Synthesis, characterization, in vitro cytotoxicity, in silico ADMET analysis and interaction studies of 5-dithiocarbamato-1,3,4-thiadiazole-2-thiol and its zinc(II) complex with human serum albumin: combined spectroscopy and molecular docking investigations

Abstract

A new ligand, 5-dithiocarbamato-1,3,4-thiadiazole-2-thiol (L) and its Zn(II) complex were synthesized and characterized using elemental analysis (CHN) and spectroscopic methods (¹H NMR, FT-IR, UV-Vis). The ligand and Zn complex were subjected to biological tests in vitro using MCF-7 breast cancer cell line. The Zn complex with IC₅₀ = 47 μM shows significant cytotoxic activity against the MCF-7 breast cancer cell line. The interaction between the compounds and Human Serum Albumin (HSA) was investigated in Tris–HCl buffer solution at pH 7.4 by means of various spectroscopic (fluorescence, UV-Vis and FT-IR) and molecular docking methods. The fluorescence data shows that L and Zn complexes quench the intrinsic fluorescence of HSA through a static quenching procedure. Binding constants (K_b) and the number of binding sites (n ∼ 1) were calculated. The distance (r) between donor (HSA) and acceptor (L or Zn complex) was obtained according to fluorescence resonance energy transfer and the alterations of HSA secondary structure induced by the compounds were confirmed by FT-IR spectroscopy. Finally, molecular docking was employed for the identification of the active site residues and their critical interactions.

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1. Introduction

The synthesis of metallodrugs containing sulfur donor ligands is on the rise, because of their resemblance to several important biomolecules, such as amino acids and vitamins.¹ Dithiocarbamates as important organosulfur compounds can coordinate with transition metals. This may be due to the small bite angle of the CSS group which can coordinate almost with all metal ions even in unusual oxidation states. Metal complexes of these ligands are used in many areas such as chemistry, biology and industry.² For example, Sn-complex of (CH₃)₂NCSS⁻ is found to possess strong anti-cancer properties.³ Also, metal complexes of dialkyl dithiocarbamates have biological properties such as anti-alkylation,⁴ antitumor^5–8 and anti-HIV.⁹ The interest in dithiocarbamate complexes of platinum has been stimulated, Manav and coworkers reported that dialkyl dithiocarbamate reduces its nephrotoxicity when it is co-administered with cisplatin.¹⁰ Moreover, it has been observed that the biological activity of certain organic compounds is enhanced through complex formation with metal ions;^11–13 the formation of a metal complex will alter the solubility and lipophilicity of the drug, resulting in changes in its pharmacokinetics, biodistribution, and biotransformation.¹⁴ Metal coordination to sulfur induces possibly the formation of a drug reserve in the cell¹⁵ and reduces renal damages.^16–22

Many physiological roles has been reported for the zinc(II) complexes (binder complexes at DNA site^23,24 radioprotective agents²⁵ tumor photosensitizers,²⁶ antidiabetic insulin-mimetic^27,28 and antibacterial or antimicrobic activities.²⁹ The role of this metal in the growth and survival of cells, its versatile coordination states and geometries preserving the same oxidation state prompted us to engineer Zn(II) complex as potential anticancer agent with low in vivo toxicity and perhaps new modes of action and cellular targets with respect to the use of structural rigid organometallic complexes as kinase^30–32 and protein–protein interaction inhibitors.^33,34 These coordination complexes have long been investigated for their ability to induce DNA cleavage, and therefore could be one possible mechanism that triggers cell death.^35–40 Molecular docking research is carried out to predict the orientation and conformation of a ligand within a protein receptor. Also, it enables us to understand protein–inhibitor interactions and the structural features of the protein's active site.⁴¹ In recent years, different classes of thiadiazole compounds have been investigated, many of which have been found to be important scaffolds with broad spectrum of pharmacological activities.^42–44 Particularly, 1,3,4-thiadiazoles are much explored for their broad spectrum of biological activities including anti-inflammatory, antiviral, antimicrobial, antidepressants, antileishmanial and anticancer.^45–47 With this background in mind, we synthesized and characterized a novel dithiocarbamate ligand containing 5-amino-1,3,4-thiadiazole-2-thiol and its Zn(II) complex using 2,2′-bipyridine as the co-ligand and evaluated in vitro their anticancer activity against MCF-7 breast cancer cell line using MTT assay. A combined spectroscopic and molecular docking study on the interaction of these compounds and human serum albumin (HSA) is also presented in this work. HSA is a major plasma protein and plays a dominant role in controlling the distribution, excretion, therapeutic efficacy and toxicity of numerous endogenous and exogenous ligands in the body.⁴⁸ Almost every compound injected into the blood encounters with a high concentration of serum albumin which is known to have a strong affinity for a variety of chemical and biochemical species. So, the exogenous ligands, such as drugs, can bind to albumins then they can be transported in the circulatory system. Hence, the investigation of binding of drugs with albumin is of interest.

