Understanding the binding interaction between methotrexate and human alpha-2-macroglobulin: Multi-spectroscopic and computational investigation

Archives of Biochemistry and Biophysics 675 (2019) 108118

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Understanding the binding interaction between methotrexate and human alpha-2-macroglobulin: Multi-spectroscopic and computational investigation

T

Mohammad Khalid Ziaa, Tooba Siddiquia, Syed Saqib Alia, Haseeb Ahsanb, Fahim Halim Khana,∗ a b

Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia, New Delhi, 110025, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Methotrexate Anticancer drug Alpha-2-macroglobulin Reactive oxygen species Antiproteinase

Methotrexate (MTX) is advised in the treatment of solid tumours, hematologic malignancies and autoimmune disorders. On reaching the circulation, 60% of MTX is bound to the proteins present in serum. Alpha-2-macroglobulin (α2M) is a plasma proteinase inhibitor with numerous functions such as binding, transportation and targeting of molecules. Our studies are the first attempt to investigate the binding interaction of pharmacologically important drug MTX, and highly abundant proteinase inhibitor- α2M. The protein functional activity assay shows 53% decrease in antiproteolytic potential of α2M upon drug interaction. The binding of MTX with α2M was studied by various biophysical methods. UV–visible absorption spectroscopy reveals hyperchromicity of α2M spectra upon drug binding. The intrinsic fluorescence spectra show quenching in fluorescence intensity of α2M and the mechanism of quenching was found to be static in nature. Far UV-CD spectra unveil slight alteration in secondary structure of α2M upon drug binding. Isothermal titration calorimetry (ITC) reveals the value of thermodynamic parameters and which affirms the binding process to be spontaneous and exothermic. Molecular docking illustrates that Asn173, Leu1298, Gly172, Lys1240, Gln1325, Ser1327, Glu913, Asn1139, Lys1236, Leu951 and Arg1297 were the key residues involved during interaction process. Molecular dynamics (MD) simulation studies suggest that MTX form a stable complex with α2M. Our study assumes importance from the fact that MTX is known to bind plasma proteins quite efficiently.

1. Introduction The evaluation of pharmacokinetics and pharmacodynamics of drugs is of extreme significance in drug therapy. Methotrexate (MTX) is an effective chemotherapeutic drug, advised in the treatment of solid tumours, hematologic malignancies and autoimmune disorders such as rheumatoid arthritis [1]. MTX is 2,4-diamino-N10-methyl propylglutamic acid of molecular weight of 454.5 g/mol [2]. It is a folic acid analogue in which NH2 group is bonded to the C4 carbon and CH3 is bonded to N10 hydrogen. MTX structure consists of three parts: pteridine ring, p-aminobenzoic acid and glutamic acid. The pKa values of MTX are 3.8, 4.8, and 5.6 [3] and its solubility in distilled water at 20 °C is 0.01 mg/ml. MTX are eliminated by the kidneys in a short period of time, resulting in a low drug concentration in the target tissues [4]. MTX have a plasma half-life of 5–8 h. The pH range for stability of MTX is 6.6–8.2 [5]. MTX is taken up by the cells and tissue which is then immediately converted to metabolites linked to glutamate (MTX-

∗

polyglutamate) [6]. The resulting complex is responsible for most of biochemical and biological activities of MTX [7]. MTX is used in the treatment of breast cancer, osteogenic sarcoma, lung cancer, bladder carcinoma, brain medulloblastoma and chronic myeloid leukemia [8,9]. Apart from a cancer chemotherapeutic agent, it is recommended for diseases such as psoriasis, multiple sclerosis, Crohn's disease, and rheumatoid arthritis [10]. The cytotoxic actions of MTX have been ascribed due to its property of inhibition of RNA, DNA and protein synthesis and release of adenosine [11]. MTX is a powerful antimetabolite for folate and competitively inhibits the enzyme dihydrofolate reductase (DHFR). DHFR is responsible for converting dihydrofolates to tetrahydrofolates and inhibition of DHFR, results in depletion of tetrahydrofolates thereby inhibiting de novo purine and pyrimidine synthesis. Subsequently, the synthesis of DNA and RNA and other metabolic reactions are interrupted as illustrated in Supplementary Fig. 1 [12,13]. Apart from DHFR, MTX also inhibits some folate-dependent enzymes causing adenosine overproduction and

Corresponding author. E-mail addresses: fahimhkhan@rediffmail.com, [email protected] (F.H. Khan).

https://doi.org/10.1016/j.abb.2019.108118 Received 11 July 2019; Received in revised form 21 September 2019; Accepted 24 September 2019 Available online 28 September 2019 0003-9861/ © 2019 Elsevier Inc. All rights reserved.

