figure 1b) ? It may be noted that the clinical kinase inhibitors target one or more of the following conformations: The DFG-Asp-in active, the DFG-Asp-out Abl/cKit like inactive, or the DFG-Asp-in c-Src/CDK like inactive conformation (Figure 1a) . (8) (9) (10) Most type-I inhibitors targeting the active conformation are promiscuous as all kinases share more or less the same conformation in their active state. Type-II kinase inhibitors targeting the inactive conformations are desirable since the kinases adapt specific inactive conformation reducing the promiscuity.

We targeted to design stable curcumin derivatives by modifying the methylenic position. The functionalization of the methylenic position in the diketone motif by 4-arylidene substituents would deter the free radical driven incorporation of oxygen in the curcumin scaffold, known to be occuring spontaneously in native curcumin at physiological pH. (11, 12) The 4-aryl substituents ( Figure 1c ) were chosen to promote non-covalent interactions with the ligand-binding pocket of the inactive form of the afore-mentioned kinases. Here in, we show that suitable functionalization of the methylenic position enhances the cellular stability and remodels the affinity towards DFG-in inactive conformation of Hck, instead of DYRK2. Hck is an attractive pharmacological target for its regulatory role in cancer development and virus infection. (13, 14) Hck expresses explicitly in myeloid and B-lymphocyte lineage cells and regulates immune receptor signaling, cell migration, proliferation, and differentiation.(15, 16) Recently, inhibition of Hck by ibrutinib in COVID-19 patients showed protection against lung injury and improved pulmonary function. (17) docking studies showed that the curcumin derivatives might have preserved the binding affinity towards DYRK2. Next, we probed, if the modified compounds have gained specificity towards the active or inactive conformations of the ligand-binding pocket. The curcumin did not distinguish between the active or inactive conformation of Src or Abl kinase domain ( Figure S1a ). The plot of relative change in docking scores of 2-5 suggest that the functionalized curcumins bind preferentially to the DFG-Asp-in cSrc/CDK like inactive conformation ( Figure S1a ). Kinase inhibitors targeted against the inactive state are desirable due to higher selectivity and potency. (18) (19) (20) The four derivatives 2-5, of which two were taken from an earlier library of 4-arylidene functionalized curcumin derivatives (21, 22) , were thus synthesized and characterized using standard analytical techniques. The synthetic method is a single step Knoevenagel condensation of curcumin with the respective aldehydes ( Figure S2a ). The curcumin (1) used in synthesis was purified by column chromatography from the crude commercial mixture.

We first evaluated the stability of 2-5 in comparison with curcumin in aqueous solution. The UV-visible data showed that the compounds 3-5 have greater solution half-lives (t 1/2 ) in pH 7.4 (1x PBS containing 5% DMSO) (Figure 1e, S2c-g and Table  S1 ) compared to 1 and 2. Next, we studied the stability of 1-5 under the kinase assay condition (Figure 1c ). The bis(2-chloroethylamine) motif bearing compound 4 is the most stable under nearphysiological conditions in the kinase assay buffer. Compound 4 also demonstrated to be stable for at least 24 h (observed period) in 8:2 v/v of 1x PBS and DMSO-d 6 in 1 H NMR ( Figure S18 ).

We determined the activity of 1-5, against DYRK2, Src, Hck, and Abl kinases (Figure 2a) using coupled kinase assay. Curcumin inhibits DYRK2 kinase activity with K i of 6 nM similar to the previous report(23) but compounds 2-5 do not (Figure 2a and S3). Besides, 1-5 also display poor activity against Abl kinase (K i > 6000 nM) in comparison to imatinib (K i > 60 nM) (24) (Figure 2a and S3 ). Compound 4 selectively inhibits Hck and Src kinase activity (Figure 2a and S3) with K i of 70 ± 11 nM and 460 ± 110 nM, respectively. Compound 1-3, 5, and imatinib showed a relatively weak inhibitory effect on Src and Hck (K i more than 10-fold compared to 4) (Figure 2a and S3). We used imatinib as a control due to its specificity towards DFG-Asp-out Abl/cKit like inactive structure (Figure 1a ). (25, 26) Unlike Src, Abl kinase can adopt all three conformations of the kinase domain ( Figure 1a ). (27) Thus, the majority of ATPcompetitive, non-covalent Src inhibitors would be promiscuous to Abl. The specific inhibition of Src and Hck over Abl by 4 suggests that it is possibly binding to an inactive conformation of the Src kinase domain. We ruled out the interaction of 4 to the DFGout inactive conformation of Src due to its relatively higher thermodynamic penalty. (24) It is exciting to find that 4 specifically inhibits Hck because of the importance of Hck in the regulation of cancer development and lung injury in COVID-19 patients. (13, 14) Therefore, we further investigated the mechanism and mode of action of 4.

