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Insilico study of anti-carcinogenic lysyl oxidase-like 2 inhibitors
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Computational Biology and Chemistry journal homepage: www.elsevier.com/locate/compbiolchem
Research Article
Insilico study of anti-carcinogenic lysyl oxidase-like 2 inhibitors Syed Aun Muhammad a , Amjad Ali b,∗ , Tariq Ismail a , Rehan Zafar b , Umair Ilyas c , Jamil Ahmad d a
Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan Riphah Institute of Pharmaceutical Sciences (RIPS) Islamabad 44000, Pakistan d Research Center of Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), Islamabad, Pakistan b c
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 13 January 2014 Received in revised form 10 March 2014 Accepted 10 March 2014 Available online xxx Keywords: LOXL2 In silico Drug designing Anti-cancer Lead molecules Triazoles
Lysyl oxidase homolog 2 (LOXL2), also known as lysyl oxidase-like protein 2 is recently been explored as regulator of carcinogenesis and has been shown to be involved in tumor progression and metastasis of several carcinomas. Therefore LOXL2 has been considered as potential therapeutic target. Doing so, its inhibitors as new chemotherapeutic lead molecules: 4-amino-5-(2-hydroxyphenyl)-1,2,4-triazol3-thione (2a) and 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol (2b) are synthesized by fusion method (refluxed at 160 ◦ C). Spectral analysis of these triazole derivatives are characterized by FTIR and NMR. Active binding sites and quality of the LOXL2 model is assessed by Ramachandran plots and finally drug–target analysis is performed by computational virtual screening tools. Compounds 2a and 2b showed optimum target binding affinity with −6.2 kcal/mol and −8.9 kcal/mol binding energies. This insilico study will add to our understanding of the drug designing and development, and to target cancer-causing proteins more precisely and quickly than before. Published by Elsevier Ltd.
1. Introduction Carcinogenesis is literally the development of cancer which can be characterized by a progression of changes at the cellular, genetic and epigenetic level and as a consequence the cells undergo abandoned cell division and form a malignant mass (Fearon and Vogelstein, 1990). During development of cancers, in common, tumor suppressor genes are silenced in cancer cells by biological enzymes which catalyze epigenetic histone modifications. Therefore these enzymes offer new therapeutic targets for anti-cancer drugs. The histone modification by lysyl oxidase homolog 2 (LOXL2) enzyme has been previously reported and its expression is shown to be up-regulated in several pathologic states and hence its contribution in development of fibrosis and cancer (Barry-Hamilton et al., 2010). In general, LOXL2 evidently over expressed in a majority of human cancers, these findings strengthened LOXL2 to be considered a putative target for cancer treatment (Jourdan-Le Saux et al., 1999). LOXL2 enzyme is considered as novel drug target that promotes the cancer metastasis and it has been reported that LOXL2
∗ Corresponding author. Tel.: +92 51 9085 6138. E-mail address: amjad [email protected] (A. Ali).
is markedly overexpressed in carcinoma relative to normal condition and associated with tumor development. The inhibition of LOXL2 target in gene knockout studies significantly inhibited the tumor growth and metastasis (Peng et al., 2009; Chang et al., 2013). Therefore, LOXL2 is likely to be an excellent therapeutic target in many cancer types (Barker and Erler, 2011) and has been selected for this study due to its potential role. Presently, it is unknown whether LOXL2 can be pharmacologically targeted for cancer treatment, nevertheless, small inhibitors molecule against histone deacetylases have been developed as an effective reagents to activate the expression of tumor suppressor gene in cancer chemotherapy, however, there is a need of chemical inhibitors that may counteract its activity (Bekircan and Gumrukcuoglu, 2005; Herranz et al., 2012). The epigenetic studies underlying tumor genesis has been proved for the identification of new therapeutic targets for cancer drugs. Nevertheless, research to develop chemotherapeutic drugs for treatment of carcinogenesis is relatively slow due to expensive techniques, uncharacterized genetic basis of cancer and increased reports of mutagenesis. Thus, there is urgent need of establishing alternative drug development strategies to encounter the situation. In this regard, in silico methods have the advantages of speed, low cost and, even more importantly, enables researchers to raise questions that would otherwise be difficult to address experimentally.
http://dx.doi.org/10.1016/j.compbiolchem.2014.03.002 1476-9271/Published by Elsevier Ltd.
Please cite this article in press as: Muhammad, S.A., et al., Insilico study of anti-carcinogenic lysyl oxidase-like 2 inhibitors. Comput. Biol. Chem. (2014), http://dx.doi.org/10.1016/j.compbiolchem.2014.03.002
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There is a progressive development in drug discovery procedures from conventional ligand based drug discovery to structural and targeted based drug designing approaches (Mdluli and Spigelman, 2006). These virtual techniques are widely used and have become vital component of drug discovery and development programs (Bajorath, 2002). The applicability of such methods in the new lead identification and optimization has served as an important tool in our quest to access novel drug like compounds (Reddy et al., 2007). Here, we have proposed new triazole derivatives as LOXL2 inhibitors that showed optimum binding affinity with minimum binding energies. These triazole compounds can inhibit the cancer cell growth by modifying this enzyme as previous studies on mice models revealed that some clinically relevant chemotherapeutic agents have anti-cancer activity by blocking the action of LOXL2 enzyme (Erler et al., 2006; Baker et al., 2013). Triazoles have been used as a pharmacore for many years in order to develop novel ligands and have been proven useful, thus gained considerable attention in drug industries because of their effective biological activities. These derivatives have been shown to possess therapeutically interesting activities such as antimicrobial, and anti-cancer (Bekircan and Gumrukcuoglu, 2005). We have synthesized and analyzed the new efficient triazole derivatives that can inhibit the LOXL2 enzyme, thereby offering new avenues for cancer research and treatment. The current study was conducted with the objectives: synthesis of new triazoles derivatives, characterization and drug likeness evaluation, and finally these drugs were computationally designed against LOXL2 enzyme to arrest metastasis. Therefore, these new triazoles derivatives can get a pharmaceutical application on future directions in anti-cancer drug development.
2. Materials and methods 2.1. Synthesis of lead compounds
2.1.3. Step 3: synthesis of 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol (Schiff base) from (2b) 1 g of 2a was dissolved in ethanol while heating on water bath. Completely dissolved equimolar quantity of benzaldehyde added and reflux was started with continuous stirring for 4 h. The completion of reaction was monitored by TLC. The mixture then cooled to form solid product. It was then filtered and recrystallized by 70% ethanol.