Archives of Biochemistry and Biophysics 675 (2019) 108118

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2.2. Methods

inducing immuno-suppression [14]. Anti-cancer drugs are known to induce apoptosis, which is a defensive mechanism, through ROS generation in human melanoma cells [15,16]. MTX also exerts its cytotoxic effects via the production of reactive oxygen species (ROS) and this prooxidant property of MTX with Cu (II) in presence of light is a critical mechanism of apoptosis induction [2,17]. ROS generation also causes oxidative harm to nuclear DNA [2]. Therefore, clinical efficacy of MTX can be attributed to multiple targets. Alpha-2-macroglobulin (α2M) is a member of plasma protein family called alpha-macroglobulins [18] and plays important role in binding, targeting and transportation of several molecules [19]. α2M is a proteinase inhibitor of broad specificity and is capable of inhibiting any proteinase very efficiently [20]. Humanα2M is a 720 kDa soluble glycoprotein composed of four identical 180 kDa subunits, each of which is encoded by a single-copy gene on chromosome 12 [21,22]. The α2M tetramer is regarded as ‘‘Dimer of Dimers’’ as it is composed of a pair of identical subunits joined by disulphide bridges and two such dimers are linked by non-covalent interactions to form a tetrameric molecule [23]. Each of its four subunits is a multidomain of 1451 residue glycosylated molecule. A single monomeric subunit of human α2M contains 129 fluorescent amino acid residues (62 Phe, 11 Trp and 56 Tyr residues) [24]. In the middle of the polypeptide chain, each subunit contains a unique sequence of amino acids termed the “bait region,” which is highly susceptible to proteolytic cleavage by almost all endopeptidases [21]. The primary site of proteolytic cleavage is located in the sequence [Arg-Val-Gly-Phe-Tyr-Glu], present in the bait region. Cleavage of this region triggers a conformational modification in the structure of α2M and consequent entrapment of the proteinase [25,26]. The five reactive sites present in α2M are the bait region, internal thiol ester, receptorbinding site, transglutaminase reactive site and the metalloprotein. The secondary structure of α2M is predominantly β-helical (60%) [24]. Human α2M have various roles in immune modulation, cancer and protection from unobstructive action of proteinases [27,28]. α2M levels were evaluated in the diagnosis of some important diseases such as prostate cancer [29]. Protein-drug interaction is a substantial motif towards the drug availability, efficiency and transport [30]. Several drugs bind reversibly with plasma proteins with moderate binding affinity and alter the protein activity and induce conformational alterations [31]. MTX in the serum is nearly 60% protein bound and its level remains relatively constant while free MTX level changes over the clinical range [32]. It is imperative to understand protein-drug interaction to have a vision on the drug delivery mode, distribution and the mechanism of action [32]. The present study was designed to explore the binding interaction of pharmacologically important drug MTX, with transporting plasma protein-α2M. The structural and conformational changes in protein were monitored using UV/Vis absorption spectroscopy, fluorescence spectroscopy and CD studies. Binding affinity constant, mode of binding and consequent binding energetic (enthalpy change, entropy change) were evaluated by fluorescence quenching studies at three different temperatures (25, 30 and 37 °C) and thermodynamic studies (ITC). Molecular modeling and molecular dynamics (MD) simulation was also performed to understand the binding process at atomic level.