We first investigated if the oligomerization of 4 could explain the mode of kinase inhibition. The oligomeric state of 1 and 4 was determined from the hydrodynamic radius measured using the dynamic light scattering in an aqueous buffer ( Figure S2 h-k) and compared with imatinib and sucrose. Unlike curcumin (1), we observed that 4 did not aggregate in solution and behaved similar to imatinib.

Selective type-II inhibitors targeted against the inactive conformation of Abl are sensitive to the phosphorylation state of the activation loop. (26, (28) (29) (30) The inhibitors that are non-selective for Src and Abl are often not sensitive to the phosphorylation of the kinase activation loop.(31) Hence, we prepared and purified unphosphorylated Src and Hck, which was uniformly phosphorylated by treating with ATP and magnesium ( Figure S4b ). Then the activity of the phosphorylated Hck (pY-Hck) and Src (pY-Src) was assayed in a time-dependent manner. The results show that 4 does not inhibit the pY-Hck and pY-Src ( Figure 2b and S4c-d) emphasizing its preference towards the inactive conformation of Hck and Src.

A hallmark of Src activation is autophosphorylation of the activation loop (29, 30, 32) , which shifts the equilibrium towards the active state. (33) (34) (35) We determined the effect of 4 on the rate of autophosphorylation of Src and Hck, and found that 4 reduces the rate of autophosphorylation by 2.5 and 500 fold, respectively (Figure 2d and S4e-f). We studied the interaction of 4 with Hck by measuring the kinetics of change in the intrinsic tryptophan fluorescence by stop-flow fluorimeter. The plot of the rate of change in tryptophan fluorescence (Figure 2c ) suggests that 4 preferentially interacts with the inactive unphosphorylated conformation of Hck. Thus, further emphasizing that the 4 shows preference to bind the inactive conformation and reduces the rate of autophosphorylation of the kinase domain.

The crystal structure of DYRK2 in complex with curcumin for the first time showed that curcumin occupies the ATP binding pocket potentially acting as a competitive inhibitor (9) . To find out if 4 could inhibit Hck and Src as an ATP-competitive inhibitor, we determined the K m (Michaelis-Menten constant) for ATP and V max (Figure 2e-f and S4f-g). Increasing concentration of 4 changes the K m without any significant change in the V max, indicating that 4 functions as a competitive inhibitor for the ATP binding site.

The above data does not rule out the possibility of covalent modification of the ligand-binding site by the potential alkylating groups in 4, which may explain the specificity towards the SFK.

The specificity of several compounds towards Src or Abl is attributed to the different sequence and structural dynamics of the P-loop. (24, 36, 37) For example, in Src the Cys277 at the P-loop and Cys400 at the β7 could be covalently modified by alkylating agents, explaining the specificity of 4 towards Src ( Figure  S5a ). In Abl kinase domain, the amino acid residues at the corresponding positions are Gln and Val, respectively, providing no scope for 4 to covalently modify them, unlike a cysteine. (38) Although the aromatic mustard motif is such that the alkylation ability would be low or absent, confirmation was required to exclude covalent modification as a possible mode of inhibition. To confirm the above we incubated the Src kinase domain and 4 in the kinase assay buffer for 1h. The resultant sample was then analyzed by HPLC under denaturing condition to determine any covalent adduct formation. We observed that the 4 treated Src kinase elutes at the same resident time as untreated Src kinase with no coelution of 4 ( Figure S5c ). The respective pure fraction of the protein sample from the HPLC was subjected to ESI-MS, which confirmed that the m/z envelope for the treated and untreated sample ( Figure S5d ) correspond to the unmodified protein. Thus, 4 is not covalently modifying the cysteine residues of Src kinase.