2. Experimental

2.1. Chemicals

2,2′-Bipyridine, zinc chloride, carbon disulfide, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Tris–HCl buffer were purchased from Merck (Germany). 5-Amino-1,3,4-thiadiazole-2-thiol and human serum albumin obtained from Aldrich (England). [Zn(bpy)Cl₂] were prepared based on what is mentioned in the literature.⁴⁹ Other used chemicals were of analytical reagent or higher purity grade. Solvents were purified prior to be used by the standard procedures. MCF-7 cell line was obtained from Pasture Institute (Tehran, Iran). All media and cell culture components were obtained from Life Technologies (USA).

2.2. Instrumentation

UV-Vis data were collected on a JASCO UV/Vis-7850 spectrophotometer in the range of 200–400 nm. ¹H NMR spectra were measured on a Bruker DRX-500 Avance spectrometer at 500 MHz, using TMS as the internal reference in DMSO-d₆. Infrared spectra (4000–400 cm⁻¹) were determined with KBr disks on a JASCO-460 plus FT-IR spectrophotometer. The HSA-binding experiments were performed separately at three temperatures 300, 310 and 318 K in Tris–HCl buffer medium. Intrinsic fluorescence measurements of HSA in the presence of dithiocarbamate ligand and Zn(II) complex were performed on a Varian spectrofluorimeter model Cary Eclipse with 1.0 cm quartz cells, the widths of both the excitation and emission slit were set as 5 nm. The excitation wavelength was set at 295 nm to selectively excite the tryptophan and tyrosine residues, and the emission spectra were recorded between 300 and 500 nm with maximum observed at 350 nm. The experiments were performed at several [drug]/[protein] molar ratios.

2.3. Synthesis and characterization

The dithiocarbamate ligand and Zn(II) complex were synthesized according to Scheme 1.

Scheme 1 Synthesis of dtc-ligand and Zn(II) complex.

2.3.1. Preparation of ligand (L). 5-Amino-1,3,4-thiadiazole-2-thiol (0.01 M) was dissolved in methanol and cooled in an ice-salt bath. Sodium hydroxide (0.01 M) was added to this solution under stirring followed by the addition of carbon disulfide (0.01 mol). The mixture was stirred for 1 h in an ice-salt bath and at room temperature for 5 h. Most of the solvents were removed under reduced pressure, and the precipitate was collected by filtration. The crude product was recrystallized from methanol/dichloromethane.

Yield: 1.42 g (61%) with a melting point of 93 °C. Anal. calcd for C₃H₂N₃S₄Na (231.12): C, 15.57; H, 0.86; N, 18.17. Found: C, 14.89; H, 0.97; N, 19.26%. IR (KBr, cm⁻¹): four characteristic stretching bands at 3133, 1610, 1060 and 755 cm⁻¹ assigned to n(N–H), n(CSS)as, ν(N–CSS) and n(CSS)s modes respectively. ¹H NMR (500 MHz, DMSO-d₆, ppm, sb = singlet broad): 13.14 (sb, 1H, H-a) and 6.62 (sb, 1H, H-b) (Fig. 1S†).

2.3.2. Preparation of Zn(II) complex. [Zn(bpy)Cl2] (1 mmol) was dissolved in methanol, to this 1 mmol of sodium 5-dithiocarbamato-1,3,4-thiadiazole-2-thiol in methanol was added by continuous stirring. This reaction mixture was refluxed for 7 h, a yellow colored solution was formed. It was then filtered and subsequently the mixture was evaporated at 35–40 °C to complete dryness. The formed zinc(II) complex was recrystallized with methanol.

