In Vitro Cytotoxicity and Interaction of Noscapine with Human Serum

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In vitro cytotoxicity and interaction of noscapine with human serum albumin: Effect on structure and esterase activity of HSA Neha Maurya, Jitendra Kumar Maurya, Upendra Kumar Singh, Ravins Dohare, Mohammad Zafaryab, M. Moshahid Alam Rizvi, Amer M. Alanazi, Azmat Ali Khan, Meena Kumari, and Rajan Patel Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00864 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019

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Molecular Pharmaceutics

In vitro cytotoxicity and interaction of noscapine with human serum albumin: Effect on structure and esterase activity of HSA Neha Maurya1, Jitendra Kumar Maurya1, Upendra Kumar Singh1, Ravins Dohare2, Md Zafaryab3, M Moshahid Alam Rizvi3, Amer M. Alanazi4, Azmat Ali Khan4, Meena Kumari5 and Rajan Patel1* 1Biophysical

Chemistry Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), New Delhi. 2Nonlinear Dynamic Laboratory, Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia (A Central University), New Delhi. 3Department of Biosciences, Jamia Millia Islamia, Central University, New Delhi. 4Pharmaceutical Biotechnology laboratory, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia. 5Biophysical Chemistry Laboratory, Department of Chemistry, IIT Delhi, Hauzkhas, New Delhi *Corresponding author. Tel.: +91 8860634100; fax: +91 11 26983409. Email address: [email protected], [email protected] (Dr. R. Patel)

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Abstract

Noscapine is effective to inhibit cellular proliferation and induced apoptosis in non-small cell, lung, breast, lymphoma and prostate cancer. It also shows good efficiency to skin cancer cell. In current work we studied the mechanism of interaction between anticancer drug noscapine (NOS) and carrier protein human serum albumin (HSA) by using a variety of spectroscopic technique (fluorescence spectroscopy, time resolved fluorescence, UV-visible, fluorescence resonance energy transfer (FRET) fourier transform infrared (FTIR), circular dichroism (CD) spectroscopy), electrochemistry (cyclic voltammetry) and computational method (molecular docking and molecular dynamic simulation). The steady-state fluorescence results showed that fluorescence intensity of HSA decreased in presence of NOS via static quenching mechanism, which involves ground state complex formation between NOS and HSA. UV-visible and FRET results also supported fluorescence result. The corresponding thermodynamic result shows that binding of NOS with HSA is exothermic in nature involving electrostatic interactions as major binding forces. The binding results were further confirmed through cyclic voltammetry approach. The FRET result signifies the energy transfer from Trp214 of HSA to the NOS. Molecular site marker, molecular docking and MD simulation results indicated that the principal binding site of HSA for NOS is site I. Synchronous fluorescence spectra, FTIR, 3D fluorescence, CD spectra and MD simulation results reveal that NOS induced the structural change in HSA. In addition, the MTT assay study on human skin cancer cell line (A-431) was also performed for NOS which shows that NOS induced 80 % cell death of the population at 320 μM concentration. Moreover, the esterase-like activity of HSA with NOS was also done to determined variation in protein functionality after binding with NOS. Key words: Noscapine, Human serum albumin, Cytotoxicity, Energy transfer, Esterase-like activity. 2 ACS Paragon Plus Environment

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1. Introduction Noscapine (NOS) is a phthalideisoquinoline alkaloid extracted from Papaver somniferum plantand generally use as a cough suppressant in human and experimental animals1-2. NOS has been gain good attention in cancer research from last few years due to its outstanding anticancer activity and low toxicity3-4. NOS is antitussive and naturally tubulin-binding compound presently undergoing phase I/II clinical trials for cancer treatment5. It has also potency to inhibit the growth of tumor cell during mitosis and induced apoptosis in lung, breast, lymphoma and prostate cancer. Due to these unique properties, it is used as an alternative chemotherapeutic drug. In this prospective past few years, many researchers have been reported the efficiency of NOS and their derivative in oncology3, 6-7. Chougule et al. showed synergistic anticancer activity of NOS with gemcitabine in lung tumor xenografts inhibition8. Joshi et al. have done the anticancer effect of NOS and found that it can interrupt tubulin dynamics and arrest mitosis9.As tubulin is a known target site for NOS, Suri et al. reported computational study on the interactions between γ-tubulin, GCP4 and NOS derivatives10. Various research reports has been shown in-vitro and in-vivo anti-tumor activity of NOS in a variety of tumor cell such as glioma, melanoma, non-small cell lung cancer, prostate, multiple myeloma, colon, ovarian and breast cancer4,

11-14.

Now a day’s NOS and their derivative have many advantages as a potential

anticancer drug still many key problems have been unrevealed in pharmacology and pharmacokinetics research. Therefore, herein we have explored different binding parameters for NOS-HSA interaction; moreover the influence of NOS on the structure of HSA was also explored. Additionally, we have also studied the NOS potency towards inhibition of skin cancer tumor growth on human skin cancer cell line (A-431).



