Isatin-Hydrazones with Multiple Receptor Tyrosine Kinases (RTKs) Inhibitory Activity and In-Silico Binding Mechanism

: Recently, we have reported a series of isatin hydrazone, two of them, namely, 3-((2,6-dichlorobenzylidene)hydrazono)indolin-2-one ( 1 ) and 3-((2-chloro-6-ﬂuorobenzylidene)hydrazono) indolin-2-one ( 2 ) having potent cytotoxicity, showing cyclin-dependent kinases (CDK2) inhibitory activity and bearing recommended drug likeness properties. Since both compounds ( 1 and 2 ) showed inhibitory activity against CDK2, we assumed it would also have multiple receptor tyrosine kinases (RTKs) inhibitory activity. Considering those points, here, above-mentioned two isatin hydrazone 1 and 2 were synthesized using previously reported method for further investigation of their potency on RTKs (EGFR, VEGFR-2 and FLT-3) inhibitory activity. As expected, Compound 1 exhibited excellent inhibitory activity against epidermal growth factor receptor (EGFR, IC 50 = 0.269 µ M), vascular epidermal growth factor receptor 2 (VEGFR-2, IC 50 = 0.232 µ M) and FMS-like tyrosine kinase-3 (FLT-3, IC 50 = 1.535 µ M) tyrosine kinases. On the other hand, Compound 2 also exhibited excellent inhibitory activity against EGFR (IC 50 = 0.369 µ M), VEGFR-2 (IC 50 = 0.266 µ M) and FLT-3 (IC 50 = 0.546 µ M) tyrosine kinases. A molecular docking study with EGFR, VEGFR-2 and FLT-3 kinase suggested that both compounds act as type I ATP competitive inhibitors against EGFR and VEGFR-2, and type II ATP non-competitive inhibitors against FLT-3.


Introduction
Cancer is not only a complex disease but it also life threatening [1]. Therefore, development of an excellent anticancer agents are very much essential, especially ones with potent biological activities, enzyme inhibitory activities and low/no toxicity [2][3][4][5] (Figure 1). Regarding enzyme inhibitory activities: (i) cyclin-dependent kinases (CDKs) are considered as a vital feature, inciting various key transitions in the cell cycle for cancer cells, in addition to instructing apoptosis, transcription and exocytosis; (ii) the epidermal growth factor receptor (EGFR) [6] kinase enzyme promote overexpression, and overexpression of certain proteins may play a role in various cancer development [7][8][9][10][11][12][13][14][15]; (iii) the vascular endothelial growth factor receptor 2 (VEGFR-2) [16,17] is highly expressed in tumor-associated endothelial cells, where it modulates tumor-promoting angiogenesis, and it is also found on the surface of tumor cells [18]; (iv) FMS-like tyrosine kinase-3 (FLT-3) is a protein found in humans and is encoded by the FLT-3 gene [19][20][21]. Mutations of the FLT-3 receptor can lead to the development of leukemia, a cancer of bone marrow hematopoietic progenitors [22][23][24][25]. The development of a cancer may be delayed or cured by inhibition of those kinase enzymes. Recently, a number of researches have been focusing on how to block EGFR kinase enzyme activity, applying synthetic organic molecules [26] such as imatinib [27], which is used in treating gastrointestinal stromal tumors (GISTs), chronic myelogenous leukemia (CML) and malignancies. Erlotinib [28], is used in the treatment of pancreatic cancer, non-small cell in the treatment of pancreatic cancer, non-small cell lung cancer (NSCLC) and several other types of cancer, but the mechanism of clinical antitumor action of erlotinib is not fully characterized. Neratinib [29][30][31], is exhibits antitumor action against EGFR, HER2, and HER4 positive carcinomas and used as an extended adjuvant treatment in adult patients with early stage HER2-overexpressed/amplified breast cancer, to follow adjuvant trastuzumab-based therapy. Sorafenib [32], is indicated for the treatment of unrespectable hepatocellular carcinoma and advanced renal cell carcinoma. Crizotinib [33][34][35], is used for the treatment of locally advanced or metastatic NSCLC that is anaplastic-lymphoma kinase (ALK)-positive as detected by a FDA-approved test. Further, many others [36][37][38][39][40][41] inhibit VEGFR-2 kinase [42][43][44][45][46][47][48][49][50][51] and FLT-3 kinase enzyme activities [25,[52][53][54][55][56][57]. However, most of them reported adverse reactions, which were edema, nausea, vomiting, muscle cramps, rash, diarrhea, fatigue and abdominal pain, etc. In our previous report [5], we have highlighted CDK2 kinase inhibitors (1 and 2), which act as potential type-II ATP competitive inhibitor, have two-fold cytotoxicity for Compound 1 (IC50 = 1.51 µM) and have similar cytotoxicity for Compound 2 (IC50 = 3.56 µM) comparing with known anticancer drug doxorubicin (IC50 = 3.1 µM) against human breast adenocarcinoma (MCF7) with recommended drug likeness properties. Those reported results for Compounds 1 and 2 motivated us to do further inhibitory activities against EGFR, VEGFR-2 and FLT-3 protein kinase enzymes with their docking simulations in order to explore the behavior of 1 and 2 within the active site of EGFR, VEGFR-2 and FLT-3 to justify its binding mechanism.