Yield: 0.198 g (43%) with a melting point of 231–234 °C. Anal. calcd for C₁₃H₁₀N₅S₄ZnCl (465.07): C, 33.54; H, 2.15; N, 15.05% found: C, 32.67; H, 2.23; N, 14.71%. Molar conductance measurement for the complex is 116.14 Ω⁻¹ mol⁻¹ cm² indicating 1 : 1 electrolytes. IR (KBr, cm⁻¹): stretching bands at 3165, 1637, 1072 and 761 cm⁻¹ assigned to n(N–H), ν(N–CSS), n(CSS)as and n(CSS)s modes respectively. ¹H NMR (500 MHz, DMSO-d₆, ppm, sb = singlet broad and m = multiple): 13.62 (sb, 1H, H-a), 6.84 (sb, 1H, H-b), 7.39–8.76 (2,2′-bipyridine protons) (Fig. 2S†). Electronic spectra of this complex exhibit four bands. The bands at 223 (log ε = 3.09), 246 (log ε = 3.11), 293 (log ε = 3.43) and 305 nm (log ε = 2.56) may be assigned to intraligand π → π* and n → π* transitions of 2,2′-bipyridine ligand as well as CSS⁻ group.

2.4. Cell culture and in vitro cytotoxicity analysis: MTT assay

Human breast cancer MCF-7 cells⁵⁰ were cultured at a seeding density of 4.0 × 104 cells per cm² into the T-50 flasks using RPMI 1640 media supplemented with 10% FBS, 100 units per mL penicillin G, and 100 mg mL⁻¹ streptomycin. The cultured cells were kept at 37 °C in a humidified CO₂ incubator during cultivation and during experiments. Cytotoxicity assay was conducted in MCF-7 cells. Cells (1 × 104 cells per well) were cultivated onto 96-well plates. At 40–50% confluency (24 h post-seeding), the cultured cells were treated with different concentrations (i.e. ranging from 6.25 to 200 μM) of Zn(II) complex and dithiocarbamate ligand Plus cisplatin as an anti-cancer drug. The concentration of Zn(II) complex was set to be equivalent to the ligand alone. The treated cells were incubated for different time frames (i.e. 24, 48, and 72 h), and then subjected to (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The media was removed and 150 mL of fresh media plus 50 mL MTT solutions (prepared as 2 mg mL⁻¹ in FBS) were added to each well and incubated for 4 h at 37 °C in a CO₂ incubator. The media was removed and the cells were washed, and then the formed formazin crystals were dissolved by adding DMSO (200 μL) and Sorenson's buffer (25 μL) to each well. The absorbance was read at 570 nm using a spectrophotometer (BioTek Instruments, Inc., Bad Friedrichshall, Germany).

2.5. Interaction with HSA

HSA solution was prepared on the basis of its molecular weight of 67 000 Da by dissolving in Tris–HCl buffer (0.1 M, pH 7.4) at the concentration of 5 × 10⁻⁵ M and stored at 4 °C. Accurate protein concentration was determined spectrophotometrically using an extinction coefficient of 35 219 M⁻¹ cm⁻¹ at 280 nm.⁵¹ Stock solutions of the ligand and complex (5 × 10⁻³ M) were prepared by dissolving them in ultra-pure water. NaCl (analytical grade, 1 M) solution was used to maintain the ionic strength of buffer at 0.1 M; pH was adjusted to 7.4 by using HCl. Working standard solution was obtained by appropriate dilution of the stock solution. For approval the energy transfer efficiency between protein and above compounds the overlap of the UV-Vis absorption spectrum of them with the fluorescence emission spectrum of HSA at 300–500 nm was estimated. In UV-Vis measurements, the spectra of L, Zn(II) complex and protein were recorded at room temperature. Also, to prevent the negative effects of dilution on peak intensity (related to UV-Vis and fluorescence measurements) the correction for dilution of working standard solution was done.