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Human serum albumin (HSA) is a principal extracellular soluble protein, involves in circulatory system and contributes extensively to the transportation, distribution and metabolism of drugs. Due to its easily availability and simple structure, HSA is utilized as model protein to interact with various ligands such as drug, free metal ions, surfactant, nanoparticle, flavonoids and natural alkaloid15-18. It binds with broad range of drugs in the body and plays a significant role in pharmacodynamics and pharmacology of the drug. Since, past two decay researchers have been extensively studied the binding mechanism of HSA with drugs19-20. Recently, Rabbani et al.21 have been explored the binding efficiency of Tolperisone hydrochloride with human serum albumin. Danesh et al.22also studied the interactions of estradiol with two carrier proteins, human serum albumin and holo-transferrin, in order to investigate the binding mechanism of carrier proteins. On the basis of X-ray crystallographic investigation, HSA is helical, monomeric protein with 585 amino acid residues and it contains three homologous domains (I–III), every domains comprised with two subdomains i.e. A and B subdomains23-24. HSA having two major drug binding site situated in hydrophobic cavities in sub-domains IIA and IIIA. Among these sites signal tryptophan located on site I (TRP214)25-26. In the present study, we used various spectroscopic and electrochemical techniques in combination with computational methods to understand the transportation and metabolisms of NOS with HSA. We have also studied the cytotoxicity of NOS on human skin cancer cell (A431). The interactions between NOS and HSA under physiological condition have been studied by steady state fluorescence, UV-visible, time resolved fluorescence, cyclic voltammetery and molecular docking. The binding constant and number of binding sites with various thermodynamic parameters such as, ΔG0 (Gibbs free energy change), ΔS0 (entropy change) and ΔH0 (enthalpy change) were calculated. FRET theory was implying to determine the distance


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between NOS and HSA. Furthermore, the conformational and micro environmental changes of HSA induce by NOS were evaluated through FT-IR spectroscopy, CD spectroscopy, 3D fluorescence, synchronous fluorescence spectroscopy and MD simulation. Additionally, the esterase like activity of HSA was also studied. This investigation may be useful and give precious information of biophysical action of NOS in human body.

Scheme1. 3D structure of noscapine. 2. Materials and methods 2.1. Materials Human serum albumin (96%), noscapine (NOS) (98%), indomethacin (99%), ibuprofen (98%) and p-nitrophenyl acetate (p-NPA) was obtained from Sigma Aldrich. The stock solution of HSA and NOS was made in phosphate buffer (10 mM, pH 7.4) and kept in 4 °C. The HSA stock concentration was calculated through UV-visible absorption spectroscopy using extinction coefficients (ε) 36600 M-1cm-1 at 280 nm26. Ultrapure Millipore water was used in all the experiments. 0.22 µm pore size Millipore filters were used to filter the buffer solution. The analytical grade reagents were utilized without further purification in all experiments. 2.2. Methods



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2.2.1. Cell culture and MTT assay The anti-tumor effect of the NOS has done using colorimetric MTT assay on human skin cancer cell line (A-431). A-431 cell line was obtained from National Curator of Cell Sciences (NCCS) Pune, India. The maintenance of cell lines were done in DMEM (Dulbecco’s modified Eagle’s medium) supplemented with 10 % fetal bovine serum, 2.5 μg/mL amphotericin B, 100 μg/mL streptomycin, and 100 units/mL penicillin. Culture were kept in incubators with 5 % CO2, 80 % humidity atmosphere at 37

0C.

More detail about MTT assay described in supporting

information. 2.2.2 Fluorescence spectroscopy The steady state fluorescence emission spectra were collected through Cary Eclipse fluorescence spectrometer (Varian, USA) attached with Peltier control temperature device. Fluorescence spectra of HSA (5µM) and different concentration of NOS (4.95 - 45.45 µM) were measured at 298, 308 and 318K in wavelength range of 295–450 nm. The excitation wavelength 280 nm is set to measure the both fluorophore of HSA (Trp and Tyr)

27.

As the inner filter effect is

affecting the spectral measurement of fluorescence and opposed to a quenching process. It occurs when the absorbing compound is added into the solution; it reduces some radiation emitted by the fluorophore. Therefore, the inner filter effect was performed to correct the measured fluorescence intensity by using absorbance NOS 280 nm and emission wavelength (342 nm) via the following equation 28-29. Fcorr = Fobsd10(A1 + A2)/2

(1)

where Fcor represents corrected fluorescence intensities and Fobsd represents the fluorescence intensities observed. A1 represent the absorbance of NOS at the excitation wavelengths and A2 represents the absorbance of the NOS at emission wavelengths. Synchronous fluorescence


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spectra pictures microenvironment of aromatic residues tyrosine (Tyr) and tryptophan (Trp) and were performed in the range of 250-310 nm. The excitation and emission wavelength intervals (∆λ) were set as 15 and 60 nm for Tyr and Trp respectively as mention in previous article

28.