Synthesis of 4-amino-5-(2-hydroxyphenyl)-1,2,4-triazol3-thione (2a) (Fig. 5A) and 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol (2b) (Fig. 5B) compounds (2a) and (2b) were synthesized according to three-steps experimental procedure.
2.1.1. Step 1: synthesis of thiocarbohydrazide (1a) Hydrazine hydrate (30 ml) was heated at reflux condenser with carbon disulfide (15 ml) in the presence of ethanol (150 ml) for about 4 h. On cooling, thiocarbohydrazide was precipitated as solid. Excess of the solvent and thiocarbohydrazide was removed by heating on water bath at their boiling points unless free from both of the solvent and unreacted hydrazine hydrate.
2.1.2. Step 2: synthesis of 4 amino-5-(2-hydroxyphenyl)-1,2,4-triazol-3-thione (2a) Equimolar quantities of thiocarbohydrazide (0.1 mol) and salicylic acid (0.1 mol) were fused at 160 ◦ C for about 2 h in an oil bath by constant stirring. TLC was used to determine the progress of reaction. After completion of reaction the mixture was cooled and filtered and recrystallized from 70% ethanol.
2.2. Structural characterization Physical properties and structural characterization of these derivatives were carried out by FTIR (Bruker Germany Alpha
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model) and the interferogram of samples were recorded in the 3600–650 cm−1 region. The NMR samples were prepared by taking 15 mg in 0.5 ml D2 O and NMR spectra were recorded on Bruker 300 MHz spectrometer equipped with 5 mm of probe head for 1 H analysis. 2.3. Lipinski’s rule of 5 Lipinski’s rule of 5 (RO5) was applied using Cheminformatic Molinspiration tool (Cheminformatics, 2013) to determine the pharmacokinetics properties (ADME/T) of these compounds. According to the RO5, the molecules must have hydrogen bond donor’s ≤5 (OH and NH groups), hydrogen bond acceptors ≤10 (N and O atoms), molecular weight <500 Da, and log P coefficient (C log P) less than 5. 2.4. Drug–targets interaction analysis 2.4.1. Preparation of ligand molecules The chemical structures of 2a and 2b derivatives were prepared by ChemBioDraw and MOL SDF format of both ligands, converted to PDBQT file using PyRx tool to generate atomic coordinates. 2.4.2. Accession of target proteins The 3D structure of LOXL2 was built on I-Tasser (Zhang, 2008) modeling server and the predicted protein models accessed from server. 2.4.3. Quality of protein model The quality of protein model was evaluated by C-score estimated by the I-Tasser server (Zhang, 2008) and Qualitative Model Energy Analysis (QMEAN) Z-score. QMEAN is a composite scoring function describing the main geometrical features of protein structures and the local geometry is analyzed by a new kind of torsion angle potential over three consecutive amino acids. QMEAN solvation potential also describes the burial status of the residues of the protein models (Benkert et al., 2008). The Ramachandran plots built by Drug Discovery Studio version 3.0 that specify low energy conformations for ϕ (phi) and (psi) along with favorable and unfavorable regions for amino acid residues. These graphs also presented the local backbone conformation and points on plot are indicating the ϕ and torsion angles of a residue. 2.4.4. Analysis of target active binding sites The active sites of target protein were analyzed using the Drug Discovery Studio version 3.0 and 3DLigandSite virtual tools (Wass et al., 2010). An active site was defined from the coordinates of the ligand in the original target protein sites. 2.4.5. Docking A computational ligand-target docking approach was used to determine structural complexes of the LOXL2 with heterocyclic triazole derivatives (2a and 2b) in order to understand the structural basis of these protein targets specificity. Initially, protein–ligand attraction were investigated for hydrophobic/hydrophilic properties of these complexes by Platinum software web server (Pyrkov et al., 2009). Finally, docking was carried out by PyRx, AutoDock Vina option based on scoring functions. The energy of interaction of these derivatives with the protein targets is assigned “grid point”. At each step of the simulation, the energy of interaction of ligand and protein was evaluated using atomic affinity potentials computed on a grid.
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Table 1 Characterization data of the compound 2a [4-amino-5-(2-hydroxyphenyl)1,2,4-triazol-3-thione] and 2b [4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol]. Compound
2a
2b
Molecular formula Color Solubility in water Solubility in ethanol melting point Form Rf value (methanol:chloroform (3:1))
C8 H8 N4 OS Yellow Not soluble Soluble on heating 120 ◦ C Crystalline 0.16
C15 H12 N4 O2 S Yellow Not soluble Soluble on heating 170 ◦ C Crystalline 0.20
3. Results 3.1. Synthesis of lead molecules Scheme 1 was employed to synthesize the effective LOXL2 inhibitors (2a and 2b) with melting point of 120 ◦ C and 170 ◦ C, respectively. These compounds were found to be soluble in ethanol on heating and TLC with Rf value 0.16 and 0.12 respectively in methanol–chloroform solvent system (Table 1).
3.2. Structural characterization 3.2.1. Spectral analysis The FTIR data of 2a and 2b confirmed the peak values of functional groups in accordance to the structures of both the compound, the peak value for OH in case of 2a observed at 3137 cm−1 while that of 2b at 3061–3100 cm−1 . The characteristic stretching vibrations of the product 2a and 2b were observed at 1591 and 1599 (C N), respectively. The absence of N H and C O absorption bands in spectra confirmed that both the compounds are formed via cyclocondensation (Table 2). The 1 H NMR spectral signals revealed that the data belongs to aliphatic and aromatic groups: for 2a compound [H NMR (DMSO, 300 MHz, 5 ppm): 7.25–7.37 (m, 4H, Ar-H), 5.37 (s, 2H, NH2 ), 10.3 (bS, 1H, 0H), 13.9 (S, 1H, SH)] and for 2b compound [7.28–7.43 (m, 8H, Ar-H), 9.1 (S, 1H, CH), 10.5 (bS, 2H, OH), 13.8 (9S, 1H, SH)].
3.2.2. Lipinski’s rule of 5 The pharmacokinetic properties (ADME/T) of 2a and 2b passed the Rule of 5 with zero violations. For 2a: the partition coefficient log P: 1.407, molecular weight: 208.46 and the number of violations: 0.0 and for 2b: the log P value: 3.397, molecular weight: 312.354 and number of violations 0.0 (Table 3).