We studied the binding kinetics of 4 with DYRK2, Hck and Abl to understand the mechanism of specificity towards Hck. We monitored the change in the intrinsic fluorescence of the protein corresponding to the pre-steady-state kinetics by titration (Figure 3 and S6). The Hck binds to 4 with approximately 10-fold faster k on rates than Abl and a K d of 50 ± 10 nM, which matches with the K i values from enzyme kinetics (Figure 3e ,g). DYRK2 did not interact with 4 ( Figure 3a ). The above result may be explained by comparing the structure of the ligandbinding-pocket in the Src DFG-Asp-in inactive structure (PDB ID: 2SRC) to the corresponding conformation of Abl kinase domain (PDB ID: 2G1T) (Figure3c). The P-loop of Abl takes a closed conformation in comparison to Src, which alters the conformational dynamics of the ligand-bindingpocket. (39) The Y253 at the P-loop of Abl may sterically block the docking of 4 to the ligand-bindingpocket. In Src, the residue at the corresponding position is substituted by F278 (Figure 3c and S5a). The Y253 is a critical residue in Abl, and its mutation to F253 causes imatinib resistance.(40) Therefore, we speculate that the different structural dynamics of the P-loop residues in the Src and Abl kinase domain determine the specificity for 4. To find out if the Y253 is responsible for selectivity, we generated the Y253F mutant Abl to comparatively study imatinib and 4. The Abl Y253F bound weakly to imatinib and did not inhibit the kinase activity (Figure 3d, f and g). Whereas 4 binds with 7-fold higher affinity (K d of 1.3 ± 0.3 µM) to the Abl Y253F and shows better inhibition compared to imatinib at a given dose (Figure 3d-g) .

The functionalization of the methylenic position in the diketone motif of curcumin by the aryl nitrogen mustard thus altered the target specificity towards Hck over DYRK2, Abl and Src. In spite of the encouraging results, the instability in the cellular environment may eclipse the potential of 4. We investigated the cellular stability exploiting the green fluorescence of 4, which allows to trace the molecule by live-cell imaging in the triple-negative-breast-cancer (TNBC) cell MDA-MB-231 ( Figure 1d ). Curcumin, as expected(1), spontaneously degraded within 1h inside the cell making it invisible (Figure S1b-d). Although the quantum yield (Table S1) of curcumin is higher than 4, the fluorescence of compound 4 was observed inside the MDA-MB-231 cell for 14h, suggesting its higher stability (Figure 1d and S1e-f). To translate the inhibitory effect of 4 to cell-based experiments, we measured the cytotoxicity on the murine macrophage RAW 264.7, MDA-MB-231,(41) colon carcinoma (HT-29), (42) and HEK 293T cell line. The comparison of IC 50 values (Figure 4a and S7a) indicate that the 4 is ten times more potent than curcumin when tested against RAW 264.7, MDA-MB-231, and HT-29 cell lines, respectively. In contrary, 4 is 10-fold less cytotoxic to HEK 293T cells (IC 50 value of 24.92 ± 1 µM). This may be because among the above four cell lines HEK293 does not overexpress SFK. DNA cross-linking assay shows that 4 do not cross-link DNA in the in-vitro assay ( Figure S7b ). We next studied if 4 disrupt the plasma membrane integrity by live-cell imaging of MDA-MB-231 cell line treated with FM4-64-FX, a lipophilic probe used for staining plasma membrane ( Figure S7c ). (43) The results support that 4 is not affecting the membrane integrity of the cells.

To evaluate if 4 inhibits Src-dependent cell migration, proliferation and apoptosis, we performed a wound-healing assay with MDA-MB-231 and probed the signalling modules regulating the respective pathways ( Figure 4 and S8). The MDA-MB-231 cells rely on Src signalling for cell migration and metastasis ( Figure 4b ). (44, 45) We observed that 4 inhibits the autophosphorylation of Src in the cell ( Figure 4d ) and significantly impaired the wound healing rates (IC 10 and IC 50 dose) ( Figure S8b -c). The migration process involves the TNF-α induced activation of Src signalling cascade and translocation of ERK1/2 to the nucleus. (46, 47) So, 4 treated MDA-MB-231 cells were activated with TNF-α and the localization of ERK1/2 to the nucleus was determined by immunofluorescence using confocal microscopy. In comparison to the untreated cells, treatment with 5µM of 4 significantly increased the cytosolic fraction of ERK1/2 even after activation with TNF-α (Figure 4ef). Comprehensively, our data suggest that 4 inhibits the Src mediated ERK1/2 signalling necessary for cell migration and proliferation.