The 3 Dimension fluorescence spectra of pure HSA and NOS-HSA complex have been also performed. The excitation wavelength from 200–250 nm was fixed, keeping the increment of 10 nm, while as the emission wavelength range was fixed from 200–500 nm. The concentrations of HSA and NOS were taken 5µM and 45µM, respectively. 2.2.3 UV-visible spectroscopy The UV-visible spectra were taken at room temperature by using Analytik Jena Specord-210 spectrophotometer equipped with Peltier control temperature device to maintain uniform temperature throughout the cell. The quartz cells with path length of 1 cm were used. The absorbance spectra of HSA (5µM) and different the concentrations of the NOS (4.95 - 45.45 µM) were measured from the wavelength range of 230-400 nm. 2.2.4 Time-resolved fluorescence spectroscopy The time-resolved spectra were collected by using time correlated single-photon counting (TCSPC) spectrometer (Horiba, Jobin Yvon, IBH Ltd, Glasgow, UK). The excitation of sample was done at 280 nm utilizing pulsed nanosecond LED. The lifetime was measured up to 10,000 counts at room temperature. Fluorescence decays data analysis was done by using IBH DAS6 decay analysis software. The fluorescence lifetime was measured using the following equation:

 f (t )   ai exp i 1  i n

  

(2)

where, αi is the pre-exponential factors and has been normalized to 1 and errors were taken up to three standard deviations. The chi-square (χ2) value was utilized to quantify the goodness of 7 ACS Paragon Plus Environment


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fitting. The calculation of average fluorescence lifetimes were done through the following equation:



a  

2

i i

(3)

 ai i

where ai represents relative contribution to the total decay and τi represents the life time of different components. 2.2.5 Cyclic voltammetry measurement The cyclic voltammetry measurements were done on Digi-Ivy three-electrode potentiostat (DY2100) equipped with bare platinum electrode (2 mm diameter, working electrode), platinum wire (counter electrode) and Ag/AgCl electrode (reference electrode), respectively. Before experiment the bare platinum electrode was ultrasonically cleaned in acetone, 0.5 M H2SO4 solution and multiple times in ultrapure water, after cleaning electrode dried in nitrogen airflow. For all experiment we utilize HSA modified platinum electrode, which is prepared by dry adsorption method developed previously30.2 µL HSA solutions (5µM) was dropped on the surface of the platinum electrode and dried overnight. Followed by washing the electrode with ultrapure water and dried with nitrogen atmosphere then use as a HSA modified electrode. 1:10 mM solution of K3Fe(CN)6/K4Fe(CN)6 was used as electrolyte in the experiments and 3 mL solution of electrolyte added in an electrochemical cell. The different concentrations of NOS (4.95 to 45.45 µM) added constantly to the electrolyte and stirred for 3 minutes and after that the reading was observed after every 2 minutes. The scan rate and scan range were set as 0.05 Vs-1 and -0.6 to 0.6 V, respectively. Before testing all the solution where pursed with pure nitrogen. 2.2.6 Circular dichroism spectroscopy


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The CD spectra equipped with a microcomputer was recorded on Jasco-715 spectropolarimeter. The instrument was calibrated with D-10-camphorsulfonic acid in presence of nitrogen atmosphere. The measurements of CD were observed in far-UV (200-250 nm) and near UV (250-310 nm) region with 0.2 nm intervals at 298 K. To maintain the uniform temperature, the thermostatically controlled cell holder equipped with Neslab RTE-110 water bath was utilized and the accuracy in temperature was ±0.10C. 10 mm path length cell was used for experiment and six accumulations were used for each scan to get better the signal-to-noise ratio. The signal of buffer solution (reference) and NOS was deducted from the CD signal. The MRE of HSA at θ222 determined by the following equation:

 222  M 0 222

(4)

10lC

where θ222 is stand for ellipticity in milli degrees at wavelength 222 nm. M0 and c are the mean residue weight and concentration of HSA. lis the path-length. The α-helical content of the HSA and HSA with NOS was evaluated using probe at 222 nm using the following equation31:

  helix 

 ( MRE 222  2340) 30300

(5)

2.2.7 FT-IR spectra spectroscopy The FT-IR measurement of pure HSA (5µM) and HSA with NOS (45µM) were taken on the Bruker Tensor 27 FT-IR spectrometer. The spectra were recorded through the ATR (attenuated total reflection) with 2 cm-1 resolution. The range was used 1400–1800 cm-1 with 128 interferograms to in order to get a good signal-to-noise ratio. The contributions of phosphate buffer and free NOS absorption were subtracted from solution. 2.2.8 Esterase like activity assay



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The influence of NOS on the esterase like activity of HSA was studied with synthetic substrate p-nitrophenyl acetate (p-NPA). UV-visible absorbance technique was used to study the enzymatic activity. HSA on interacting with p-NPA released p-nitrophenyl. The absorbance of released product was recorded at 400 nm32. The reaction mixture has 100-700 μM p-nitrophenyl acetate, HSA:NOS molar ratio (1:0, 1:1, 1:2, 1:5) where concentration of HSA is 5µM at 37 0C and 7.4 pH. A molar extinction coefficient (ε) was taken as 17700 M-1 cm-1 to calculate the concentration of p-nitrophenol. The initial slop of the absorbance graph between 0-2 minute was utilizing to calculate the initial reaction velocities (ν0). All kinetic parameter of pure HSA and NOS-HSA interaction were calculated through following Michaelis-Menten equation by fitting the initial rate:

v0 

Vmax [ S ] K m  [S ]

(6)

where ν0 is initial velocity, Vmax shows as maximum velocity, Km stand for Michaelis-Menten constant and [S] represents molar concentration of p-NPA, respectively. Kcat was calculated by employing the follow equation:

Vmax  K cat [ E ]

(7)

where [E] is the HSA concentration. 2.2.9 In-Silico Studies The in-silico studies (molecular docking and molecular dynamic simulation) have been done to explore the interaction and binding site between NOS and HSA. The crystal structure of HSA (PDB ID: 1AO6) was retrieved from Protein Data Bank (PDB) and 3D structure of NOS was obtained by PubChem and its structure was optimized using Discovery Studio 2.5. The more detail information about technique is mentioned in the supporting information section. 3. Results and discussion 10 ACS Paragon Plus Environment

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3.1. Cytotoxicity studies The MTT assay was used to demonstrate the cytotoxicity of NOS. The results indicate that NOS showed dose dependent cytotoxicity with increasing concentrations (10-320 μM) on human skin cancer cell line (A-431). The concentration of NOS for cytotoxicity and in-vivo study were selected according to the reported literature

6, 33.