a) Purifications of α2M Purification of human α2M from blood was performed on the basis of the previous work [33] standardized in our laboratory. Fresh outdated/discarded human blood was collected from the Blood Bank, Jawaharlal Nehru Medical College and Hospital, Aligarh. Human plasma was isolated from the blood by centrifugation. Afterwards, ammonium sulphate fractionation was performed and the fraction which precipitates between 20% and 40% saturation was dialysed against phosphate buffer (1000 ml) of 50 mM, pH 7.5. The sample was further loaded on to a gel filtration chromatography column packed with sephacryl-S-300 HR and fractions of 3 ml were procured. The fractions showing inhibitory activity against trypsin were pooled and concentrated. 5% non-denaturing PAGE [34] using tris–glycine buffer (pH 8.3) was performed to check electrophoretic homogeneity of α2M. 10 μg of protein was loaded onto wells and allowed to run. The gel was stained with 0.15% coomassie brilliant blue R-250 for 30 min, then washed and destained. Electrophoretically pure α2M shows a single band in PAGE and was stored at 4 °C [35]. b) Sample preparation MTX stock solution (1 mM) was prepared in DMSO (dimethyl sulphoxide) and was further diluted using sodium phosphate buffer (50 mM, pH 7.5) to obtain desired (5–40 μM) drug concentration. Purified human α2M (10 μM) was incubated against varying concentrations (5–40 μM) of the drug for 1 h at 37 °C before performing any spectroscopic measurements. Experiments were performed in triplicates before reporting the final result. c) Assay of α2M antiproteinase activity α2M (10 μM) was incubated with varying concentration of MTX (5–40 μM) for 1 h. The antiproteinase activity of α2M pre-incubated with MTX was determined by the procedure described by Garnot [36]. The protocol mainly exploits the ability of α2M to shield the amidolytic activity of trypsin from an excess of soybean trypsin inhibitor (STI). Briefly, appropriate amount of α2M (10 μM) was incubated with three fold molar excess of trypsin for 15 min at 37 °C. After trypsin incubation, 100 μl of STI was added and incubated again for 15 min at 37 °C. BAPNA (2 ml) was used as a substrate of trypsin and was added and incubated in the reaction mixture for 30 min. The absorbance was then recorded at 410 nm. The activity of native α2M (not treated with MTX) was taken as reference. Control containing trypsin and drug only did not show any loss of trypsin activity. d) UV/Visible spectroscopy The UV–visible spectra were obtained using PerkinElmer Lambda 25 double beam UV–visible spectrophotometer. UV/Visible spectroscopy is based on the absorption of light by the sample in a given wavelength range. The chromophores (tryptophan, tyrosine and phenylalanine) present in the protein are responsible for the absorption of light by the sample. Tryptophan and tyrosine dominates the absorption spectrum with their absorption maximum at 280 nm and 274 nm whereas phenylalanine exhibits a weak absorption maximum at 257 nm. The absorbance was recorded in the wavelength range of 250–350 nm with a scanning speed of 1000 nm/min in a cuvette of cell length 10 mm. Corresponding drug solution without α2M were used as blanks and subtracted.

2. Materials and methods 2.1. Materials N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), phenylmethylsulphonylflouride (PMSF), soya bean trypsin inhibitor (STI), trypsin, sephacryl S300HR and methotrexate were acquired from Sigma-Aldrich, Chemical Co. (St. Louis, MO). Chemicals for electrophoresis and calibration were procured from Merck were analytical grade commercially available.

e) Intrinsic fluorescence measurements The fluorescence spectroscopy was performed on a Shimadzu RF2

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5301 spectrophotometer (Tokyo, Japan). Proteins possess three intrinsic fluorophores which are tryptophan, tyrosine and phenylalanine of which phenylalanine contribution to protein fluorescence is negligible. Tyrosine also has lower quantum yield and when it is ionised its fluorescence is totally quenched. Intrinsic protein fluorescence is thus determined by tryptophan fluorescence. Intrinsic fluorescence was measured by exciting the protein solution at a low wavelength of 280 nm and collecting the emission spectra at a higher wavelength range of 300–400 nm. Both the slits for excitation and emission were fixed at 5 nm. Spectra were recorded at three different temperatures, viz, 25 °C, 30 °C and 37 °C. Suitable blanks corresponding to the MTX concentration were subtracted to correct the background fluorescence. The emission spectrum of MTX alone was also taken to preclude the probability of fluorescence by drug. The fluorescence data were analyzed and quenching constant (Ksv) was calculated by the Stern-Volmer equation equation (Eq. (1)) [37].

Fo = Ksv [Q] + 1 = kqτo [Q] + 1 F

(Eq. 1)

Fig. 1. Absorption spectra of α2M in absence and presence of MTX. Purified human α2M (10 μM) was incubated with increasing concentration of MTX (5–40 μM) at 37 °C for 1 h.

where, Fo and F are the fluorescence intensities in the absence and presence of MTX respectively, Ksv is the Stern–Volmer quenching constant, [Q] is the concentration of MTX, kq is the bimolecular rate constant of the quenching reaction and τo is the average integral fluorescence life time of tryptophan which is 10−8 s [38,39]. Furthermore, the binding constant (Kb) and the stoichiometry (n) of binding were obtained by modified Stern-Volmer equation (Eq. (2)) [40].

log ⎡ ⎣

Fo − F ⎤ = logKb + nlog [Q] F ⎦

VPC-ITC Micro Cal (GE Healthcare, USA) was used to determine the binding energetic such as entropy change, enthalpy change, Gibbs free energy change, number of binding sites and binding affinity during α2M-MTX interaction. Air bubbles were removed from the samples by degassing in a thermovac unit equipped with the instrument [43]. The sample cell was loaded with α2M (1 μM), reference cell was brimmed with 50 mM sodium phosphate buffer and the syringe was fill up with MTX solution (40 μM). Multiple injections of MTX were made into sample cell containing α2M. Subsequent titrations were carried out and stirring speed was fixed at 307 rpm. Each injection was made over 20s with an interval of 180s between successive injections. The ORIGIN® software was used to analyze the titration curves. Heat of dilution for the drug were determined by control experiments and subtracted from the integrated data before curve fitting.

(Eq. 2)

whereas the change in free energy (ΔG°) was calculated from GibbsHelmholtz equation (Eq. (3)).