Table 2 FTIR spectral analysis for amino-triazole derivatives. Compound
FTIR (cm−1 )
2a [4-Amino-5-(2hydroxyphenyl)-1, 2, 4-triazol-3-thione]
3137 cm−1 stretching (O H), 2950–3000 (N H), 1591–1599 stretching (N C), 1820–1760 (C O, N H), 1562–1598 cm−1 ( C N), 1313–1365 cm−1 ( C N), 1257 (C C), stretching ( C O ) 3061–3100 cm−1 stretching (O H), 2950–3000 (N-H), 1591–1599 stretching (N C), 1760 (C O), 1562–1598 cm−1 ( C N), 1313–1365 cm−1 ( C N), 1257 (C C), stretching ( C O )
2b [4-(2-Hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3thiol]
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Fig. 1. Top five predicted protein models by I-Tasser from A–E. C-score that reveals the strength of the predicted model and the model with the highest score (first one) is the most likely to exist.
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Table 3 Drug likeness properties of 2a and 2b molecules using Lipinski’s rule of 5. RO5a (pharmacokinetic properties)
Ligand (2a)
Ligand (2b)
MiLogP TPSA natoms MW nON nOHNH nviolation nrotb volume
1.407 76.969 14.0 208.246 5 3 0 1 169.473
3.397 83.538 22.0 312.354 6 2 0 3 260.871
a
RO5: rule of 5.
3.3. Drug–targets interaction 3.3.1. Protein models analysis The quality of the model (LOXL2, Fig. 1) was estimated by Cscore and highest score was observed for protein model 1 (C-score: −1.00), while QMEAN for this model was −6.30 (Fig. 2). Analysis of Ramachandran plots revealed that about 90% residues of almost all predicted protein models (␣-helices) in the most favored regions while less than 2% were in the disallowed region. The Ramachandran plot is indicating low energy conformations for ϕ (phi) and (psi), the graphical representation of the local backbone conformation of each residue of our target protein is shown in (Fig. 3). 3.3.2. Active binding site analysis The active binding sites are figure out on to the surface of LOXL2 protein using virtual tools, which revealed that the probable sites:
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Table 4 Energy and RMSD values obtained during docking analysis of 2a as ligand molecule and LOXL2 as target protein. Complex
Binding affinity
RMSD/UBa
RMSD/LBa
LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2
−6.2 −6.1 −6.1 −6 −6 −6 −5.9 −5.9 −5.7
0 2.869 23.158 23.947 23.368 20.95 23.922 16.868 18.804
0 1.798 22.755 23.321 22.836 20.279 23.074 16.358 17.953
2a 2a 2a 2a 2a 2a 2a 2a 2a
a RMSD/UB: root mean square deviation/upper bond; RMSD/LB: root mean square deviation/lower bond.
ASP (average distance: 0.33), LEU (average distance: 0.51), VAL (average distance: 0.30), MET (average distance: 0.42) and ALA (average distance: 0.49) (Figs. 4 and 5). 3.3.3. Hydrophobic, hydrophilic and docking analysis The distribution of hydrophobic/hydrophilic properties of LOXL2 and its binding site projected onto the surface of the ligand, and their complementarity is analyzed and shown in Fig. 6, where it has been observed that aromatic rings are less hydrophilic. These stacking interactions are used to rank the molecular docking and matching between target and drugs. Considering hydrophobic properties of these interacting molecules (LOXL2 target and triazoles) using the concepts of molecular hydrophobicity potential was calculated and compared by PLATINUM virtual tool. These hydrophobicity maps are used to analyze the progress of hydrophobicity clusters on the membrane surface along the molecular dynamics run and the features of interface between the membrane and membrane-binding molecule (Fig. 7). The docking of target proteins with synthesized compounds (2a and 2b) using AutoDock procedure revealed that all the computationally predicted lowest energy complexes of LOXL2are stabilized by intermolecular hydrogen bonds and stacking interactions. It is found that A, SA, OA, HD, N are the ligand atoms involved in docking with the enzymes (Figs. 8 and 9). The AutoGrid model presented the most energetically favorable binding mode of 2a and 2b to these enzymes. Analysis of different choices for combining structures into a single representative energy grid is performed in this study. These inhibitors are docked into the generated combined grids and the RMSD from native pose and the binding energy are evaluated and it is observed that the weight averaged grids performs the best. The ligands (2a and 2b) showed the best interaction with target proteins based on the RMSD values. Beside RMSD clustering, AutoDock Vina has also calculated the binding free energies of these interactive molecules to find the best binding mode. During these interaction procedures, the hydrogen bond between these molecules and protein targets are most important, i.e., it can decide the binding strength and the conformation of ligands. The calculated final docked energies for 2a is −6.2 kcal/mol and for 2b is-8.9 kcal/mol. The energy information of these docking results along with RMSD values are also listed in Tables 4 and 5. Docking results revealed that these ligands molecules can enter the substrate-binding region of the active site. Finally, these results demonstrated clearly that 2a and 2b molecules accurately interact with LOXL2 protein target. 4. Discussion
Fig. 2. The quality of the model was estimated by Z-score QMEAN which was −6.30. (A) Z-score with various aspects of the protein molecule [red indicates worse while blue indicates better]. (B) Comparison with protein size and normalized QMEAN Z-score of non-redundant set of protein structures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
In this study, we synthesized amino triazoles as inhibitors which reveal significant binding affinity to human LOXL2 under computational docking analysis. LOX expression is often upregulated in many types of cancer including breast, lungs, head and neck tumors (Erler et al., 2006) and due to the
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Fig. 3. These Ramachandran plots of top five predicted protein models (A–E) for LOXL2 constructed by Drug Discovery Studio version 3.0 indicated that whether the amino acid residues occur in the “favored region” or “disallowed region” of the plot. For a good protein model there must be ≥90% amino acid residues in the most favored region or <2% in the disallowed region of the plot.
fact this enzyme serves as good therapeutic target. As there is scarcity in treating metastasis due to LOXL2 activity, consequently this study add significant advances in describing the synthesis of new chemotherapeutic agents and emphasizing on new
treatments that would block the LOXL2 over expression activity. The anti-cancer 3-amino triazoles derivatives were synthesized at 160 ◦ C by reaction of thiocarbohydrazide with salicylic acid to
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Fig. 4. Active binding site analysis of LOXL2 protein target with binding cavity contains amino acids residues ASP, LEU, VAL, MET and ALA.
form 4-amino-5-(2-hydroxyphenyl)-1,2,4-triazol-3-thione (2a) and 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4triazole-3-thiol (2b). Similarly, in 2010, Kocyigit-Kaymakcioglu al. (2013) synthesized antibacterial 4-amino-5-(1et phenylethyl)-2,4-dihydro-3H-1, 2,4-triazole-3-thione by reacting
thiocarbohydrazide with 2-phenylpropanoic acid in an oil-bath at 130 ◦ C for 2 h and prepared Schiff bases by reacting equimolar amounts of 4-aminotriazole and substituted aldehydes in ethanol over 45 min. Due to structural properties, these 3-amino triazoles are important as potential biologically active compounds and
Fig. 5. Molecules 2a (A) and 2b (B).