As shown in Figure1, NOS induced 80 % cell

death of the population at 320 μM concentration. The IC50 value (concentration of drug at which 50% of cell death occurs) was calculated through plots of cell viability (%) versus concentration of NOS. IC50 of NOS was determined 71.42 μM for human skin cancer cell line (A-431).The cytotoxicity of NOS were well studied on numerous cancer cell lines, such as breast cancer (MCF-7), lung cancer (A549), thymocyte cells, and prostate cancer, proving that NOS is a potency to induce tumor cell7, 12. This study clearly shows that NOS have cytotoxicity effect to human skin cancer cell line (A-431), hence NOS may be utilized for the treatment of skin cancer. 3.2. HSA-NOS interaction 3.2.1. Steady state fluorescence study The emission profile of NOS interaction with HSA shows a significant change in the florescence spectra. The HSA shows a strong emission peak at 343 nm in aqueous buffer, which indicates the surrounding environment of Trp residue is hydrophobic in nature28,

34.

Since intrinsic

fluorescence of HSA is very sensitive to its local environment, therefore it can be used as a probe to study the interaction of protein with an exogenous ligand or drug molecule. Figure 2 shows that on increasing the NOS concentration, the fluorescence intensity of HSA decrease continuously with a significant blue shift from 343 to 335 nm which suggests that Trp is located at or close to the drug binding site. The above finding is analyse in terms of following assumptions (a) the internalization of Tryptophan (fluorophore) structure 34and (ii) NOS induced



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structural changes in HSA, which shows more hydrophobic interaction between tryptophan and the hydrophobic moieties of the NOS. The hypsochromic shift in emission spectrum of HSA with NOS suggests that microenvironment of Trp214 highly hydrophobic as compared to that in the absence of NOS. The binding isotherm has been used for the study of HSA-NOS interaction. The fraction of protein (α) bound to the NOS has been calculated by the given equation.



I obs  I 0 I max  I 0

(8)

where I0= fluorescence intensity of HSA in absence of NOS, Iobs = fluorescence intensity at any NOS concentration, and Imax= fluorescence intensity at saturation binding condition35. The binding isotherm for NOS-HSA interaction is shown in Figure3. There are two breakpoints denoted by C1 and C2as shown in Figure 3, which clearly confirms that the binding of NOS to HSA takes place stage by stage in sequential manner. HSA has some highly energetic sites in their native state which are available for binding to the NOS. The binding on these sites are highly specific at lower concentration. The reduction in fluorescence intensity up to region I(C1) indicates that the binding of NOS with HSA occur due to the change in the microenvironment of Trp residue in HSA which is highly energetic sites in the protein 36. The further increases in the concentrations of NOS, the folded structure of protein opens, and it loses a part of its secondary structure, at this stage the NOS bind with protein in a cooperative manner, region II(C2).In the region III, the fluorescence intensity of protein goes to be saturated with NOS concentration and binding may not be consequential. The fluorescence results suggested that NOS binds with the fluorophore of HSA and changes its microenvironment towing to the occurrence of the slight conformational changes in HSA. This result also suggested that NOS does give any significant effect on the stability of HSA. 12 ACS Paragon Plus Environment

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3.2.2. Time resolved fluorescence study The biexponential decay of HSA in aqueous buffer is explained by the rotameric model of Tryptophan. The Trp exists in the three possible conformations in aqueous buffer which are shown in the following scheme 2.

NH3+

CO2-

H

H

H

H

H

H

NH3+

H

CO2-

A

+

H CO2-

H3N

HN

HN

HN

H

B

C

Scheme2. Structures of different conformation of tryptophan The biexponential decay of Trp in originates from the conformations A, B and C. This biexponential decay comprises by two component one is faster component which is believe to represent by conformer C whereas other is slower component, represent by conformer A and B which are readily inter-converting to each other. The faster conformer shows difficulty to convert into either form on the nanosecond time scale37. The indole ring of Trp exist in slightly puckered conformation and in this where reduction of the delocalization of π-electrons occurs through the indole ring37-38. However, in planner conformation the delocalization of nitrogen lone pair of indole is occur with the aromatic system. When a quencher molecule interact with the indole ring of Trp, it perturb its planarity which altered the microenvironment of Trp and results in the decrease of fluorescence lifetime38.