2.6. Molecular docking study

Molecular docking was carried out using smina⁵² which uses the AutoDock Vina⁵³ scoring function by default. The default AutoDock Vina scoring function includes three steric terms, a hydrogen bond term, hydrophobic term, and torsion count factor. However, a larger space of energetic terms were considered in the design of AutoDock Vina. In addition to the Gaussian, repulsion, hydrogen bonding, and hydrophobic terms that compose the default scoring function, there are an assortment of simple property counts, an electrostatic term, an AutoDock 4 desolvation term,⁵⁴ a nonhydrophobic contact term, and a Lennard-Jones 4–8 van der Waals term. For scoring purposes, only heavy atom interactions between the ligand and protein are considered (when docking, intramolecular heavy-atom interactions are also used). All these terms are made available and fully parametrizable in smina. The 3D X-ray structure of human serum albumin was taken from the protein data bank (PDB) encoded 1O9X. Ligand and complex files were provided using AutoDock Tools. For the recognition of the binding sites in HSA, blind docking was carried out with setting of grid size to 90, 68, and 68 along x, y, and z axes with a grid spacing of 1 Å after assigning the protein and probe with Kollman charges. The grid center was set at 20, 70, and 10 Å. All other parameters were default settings. Smina was run with default settings, which samples nine ligand conformations using the Vina docking routine of Monte Carlo stochastic sampling. For each of the docking cases, the lowest energy docked conformation, according to the smina scoring function, was selected as the binding mode. The output from smina was presented with BIOVIA Discovery Studio client 2016.⁵⁵ The interacting energies between each amino acid with the best pose of docked ligand and complex into HSA binding site were calculated by Molegro Molecular Viewer 2.5 (MMV) (http://www.molegro.com/mmv-product.php).

3. Results and discussion

3.1. In vitro effects of L and Zn complex on MCF-7 cell line

It is known that thiadiazole compounds have promising anticancer activity.^56,57 Accordingly, we selected MCF-7 cell lines to evaluate cytotoxic effect of Zn(II) complex and dithiocarbamate ligand compared with a known complex anticancer drug such as cisplatin. Having used the MTT assay, we observed a significant difference of cytotoxicity between the MCF-7 cells treated with Zn(II) complex and dithiocarbamate ligand (about 80% toxicity for Zn(II) complex after 72 h, Fig. 1C). Also, in MCF-7 cells, IC₅₀ values of Zn(II) complex, dithiocarbamate ligand and cisplatin were evaluated 47 ± 3.3, 168 ± 5.9 and 14 ± 2.1 μM, respectively. As shown in Fig. 1A–C, the cytotoxic effect of above compounds seems to be time and concentration dependent similar to that of cisplatin. Probably, the more ability of Zn(II) complex in protein binding compared to dithiocarbamate ligand can be effective on noticeable cytotoxic effects of complex. Also, recently Gao et al. have reported that the introduction of phenyl in the bpy ligand resulted in a slight increase in the anticancer activity of Zn(II) complex with IC₅₀ values ranging from 20–40 μM.⁵⁸ Actually, the more ability of Zn(II) complex compared to dithiocarbamate ligand not only is dependent on the nature of metal center but also strongly is related to the structure of heterocyclic aromatic ligand.

Fig. 1 Cytotoxic (MTT assay) study of dtc-ligand, Zn(II) complex and cisplatin. Data represents mean (n = 5) + SD. (A) Treated cells after 24 h. (B) Treated cells after 48 h. (C) Treated cells after 72 h.

3.2. In silico ADME and toxicity risk assessment test analysis of ligand and complex

The most important strategy of pharmaceutical industry is to predict the molecular properties for absorption, distribution, metabolism, excretion and toxicity (ADMET).⁵⁹ This information helps the chemist to ameliorate the pharmacokinetic features of compounds. Drug-likeness or druggability of molecules was assessed based on Lipinski rule of five as suggested by Christopher A. Lipinski.⁶⁰ According to Lipinski's ‘rule-of-five’ drugs should have a molecular weight of ≤500 Da, a log P ≤ 5, hydrogen bond donor ≤ 5 and hydrogen bond acceptor sites (N and O atoms) ≤ 10 that they have strong absorption. Bioavailability, reflecting the drug proportion in the circulatory system, is a significant index of drug efficacy. Screening for absorption ability is one of the most important parts of assessing oral bioavailability; therefore, it is crucial in ADMET profiling. Topological polar surface area (TPSA) was calculated. It should be ≤140 Ų of a molecule which correlates well with the passive molecular transport through membranes.⁶¹ TPSA is closely related to hydrogen bonding and contain the information about log P. Because of the presence of phospholipid bilayer, high lipophilicity (log P) of a compound is favourable for efficient permeability. The predicted log S for the ligand and complex showed that these molecules possessed intermediate aqueous solubility (aqueous solubility in mol dm⁻³ should be −6.5 to 0.5). Moreover, toxicity risk assessment test on the ligand and complex was performed to evaluate any kind of toxicity risk such as mutagenicity, tumorigenicity, irritant effects and reproductive effects. Computational prediction model provides a cheap and fast way to assess the potential for ADMET properties of candidate drugs. All of the properties were calculated for ligand and complex using the OSIRIS Dataworior 4.1.1 program.⁶² All of the descriptors that mentioned above are in accepted range. They are listed in Table 1S.† Also, the results of toxicity risk assessment test (Table 1S†) indicate that none of the compounds a risk of mutagenicity, tumorigenicity, irritation or reproductive toxicity.