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Fig. 6. Visualization where 3-windows show the distribution of hydrophobic/hydrophilic properties of LOXL2 in X-ray position (left) and exerted by protein surroundings (middle), and their complementarity (right). The structures are the X-ray models from I-Tasser server. The screenshot was produced using the online visualization Jmol applet as implemented in the PLATINMUM web-service (A) 2a and (B) 2b.
they have been reported to possess antimicrobial and anti-cancer (Bekircan and Gumrukcuoglu, 2005; Colanceska-Ragenovic et al., 2001; Murti et al., 2011) activities. These compounds 2a and 2bare found soluble in ethanol on heating with relevant flow rate of Rf :
0.16 and Rf : 0.12 respectively in methanol–chloroform solvent system. The molecular weight, partition coefficients and other pharmacokinetics properties of these molecules have passed the Lipinski’s rule of 5 with zero violations (Zaid et al., 2010).
Fig. 7. Hydrophobicity map for upper layer of LOXL2 target protein membrane, position of the protein is projected along the Z-axis (A) 2a (B) 2b.
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Fig. 8. Various views of docking interaction steps of 2a ligand molecule. (A) The AutoGrid dimensions between ligand and LOXL2 target protein atoms (A, HD, OA, and N) are: grid center X: 18.0175, Y: 23.2912, Z: 13.5997 with dimension (Angstrom) X:Y:Z: 25. (B) Confirmation and pose of 2a ligand molecule with protein target. (C) Binding site atoms of LOXL2 amino acids residues around ligand. (D) Solid surface slab around ligand atoms. (E) Interaction of amino acid residues ASP, MET, GLY, ILE, with ligand atoms (carbon and oxygen atoms).
The spectral data of these azole heterocyclic molecules confirmed the peak values of functional groups in accordance to the structures of both compounds. The absence of N H and C O absorption bands in spectra shows that these compounds were formed via cyclocondensation while the formation of triazole
ring by FTIR peaks has been confirmed that the characteristic absorption bands of >C N and C N of triazole ring appeared at 1562–1598 cm−1 and 1313–1365 cm−1 respectively (Pyrkov et al., 2009). Similarly, the 1 H NMR spectral signals revealed that the data belongs to aliphatic and aromatic groups. The absence of C S, N H
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Fig. 9. Various views of docking interaction steps of 2b ligand molecule. (A) The AutoGrid dimensions between ligand and LOXL2 target protein atoms (A, HD, OA, and N) are: grid center X: 18.0175, Y: 23.2912, Z: 13.5997 with dimension (Angstrom) X:Y:Z: 25. (B) Confirmation and pose of 2b ligand molecule with protein target. (C) Binding site atoms of LOXL2 amino acids residues around ligand. (D) Solid surface slab around ligand atoms. (E) Interaction of amino acid residues ASP, MET, GLY, ILE, with ligand atoms (carbon and oxygen atoms).
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Table 5 Energy and RMSD values obtained during docking analysis of 2b as ligand molecule and LOXL2 as target protein. Complex
Binding affinity
RMSD/UBa
RMSD/LBa
LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2
−8.9 −8.7 −8.4 −8.3 −8.1 −8.1 −7.8 −7.7 −7.6
0 2.38 3.368 3.014 3.058 3.733 22.333 4.533 22.02
0 1.517 1.83 1.834 2.548 2.612 21.933 2.71 20.879
2b 2b 2b 2b 2b 2b 2b 2b 2b
a RMSD/UB: root mean square deviation/upper bond; RMSD/LB: root mean square deviation/lower bond.
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stabilized by intermolecular interactions. These newly synthesized triazole derivatives can be clinically investigated for establishing safe doses. Conflict of interest The authors confirm that this article content has no conflicts of interest. Source of funding None declared. Ethical approval
and presence of S-H absorption established that the triazole ring in compound are in thiol form (Prakash et al., 2004; Sztanke et al., 2006; Liu et al., 2008). Previous clinical investigation showed that inhibition of LOXL2 activity has been established to eradicate metastases in mice models (Erler et al., 2006) which emphasize to develop new anti-LOXL2 inhibitors for the treatment of cancers. Furthermore, tumor growth inhibition properties of these heterocyclic triazoles have been previously reported (Bekircan and Gumrukcuoglu, 2005; Murti et al., 2011). Therefore, in silico association between LOXL2 enzyme and these new triazole derivatives have been positively analyzed and reported for the first time. This study is based on such protocols by synthesizing the new LOXL2 inhibitors. Five protein models of LOXL2 enzyme have been obtained using I-Tasser server and quality of the models were estimated based on C-score (Zhang, 2008), QMEAN value (Benkert et al., 2008) and Ramachandran plot. Among five, first protein model was selected due to best C-score (−1.00) and QMEAN (−6.30) values while Ramachandran plot revealed that about 90% residues of almost all predicted protein models (␣helices) were found to be in the most favored regions while less than 2% were in the disallowed region. The binding residues that define the coordinates of the ligand (Gohlke and Klebe, 2002) on to the surface of LOXL2 protein were analyzed confirming that these ligands molecules can enter the substrate-binding region of the protein active sites. The docking interaction of target protein with synthesized compounds (2a and 2b) based on conformation and scoring functions (Kitchen et al., 2004) showed stable complexes with LOXL2 with lowest binding energies. The AutoGrid Model presented the most energetically favorable binding mode of 2a and 2b to this enzyme and the calculated final docked energies for 2a: −6.2 kcal/mol and for 2b: −8.9 kcal/mol. These results exposed that 2a and 2b molecules accurately interact with LOXL2 protein target. 5. Conclusion No doubt the availability of computational and in silico approaches is the landmarks in drug discovery process. The virtual screening and the docking analysis have provided a remarkable approach for accessing the lead molecules to be used as drug ligands and to understand the protein–ligand binding affinity. Lysyl oxidase like 2 proteins (LOXL2) is important enzyme that has been reported in the up-regulation and proliferation of tumors and considered as a good therapeutic target for the treatment of breast, head and neck cancer. Due to shortage of chemotherapeutic agents, new triazole derivatives (2a and 2b) are synthesized and their interaction with LOXL2 is evaluated. This analysis revealed that all the computationally predicted lowest energy complexes of these enzymes are
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Contents lists available at ScienceDirect
Computational Biology and Chemistry journal homepage: www.elsevier.com/locate/compbiolchem
Research Article
Insilico study of anti-carcinogenic lysyl oxidase-like 2 inhibitors Syed Aun Muhammad a , Amjad Ali b,∗ , Tariq Ismail a , Rehan Zafar b , Umair Ilyas c , Jamil Ahmad d a
Department of Pharmacy, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad 44000, Pakistan Riphah Institute of Pharmaceutical Sciences (RIPS) Islamabad 44000, Pakistan d Research Center of Modeling and Simulation (RCMS), National University of Sciences and Technology (NUST), Islamabad, Pakistan b c
a r t i c l e
a b s t r a c t
i n f o
Article history: Received 13 January 2014 Received in revised form 10 March 2014 Accepted 10 March 2014 Available online xxx Keywords: LOXL2 In silico Drug designing Anti-cancer Lead molecules Triazoles
Lysyl oxidase homolog 2 (LOXL2), also known as lysyl oxidase-like protein 2 is recently been explored as regulator of carcinogenesis and has been shown to be involved in tumor progression and metastasis of several carcinomas. Therefore LOXL2 has been considered as potential therapeutic target. Doing so, its inhibitors as new chemotherapeutic lead molecules: 4-amino-5-(2-hydroxyphenyl)-1,2,4-triazol3-thione (2a) and 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol (2b) are synthesized by fusion method (refluxed at 160 ◦ C). Spectral analysis of these triazole derivatives are characterized by FTIR and NMR. Active binding sites and quality of the LOXL2 model is assessed by Ramachandran plots and finally drug–target analysis is performed by computational virtual screening tools. Compounds 2a and 2b showed optimum target binding affinity with −6.2 kcal/mol and −8.9 kcal/mol binding energies. This insilico study will add to our understanding of the drug designing and development, and to target cancer-causing proteins more precisely and quickly than before. Published by Elsevier Ltd.