General
Solvents and chemicals were reagent grade (Sigma-Aldrich, St. Louis, MO, USA) and were used without further purification. Electrothermal IA9100 (Stone, Stafforshire, ST15 OSA, UK) equipment were used to measure the melting points of synthesized product. IR spectra were taken on a Perkin Elmer FT-IR Spectrum BX device (Ayer Rajah Crescent, Singapore). The 600 MHz Bruker NMR spectrometer (Reinstetten, Germany) was used for taking NMR (1H & 13C) spectra. An Agilent 6410 QQQ mass spectrometer was used to take mass of the synthesized compounds (Agilent Technologies, Palo Alto, CA, USA).

General
Solvents and chemicals were reagent grade (Sigma-Aldrich, St. Louis, MO, USA) and were used without further purification. Electrothermal IA9100 (Stone, Stafforshire, ST15 OSA, UK) equipment were used to measure the melting points of synthesized product. IR spectra were taken on a Perkin Elmer FT-IR Spectrum BX device (Ayer Rajah Crescent, Singapore). The 600 MHz Bruker NMR spectrometer (Reinstetten, Germany) was used for taking NMR (1H & 13C) spectra. An Agilent 6410 QQQ mass spectrometer was used to take mass of the synthesized compounds (Agilent Technologies, Palo Alto, CA, USA).

Calculation of the IC 50 Values
Calculation of the IC 50 values of EGFR, VEGFR-2 and FLT-3 inhibitory assays was performed using linear equation (y = mx + c) for log concentration vs. percentage of inhibition, and detailed results were inserted in the supporting material.

In Vitro EGFR/VEGFR-2 Inhibitory Activity
The EGFR/VEGFR-2 assay kit was designed to measure EGFR/VEGFR-2 inhibitory activity for screening and profiling applications, using Kinase-Glo ® MAX as a detection reagent. The EGFR/VEGFR-2 assay kit comes in a convenient 96-well format, with enough purified recombinant EGFR/VEGFR-2 enzyme, EGFR/VEGFR-2 substrate, ATP and kinase buffer 1 for 100 enzyme reactions. The assay was performed according to the protocol supplied from the EGFR/VEGFR-2 kinase assay kit #40321 (BPS Bioscience, San Diego, CA, USA) and #40325 (BPS Bioscience, San Diego, CA, USA), respectively [58,59]. The EGFR/VEGFR-2 activity at a single dose concentration of 10 µM was performed, where the Kinase-Glo MAX luminescence kinase assay kit (Promega#V6071) was used. The compounds were diluted in 10% DMSO and 5 µL of the dilution was added to a 50 µL reaction so that the final concentration of DMSO was 1% in all of the reactions. All of the enzymatic reactions were conducted at 30 • C for 40 min. The 50 µL reaction mixture contained 40 mM Tris, pH 7.4, 10 mM MgCl 2 , 0.1 mg/mL BSA, 1 mM DTT, 10 mM ATP, kinase substrate and the enzyme EGFR/VEGFR-2. After the enzymatic reaction, 50 µL of Kinase-Glo ® MAX luminescence kinase assay solution was added to each reaction and the plates were incubated for 5 min at room temperature. Luminescence signal was measured using a Bio Tek Synergy 2 microplate reader (For details, please see Table S1, raw data of enzyme assay for Compounds 1 and 2 is given in the Supplementary Materials file).

In Vitro FLT-3 Inhibitory Activity
The FLT-3 assay kit (FLT-3 kinase assay, Promega Corporation, Madison, WI, USA) was designed to measure FLT-3 activity for screening and profiling applications, using ADP-Glo TM as a detection reagent. The FLT-3 kinase-glo assays were carried out in 96-well plates at 30 • C for 4 h and tested compound in a final volume of 50 µL [55,60]. Enzyme, substrate, ATP and Compounds 1 and 2 were diluted in tyrosine kinase buffer. A total 1 µL of Compounds 1 or 2 in 5% DMSO, 2 µL of enzyme (defined from Table 1), 2 µL of substrate/ATP mixture was added to the wells of 96 low volume plate and incubated at room temperature for 120 min (For details, please see Table S1. raw data of enzyme assay for Compounds 1 and 2 is given in the Supplementary Materials file).