3.3. Fluorescence quenching mechanism of HSA by L and Zn complex

The fluorescence of HSA comes from tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) residues. The inherent fluorescence of HSA is almost due to tryptophan alone, because phenylalanine has a very low quantum yield and the fluorescence of tyrosine is almost totally quenched if it is ionized or if it is near an amino group, a carboxyl group, or a tryptophan.^63,64 That is, the change of intrinsic fluorescence intensity of HSA was due to Trp residue when small molecules bound to HSA. Fig. 2 shows the fluorescence emission spectra of HSA in the absence and presence of Zn(II) complex and L (inset) at different concentrations. The fluorescence intensities of HSA decreased gradually with the increasing concentration of Zn complex and L which indicated that both these compounds interacted with HSA.

Fig. 2 Effect of Zn(II) complex on the fluorescence spectrum of HSA at 310 K, [HSA] = 15 μM, [complex] = 0–145 μM (inset for L, [L] = 0–176 μM).

The decrease fluorescence intensity of fluorophore (called as quenching) can be induced by a variety of molecular interactions.⁶⁵ The mechanisms of quenching are usually classified as either dynamic (collisional encountering between the fluorophore and quencher) or static (formation of a non-fluorescent ground state complex).⁶⁶

The two types of quenching mechanism can be distinguished by the temperature dependent behaviour of their binding constants. The binding constants increase with increasing temperature for dynamic quenching, while the reverse effect is observed for static quenching. In order to determine the quenching mechanism, the fluorescence data were analyzed by the Stern–Volmer eqn (1)

F₀/F = 1 + K_qτ₀[Q] = 1 + K_sv[Q] (1)

where, F and F₀ are fluorescence intensity, with and without quenching reagent respectively, K_q is the maximum scatter collision quenching constant, τ₀ is the average fluorescence lifetime (10⁻⁸ s) of biomacromolecules, K_sv is the Stern–Volmer quenching constant, and [Q] is the concentration of quencher. The values of K_sv and K_q at different temperatures are shown in Table 1. As shown in Table 1 and Fig. 3, the quenching constant K_sv decreases with increasing temperature and the values of K_q are much greater than the maximum diffusion collision quenching rate constant of various quenchers with the biomacromolecules (2 × 10¹⁰ mol⁻¹ s⁻¹),⁶⁷ indicating that the fluorescence quenching of HSA by Zn complex and L are caused mainly by the static quenching but not a dynamic one.

Table 1 Stern–Volmer quenching constants for the interaction of L or Zn(II) complex with HSA

Compound	T (K)	Ksv (104 M^−1)	Kq (1012 M^−1 s^−1)
L	302	0.84	0.84
	310	0.71	0.71
	318	0.62	0.62
[Zn(bpy)(L)]Cl	302	7.11	7.11
	310	6.63	6.63
	318	4.72	4.72

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Fig. 3 Stern–Volmer plots for the quenching of HSA by Zn complex (inset for L) at different temperatures and pH 7.4.

3.4. Binding constant and binding sites

Several models are given in literature for the determination of the binding parameters, the number of sites (n) and the binding constants (K). Generally, all the models start from the Scatchard equation⁶⁸ for a single class of n independent binding sites, eqn (2):where ν represents the ratio of the bound ligand, [L_b] to the total protein [P_t]:where [L_t], [L_f] are the total and free concentrations of the ligand, respectively. In some models of Scatchard equation [L_f] is replaced by [L_t], approximation that is not always valid. In following equation this approximation is avoided.