1. Introduction Carcinogenesis is literally the development of cancer which can be characterized by a progression of changes at the cellular, genetic and epigenetic level and as a consequence the cells undergo abandoned cell division and form a malignant mass (Fearon and Vogelstein, 1990). During development of cancers, in common, tumor suppressor genes are silenced in cancer cells by biological enzymes which catalyze epigenetic histone modifications. Therefore these enzymes offer new therapeutic targets for anti-cancer drugs. The histone modification by lysyl oxidase homolog 2 (LOXL2) enzyme has been previously reported and its expression is shown to be up-regulated in several pathologic states and hence its contribution in development of fibrosis and cancer (Barry-Hamilton et al., 2010). In general, LOXL2 evidently over expressed in a majority of human cancers, these findings strengthened LOXL2 to be considered a putative target for cancer treatment (Jourdan-Le Saux et al., 1999). LOXL2 enzyme is considered as novel drug target that promotes the cancer metastasis and it has been reported that LOXL2
∗ Corresponding author. Tel.: +92 51 9085 6138. E-mail address: amjad [email protected] (A. Ali).
is markedly overexpressed in carcinoma relative to normal condition and associated with tumor development. The inhibition of LOXL2 target in gene knockout studies significantly inhibited the tumor growth and metastasis (Peng et al., 2009; Chang et al., 2013). Therefore, LOXL2 is likely to be an excellent therapeutic target in many cancer types (Barker and Erler, 2011) and has been selected for this study due to its potential role. Presently, it is unknown whether LOXL2 can be pharmacologically targeted for cancer treatment, nevertheless, small inhibitors molecule against histone deacetylases have been developed as an effective reagents to activate the expression of tumor suppressor gene in cancer chemotherapy, however, there is a need of chemical inhibitors that may counteract its activity (Bekircan and Gumrukcuoglu, 2005; Herranz et al., 2012). The epigenetic studies underlying tumor genesis has been proved for the identification of new therapeutic targets for cancer drugs. Nevertheless, research to develop chemotherapeutic drugs for treatment of carcinogenesis is relatively slow due to expensive techniques, uncharacterized genetic basis of cancer and increased reports of mutagenesis. Thus, there is urgent need of establishing alternative drug development strategies to encounter the situation. In this regard, in silico methods have the advantages of speed, low cost and, even more importantly, enables researchers to raise questions that would otherwise be difficult to address experimentally.
http://dx.doi.org/10.1016/j.compbiolchem.2014.03.002 1476-9271/Published by Elsevier Ltd.
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There is a progressive development in drug discovery procedures from conventional ligand based drug discovery to structural and targeted based drug designing approaches (Mdluli and Spigelman, 2006). These virtual techniques are widely used and have become vital component of drug discovery and development programs (Bajorath, 2002). The applicability of such methods in the new lead identification and optimization has served as an important tool in our quest to access novel drug like compounds (Reddy et al., 2007). Here, we have proposed new triazole derivatives as LOXL2 inhibitors that showed optimum binding affinity with minimum binding energies. These triazole compounds can inhibit the cancer cell growth by modifying this enzyme as previous studies on mice models revealed that some clinically relevant chemotherapeutic agents have anti-cancer activity by blocking the action of LOXL2 enzyme (Erler et al., 2006; Baker et al., 2013). Triazoles have been used as a pharmacore for many years in order to develop novel ligands and have been proven useful, thus gained considerable attention in drug industries because of their effective biological activities. These derivatives have been shown to possess therapeutically interesting activities such as antimicrobial, and anti-cancer (Bekircan and Gumrukcuoglu, 2005). We have synthesized and analyzed the new efficient triazole derivatives that can inhibit the LOXL2 enzyme, thereby offering new avenues for cancer research and treatment. The current study was conducted with the objectives: synthesis of new triazoles derivatives, characterization and drug likeness evaluation, and finally these drugs were computationally designed against LOXL2 enzyme to arrest metastasis. Therefore, these new triazoles derivatives can get a pharmaceutical application on future directions in anti-cancer drug development.
2. Materials and methods 2.1. Synthesis of lead compounds
2.1.3. Step 3: synthesis of 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol (Schiff base) from (2b) 1 g of 2a was dissolved in ethanol while heating on water bath. Completely dissolved equimolar quantity of benzaldehyde added and reflux was started with continuous stirring for 4 h. The completion of reaction was monitored by TLC. The mixture then cooled to form solid product. It was then filtered and recrystallized by 70% ethanol.
Synthesis of 4-amino-5-(2-hydroxyphenyl)-1,2,4-triazol3-thione (2a) (Fig. 5A) and 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol (2b) (Fig. 5B) compounds (2a) and (2b) were synthesized according to three-steps experimental procedure.