In-Silico Binding Mechanism
Molecular docking was carried out using the Protein Data Bank (PDB) structures corresponding to the EGFR, VEGFR-2 and FLT-3 protein kinases and were downloaded from the RCSB PDB database (https://www.rcsb.org/, accessed on 5 December 2020) in PDB format. The PDB IDs used for EGFR, VEGFR-2 and FLT-3 protein kinases were 6DUK, 3VHE and 6JQR, respectively. Protein and compounds structures were energy minimized, refined and prepared for docking study by Schrödinger Maestro (Version 2018-4). OLPS3 force field and extra precision (XP) docking protocol was selected to generate induced fit Appl. Sci. 2021, 11, 3746 4 of 12 docking scores which was explained in the established procedure [5,[61][62][63][64][65][66]. Van der Waals scaling factor and partial charges cutoff were selected to be 0.85 and 0.15 respectively for ligand molecules. The docking cutoff value was fixed at −10.00 kcal/mole for the screening of best poses of the docked compounds for subsequent processing.

EGFR, VEGFR-2 and FLT-3 Protein Kinase Inhibitory Activities of 1 and 2
Inhibitory activity results of Compounds 1 and 2 against EGFR, VEGFR-2 and FLT-3 protein kinases are summarized in Table 1.
As summarized in Table 1, 1 and 2 exhibited excellent inhibitory activity against EGFR, VEGFR-2 and FLT-3 comparing control drugs.

Overall Structural Arrangement of the Kinase Domain of EGFR, VEGFR-2 and FLT-3 Protein Kinases
Similar to the universal kinase domain conformations of numerous protein kinases, EGFR, VEGFR-2 and FLT-3 also exhibit a common kinase domain conformation ( Figure 2) [67][68][69]. The main domain organization involves an N-terminal lobe which contains the nucleotide binding loop with its core anti-parallel β-sheets and a C-terminal lobe which comprises the activation loop and catalytic loop. The Nand C-terminal lobes are connected by a short linker. To find the conserved regions in the kinase domain of the three kinases, multiple sequence alignment was done which showed 6 highly clustered conserved residues (Figure 3).

In-Silico Binding Mechanism Analysis
Molecular docking analysis of Compound 1 with EGFR kinase domain showed several important interactions with ATP binding site residues as well as DFG motif residues, which is important for inhibition of EGFR kinase. Interactions involved hydrogen bonding interaction with ATP binding site and DFG motif residue Phe856 and π-anion interaction with Asp855 ( Figure 4A,B). Binding analysis of Compound 2 with kinase domain of EGFR kinase revealed that, it formed hydrogen bond with Phe856, π-anion interaction with Asp855, π-sulfur interaction with Met790, π-alkyl interactions with Met766, Leu777, Leu747 and Leu858 ( Figure 4C,D). Among them, Asp855 and Phe856 involved in the ATP binding site and DFG motif.

In-Silico Binding Mechanism Analysis
Molecular docking analysis of Compound 1 with EGFR kinase domain showed several important interactions with ATP binding site residues as well as DFG motif residues, which is important for inhibition of EGFR kinase. Interactions involved hydrogen bonding interaction with ATP binding site and DFG motif residue Phe856 and π-anion interaction with Asp855 ( Figure 4A,B). Binding analysis of Compound 2 with kinase domain of EGFR kinase revealed that, it formed hydrogen bond with Phe856, π-anion interaction with Asp855, π-sulfur interaction with Met790, π-alkyl interactions with Met766, Leu777, Leu747 and Leu858 ( Figure 4C,D). Among them, Asp855 and Phe856 involved in the ATP binding site and DFG motif. Multiple sequence alignment of PDB sequences from EGFR, VEGFR-2 and FLT-3 protein kinase used for docking study (red boxes implicate cluster of conserved residues where blue box represents ac-tivation loop with DFG motif). Here, asterisk "*" represents conserved residues in all three kinase domains; colon ":" shows at least two residues are conserved; and period "." indicates non-conserved residues.  Docking analysis of Compound 1 with VEGFR-2 kinase domain revealed 2 hydrogen bond interactions with Cys919 and Glu917, π-π T shaped interactions with Leu840, Val848, Ala866, Lys868, Val899, Val916, Leu1035, Cys1045 and Phe1047 as well as van dar Walls interaction with Phe918 ( Figure 5A,B). Compound 2 similarly made 2 hydrogen bond interactions with the gate keeper residue Cys919 and π-π T shaped interaction with DFG motif residue Phe1047. Additionally, it formed several π-alkyl interactions as Compound 1 with the similar residues except Lys868 ( Figure 5C,D). Binding mechanism of Compound 1 with FLT-3 kinase domain involved hydrogen bond interaction with Cys694, π-π stacked binding with Tyr693, several π-alkyl interactions with Leu616, Val624, Ala642, Lys644, Val675 and Leu818. One π-sulfur interaction was observed with Cys828 ( Figure 6A,B).