The slope of the linear plot of log(F₀ − F)/F vs. log(1/([L_t] − (F₀ − F) × [P_t]/F₀)) gives the number of sites and the intercept with the ordinate is the product n × log K (Fig. 4). The values of n and K are included in Table 2 and as can be seen from results the binding constants of the interaction between Zn(II) complex and HSA is more than that of dithiocarbamate ligand, which means that Zn(II) complex has stronger ability to bind with HSA. In fact, the coordination of ligand to metal centre and the creation of a larger molecular size than the alone ligand plays significant role in the binding between Zn complex and HSA. The large size drug molecule may have larger hydrophobic area which can interact with hydrophobic surface on the protein molecule. The results illustrate that the number of binding sites n is approximately equal to unity indicating that in each case the dithiocarbamate compound is located in one binding site.

Fig. 4 Linear segment fitting plot for the HSA–Zn complex system to eqn (4) at different temperatures; [HSA] = 15 μM, [complex] = 0–145 μM (inset for HSA–L system, [L] = 0–176 μM). λ_ex = 295 nm.

Table 2 Binding and thermodynamic parameters of HSA interaction with L and Zn(II) complex

Compound	T (K)	Kb (104 M^−1)	n	ΔG (kJ mol^−1)	ΔH (kJ mol^−1)	ΔS (J mol^−1 K^−1)
L	302	0.60	1.09	−21.92	−4.34	58.02
	310	0.58	1.05	−22.39
	318	0.55	0.98	−22.85
[Zn(bpy)(L)]Cl	302	7.71	1.49	−28.32	−25.80	8.31
	310	5.81	1.52	−28.38
	318	4.45	0.96	−28.45

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3.5. Energy transfer between dtc-compounds and HSA

The overlap of the UV-Vis absorption spectrum of L and Zn(II) complex with the fluorescence emission spectrum of HSA is shown in Fig. 3S.† The efficiency of energy transfer in biochemistry can be used to evaluate the distance between the ligands and the fluorophores in the protein. The rate of energy transfer depends on the extent of the overlapping of the donor emission spectrum with the acceptor absorption spectrum, the relative orientation of the donor and acceptor transition dipoles, and the distance between these molecules. The distance from the tryptophan residue (donor) to the bound drug (acceptor) in HSA can be calculated according to the Forster's theory. The energy transfer efficiency E and the distance between the acceptor and donor r can be defined as the following equations:^69–71

E = R₀⁶/(R₀⁶ + r₀⁶) = 1 − F/F₀ (5)

R₀ is the distance at which the transfer efficiency equals to 50%, is given by the eqn (6):

R⁶₀ = 8.8 × 10⁻²⁵ K² N⁻⁴ ΦJ (6)

where K² is the spatial orientation factor describing the relative orientation in space of the transition dipoles of the donor and acceptor, N is the refraction index for the medium, Ф is the fluorescence quantum yield of the donor in the absence of the acceptor and J is the overlap integral between the donor fluorescence emission spectrum and the acceptor absorption spectrum. J can be given by:

J = (∑F(λ)ε(λ)λ⁴Δλ)/(∑F(λ)Δλ) (7)

where F(λ) is the fluorescence intensity of the fluorescence donor at wavelength λ, ε(λ) is the molar absorption coefficient of the acceptor at wavelength λ and its unit is l mol⁻¹ cm⁻¹. In the present case, K² = 2/3, n = 1.36, Φ = 0.15.^72,73 From eqn (5)–(7), we could calculate that J = 2.86 × 10⁻¹⁴ cm³ L mol⁻¹, E = 0.127, R₀ = 3.42 nm and r = 4.70 nm for Zn complex–HSA system and J = 4.72 × 10⁻¹⁴ cm³ L mol⁻¹, E = 0.144, R₀ = 03.71 nm and r = 5.01 nm for L–HSA system. In both cases, the larger value of r compared to that of R₀ also revealed the presence of static type of quenching mechanism.⁷⁴

3.6. UV-visible absorption spectral studies

UV-visible absorption spectroscopy technique can be used to explore the structural changes in HSA and to investigate protein–ligand complex formation.⁷⁵ Herein, we have recorded the absorption spectra of the HSA at different concentration of above compounds (L and Zn complex) to confirm the interaction of HSA to L and Zn complex. HSA has two absorption peaks that strong absorption located at 230 nm, which reflects the framework conformation of protein and another weak absorption at 280 nm due to the aromatic amino acid. Fig. 5 shows the absorption spectra of HSA in the absence and presence of the above mentioned drugs. In the dynamic quenching mechanism, the UV-Vis spectrum of the HSA molecule should not change. In the static quenching, HSA forms a complex with the drug, causing the change in the UV-Vis spectrum of HSA.⁷⁶ As can be seen in Fig. 5, with the addition of Zn complex and L into HSA solution, the intensity of the UV-Vis spectrum of HSA at 280 nm increased continuously. Also, the intensity of the peaks at 230 nm got decreased with slight shift towards longer wavelengths. This observation clearly indicated the interaction between HSA and above mentioned compounds and change in protein conformation.