2.1.1. Step 1: synthesis of thiocarbohydrazide (1a) Hydrazine hydrate (30 ml) was heated at reflux condenser with carbon disulfide (15 ml) in the presence of ethanol (150 ml) for about 4 h. On cooling, thiocarbohydrazide was precipitated as solid. Excess of the solvent and thiocarbohydrazide was removed by heating on water bath at their boiling points unless free from both of the solvent and unreacted hydrazine hydrate.
2.1.2. Step 2: synthesis of 4 amino-5-(2-hydroxyphenyl)-1,2,4-triazol-3-thione (2a) Equimolar quantities of thiocarbohydrazide (0.1 mol) and salicylic acid (0.1 mol) were fused at 160 ◦ C for about 2 h in an oil bath by constant stirring. TLC was used to determine the progress of reaction. After completion of reaction the mixture was cooled and filtered and recrystallized from 70% ethanol.
2.2. Structural characterization Physical properties and structural characterization of these derivatives were carried out by FTIR (Bruker Germany Alpha
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model) and the interferogram of samples were recorded in the 3600–650 cm−1 region. The NMR samples were prepared by taking 15 mg in 0.5 ml D2 O and NMR spectra were recorded on Bruker 300 MHz spectrometer equipped with 5 mm of probe head for 1 H analysis. 2.3. Lipinski’s rule of 5 Lipinski’s rule of 5 (RO5) was applied using Cheminformatic Molinspiration tool (Cheminformatics, 2013) to determine the pharmacokinetics properties (ADME/T) of these compounds. According to the RO5, the molecules must have hydrogen bond donor’s ≤5 (OH and NH groups), hydrogen bond acceptors ≤10 (N and O atoms), molecular weight <500 Da, and log P coefficient (C log P) less than 5. 2.4. Drug–targets interaction analysis 2.4.1. Preparation of ligand molecules The chemical structures of 2a and 2b derivatives were prepared by ChemBioDraw and MOL SDF format of both ligands, converted to PDBQT file using PyRx tool to generate atomic coordinates. 2.4.2. Accession of target proteins The 3D structure of LOXL2 was built on I-Tasser (Zhang, 2008) modeling server and the predicted protein models accessed from server. 2.4.3. Quality of protein model The quality of protein model was evaluated by C-score estimated by the I-Tasser server (Zhang, 2008) and Qualitative Model Energy Analysis (QMEAN) Z-score. QMEAN is a composite scoring function describing the main geometrical features of protein structures and the local geometry is analyzed by a new kind of torsion angle potential over three consecutive amino acids. QMEAN solvation potential also describes the burial status of the residues of the protein models (Benkert et al., 2008). The Ramachandran plots built by Drug Discovery Studio version 3.0 that specify low energy conformations for ϕ (phi) and (psi) along with favorable and unfavorable regions for amino acid residues. These graphs also presented the local backbone conformation and points on plot are indicating the ϕ and torsion angles of a residue. 2.4.4. Analysis of target active binding sites The active sites of target protein were analyzed using the Drug Discovery Studio version 3.0 and 3DLigandSite virtual tools (Wass et al., 2010). An active site was defined from the coordinates of the ligand in the original target protein sites. 2.4.5. Docking A computational ligand-target docking approach was used to determine structural complexes of the LOXL2 with heterocyclic triazole derivatives (2a and 2b) in order to understand the structural basis of these protein targets specificity. Initially, protein–ligand attraction were investigated for hydrophobic/hydrophilic properties of these complexes by Platinum software web server (Pyrkov et al., 2009). Finally, docking was carried out by PyRx, AutoDock Vina option based on scoring functions. The energy of interaction of these derivatives with the protein targets is assigned “grid point”. At each step of the simulation, the energy of interaction of ligand and protein was evaluated using atomic affinity potentials computed on a grid.
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Table 1 Characterization data of the compound 2a [4-amino-5-(2-hydroxyphenyl)1,2,4-triazol-3-thione] and 2b [4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3-thiol]. Compound
2a
2b
Molecular formula Color Solubility in water Solubility in ethanol melting point Form Rf value (methanol:chloroform (3:1))
C8 H8 N4 OS Yellow Not soluble Soluble on heating 120 ◦ C Crystalline 0.16
C15 H12 N4 O2 S Yellow Not soluble Soluble on heating 170 ◦ C Crystalline 0.20
3. Results 3.1. Synthesis of lead molecules Scheme 1 was employed to synthesize the effective LOXL2 inhibitors (2a and 2b) with melting point of 120 ◦ C and 170 ◦ C, respectively. These compounds were found to be soluble in ethanol on heating and TLC with Rf value 0.16 and 0.12 respectively in methanol–chloroform solvent system (Table 1).
3.2. Structural characterization 3.2.1. Spectral analysis The FTIR data of 2a and 2b confirmed the peak values of functional groups in accordance to the structures of both the compound, the peak value for OH in case of 2a observed at 3137 cm−1 while that of 2b at 3061–3100 cm−1 . The characteristic stretching vibrations of the product 2a and 2b were observed at 1591 and 1599 (C N), respectively. The absence of N H and C O absorption bands in spectra confirmed that both the compounds are formed via cyclocondensation (Table 2). The 1 H NMR spectral signals revealed that the data belongs to aliphatic and aromatic groups: for 2a compound [H NMR (DMSO, 300 MHz, 5 ppm): 7.25–7.37 (m, 4H, Ar-H), 5.37 (s, 2H, NH2 ), 10.3 (bS, 1H, 0H), 13.9 (S, 1H, SH)] and for 2b compound [7.28–7.43 (m, 8H, Ar-H), 9.1 (S, 1H, CH), 10.5 (bS, 2H, OH), 13.8 (9S, 1H, SH)].
3.2.2. Lipinski’s rule of 5 The pharmacokinetic properties (ADME/T) of 2a and 2b passed the Rule of 5 with zero violations. For 2a: the partition coefficient log P: 1.407, molecular weight: 208.46 and the number of violations: 0.0 and for 2b: the log P value: 3.397, molecular weight: 312.354 and number of violations 0.0 (Table 3).