Activation of ERK1/2 promotes cell survival by enhancing the stability and activity of Bcl-2, an anti-apoptotic molecule that regulates the intrinsic apoptotic pathway. (48, 49) Many oncogene-targeting molecules directly or indirectly inhibit ERK1/2 signalling to cause Bcl-2 degradation and cell death.(49) Therefore, we examined if inhibition of ERK1/2 destabilizes Bcl-2 and activates intrinsic apoptotic pathway (Figure 4b ). Destabilization of Bcl-2 will release cytochrome c from mitochondria, subsequently activating Caspases3 causing apoptosis. (50) (51) (52) We observed that 4 activates the mitochondria-mediated apoptotic pathway by decreasing Bcl-2 level in the cell, leading to release of cytochrome C from mitochondria and activation of Caspase3 ( Figure 4g ). 4 does not affect the Bid expression level suggesting that 4 induce apoptosis through an intrinsic apoptotic pathway. Together our data indicate that 4 interferes with multiple SFK mediated signalling pathways regulating cell proliferation and apoptosis.

The upregulation of SFK in macrophage cells lead to tumorigenesis by activating diverse signalling modules, causing increase in angiogenesis, cell polarization, migration, and cell adhesion ( Figure  5a ). (39, 53, 54) We investigated if inhibition of SFK by 4 in macrophages inhibit downstream signalling involved in tumorigenesis (53) 

All chemicals and solvents were purchased from commercial sources. Solvents were distilled and dried prior to use by standard procedures (55) . Curcumin was purchased either from Sigma-Aldrich or Carbosynth Ltd. Curcumin obtained from commercial sources were further purified using column chromatography to get rid of curcuminoid mixtures. MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide)] (USB), cell growth media and their supplements were purchased from Gibco. Abl kinase domain was purchased from Addgene, USA (Addgene plasmid # 79727) (56) . The details for the list of antibodies used in this study is provided in Table S1 . All the solvents used for spectroscopy and lipophilicity measurements are of spectroscopy grade and purchased from Merck. UV-Visible measurements were done using either Agilent Technologies Cary 300 Bio or Perkin Elmer Lambda 35 spectrophotometer. The fluorescence measurements were done using a Horiva-Yovon FluoroMax Plus spectrophotometer. The stopped flow measurements were performed in a SFM2000 bioLogic spectrophotometer. FT-IR spectra were recorded using Perkin-Elmer SPECTRUM RX I spectrometer in KBr pellets. NMR spectra were recorded using either JEOL ECS 400MHz or Bruker Avance III 500MHz spectrometer at ambient temperature and calibrated using residual undeuterated solvents as internal reference. All 13 C spectra reported are proton decoupled. The chemical shifts (δ) are reported in parts per million (ppm). Elemental analyses were performed on a Perkin-Elmer CHN analyser (Model 2400). Electro-spray ionisation mass spectra of compounds were recorded in positive mode electrospray ionisation using a Q-ToF Bruker maXis II™ instrument. All the compounds were kept in dark after purification and stored in refrigerator.

Syntheses of the four curcumin derivatives (2-5) were conducted using scheme in Figure S2a following modified procedure reported elsewhere.(5) Briefly, 1.0 mmol of curcumin and 2 mmol. of appropriate aromatic aldehyde was dissolved first in pre-warmed toluene at 100°C. Next into this hot mixture, catalytic amount of piperidine (0.05 mmol) and acetic acid (0.08 mmol) were added. The solution was then heated to reflux at 140°C using a dean-stark apparatus. Resulting water as a byproduct of reaction was eliminated by anhydrous Na 2 SO 4 in the apparatus. The reaction was continued up to 18-20 h under dark. After completion of reaction the whole solution was evaporated out under reduced pressure. Pure product was isolated using silica gel column chromatography (ethyl acetate-hexane mixture as eluent). Figure S14 -S17).