ΔG = RTlnKb

(Eq. 3)

where R (1.987 cal mol−1 K−1) is gas constant and T is the absolute temperature (310K). f) Synchronous fluorescence measurements

i) Molecular docking

The synchronous fluorescence spectra were used to further investigate the effect of MTX interaction in changing the micro-enviornment of fluorophores. The fluorescence properties of a molecule depend on the molecule itself as well as the surrounding environment. Any shift in emission maxima occur as a result of changes in fluorophore environment and predict the alterations in polarity around tryptophan and tyrosine residues [41]. The excitation and emission monochromators were scanned simultaneously in synchronous fluorescence. When the wavelength interval between excitation and emission (Δλ) was set as 60 nm, the synchronous fluorescence describes about perturbation in microenvironment in the vicinity of tryptophan [42]. When the wavelength interval was set as 15 nm, it gives information about any change in microenvironment around tyrosine [40]. The excitation wavelength was set at 240 nm whereas the emission range was 255–400 nm for tyrosine residues and 300–400 nm for tryptophan.

Molecular docking deciphers the sites of interaction between the protein and the drug. Autodock 4.2 and Autodock tools utilizing Lamarckian genetic Algorithm were opted to determine the binding mode of MTX. The three dimensional structure of α2M was downloaded from RCSB protein data bank (PDB ID:4acq) [44]. PDB file of MTX was downloaded by RCSB protein data bank. During the process out of all conformers of the drug, the conformer with the lowest binding energy was opted for docking. AutoGrid was run to generate the grid box with grid dimensions X = 96 Å, Y = 76 Å, Z = 108 Å and centre of the grid was X = 58.303 Å, Y = 89.08 Å, Z = 110.446 Å. Water molecules and ions present were removed and hydrogen atoms were added. The protein- α2M was set to be stiff and rigid. Discovery studio 3.5 was used for recognition of residues involved in binding. Ligplot was used to obtain 2D surface model of α2M depicting the amino acid residues involved in MTX interaction [45]. To further investigate the mechanism of MTX binding to α2M, the preferential binding mode of α2M with MTX was determined by 10 ns MD simulations based on the docking results.

g) Circular dichroism spectroscopy CD spectra were recorded on a JASCO CD J-815 spectropolarimeter in the far-UV range (190–260 nm) by using a quartz cuvette of 1 mm path length. The concentrations of MTX used were 20 μM and 40 μM. Analysis of CD spectra were carried out to elucidate alterations in secondary structure of α2M due to interaction with the drug-MTX [43]. Background spectra of MTX solution was subtracted from the spectra of protein-drug complex.

j) Molecular dynamics simulation The protein-α2M docked complex with drug-MTX was selected for molecular dynamics simulation to check the stability of drug within the protein pocket. Desmond v3.2 software was used for MD simulation [46]. The protein- α2M was pre-processed by deleting water molecules with HET ligands (NAG, MAN). It has 1270 amino acids in single chain. The docked complex of MTX at site of α2M was taken as an initial

h) Isothermal titration calorimetry

3

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Fig. 2. (A) Intrinsic fluorescence spectra of α2M in absence and presence of MTX. Purified human α2M (10 μM) was incubated with increasing concentration of MTX (5–40 μM) at 37 °C for 1 h (B) Stern-Volmer plot (C) modified Stern-Volmer plot of fluorescence quenching of α2M by MTX at three different temperatures i.e. 25,30 and 37 °C (D) Van't Hoff plot for temperature dependence of Kb.

conformation for performing MD simulation using OPLS2005 force field [47–49]. The system preparation was done by adding SPC (Simple point charge) water molecules in triclinic box. The simulation box contains 163353 atoms. The final MD run was set to 10 ns simulation time. MD simulation was carried in the isobaric-isothermal ensemble (NPT: 300 K and 1 bar). Remaining parameters were taken with default values. In the MD simulation, a time step of 2 fs was used, and the trajectory coordinates were recorded for every 5 ps. Maestro was utilized for analyzing the trajectories and calculating RMSDs from initial structures.

Table 1 Binding parameters of α2M-methotrexate interaction obtained from fluorescence experiment at three different temperatures i.e. 298, 303 and 310K. Temp (K)

Ksv (M−1)

Kq (M−1s−1)

Kb (M−1)

n

ΔG (Kcal mol−1)

298 303 310

4.15 × 104 3.26 × 104 2.93 × 104

4.15 × 1012 3.26 × 1012 2.93 × 1012

5.42 × 104 4.12 × 104 3.87 × 104

1.10 1.10 1.10

−6.593 −6.550 −6.507

*R2 for all the values ranges from 0.98 to 0.99.

k) Statistical Analysis Data in results were shown as the mean and the standard deviation (S.D.) values, n = 3 representing as the number of independent experiments.