Table 2 FTIR spectral analysis for amino-triazole derivatives. Compound
FTIR (cm−1 )
2a [4-Amino-5-(2hydroxyphenyl)-1, 2, 4-triazol-3-thione]
3137 cm−1 stretching (O H), 2950–3000 (N H), 1591–1599 stretching (N C), 1820–1760 (C O, N H), 1562–1598 cm−1 ( C N), 1313–1365 cm−1 ( C N), 1257 (C C), stretching ( C O ) 3061–3100 cm−1 stretching (O H), 2950–3000 (N-H), 1591–1599 stretching (N C), 1760 (C O), 1562–1598 cm−1 ( C N), 1313–1365 cm−1 ( C N), 1257 (C C), stretching ( C O )
2b [4-(2-Hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4-triazole-3thiol]
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Fig. 1. Top five predicted protein models by I-Tasser from A–E. C-score that reveals the strength of the predicted model and the model with the highest score (first one) is the most likely to exist.
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Table 3 Drug likeness properties of 2a and 2b molecules using Lipinski’s rule of 5. RO5a (pharmacokinetic properties)
Ligand (2a)
Ligand (2b)
MiLogP TPSA natoms MW nON nOHNH nviolation nrotb volume
1.407 76.969 14.0 208.246 5 3 0 1 169.473
3.397 83.538 22.0 312.354 6 2 0 3 260.871
a
RO5: rule of 5.
3.3. Drug–targets interaction 3.3.1. Protein models analysis The quality of the model (LOXL2, Fig. 1) was estimated by Cscore and highest score was observed for protein model 1 (C-score: −1.00), while QMEAN for this model was −6.30 (Fig. 2). Analysis of Ramachandran plots revealed that about 90% residues of almost all predicted protein models (␣-helices) in the most favored regions while less than 2% were in the disallowed region. The Ramachandran plot is indicating low energy conformations for ϕ (phi) and (psi), the graphical representation of the local backbone conformation of each residue of our target protein is shown in (Fig. 3). 3.3.2. Active binding site analysis The active binding sites are figure out on to the surface of LOXL2 protein using virtual tools, which revealed that the probable sites:
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Table 4 Energy and RMSD values obtained during docking analysis of 2a as ligand molecule and LOXL2 as target protein. Complex
Binding affinity
RMSD/UBa
RMSD/LBa
LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2
−6.2 −6.1 −6.1 −6 −6 −6 −5.9 −5.9 −5.7
0 2.869 23.158 23.947 23.368 20.95 23.922 16.868 18.804
0 1.798 22.755 23.321 22.836 20.279 23.074 16.358 17.953
2a 2a 2a 2a 2a 2a 2a 2a 2a
a RMSD/UB: root mean square deviation/upper bond; RMSD/LB: root mean square deviation/lower bond.
ASP (average distance: 0.33), LEU (average distance: 0.51), VAL (average distance: 0.30), MET (average distance: 0.42) and ALA (average distance: 0.49) (Figs. 4 and 5). 3.3.3. Hydrophobic, hydrophilic and docking analysis The distribution of hydrophobic/hydrophilic properties of LOXL2 and its binding site projected onto the surface of the ligand, and their complementarity is analyzed and shown in Fig. 6, where it has been observed that aromatic rings are less hydrophilic. These stacking interactions are used to rank the molecular docking and matching between target and drugs. Considering hydrophobic properties of these interacting molecules (LOXL2 target and triazoles) using the concepts of molecular hydrophobicity potential was calculated and compared by PLATINUM virtual tool. These hydrophobicity maps are used to analyze the progress of hydrophobicity clusters on the membrane surface along the molecular dynamics run and the features of interface between the membrane and membrane-binding molecule (Fig. 7). The docking of target proteins with synthesized compounds (2a and 2b) using AutoDock procedure revealed that all the computationally predicted lowest energy complexes of LOXL2are stabilized by intermolecular hydrogen bonds and stacking interactions. It is found that A, SA, OA, HD, N are the ligand atoms involved in docking with the enzymes (Figs. 8 and 9). The AutoGrid model presented the most energetically favorable binding mode of 2a and 2b to these enzymes. Analysis of different choices for combining structures into a single representative energy grid is performed in this study. These inhibitors are docked into the generated combined grids and the RMSD from native pose and the binding energy are evaluated and it is observed that the weight averaged grids performs the best. The ligands (2a and 2b) showed the best interaction with target proteins based on the RMSD values. Beside RMSD clustering, AutoDock Vina has also calculated the binding free energies of these interactive molecules to find the best binding mode. During these interaction procedures, the hydrogen bond between these molecules and protein targets are most important, i.e., it can decide the binding strength and the conformation of ligands. The calculated final docked energies for 2a is −6.2 kcal/mol and for 2b is-8.9 kcal/mol. The energy information of these docking results along with RMSD values are also listed in Tables 4 and 5. Docking results revealed that these ligands molecules can enter the substrate-binding region of the active site. Finally, these results demonstrated clearly that 2a and 2b molecules accurately interact with LOXL2 protein target. 4. Discussion
Fig. 2. The quality of the model was estimated by Z-score QMEAN which was −6.30. (A) Z-score with various aspects of the protein molecule [red indicates worse while blue indicates better]. (B) Comparison with protein size and normalized QMEAN Z-score of non-redundant set of protein structures. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
In this study, we synthesized amino triazoles as inhibitors which reveal significant binding affinity to human LOXL2 under computational docking analysis. LOX expression is often upregulated in many types of cancer including breast, lungs, head and neck tumors (Erler et al., 2006) and due to the
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Fig. 3. These Ramachandran plots of top five predicted protein models (A–E) for LOXL2 constructed by Drug Discovery Studio version 3.0 indicated that whether the amino acid residues occur in the “favored region” or “disallowed region” of the plot. For a good protein model there must be ≥90% amino acid residues in the most favored region or <2% in the disallowed region of the plot.
fact this enzyme serves as good therapeutic target. As there is scarcity in treating metastasis due to LOXL2 activity, consequently this study add significant advances in describing the synthesis of new chemotherapeutic agents and emphasizing on new
treatments that would block the LOXL2 over expression activity. The anti-cancer 3-amino triazoles derivatives were synthesized at 160 ◦ C by reaction of thiocarbohydrazide with salicylic acid to
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Fig. 4. Active binding site analysis of LOXL2 protein target with binding cavity contains amino acids residues ASP, LEU, VAL, MET and ALA.
form 4-amino-5-(2-hydroxyphenyl)-1,2,4-triazol-3-thione (2a) and 4-(2-hydroxybenzalidine) amine-5-(2-hydroxy) phenyl-1,2,4triazole-3-thiol (2b). Similarly, in 2010, Kocyigit-Kaymakcioglu al. (2013) synthesized antibacterial 4-amino-5-(1et phenylethyl)-2,4-dihydro-3H-1, 2,4-triazole-3-thione by reacting
thiocarbohydrazide with 2-phenylpropanoic acid in an oil-bath at 130 ◦ C for 2 h and prepared Schiff bases by reacting equimolar amounts of 4-aminotriazole and substituted aldehydes in ethanol over 45 min. Due to structural properties, these 3-amino triazoles are important as potential biologically active compounds and
Fig. 5. Molecules 2a (A) and 2b (B).