Discussion
The structures of the synthesized Compounds 1 and 2 were confirmed using IR, NMR ( 1 H and 13 C), mass spectral data and physical properties, and compared with those reported values [5].

Discussion
The structures of the synthesized Compounds 1 and 2 were confirmed using IR, NMR ( 1 H and 13 C), mass spectral data and physical properties, and compared with those reported values [5].
In our previous study [5] we have reported that, Compounds 1 and 2 inhibited CDK2 kinase in an ATP dependent manner and acted as type II inhibitor by lacking DFG motif interaction in the activation loop. Interaction with DFG motif residues is crucial for an inhibitor to define as active or inactive state kinase inhibition. Similarly, in this experiment, the interaction of Compounds 1 and 2 with EGFR, VEGFR-2 and FLT-3 protein kinases was evaluated by molecular docking analysis.
Since Compound 1 with EGFR kinase domain showed several important interactions and involved hydrogen bonding interactions with ATP binding site and DFG motif residue, thus, it can be said that, Compound 1 might act on active kinase by interacting with DFG motif residues. It also formed several π-π stacked and π-π T shaped interactions with Met766, Met790, Phe723, Leu747, Leu777, Leu788, Ile759, Leu858 and Leu861. Compound 2 with kinase domain of EGFR kinase formed hydrogen bond. Among the hydrogen bonds, Asp855 and Phe856 involved in the ATP binding site and DFG motif, thus, Compound 2 can act as type I inhibitor by active against EGFR kinase. The interactions of both compounds (1 and 2) with EGFR kinase showed ATP competitive inhibition and thus support previous experiments [71,72]. Compound 1 with VEGFR-2 kinase domain revealed two hydrogen bond interactions with Cys919 and Glu917, π-π T shaped interactions with many residues, as well as van dar Walls interaction, therefore, it was clear from the analysis that, Compound 1 can interact with the DFG motif residue Phe1047 of the ATP binding site Cys919 of the hinge region which also act as a gate keeper residue. Compound 2 similarly made two hydrogen bond interactions with the gate keeper residue, and additionally, it formed several π-alkyl interactions as Compound 1 with the similar residues except Lys868. The interactions of Compounds 1 and 2 with the kinase domain is consistent with the previous docking results which showed similar interaction of the synthesized compounds [73,74]. Therefore, the docking result of Compounds 1 and 2 with the kinase domain of VEGFR-2 showed similar fashion of interactions involving both DFG motif and hinge region (which comprise the ATP binding site) interaction could possibly make them type I ATP competitive inhibitor (DFG motif interaction implies active state of kinase) against VEGFR-2 kinase. The interactions of Compound 1 and Flt-3 kinase domain involve hydrogen bond. Previously published co-crystal structure of FLT-3 with Quizartinib showed that, interactions with Phe830 in the DFG motif and Phe691 in the hinge region would be crucial for inhibition of the active kinase [75]. However, Compound 1 lacked these interactions. The interactions between Compound 2 and Flt-3 kinase domain lack active state kinase residues. However, it showed several hydrogen and ionic bond interactions similar to Compound 1 except Glu692. It lacked interaction with Tyr693 compared to Compound 1. Finally, from the docking result of Compounds 1 and 2 with FLT-3 kinase domain it can be concluded that both compounds lacked active state kinase domain interactions and lacked interactions with hinge region of the ATP binding site and can be considered as type II inhibitor in case of FLT-3 kinase but not as ATP competitive inhibitor.

Conclusions
In conclusion, isatin hydrazones 1 and 2 exhibited excellent inhibitory activity against EGFR, VEGFR-2 and FLT-3 protein kinases. Binding mechanism analysis by molecular docking study of 1 and 2 revealed that, both compounds acted as type I ATP competitive inhibitor against EGFR and VEGFR-2 kinase by interacting with DFG motif and hinge region of ATP binding site. However, they lacked important interactions with ATP binding site residues of FLT-3 kinase thus might act as type II ATP non-competitive inhibitor.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/app11093746/s1, Figure S1: Calculation of the IC 50 Values of Protein Kinase Enzyme EGFR for Compound 1, Table S1: Enzyme assays for Compounds 1 and 2 and Experimental Data for