3. Result and discussion 3.1. Effect of MTX on α2M antiproteinase activity The trypsin inhibitory assay was performed in order to monitor the inhibitory activity of human α2M in the presence of MTX. Native α2M exhibit maximum activity and was taken as reference. Upon interaction with increasing concentration of MTX (5–50 μM), α2M activity decreases gradually. At 25 μM of MTX, 57% of antiproteolytic activity was left in the protein. On further increasing the MTX concentration to 50 μM, protein activity declines to 35% of its original activity as shown in Supplementary Fig. 2. This incessant reduction in the inhibitory activity of α2M was due to interaction with increasing concentration of MTX. Even low concentration of MTX (micromoles) lowers the activity of α2M efficiently. Control having trypsin and drug only did not reveal any loss of trypsin activity. The reduced functional activity might be due to the structural and conformational alteration of α2M which ultimately suggests the transition of native α2M. The transition of native

Fig. 3. Far-UV CD spectra of native α2M and MTX treated α2M. Purified human α2M was incubated with 20 μM and 40 μM of MTX at 37 °C for 1 h.

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hyperchromic shift in absorption intensity upon addition of drug (10–50 μM) implies structural change in α2M upon interaction with MTX, thus corroborating the formation of α2M-MTX complex. Thus, it can be concluded that the increment in the absorption intensity could be due to the formation of α2M-MTX complex. UV spectra of drug alone were also taken to rule out possible contribution from MTX. The UVabsorption spectra of MTX and the difference spectra of α2M-MTX complex shows no overlapping between the spectra, thus confirming the formation of ground state complex formation between α2M and MTX. Similar findings of increase in absorption intensity were reported by Rehan et al. during the interaction of anticancer drug clofarabine with human serum albumin (HSA) [50]. 3.3. Intrinsic fluorescence measurement Intrinsic fluorescence spectroscopy measures the cumulative fluorescence of individual fluorophores of the protein. Aromatic amino acid residues (Tyr, Trp, and Phe) are the key residues responsible for imparting fluorescence. Fluorescence intensity measurement technique detects the conformational changes within the protein [43,52]. The intrinsic fluorescence of α2M was measured in presence of increasing concentrations (5–40 μM) of MTX. As illustrated in Fig. 2(A), α2M exhibits a strong fluorescence emission peak at 340 nm which progressively quenches on interaction with MTX. This decrease in fluorescence intensity upon addition of MTX suggests binding of α2M with MTX. The decrease influorescence intensity can be due to alterations in the microenvironment around fluorophores. The quenching of fluorescence intensity was analyzed according to the Stern–Volmer equation (1) at three different temperatures viz. 25, 30 and 37 °C [50]. A linear dependence between Fo/F and molar concentration of the MTX (1:1) (Fig. 2(B)) confirms the occurrence of single quenching mechanism which can either be static or dynamic [53]. Dynamic quenching is due to collisional encounter between the fluorophore and the ligand whereas static quenching is the result of ground state complex formation between the fluorophore and ligand [38,53]. To determine the type of quenching occurring in MTX-α2M system, Stern-Volmer quenching constant (Ksv) and bimolecular quenching constant (Kq) were evaluated using Stern-Volmer equation (1) at three different temperatures. In static quenching, Ksv decreases with an increase in temperature due to complex formation, which undergoes dissociation on increasing the temperature, however, in dynamic quenching, Ksv increases with temperature as in this case higher temperature results in faster diffusion of quencher and hence larger extent of collisional quenching [40,54]. As can be seen from Table 1, Ksv value decreases with increase in temperature which reflects the static nature of quenching process rather than dynamic [54]. Furthermore, the value of Kq for MTX-α2M interaction was found to be 100 times higher than the maximum scatter collision quenching constant of various quenchers with biopolymers (2 × 1010 M−1s−1) [40,50]. This further indicates that the quenching mechanism is static, and is due to ground state complex formation between the fluorophore and the drug [54]. Similar reasoning of static mechanism of fluorescence quenching has been documented for the interaction of MTX with HSA [55]. The interaction of other anti-cancer drugs such as cytosine β-D arabinofuranoside [56] and busulfan [57] with HSA also reports similar results. The binding constant (Kb) values, obtained from modified SternVolmer equation (2) were found to decrease with increase in temperature suggesting that higher temperature leads to less stable complex formation. Fig. 2(C) and (D) represent modified Stern-Volmer plot at three different temperatures and van't Hoff plot for temperature dependence of Kb. The stoichiometry of binding was almost equal to unity demonstrating that there is one independent class of binding site for MTX on α2M. The change in free energy (ΔG), calculated from Gibbs-Helmholtz equation (3) at three different temperatures (25, 30 and 37 °C) gives a negative value (Table 1) of free energy change which