Fig. 5 Absorption spectra of HSA (15 μM), with various amounts of Zn complex (0–90 μM), inset for L (0–165 μM) at 310 K.

3.7. FT-IR spectroscopy

To further understand the structural alternations of HSA induced by the binding of above drugs to HSA, FT-IR spectroscopy were performed on HSA and Zn complex–HSA and L–HSA systems (Fig. 6). The spectrum in Fig. 6A was obtained by subtracting the absorption of the HSA solution from the spectrum of the Tris–HCl. The spectrum in Fig. 6B was obtained by subtracting the absorption of the drug-free form from that of the drug-bound form. Infrared spectra of HSA exhibit a number of the amide bands, which represent different vibrations of the peptide moiety. The amide I peak was observed in the range 1600–1700 cm⁻¹ (mainly C=O stretch) and the amide II band in the region 1500–1600 cm⁻¹ (C–N stretch coupled with N–H bending mode). It is well known that the amide bands have a relationship with the secondary structure of HSA, and amide I is more sensitive than amide II to change of secondary structure of HSA. As shown in Fig. 6 the peak positions of amide I bands were shifted from 1653.1 cm⁻¹ to 1644.71 cm⁻¹ for Zn complex and from 1652.20 to 1647.35 cm⁻¹ for L, indicating that the secondary structure of the HSA changed in the presence of these drugs.

Fig. 6 FT-IR spectra and different spectra of HSA in aqueous solution: (A) FT-IR spectrum of HSA; (B) FT-IR difference spectrum of HSA obtained by subtracting the spectrum of the Zn complex-free form from that of the Zn complex-bound form in Tris–HCl buffer, pH 7.4, at room temperature in the region of 1750–1400 cm⁻¹ ((2) for dithiocarbamate ligand), [HSA] = 10 μM, [Zn complex] = 8 μM and [L] = 8 μM.

3.8. Molecular docking analysis

Molecular docking method is an attractive method to understand the ligand–protein interactions which can confirm our experimental results. The docking programs were employed in two processes of the research. First, their use prior to experimental screening can be considered as powerful computational filters to reduce work and cost needed for the development of effective medicinal compounds. Second, when they are used after experimental screening, they can help in better understanding of bioactivity mechanisms. Albumin architecture consists of a single chain, 585 amino acid residues organized in three homologous domain (I, II, III), each of which contains two subdomains (A and B).^77,78 According to the conventional view based on Sudlow's classification, drug ligands of HSA are accommodated at two main binding sites located in subdomain IIA (site IIA) and IIIA (site IIIA), respectively.^77,79 These subdomains are predominantly helical and extensively cross-linked through several disulfide bridges, with one tryptophan residue (Trp214) in subdomain IIA.⁸⁰ It is suggested that the principal regions of drug binding to HSA are situated in hydrophobic cavities in subdomains IIA and IIIA. A third binding pocket within subdomain IB (site IB) has recently been identified as the primary binding site of a bilirubin photoisomer,⁸¹ hemin,^82,83 a sulphonamide derivative. Carter and co-workers have claimed that subdomain IB (site IB) is the third major drug-binding region of HSA, the general ligand binding importance of which has not been recognized previously.^84–86 To identify the dithiocarbamate ligand and Zn(II) complex binding site on HSA, blind docking was carried out. Allowing to smina output, 9 conformations for ligand and complex were achieved; they ranked top conformers for ligand and complex based on affinity score show in Table 2S.† The docking results as shown in Fig. 7A demonstrated that the best binding mode binding of ligand is located within subdomain IB. On the other hand the complex prefers lying in subdomain IIA of HSA.

Fig. 7 The docking results of ligand and complex with 3D structure of HSA. (A) The binding site of ligand and complex in HSA. Ligand and complex are showed in ball-and-stick form. (B) The conformation of ligand in the binding site of HSA. (C) The conformation of complex in the binding site of HSA.