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Figure S1 shows the decay profiles of HSA with different concentrations of NOS. As shown in FigureS1, the addition of NOS reduces the lifetime of Trp in protein. In the biexpotinal decay of the HSA, the individual lifetime was relate to respective contribution from the faster (amplitude ∼ 32%) and slower component (amplitude ∼ 68%) of Trp. The amplitude of the slower component was observed twice as obtained for the faster component (Table 1). The slower relates to conformer A/B while the faster component results from conformer C. An increase in faster component ( ∼ 45%) was observed while a decrease in slower component ( ∼ 54%) was observed. The observation infers loss of the secondary structure and the possibility of interconversion from C to A or B (α2 in Table 1)32, 38. Moreover, due to the improved planarity of the ring, the rotation of Trp indole ring was facilitated in the excited state. This may be due to enhanced the possibly of interconversion of Trp conformer to more stable rotamer C as the amplitude of the shorter component increases (α1 in Table 1)32. It is however disputed that the non-radiative decay due to transfer of charge from indole ring to nearby substituents in Trp 214 are accelerated which results in decreased life time37, 39. 3.2.3. UV-visible spectra The UV-visible spectroscopy utilized to study the structural changes of HSA and to investigate the complex formation between NOS and HSA

40.

The absorbance of HSA (5 M) with

increasing concentration of NOS (4.95-45.45 µM) are shown in Figure S2. As shown in Figure S2, the absorbance spectrum of HSA increases up on adding NOS with small red shift. This outcome suggested the complex formation between NOS and HSA at ground state level. 3.3 Binding properties of NOS–HSA 3.3.1. Mechanism of binding


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Fluorescence quenching is very useful tool to provide information about specific ligand binding mechanism in macromolecules. Usually, two type of quenching mechanism was involved in protein ligand binding. It can be either static due to the ground state complex formation or dynamics which occurs through collision process in the excited state. The quenching mechanism is of two types, static and dynamic and classify on the behalf of observed Ksv values at different temperatures. Dynamic quenching is characterized by fast diffusion with enhanced Ksv at elevated temperature result of low complex stability, while in case of static quenching, Ksv was reduce because of the complex stability sustained by intermolecular forces and providing strength to hydrophobic effect41.Therefore, the fluorescence experiment were employed at different temperatures (298 K, 303 K, 308 K). The Stern–Volmer quenching constant, Ksv was calculated by using following equation42: F0  1  K SV [Q]  1  k q τ 0 [Q] F

(9)

where Ksv is the Stern–Volmer quenching constant, F0 is fluorescence intensity of HSA and F is the intensity of HSA in presence of NOS, [Q] is the concentration of NOS, kq stand for the quenching rate constant and τ0 represents as the average life time of biomolecule, which is 10-8 s 43.

Occasionally the Stern-Volmer plots show negative divergences from the linearity due to

various sub-population of tryptophan residues in protein with different Ksv values44-46. However, in our case, the linear Stern-Volmer plot was observed for NOS-HSA interaction. Figure S3 shows the linear Stern–Volmer plot of NOS-HSA system at different temperatures. From Table S1, the values of Ksv decreases with increase in temperatures suggest the involvement of static quenching between NOS and HSA. It was also reported that the maximum collision rate constant of various quenchers with the biopolymer is 2 × 1010 (L mol-1 s-1)

47.

In the current study the



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value of kq was observed higher than 2 x 1010 L mol-1 s-1which again describes the participation of static quenching mechanism in NOS-HSA system. 3.3.2 Binding parameters The following double-logarithmic equation (10) was utilized to measure the binding constant (Kb) and number of binding site (n) for NOS-HSA interaction system: log

F0  F  logK b  nlog[Q] F

(10)

where, F0 is intensities of fuorophore in absence of quencherand F is fuorescence intensities of fuorophore in the presence of quencher. Kb represent as the binding constant and n correspond to the number of binding site for the NOS-HSA system. From equation 10, a linear plot of log F0-F/F versus log NOS at three different temperatures was observed (Figure S4 A). The slope of the plot provides the number of binding sites (n) while intercept provide the values of binding constant, Kb at different temperatures. The values of binding constant at different temperature were shown in Table 2. From the Table 2, the value of n was close to 1 which suggested 1:1 binging of NOS with HSA. Previous studies have investigated ligand binding to serum albumin and have reported in order of 103 to 105 M-1

15.For

NOS-HSA system the binding order was also found in the similar range i.e. 1.77 x 104 M − 1 at 298K.The higher magnitude of Kb value also suggested that at in-vivo condition NOS can bind effectively with HSA at the same temperature condition48-50. In addition, from the Table.2, it can be seen that the Kb also reduces with increasing temperature due to the instability of the NOSHSA complex at higher temperature51. 3.3.3 Thermodynamic parameters and binding forces The non-covalent week interactions that are generally involved in ligand-protein binding are hydrogen bonding, van der Walls forces, electrostatic and hydrophobic interactions.

52.

These 16

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acting forces can be explained using the sign and magnitude of different thermodynamic parameters like ΔH0 (enthalpy change), ΔS0 (entropy change) and ΔG0 (Gibbs free energy change). According to Ross and Subramanian, if the values of both ΔH0 and ΔS0 are positive then the hydrophobic interactions plays major roles in the binding mechanism while negative sign shows binding occurs through van der Waals and hydrogen bond interactions. Finally, the positive ΔS0 and negative or small positive ∆H0 represented the involvement of electrostatic interaction 53. The values of ∆H0 and ∆S0 for NOS-HSA system can be evaluated from the vant’t Hoff plot (Figure S4 B) of lnK versus 1/T by using the flowing equation54: lnK  

ΔH 0 ΔS 0  RT R

(11)

where K, R and T represents as binding constant, gas constant and experimental temperature respectively. Further, the free energy change (∆G0) was calculated as follow relationship: ΔG 0  ΔH 0  TS 0