Kinase domain of Src (amino acid residues 254-535) and Hck (amino acid residues 166-445) were gifted from Markus Seeliger. The Abl (amino acid residues 229-460), and DYRK-2 (amnio acid residues 146-540) plasmids were a gift from John Chodera, and Nicholas Levinson, and Markus Seeliger (Addgene plasmid # 79727). The Abl, Src and Hck kinase domain were co-expressed with the YopH phosphatase and Trigger factor in E. coli BL21(DE3) and purified as described previously. (56, 58) The DYRK2 was co-expressed with λ-phosphatase and Trigger factor, and purified as described previously. (3, 56) Briefly, the bacterial cells were grown to an OD 600 of 1.2 at 37 °C before induction with 1mM IPTG for 16 h at 18 °C. For purification of Src, Hck and Abl kinase domain, cells were harvested and resuspended in buffer-A (50mM Tris-HCl, pH 8.0, 500mM NaCl, 25mM imidazole, 5% glycerol). Cells expressing DYRK2 kinase domain was resuspended in lysis buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 10 mM imidazole,5% glycerol and 5 mM β-mercaptoethanol). Cells were lysed by using cell-homogenizer or sonication. Protein was purified by using nickel-affinity chromatography. The Nterminal His tag in Abl and Hck kinase domain was removed by dialysing overnight in Buffer-C (20mM Tris,100mM NaCl, 1mM DTT, 5% glycerol) containing Tev-protease. The YopH phosphatase and other contaminants were removed by anion-exchange chromatography followed by size exclusion chromatography in the gel filtration buffer, (for Src, Hck, and Abl kinase 50mM Tris-HCl, pH8.0, 100mM NaCl, 5% glycerol, 1mM DTT was used) or (DYRK kinase, 25 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM DTT was used). The purified protein was concentrated and stored at −80 °C.

Activity of Src, Hck and Abl kinase was monitored by measuring the rate of phosphorylation of a Src specific peptide (EAIYAAPFAKKK), and for DYRK2 Woodtide (KKISGRLSPIMTEQ) was used. (3, 59) Reaction was continuously monitored by measuring the rate of oxidation of NADH to NAD in a buffer containing 10mM MgCl 2 , 2.2 mM ATP, 100mM Tris (pH 8.0), 1mM Phosphoenolpyruvate (PEP), 0.6mg/mL NADH, 75 U/mL Pyruvate Kinase, 105 U/mL lactate dehydrogenase and 0.5 mM substrate peptide.(60) For DYRK2, 1mM ATP was used in each reaction. All the reactions were initiated by the addition of kinase at a final concentration of 35-100nM. Decrease in NADH absorbance at 340nM was monitored for 40 min at 25°C. All the compounds were dissolved in DMSO and diluted in the gel filtration buffer. To determine the effect of curcumin and its derivatives, the enzyme and the compounds were incubated at 25°C for 15 min before initiating the kinase reaction. Inhibitory constant (K i ) was calculated from IC 50 according to the following relationship(61):

where [ATP] is the concentration of ATP in the assay and K m is the Michaelis constant for ATP. K m for ATP was determined for the Src kinase domains to be 99 µM and 210 µM in absence and in presence of 0.3% DMSO, respectively. The data analysis and curve fitting were done with GraphPad Prism 5 ® Ver 5.03. The V max and K m was determined by fitting Michaelis-Menten curve to the rate of phosphorylation measured at indicated ATP concentration.

The autophosphorylated Src or Hck kinase domain was prepared by incubating 10 µM the kinase with 1mM ATP and 10 mM MgCl 2 for 2 h at 25°C, as described previously.(62)

The rate of autophosphorylation was analysed by western blot using total anti-phosphotyrosine antibody. 250 nM of Src or Hck kinase domain was incubated with 15 µM Compound 4 or with DMSO at 25°C for 15 min. The Src autophosphorylation was initiated by adding 1mM ATP, 10mM MgCl 2 and 1mM Na 3 VO 4 at 25°C.(62) The reaction was stopped at indicated time points using 25µL of 2X SDS-PAGE loading buffer. Level of phosphorylation and amount of Src Kinase domain loaded was detected by anti-phosphotyrosine antibody (pY)(Abcam) and anti-His antibody (Biobharati), respectively. The amount of Hck loaded in each well was determined by coomassie stain. Rate of phosphorylation was determined from the plot of normalised intensity of anti-phosphotyrosine antibody (pY): amount of protein loaded in respective wells against time. The Image J software was used to determine the intensity from the blot(63). Rate of autophosphorylation was calculated from linear curve-fitting.

Cytotoxicity of 1 and 4 was determined against MDA-MB-231, HT-29 cells, RAW 264.7 and HEK 293T cell lines using MTT assay. In brief, cells were seeded in 96 well plate at a cell density of 6 × 10 6 cells per well in DMEM supplemented with 10% FBS (200 µL per well), antibiotics (100 units mL -1 penicillin and 100 µg mL -1 streptomycin) incubated at 37°C and maintained in a 5% CO 2 environment. RAW 264.7 cell line was grown in RPMI media supplemented with 10% FBS, antibiotics (100 units mL -1 penicillin and 100 µg mL -1 streptomycin). After 48 hours of growth, the media was removed and replaced with fresh media. The compounds were dissolved in DMSO (molecular biology grade) and then diluted with growth media before adding to the cell culture at the desired concentration. In all the wells final concentration of DMSO was within 0.2%. Cells were then incubated with 1 or 4 for another 48 h. After the incubation is over, the drug-containing media was removed and wells replenished with fresh media (200 µL) followed by treatment with 20 µL of 1 mg/mL MTT dissolved in PBS (pH 7.2). After 3 hours of incubation, media was removed and 200 µL of DMSO was added to each well. The inhibition of cell growth induced by the tested complexes was detected by measuring the absorbance of each well at 595 nm using SpectraMax M2e plate reader. The data was plotted and IC 50 calculated using GraphPad Prism 5® Ver 5.03. Each experiment was performed in triplicate, and average data with standard deviations are reported.