Fig. 4. (A)Thermogram(B) binding isotherm for the titration of MTX with α2M at 37 °C. The molar concentration of α2M in the sample cell was 10 μM. MTX molar concentration in the syringe was 40 μM. Reference cell was loaded with 50 mM sodium phosphate buffer (pH 7.4). Table 2 Thermodynamic and binding parameters for the interaction of MTX with α2M obtained from ITC experiment at 310K. Parameters

Value

Stoichiometry (n) Binding constant (Kb) Enthalpy change (ΔH) Entropy change (TΔS) Gibbs free energy change (ΔG)

1.12 ± 0.02 (3.62 ± 0.09) × 104 M−1 −2.69 ± 0.09 kcalmol-1 3.60 ± 0.08 kcalmol-1 −6.29 ± 0.12 kcalmol-1

α2M to non-native form decreases the possibility of α2M to bind the active site of the proteinase. Drug-protein interaction can disrupt the normal functioning of proteins [50]. 3.2. UV/visible absorption measurements The UV/visible absorption spectroscopy is a consistent technique to monitor perturbations caused by the binding of drug to protein. The perturbations induced in protein on binding with drug are symptomatic of conformational alterations. Aromatic amino acids (Trp, Phe, Tyr) are responsible for the characteristic absorption of the proteins, exclusively at 280 nm [50,51]. Here, the UV spectrum of α2M was monitored in the presence of increasing concentrations of MTX after incubating for 1 h. It is apparent from Fig. 1 that the maximal absorption of sheep α2M was at 280 nm and onincreasing concentration of MTX (5–40 μM), absorption intensity increases regularly with no shift in peak position. The 5

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Fig. 5. Molecular docking of MTX with human α2M (PDB Id: 4acq). Surface model of human α2M depicting the pocket where the drug binds to α2M.

and hence changes in CD spectra are quite evident after binding of drug to the protein. The secondary structure of α2M is predominantly β-helical (nearly 60%) [24,26]. CD spectrum of native human α2M shows a positive peak at 195 nm and a defined negative peak at 215 nm [51]. However, when α2M was incubated with 20 μM MTX, the α2M spectrum showed a decrease in negative ellipticity as shown in Fig. 3. On further increasing the concentration of MTX to 40 μM, negative ellipticity decreases further. Our observation of decrease in negative ellipticity points to that fact that MTX decreases the β-helical content of α2M. The decrease in ellipticity of α2M in presence of MTX as compared to native α2M clearly suggests some sort of conformational changes in α2M induced by MTX. Using K2D2 software, the helicity of proteins were calculated [61]. At 1:2 protein to drug molar ratio, the β-helicity was found to decrease from 57.25 ± 1.05% to 55.35 ± 1.75% for α2M and at 1:4 protein to drug molar ratio, the helicity decreases from 57.25 ± 1.37% to 53.60 ± 1.28% for α2M. This indicates that MTX interaction decreases the β-helical content of α2M secondary structure. Drug-protein binding likely alters the intermolecular forces liable for sustaining the secondary structures leading to conformational alterations in protein. Studying conformational alterations in the large protein especially α2M, is significant because conformation governs the functional status of protein [62]. The observed change in the secondary structure of α2M can also affect its physiological functions.

Fig. 6. MD simulation ofα2M with MTX for 10 ns. RMSD of protein backbone and MTX for 0–10 ns.

represents a spontaneous occurrence of the reaction. Synchronous fluorescence measurements provide information about alteration in micro-environment around aromatic moieties upon drug interaction. The shift in emission maxima predicts the alterations in polarity around tryptophan and tyrosine [41]. Synchronous fluorescence spectra of α2M with varying MTX concentration (5–40 μM) were shown in Supplementary Figs. 3(A and B). A subtle red shift in the peak (from 340 to 345 nm) along with reduction in fluorescence intensity was observed (when Δλ = 60 nm) which signifies change in the microenvironment around tryptophan residue [58]. This shift implies that MTX interaction drives the tryptophan residues of α2M from the nonpolar environment to a more polar environment hence reducing the hydrophobicity [59,60]. However addition of MTX did not causes any shift but a decrease in fluorescence intensity was observed (when Δλ = 15 nm). Hence, it was concluded that tryptophan plays an important role during fluorescence quenching of α2M and signified that MTX approaches the tryptophan more than the tyrosine. This was further supported by the computational data.