The best-docked conformation of the ligand associated to affinity −4.3 kcal mol⁻¹ is shown in Fig. 7B with the labeled key residues. Detailed analysis of the binding mode of the best docked pose of ligand shows that a pi–pi stacked between the TYR140 and thiadiazole of the ligand. There is a conventional hydrogen bond between the adjacent O atom of the hydroxyl group of TYR140 and H atom of the ligand in agreement with our conclusion of thermodynamic analysis. Also, a pi–sulfur interaction observes with TYR140. The result shows that the ligand interacts with ARG144 residue through one hydrogen bond between N atom of ligand and the H atom of the alkyl group of ARG144 in agreement with our conclusion of thermodynamic analysis. Other interaction of ARG144 is a pi–alkyl interaction with the ligand. ARG 145 and ligand interact via pi-cation and pi-donor. As it can be seen, van der Waals interactions with surrounding receptor amino acids such as PRO35 and GLU141 with the hydrophobic part of ligand represent in Fig. 7B.

The lowest binding energy docking conformation of the complex associated to affinity −8.1 kcal mol⁻¹ is shown in Fig. 7C with the labeled key residues and a hydrophobicity surface for a more detailed discussion. Detailed analysis shows that some pi–alkyl interactions between the TRP214, PHE211, LYS199, LYS195 and complex. The complex interacts a pi-cation with TRP214 and an alkyl interaction with LEU481. To confirm our conclusion of thermodynamic analysis, from the docking results we can observe the complex interacts with LYS195 residue through one hydrogen bond between S atom of ligand and the H atom of the hydroxyl group of LYS195. GLN196 with the hydrophobic part of ligand has a van der Waals interaction.

2D schematic interaction model of ligand and complex (Fig. 4S(A and B)†) was used to investigate and clarify the interactions between ligands and receptor. Herein, our goal was to compare affinity rankings of ligand and complex to dock into a unique receptor. The analysis presents that affinity of complex is more negative than the ligand (Table 2S†), because complex possess more interactions with HSA in comparison to ligand. We can conclude docking results in accordance with the spectroscopic techniques, which can provide reliable and quick information on the capability of new drugs to interact with HSA. Furthermore, the interaction energy between the ligand and complex with amino acid residues are listed in Tables 3 and 4, respectively.

Table 3 Interaction energy between the ligand and responsive amino acid residues in molecular docking

Amino acid residues	Interaction energy kJ mol^−1
Arg144	−7.94327
Arg145	−12.143
Gln32	−0.42786
Gln33	−1.47119
Glu141	−7.39741
Leu112	−0.57729
Leu115	−0.79441
Tyr140	−10.6059

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Table 4 Interaction energy between the complex and responsive amino acid residues in molecular docking

Amino acid residues	Interaction energy kJ mol^−1
Ala210	−0.47965
Asp451	−0.38084
Gln196	−5.53304
Glu450	−1.89213
Leu198	−0.71778
Leu481	−7.83379
Lys195	−16.1364
Lys199	−13.7788
Phe211	−5.79494
Ser192	−2.18260
Ser202	−3.04000
Ser454	−1.97316
Trp214	−36.3246

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4. Conclusion

In the present study, 5-dithiocarbamato-1,3,4-thiadiazole-2-thiol (L) and zinc(II) complex [Zn(L)(bpy)]Cl were synthesized and characterized by spectroscopic methods. The dithiocarbamate ligand and Zn(II) complex were screened in vitro for their anticancer activity against MCF-7 breast cancer cell line using MTT assay. The result showed that dithiocarbamate ligand did not elicit noticeable cytotoxic effects in comparison with Zn(II) complex even after 72 h. The interaction between above mentioned compounds and HSA was investigated employing different spectroscopic (fluorescence, UV-Vis, FT-IR) and molecular docking techniques. The experimental results indicated that L and Zn(II) complex bind to HSA with moderate affinity and the intrinsic fluorescence of HSA was quenched through static quenching mechanism. The binding parameters were calculated. Furthermore, UV-Vis and FT-IR evidences show that the secondary structure of HSA was changed after above compounds were bound to HSA. Among the investigated compounds, Zn(II) complex has stronger ability to bind with HSA than L. Indeed, the coordination of dithiocarbamate ligand to Zn(II) centre can create such ability.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra17322e

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