MDA-MB-231 cells were grown to confluence in 6well plates in DMEM medium supplemented with 10% FBS at 37°C in a 5% CO 2 atmosphere. A single wound generated in each well by scratching gently with a sterile pipette tip. Then cells washed twice with PBS (pH 7.2) followed by addition of fresh DMEM media containing 10% FBS. The respective IC 10 (100 nM and 600 nM) and IC 50 

The compound stability was determined both in vitro and in a cell-based assay. The solution stability of 1-5 was determined by dissolving the compounds in 5% DMSO containing 1× PBS of pH 7.4 and measuring the absorbance for 24 h at 25°C. The stability of 1-5 was also determined in the kinase assay buffer (50mM Tris-HCl, pH 8.0/100mM, NaCl/5%, glycerol/1mM DTT) using 5% DMSO for the period of kinase assay (1 h).

The stability of the compound in cells was determined by recording the fluorescence images of 1 or 4 treated cells for 14 h. MDA-MB-231 cells were seeded in a glass-bottomed 35 mm Petri dish (Corning) at a density of 0.2 × 10 6 cells per plate. At 80% confluency cells were treated with 10 µM of 1 or 4 as described previously in the cytotoxicity assay. Cells were allowed to grow for one hour and washed with pre-warmed 1X PBS and opti-MEM was added. Live-cell images of the cells were recorded at 1h, 2h, 3h, 4h, 5h, 6h, 10h and 14h time point using a Leica confocal microscope. The fluorescence intensity for each cell was measured and analysed by using the ImageJ software. For each time interval, background fluorescence of cells were subtracted from the control cells. The fluorescence intensity per µm 2 for 15 cells were plotted against time; the error bar represents the standard deviation within the data set.

In gel fluorescence assay was used to determine any covalent modification. 10 μM Src-KD was incubated with 1 and 4 at 1:1 for 25 min at room temperature. Samples were run in 8% Native-PAGE at 4°C. Gel fluorescence was checked at 400-500 nm and then stained with Coomassie brilliant blue.

HPLC and mass spectrometry HPLC was done by using C18 reverse phase HPLC column. Control sample was prepared by incubating 0.5mg/ml Src-KD with 5M Guanidinium chloride (Gu.HCl) for 10 min. To determine if 4 is covalently modifying the Src kinase domain, 0.5 mg/ml Src was incubated with an equimolar ratio of 4 for 1 h and then the sample was treated with 5M Gu.HCl for 10 min. 100 μL of each sample was injected into the column that was pre-equilibrated with 5% CH 3 CN + 95% water + 0.1% TFA. Protein was eluted by a linear gradient of 10-90% CH 3 CN+0.1% TFA. The elution of protein and 4 was monitored by measuring the absorbance at 280 and 416 nm, respectively. The pure eluted fractions were used for ESI-MS mass spectrometry (Bruker maXis II™ instrument). 

Molecular docking was carried out using Autodock vina 4.0(64).The structure of 4 was optimised in Gaussian09 software package (Gaussian 09, Revision A.02, Gaussian, Inc., Wallingford CT, 2016). Optimisation of 4 was performed at the DFT level of theory with B3LYP function and 6-31G(d,p) basis set (65) . Orbitals were defined as restricted during calculations. The conductor-like polarisable continuum model (CPCM) was used with water as the solvent during optimisation(66) (67) (68). Structure of Src and Abl kinases were used from the protein data bank (PDB ID: 2SRC, 2OIQ, 4MXO, 1OPL, 1OPJ, 2G1T). Gaussian optimised structures of 1-5 were used as the ligand input files. Kinase structures were further optimised, and polar hydrogens were assigned by OPLS (Optimised potential for liquid simulations) 3 force field to rectify the molecular geometries to get least energy conformations required for docking.All the water molecules (except those present in the binding site) were optimised for hydrogen bonding network using OPLS3, corresponding to pH 7(69).The receptor grid box was generated using autodock tools, surrounding ATP binding domain of the respective kinase structures ( Table 1) 