3.5. Isothermal titration calorimetry analysis The thermodynamic parameters such as change in entropy (ΔS), change in enthalpy (ΔH), Gibbs free energy (ΔG), number of binding sites (N) and binding affinity constant (K) for the interaction of MTX with α2M were obtained from ITC at 37 °C.A representative calorimetric titration profile is shown in Fig. 4(A), every peak of binding isotherm symbolizes a single round of injection of MTX into the α2M solution. Fig. 4(B) shows the profile of heat liberated per injection as a function of molar ratio of MTX into α2M. The thermodynamic data (association constant and enthalpy change) were directly obtained after the best fitting for the integrated heats using single set of binding model with lowest chi square value summarized in Table 2. The calorimetric data was subtracted for the corresponding blank measurements. The 1:1 binding stoichiometry of MTX to α2M with a binding constant value of 3.62⨉104 M−1 at 37 °C indicates moderate and specific interaction. The intermolecular forces involved during the interaction of MTX with α2M were determined by the rules of Ross and Subramanian [63]. Ross and Subramanian stated that negative values of ΔH (−2.69

3.4. Circular dichroism measurements Circular dichroism is primarily employed to determine variations in protein secondary structure during protein-drug interaction. The conformation of protein usually gets altered once drug binds to the protein. This altered protein conformation corresponds to altered CD spectra 6

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Fig. 7. (A) Bar chart showing the involvement of various interactions between α2M and MTX. The stacked bar charts are normalized over the course of the trajectory and the values over 1.0 represent the participation of amino acid residue in more than one kind of interactions with the ligand. Green = hydrogen bond, blue = water interaction, magenta = ionic interaction, violet = hydrophobic interaction. (B) The interaction fraction shows contacts which are constant between 8 and 15 contacts during 10 ns simulation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

kcalmol-1) suggests the reaction was exothermic and possibly involves hydrogen bonds [64]. A positive value of ΔS (3.60 kcal mol−1) was symptomatic of hydrophobic interactions and was a strong indication that water molecules have been excluded from the binding site interface [65,66]. Furthermore, the negative value of ΔG (−6.29 kcal mol−1) signifies that the reaction was spontaneous and thermodynamically favorable.The binding parameters obtained from ITC at 37 °C were comparable to that determined from spectroscopic studies.

to measure the average change in displacement of a selection of atoms. Fig. 6 shows RMSD of protein backbone and MTX after MD simulation with respect to the initial original frame. The protein backbone atoms show some fluctuations in an initial simulation time (0–2nsec), the RMSD lie between 4.2 and 4.8 Å. After 2 ns simulation time, the RMSD remain constant between 2.4 and 3.0 Å till end of the simulation studies. This indicates that protein is stable throughout simulation and showed steady state dynamics. The protein-drug complex was stabilized only after 2 ns of simulation. Similarly, RMSD of drug shows stability in active pocket of protein throughout simulation time between 0.3–0.8 Å RMSD. No major fluctuations in drug were observed during simulation (Fig. 6). This depicts the both protein and drug RMSD are stable throughout simulation time. α2M shows strong binding with drug, during simulation. Following residues mainly shows > 90% contacts: Asn173, Thr1239, Ser957, Gly956, Asp953, Tyr1264, Lys1236, Gly952 and Gly172 (Fig. 7(A)). The interaction fraction shows constant contacts between 8 and 15 contacts during 10 ns simulation in Fig. 7(B). Active pocket residues which are involved in protein drug interaction during simulation provided in Supplementary Fig. 5. Tryptophan 1237 is present in close vicinity to MTX molecule (Supplementary Fig. 5) and hydrophobic interactions were the main forces involved during MTXα2M interaction as shown by docking studies. Synchronous fluorescence also confirms that MTX alters the micro-environment around tryptophan residues of α2M and decrease the hydrophobicity around it. Hence, computational data corroborates synchronous fluorescence analysis.

3.6. Molecular docking study of α2M − MTX interaction Molecular modeling was performed to predict the binding sites of the drug within the proteins polypeptides. Molecular docking is a type of bioinformatics tool which involves the interaction of two or more molecules to give the stable adduct. Depending upon binding properties of ligand and target, it predicts the three-dimensional structure of any complex. Molecular docking was performed with a monomer of α2M and grid size was reduced. Result suggests that MTX primarily interacts with the monomer of α2M (Fig. 5). Asn173, Leu1298, Gly172, Lys1240, Gln1325, Ser1327, Glu913, Asn1139, Lys1236, Leu951 and Arg1297 were the key residues involved during interaction process and illustrated in Supplementary Fig. 4. The information acquired from the docking can be used to suggest the binding energy and stability of complexes. The interaction forces prevailing in MTX-α2M interaction were the hydrogen bonding and hydrophobic interactions. Binding energy supports the spontaneity of the reaction and was found to be −6.41 kcal/mol. The binding site of MTX is different from the site of binding reported for other anticancer drugs from our laboratory [37,51] and is primarily surrounded by both polar amino acids.