(12)

Thermodynamic parameters calculated for NOS and HSA are listed in Table 2. The positive value of ∆S0 and the negative value of ∆H0 suggest the involvement ofthe electrostatic interactions for NOS-HSA binding. These thermodynamic values indicated that NOS-HSA binding process is exothermic and entropic driven. Also, the negative ∆G0 value means the interaction of NOS with HSA was spontaneous in nature. 3.3.4 Molecular displacement for site-specific binding of NOS Primary sites in HSA are Sudlow’s I and II located in subdomains II and III. To determine these specific binding pocket of HSA for NOS binding, we used two site marker indomethacin (site I marker) and ibuprofen (site II marker) for competitive binding between NOS and site-specific markers. The steady state fluorescence spectra of HSA (5µM) were measured with the both sitemarker as a function of different concentration of NOS (4.95-45.45 µM). As shown in Figure 17 ACS Paragon Plus Environment


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4A, with indomethacin, the maximum emission wavelength of HSA was shifted towards higher wavelength and the intensity was also significantly higher in comparison of HSA (pure) spectra. On the other hand, in presence of ibuprofen, the fluorescence intensity of HSA was slightly decreased without any shifting in the maximum fluorescence emission wavelength (Figure 4B). On the addition of NOS into HSA-site marker system, the fluorescence intensity of HSA decreased gradually for the site-specific markers (indomethacin or ibuprofen) and a very little blue shift in maximum emission wavelength of HSA was recorded with indomethacin. The result signifies substitution of NOS from site I by indomethacin. This site is associated with the hydrophobic cavity in subdomain II A involving residue Trp214. This result was further validated by docking and MD result (section3.7). 3.4Cyclic voltammetry measurements Cyclic voltammetry is also a very helpful technique to investigate the protein-drug interaction under the physiological conditions55-56. Therefore, herein we have been utilized cyclic voltammetry to study the interaction between NOS and HSA at platinum electrode to support our spectroscopic result. The HSA and NOS are non-electrochemical active compound, prior to experiments HSA was fixed on platinum electrode surface. Afterwards electrochemical behavior of this modified electrode was checked using K3Fe(CN)6/K4Fe(CN)6 electrolyte probe and then different concentration of NOS was also administered in K3Fe(CN)6/K4Fe(CN)6 electrolyte solution. As shown in Figure5A, a good electrochemical response with reversible redox peak of K3Fe(CN)6/K4Fe(CN)6observe on both bare platinum electrode and HSA-modified platinum electrode. In contrast, the redox potential peak was shifted, and potential current of modified HSA was decreased, this result showing great deviation occurred on the surface of the platinum


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electrode. The difference of separation between redox peak potential increased to 0.17 V from 0.13 V. Moreover, the redox current also decreased from 0.0035 A to 0.0019A. The different values obtained signifies decrease of both the electron transfer rate of K3Fe(CN)6/K4Fe(CN)6 and the conductivity of the modified platinum electrode. As well, the HSA modified platinum electrode could be reprocessing with the constant redox peak potentials and redox peak currents and indicating that HSA immobilize on platinum electrode surface. The HSA-modified platinum electrode was utilized as the working electrode and K3Fe(CN)6/K4Fe(CN)6 as electrolyte solution for determined the interaction between NOS and HSA. When we added NOS in this system, a significant decrease in the redox peak currents (0.0019 A to 0.0016 A) and a slight shift in the redox peak potentials (0.17 V to 0.24 V) were found. As NOS was added in to the electrolyte solution,it interacted with HSA and resulted HSA film denser, making the migration of K3Fe(CN)6/K4Fe(CN)6ions through the film harder. This result caused the reduction in redox peak current.Upon addition of NOS, shifting in redox peak potential and decreasing in redox peak current confirm the interaction between HSA and NOS that reduced the diffusion coefficient of K3Fe(CN)6/K4Fe(CN)6 subsequently. Additionally, the reduction in the redox peak current with increasing concentration of NOS indicated that that the decrease in redox peak current is dependent on NOS concentration. To determine the binding equilibrium constant, we plot the curve between reciprocal of the current drop vs the reciprocal of the NOS concentration using flowing Langmuir equation55: 1 1 1   I P I P max I P max K a c

(13)

where, Ka stand for the binding constant of NOS-HSA system. ΔIp and ΔIp max is the current drop and maximum current drop, respectively. c correspond to the concentration of NOS. Figure5 B shows a good linear relationship and equilibrium constant, Ka was calculated through the slope 19 ACS Paragon Plus Environment


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of the curve. The value of the Ka was to be found 9.6 x104 L M-1, which is near to the value obtained from spectroscopic results i.e. 1.77 x 104 M −1. The cyclic voltammetry results suggest the complex formation and strong binding between NOS and HSA and this result in complement with our spectroscopic results. 3.5. Fluorescence resonance energy transfer FRET is a non-radiative energy transfer mechanism of between two closely spaced flurophore, one of which behaves a donor and the other one as acceptor. 57. The distance between ligand and protein can be calculated in accordance with Förster theory of fluorescence resonance energy transfer (FRET). FigureS5 shows the overlapping of spectrum of the fluorescence emission and absorption of spectra of HSA (5µM) and NOS (5µM), respectively. As per FRET theory58, the efficiency of energy transfer depends on: (a) Overlapping area between the florescence and UV spectra of acceptor and donor molecule. (b) Distance between the both flurophores involved i.e. donor and acceptor. (c) Orientation of transition dipole of acceptor and donor 59. The following equation computes the efficiency of energy transfer and distance between donor and acceptor 60:

E  1

R6 F  6 0 6 F0 R0  r

(14)

where F represents fluorescence intensities of HSA with NOS and F0 represents the fluorescence intensities of HSA. R0 is the critical distance at 50% transfer efficiency and r stand for the distance between donor (HSA) and acceptor (NOS). The following relation was used to calculate R061: R06  8.8  10 25  2 n 4J

(15)

where N stands for the refractive index of medium and k2 is the spatial orientation factor and Φ stand for fluorescence quantum yield of the donor. J represents the overlap integral of the


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fluorescence emission spectrum of the donor and the absorption spectrum of the acceptor. J is calculated by following relation60:

 F      J  F   4

(16)

where F(λ) represents the corrected fluorescence intensity of the donor and ε(λ) represents the molar extinction coefficient of the acceptor at wavelength, λ. Using the values of κ 2 = 2/3, n = 1.336 and Φ = 0.14, the values of E, J, R0 and r were calculated to be 0.307, 1.25 x 1015 cm3 L mol-1, 1.78 nm and 2.04 nm, respectively. The results show less than 8 nm distances between the HSA and NOS during interaction. This show high efficacy of energy transfer mechanism operating52 and formation of ground state complex

62-63.

Analysed

data is also supported with our UV-visible results and steady state fluorescence measurement. 3.6 Conformational change of HSA binding on NOS 3.6.1 Synchronous fluorescence spectra It is a technique that unravels invaluable information about the microenviroment of aromatic amino acid. Synchronous fluorescence provide many advantages such as simplification in spectra, reduction of spectral bandwidth and evading different perturbing effects

64.

The

synchronous spectra is performed by scanning the excitation and emission monochromators simultaneously at constant wavelength interval (Δλ), where information in the vicinity reveal for Tyr (Δλ = 15) or Trp (Δλ = 60), respectively

65.

The shift in Trp and Tyr maximum emission

wavelength can provide information about polarity of their surroundings and conformation changes of the protein 66. Therefore, synchronous spectra utilized to confirm the effect of NOS on HSA microenvironment, are shown in Figure S6 with Δλ = 15 nm and Δλ = 60 nm. These results signify that with increasing concentration of NOS, Trp residue shows stronger quenching (30%) then Tyr residue (19 %). The quenching of Trp intensity with NOS concentration is 21 ACS Paragon Plus Environment


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mainly due to the reduced emission from Trp 214 residue. The red shift in emission peak 2 nm (280 to 282) is contributed by the increasing polarity in microenvironment of Trp 214. The red shift and decreasing intensity also relates towards the altered conformation of HSA under influence of NOS binding to it

67.

However, the microenvironment vicinity of Tyr residues did

not show obvious changes during the binding of NOS. 3.6.2. FT-IR spectroscopic studies: FT-IR spectroscopic is a powerful and effective method for the analysing the various content of protein secondary structure. The two major frequencies in FTIR range, characteristic of secondary structure includeamide I and amide II with frequencies 1650 cm-1 and 1550 cm-1, respectively. Carbonyl stretching (>C=O) is responsible for its vibrational frequency of amide I band and vibration of amide II originates because of the deformation of N-H and stretching of CN frequency present in HSA55. Among of these, amide I mainly contributed in the secondary structure of the protein.The change in secondary structure of HSA was analysed with the help of shift in band upon the addition of NOS. The spectra of HSA and NOS-HSA complex were recorded to explore the change in shift of band. The results are shown in FigureS7. Amide I peak for pure HSA was found approx at 1644 cm, while the peak of HSA-NOS complex was shifted to 1640 cm which conformed that NOS induces the conformational change in HSA. Also, the change in both the amide peaks suggested that NOS show possible interaction with C=O, C–N and N–H groups of the HSA that alters the secondary structural conformation. This result is further confirmed by CD measurements. 3.6.3. 3D Emission spectral studies: To investigate the conformational alteration of HSA upon binding of NOS, 3D-Emission spectroscopy or excitation-emission matrix spectroscopy is was utilized68. It provides depth


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information about the structural configuration of proteins and observes the alteration in microenvironment of aromatic amino acids and peptide backbone of a protein upon binding with ligand. The 3D fluorescence spectra of the pure HSA and NOS-HSA complex are presented in the Figure S8 and corresponding analysis summarized in Table S2. From the Figure S8, four peaks could be observed in 3D emission spectrum of HSA. The peak a and peak b are resulted due to first ordered (λex =λem) and second ordered (2λex =λem) Rayleigh scattering peak, respectively. It was clearly seen in Figure 7 that, both Rayleigh scattering peaks intensities were decreased in NOS-HSA complex where scattering effect was weakened due to the complex formation between NOS and HSA69. Additionally, two fluorescence peaks were also observed, peak I and peak II in Figure 13, where peak Iresulted due to the π–π* transition of polypeptide backbone structures of HSA and peak IIrepresented fluorescence spectral behavior of tryptophan and tyrosine residues56, 70. As listed in Table S2, the intensity of peak I decreased from 200 to 145without any change in stokes shift signifying disturbance of the polypeptide backbone structure of HSA after binding of NOS. Furthermore, intensity of peak II decreased from 145 to 98 with slight increase in stokes shift (2 nm) upon binding with NOS, suggested that the binding of NOS with HSA contribute to change the polarity and hydrophobicity around the Trp residue of HSA. This result confirmed that the binding of NOS to HSA results to unfold the polypeptide backbone of the protein. 3.6.4. Circular Dichroism study The circular dichroism (CD) spectroscopy is most popular quantitative tool to investigate the protein conformation and effect of ligand on the overall secondary structure of protein

71.