The nuclear translocation of ERK1/2 and STAT3 was determined by confocal microscopy of activated MDA-MB-231 and RAW 264.7 lines respectively. The cells were grown on coverslip upto a confluency of 90% and treated with 1 μM and 5 μM 4 for 6 h. After incubation, cells were washed with PBS and activated. The MDA-MB-231 and RAW 264.7 cell lines were activated using 50 nM TNFα and 100 nM LPS for 30 minutes and 8 h, respectively. After activation cell were washed three times with 1PBS and fixed with 4% paraformaldehyde for 30 min. The cells were then permeabilised with 0.2% Triton X-100 for 20 min and blocked in 3% bovine serum albumin for 1 h. The cells were incubated with respective primary antibodies (STAT3 and ERK1/2) in blocking buffer for overnight. After washing three times with PBS containing 0.1% Tween-20, cells were incubated with secondary antibody conjugated with alexa fluor 562 and phalloidin (Invitrogen) for 2 h. Finally, coverslips were mounted with DAPI (4′,6diamidino-2-phenylindole) and imaged under Confocal laser scanning microscope (Leica TCS SP8).

In cell activity of Src kinase and its downstream signalling molecules like ERK1/2 and STAT3 in response to 4 was determined by probing the phosphorylation level the of respective immunoprecipitated proteins from MDA-MB-231 and RAW 264.7 cell line. MDA-MB-231 cells were grown upto 70% confluency, and then treated with 1 -10 μM of 4 or DMSO for 6 h. After incubation cells were activated as described previously. Activated cells were harvested, and lysed with RIPA lysis buffer (25 mM Tris/HCl pH 7.4, 1mM EDTA, 100mM NaCl, 1% Nonidet P40,1% Triton-x100 supplemented with 0.1 mM vanadate and a proteaseinhibitor cocktail (5mg/L leupeptin, 0.1 mM phenylmethylsulphonyl fluoride, 2mM Benzamidine). Cell lysate was sonicated and cleared by centrifuging at 15,000 x g for 10 minutes. The supernatant was incubated with anti-Src, anti-ERK1/2 or anti-STAT3 antibody for overnight at 4°C. After overnight incubation, the antibody bound protein solution was further incubated with 30 µL protein G beads for 2 h. The beads were washed 4 times with ice cold PBS supplemented with 2mM Na 3 VO 4 . The samples were boiled with Laemmli sample buffer and ran in SDS-PAGE. Following SDS-PAGE, lysates were transferred onto nitrocellulose paper and blocked with 5% skimmed milk in TBST (0.1% tween 20) for 2 h at room temperature. For detecting the level of phosphorylation, blocked membranes were incubated with total anti-pY antibody. To detect respective loading control, membranes were incubated with anti-Src, anti-ERK1/2 or anti-STAT3 for overnight at 4°C followed by 2h incubation with HRP conjugated antimouse or anti-rabbit secondary antibody.

MDA-MB-231 cells were grown in DMEM/Ham's F12 (50:50) media supplemented with 10% FBS and antibiotics at 37°C in 5% CO2. To test the effect of 4 on the protein marker regulating the apoptotic pathway, the expression level of essential proteins like Cytochrome C, caspase3, Bcl2, and Bid were observed. Cells were grown up to 90% confluency and then treated with 10 and 20 μM of 4 or DMSO vehicle for 12 h. The cells were lysed, and the protein samples were resolved by running through 6% SDS-PAGE. The protein was transferred onto nitrocellulose paper and blocked with 5% skimmed milk for 2 h at room temperature. The membrane was then incubated overnight at 4°C with respective primary antibodies. The expression level of indicated proteins were detected with HRP conjugated anti-rabbit secondary antibody. The normalized protein expression level was determined from the densitometric analysis of the respective blot using the program Image J. 

The interaction between the kinase domain and 4 was studied by measuring the kinetics of change in intrinsic tryptophan fluorescence of the kinase domain using a fluorimeter fitted with stop flow attachment at room temperature. In all the experiments, λ ex and λ em was fixed at 290 and 350nm, respectively.