4. Conclusion Present study explores the binding and interaction of widely used anticancer drug-MTX with major human proteinase inhibitor-α2M. Binding of protein to drug have significant impact on pharmacokinetic behavior of drug [66]. Our study investigated the binding behavior of MTX with antiproteinase-α2M present abundantly in human plasma. Result of functional activity assay reveals significant loss in antiproteolytic potential of α2M on exposure to increasing concentration of MTX (5–40 μM). UV/Vis absorption spectroscopy showed marked

3.7. Molecular dynamics simulation studies MD simulation was employed to determine the stability of MTXα2M complex under physiological conditions as predicted by docking. The initial conformation of α2M bound with MTX was used for MD simulation for 10 ns. The Root Mean Square Deviation (RMSD) is used 7

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increase in absorbance due to complex formation between the drug and protein. Intrinsic fluorescence spectra reveal quenching in a dose dependent manner (5–40 μM) and the quenching mechanism was found to be static in nature. Synchronous fluorescence reveals alteration in the micro-environment of tryptophan residues thus decreasing hydrophobicity around them. Changes in the CD spectra confirm perturbation in secondary structural conformation of α2M due to drug binding. Molecular docking studies disclose that Asn173, Leu1298, Gly172, Lys1240, Gln1325, Ser1327, Glu913, Asn1139, Lys1236, Leu951 and Arg1297 were the principal residues in α2M monomer involved in interaction with MTX. The forces of interaction were hydrophobic forces and hydrogen bonding. MD simulation studies illustrated that protein and drug were stable throughout 0–10 ns simulation time. Overall, our work provides a better understanding of the binding of MTX with α2M in vitro, which is worthy to have an understanding of the binding and transport process of therapeutic molecule in the body. This study assumes importance from that fact that MTX is most common drug, advised in treatment of cancer as well as other diseases [8–10] and whose major portion is known to bind protein [32]. A damaged antiproteinase is likely to have multidimensional effect on the proteinase–antiproteinase balance of the body and hence its ramification in the etiology and pathogenesis of variety of diseases. Declaration of competing interest None Acknowledgements Facilities provided by Department of Biochemistry, Aligarh Muslim University, Aligarh are gratefully acknowledged. Authors wish to thank the Department of Science and Technology (DST-FIST-1715) and University Grants Commission, New Delhi, Government of India for the financial support. M. K. Zia is highly thankful to UGC-MANF for senior research fellowship (SRF). T. Siddiqui is thankful to Department of Biotechnology (DBT), New Delhi, for senior research fellowship (SRF). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.abb.2019.108118. References [1] W.T. Purcell, D.S. Ettinger, Novel antifolate drugs, Curr. Oncol. Rep. 5 (2003) 114–125. [2] S. Chibber, I. Hassan, M. Farhan, I. Naseem, In vitro pro-oxidant action of methotrexate in presence of white light, J. Photochem. Photobiol. B Biol. 104 (2011) 387–393. [3] L.K. Rahman, S.R. Chhabra, The chemistry of methotrexate and its analogues, Med. Res. Rev. 8 (1988) 95–155. [4] M.P. Iqbal, Accumulation of methotrexate in human tissues following high-dose methotrexate therapy, J. Pak. Med. Assoc. 48 (1998) 341–343. [5] D.C. Chatterji, J.F. Gallelli, Thermal and photolytic decomposition of methotrexate in aqueous solutions, J. Pharm. Sci. 67 (1978) 526–531. [6] L. Genestier, R. Paillot, L. Quemeneur, K. Izeradjane, J.P. Revellard, Mechanisms of action of methotrexate, Immunopharmacology 47 (2000) 247–257. [7] J.C. Panetta, A. Wall, C.H. Pui, M.V. Relling, W.E. Evans, Methotrexate intracellular disposition in acute lymphoblasticleukemia: a mathematical model of glutamyl hydrolase activity, Clin. Cancer Res. 8 (2002) 2423–2429. [8] W.A. Bleyer, The clinical pharmacology of methotrexate: new applications of an old drug, Cancer 41 (1978) 36–51. [9] J. Grim, J. Chladek, J. Martinkova, Pharmacokinetics and pharmacodynamics of methotrexate in non-neoplastic diseases, Clin. Pharmacokinet. 42 (2003) 139–151. [10] S.S. Abolmaali, A.M. Tamaddon, R. Dinarvand, A review of therapeutic challenges and achievements of methotrexate delivery systems for treatment of cancer and rheumatoid arthritis, Cancer Chemother. Pharmacol. 71 (2013) 1115–1130. [11] D.C. Phillips, K.J. Woollard, H.R. Griffiths, The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species, Br. J. Pharmacol. 138 (2003) 501–511. [12] G. Budzik, L. Colletti, C. Faltynek, Effects of methotrexate on nucleotide pools in normal T cells and the CEM T cell line, Life Sci. 66 (2000) 2297–2307.

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