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Fig. 6. Visualization where 3-windows show the distribution of hydrophobic/hydrophilic properties of LOXL2 in X-ray position (left) and exerted by protein surroundings (middle), and their complementarity (right). The structures are the X-ray models from I-Tasser server. The screenshot was produced using the online visualization Jmol applet as implemented in the PLATINMUM web-service (A) 2a and (B) 2b.
they have been reported to possess antimicrobial and anti-cancer (Bekircan and Gumrukcuoglu, 2005; Colanceska-Ragenovic et al., 2001; Murti et al., 2011) activities. These compounds 2a and 2bare found soluble in ethanol on heating with relevant flow rate of Rf :
0.16 and Rf : 0.12 respectively in methanol–chloroform solvent system. The molecular weight, partition coefficients and other pharmacokinetics properties of these molecules have passed the Lipinski’s rule of 5 with zero violations (Zaid et al., 2010).
Fig. 7. Hydrophobicity map for upper layer of LOXL2 target protein membrane, position of the protein is projected along the Z-axis (A) 2a (B) 2b.
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Fig. 8. Various views of docking interaction steps of 2a ligand molecule. (A) The AutoGrid dimensions between ligand and LOXL2 target protein atoms (A, HD, OA, and N) are: grid center X: 18.0175, Y: 23.2912, Z: 13.5997 with dimension (Angstrom) X:Y:Z: 25. (B) Confirmation and pose of 2a ligand molecule with protein target. (C) Binding site atoms of LOXL2 amino acids residues around ligand. (D) Solid surface slab around ligand atoms. (E) Interaction of amino acid residues ASP, MET, GLY, ILE, with ligand atoms (carbon and oxygen atoms).
The spectral data of these azole heterocyclic molecules confirmed the peak values of functional groups in accordance to the structures of both compounds. The absence of N H and C O absorption bands in spectra shows that these compounds were formed via cyclocondensation while the formation of triazole
ring by FTIR peaks has been confirmed that the characteristic absorption bands of >C N and C N of triazole ring appeared at 1562–1598 cm−1 and 1313–1365 cm−1 respectively (Pyrkov et al., 2009). Similarly, the 1 H NMR spectral signals revealed that the data belongs to aliphatic and aromatic groups. The absence of C S, N H
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Fig. 9. Various views of docking interaction steps of 2b ligand molecule. (A) The AutoGrid dimensions between ligand and LOXL2 target protein atoms (A, HD, OA, and N) are: grid center X: 18.0175, Y: 23.2912, Z: 13.5997 with dimension (Angstrom) X:Y:Z: 25. (B) Confirmation and pose of 2b ligand molecule with protein target. (C) Binding site atoms of LOXL2 amino acids residues around ligand. (D) Solid surface slab around ligand atoms. (E) Interaction of amino acid residues ASP, MET, GLY, ILE, with ligand atoms (carbon and oxygen atoms).
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Table 5 Energy and RMSD values obtained during docking analysis of 2b as ligand molecule and LOXL2 as target protein. Complex
Binding affinity
RMSD/UBa
RMSD/LBa
LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2 LOXL2
−8.9 −8.7 −8.4 −8.3 −8.1 −8.1 −7.8 −7.7 −7.6
0 2.38 3.368 3.014 3.058 3.733 22.333 4.533 22.02
0 1.517 1.83 1.834 2.548 2.612 21.933 2.71 20.879
2b 2b 2b 2b 2b 2b 2b 2b 2b
a RMSD/UB: root mean square deviation/upper bond; RMSD/LB: root mean square deviation/lower bond.
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stabilized by intermolecular interactions. These newly synthesized triazole derivatives can be clinically investigated for establishing safe doses. Conflict of interest The authors confirm that this article content has no conflicts of interest. Source of funding None declared. Ethical approval
and presence of S-H absorption established that the triazole ring in compound are in thiol form (Prakash et al., 2004; Sztanke et al., 2006; Liu et al., 2008). Previous clinical investigation showed that inhibition of LOXL2 activity has been established to eradicate metastases in mice models (Erler et al., 2006) which emphasize to develop new anti-LOXL2 inhibitors for the treatment of cancers. Furthermore, tumor growth inhibition properties of these heterocyclic triazoles have been previously reported (Bekircan and Gumrukcuoglu, 2005; Murti et al., 2011). Therefore, in silico association between LOXL2 enzyme and these new triazole derivatives have been positively analyzed and reported for the first time. This study is based on such protocols by synthesizing the new LOXL2 inhibitors. Five protein models of LOXL2 enzyme have been obtained using I-Tasser server and quality of the models were estimated based on C-score (Zhang, 2008), QMEAN value (Benkert et al., 2008) and Ramachandran plot. Among five, first protein model was selected due to best C-score (−1.00) and QMEAN (−6.30) values while Ramachandran plot revealed that about 90% residues of almost all predicted protein models (␣helices) were found to be in the most favored regions while less than 2% were in the disallowed region. The binding residues that define the coordinates of the ligand (Gohlke and Klebe, 2002) on to the surface of LOXL2 protein were analyzed confirming that these ligands molecules can enter the substrate-binding region of the protein active sites. The docking interaction of target protein with synthesized compounds (2a and 2b) based on conformation and scoring functions (Kitchen et al., 2004) showed stable complexes with LOXL2 with lowest binding energies. The AutoGrid Model presented the most energetically favorable binding mode of 2a and 2b to this enzyme and the calculated final docked energies for 2a: −6.2 kcal/mol and for 2b: −8.9 kcal/mol. These results exposed that 2a and 2b molecules accurately interact with LOXL2 protein target. 5. Conclusion No doubt the availability of computational and in silico approaches is the landmarks in drug discovery process. The virtual screening and the docking analysis have provided a remarkable approach for accessing the lead molecules to be used as drug ligands and to understand the protein–ligand binding affinity. Lysyl oxidase like 2 proteins (LOXL2) is important enzyme that has been reported in the up-regulation and proliferation of tumors and considered as a good therapeutic target for the treatment of breast, head and neck cancer. Due to shortage of chemotherapeutic agents, new triazole derivatives (2a and 2b) are synthesized and their interaction with LOXL2 is evaluated. This analysis revealed that all the computationally predicted lowest energy complexes of these enzymes are
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