For the fluorescence kinetics measurements the kinase domain at 100 nM concentration in 50 mM Tris (pH 8.0) buffer (containing 100 mM NaCl, 5% glycerol, 1 mM DTT) was titrated against indicated concentration of 4. To compare the kinetic transient of Abl-wt and Y253F-Abl in response to imatinib, 100 nM protein was mixed with 15 µM imatinib. Each transient was measured over 5ms interval with 6000-12000 time points and repeated four times. Data were fit to the following first-order kinetic equation

The observed rate constants were plotted against the concentration of 4 and fitted to a linear function to determine the binding rate constant (k on ) and dissociation rate constant (k off ) from the slope and the intercept, respectively. Binding constant (K d ) was derived from the ratio of k off and k on .

DLS measurements were performed at 30° c using Malvern Zetasizer Nano ZS. Each measurement correlation time of 15s per run was set, and ten runs per sample were collected. To check aggregation of 1 and 4, 15µM of each compound was resuspended in assay buffer (50mM Tris, 100mM NaCl, 5% glycerol and 1mM DTT) and incubated for 30 mins at room temperature. Each sample was passed through 0.2 µm filters before measuring with DLS. 30% (W/V) sucrose solution was under the same condition was used as a monomer control. (72) The imatinib was used as a reference sample for comparative study. The experimental results were analysed using built-in Zetasizer software. During the calculation of hydrodynamic radius, a dispersant viscosity of 0.79 mPa and a refractive index of 1.33 was used.

MDA-MB-231 cells were seeded in a glass-bottomed 35 mm petri dish at a density of 0.5 × 10 6 cells per plate. At 70-80 % confluency cells were treated with 15μM of 1 and 4 as described previously in the cytotoxicity assay. After 6 h incubation, cells were washed with ice-cold Hanksʼ balanced salt solution (HBSS) without magnesium or calcium and immediately emersed with ice-cold FM4-64-FX staining solution (5 μg/ml) for 1 minute on ice and immediately washed with HBSS. (43) The live-cells were imaged using LEICA DM I8 confocal microscope at λ ex / λ em of 565 nm/ 744, and 480 nm/ 550 nm for the FM4-64-FX lipophilic dye and 4 (or 1), respectively.

In summary, we have designed a fluorescent, curcumin based Hck inhibitor of nanomolar affinity and enhanced cellular stability. Hck inhibitors are shown to be useful in treating leukemia, management of viral induced hyperimmune reactions (cytokine storm) and various autoimmune diseases. Almost all kinase inhibitors exhibit some degree of promiscuity. However, it is well understood that to reduce promiscuity small molecules are designed to target the inactive conformations. Our biochemical studies suggest that the 4-aryl nitrogen-mustard curcumin derivative, 4, selectively targets the Hck over Abl, DYRK2 or Src kinase domain. Most importantly the selective targeting of the DFG-in inactive conformation of Hck prevents the structural transition of the kinase domain to the active state, which suggests that 4 may be less promiscuous. Cellular studies show that the cytotoxicity of 4 is predominantly due to inhibition of SFK mediated cell signalling. This work widens the horizon of the fluorescent curcumin scaffold, through 4-arylidene modification at the methylenic position, to design kinase inhibitors with enhanced stability and remodelled target specificity. funding and infrastructural facilities of IISER Kolkata. The work presented in this manuscript is extramurally funded by SERB, Government of India vide project no. EMR/2107/002324, ECR/2015/000142 and DBT Ramalingaswamy Fellowship (BT/RFF/Re-entry/14/2014) to RD. We thank Dr. Arnab Gupta for helping us with the confocal microscopy, a facility funded by DBT Wellcome Trust to AG. SB, MPC thanks CSIR and SR thanks INSPIRE for their respective research fellowships. We are thankful to DST-FIST (SR/FST/LS-II/2017/93) for the analytical biology facility at IISER Kolkata.

Author Contributions ‡ These authors contributed equally. The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript. AM helped in the design, SB and SR synthesized and characterized the curcumin derivatives. SB and SR performed the stability studies. In vitro cytotoxicity was done by SB, SR and MPC. RD, MPC and SB designed the biochemical and cell-based experiments. IB and MPC performed the confocal microscopy.

The authors declare that they have no conflicts of interest with the contents of this article.

The Supporting Information contains FigureS1-S17.

All the relevant data are contained within this article and in the supporting information. 

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The authors thank Professor John Kuriyan at the University of California, Berkeley and Prof. Markus Seeliger at Stonny Brook University for the Src, Yop and Hck constructs. The authors thank the research