Therefore, we performed far-UV CD measurements for HSA with different concentration of NOS (Figure 6A). The far-UV CD spectra of HSA exhibits two negative peaks at 208 and 222



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nm illustrate a characteristic of the α-helical structure which observe due to π-π* and n-π* electronic transfer of -helical structure in protein72. As shown in figure, CD signal of HSA was increase at all wavelengths with the addition of NOS, signifying decrees in -helical structure of the protein. The -helical content of pure HSA was found to be 67 % which was good agreement with the previous reported result having major helical content in its native structure (60−67%) 64, 73.

After the addition of NOS (2.45-23.80 µM), -helical content was decreases up to46%. The

decrease in helical structure was observed with addition of suggested alteration in the secondary structure of HSA, as shown in FTIR data analysis. It is a known characteristic, the secondary structure of protein directly related to its biological activity74. On this prospect, our CD result also signify that the decrease of the functional activity (esterase activity section 3.6) of HSA upon the interaction with higher concentration of NOS. Reduced structure after addition of NOS to the HSA may be due to the reach of NOS to hydrophobic pocket of HSA that destroys the Hbonding network of HSA. The results signify change in conformation of HSA towards different conformation that alters its binding behavior and affinity for same class of drugs. We also analyzed near-UV CD spectra (269-310 nm), to determine the tertiary structure of pure HSA and with NOS (Figure6B). The near-UV CD spectroscopy is an insightful technique for examining slight structural perturbations and tertiary structural organization of the protein by identifying asymmetry in the moiety of aromatic amino acid residues. Near-UV CD spectra of native HSA characterize two minima around 262 nm and 290, which are attributing for the disulfide and aromaticchromophores75. In near-UV spectra the sharp and weak bands are characteristic wavelength for Trp, Tyr and Phe. A sharp band at 290 and 305 nm corresponds to Trp, while a band around 272 to 280 nm originates due to Tyr. The weak bands around originate round 255 to 270 nm corresponds to Phe

70.

The addition of NOS to HSA increases ellipticity 24


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260 to 280 region for lower concentration, while further addition decreases the ellipticity at 280 to 295 region. This decrease signifies the disruption of tertiary interaction of HSA and loss of structure of HSA, with possible alteration around TRP residues of HSA. 3.7. Esterase-like activity of HSA HSA used as transport protein for carrying drug, therefore the effect of the NOS on the catalytic functions of HSA such as esterase-like activity was performed. The functional activity of HSA was monitoring by the absorbance of the liberated product, p-nitrophenol at 405 nm76. The two vital amino acid residues Arg-410 and Tyr-411 of HAS situated in site II (subdomain IIIA) shows catalytic cleavage mechanisms and contributing in esterase-like activity of HSA. In catalytic cleavage mechanism Tyr-411 is the first amino acid residue of HSA, which speedily acetylated by p-NPA77. The esterase-like activities of HSA inhibit by the variety of the drugs which is due to the binding sites of drugs with the substrate (p-NPA). The Figure7A shows the relative modulation of HSA activity in the presence of different molar ratio of HSA:NOS (1:0, 1:1, 1:2, 1:5). It is observed that at lower concentration (5µM) of NOS increases relative esterase activity of HSA (137 %), while 66% of the activity is retained at 25µM. This result shows that NOS increases HSA esterase activity with positive cooperation at lower concentration but at higher concentration it shows reduction in esterase activity which may be attributed to the perturbation of the protein. Additionally, the kinetic parameters (Km, Vmax and kcat) of p-NPA hydrolysis at different molar ratio of HSA:NOS were evaluated by fitting initial velocity against different p-NPA concentration (100-700 µM) using Michaelis-Menten equation (Figure7B) and Lineweaver-Burk plot (Figure7C). Relative activity and kinetic parameters for different molar ratio of HSA:NOS are listed in Table 3.It was clearly seen that at higher concentration of NOS competitive inhibition occurs i.e., Km alter while Vmax remains almost unchanged. This signifies



high affinity of NOS for HSA to occupy its catalytic site

Page 26 of 46

31, 78.

This indicates that NOS binds

with HSA at active site where substrate generally occupies. The progressively increase in Km value and decrease in catalytic efficiency (kcat/Km) signify that NOS inhibits the HSA functionality at higher concentration in competitive manner79. The binding of NOS triggers the conformational change in HSA. It increases its catalytic efficiency and majorly the Km. The obtained results were in agreement to the above mentioned techniques. 3.8. Insilco studies 3.8.1 Molecular docking Binding site recognition is very important to expose true binding mechanisms as well as interaction between drug and protein. To characterize this, molecular docking and molecular dynamic simulation are preeminent theoretically prediction approaches to investigate binding sites for NOS on HSA at molecular level and validate experimental result80. Among 10 conformations generated the lowest binding free energy was utilized for further analysis and MD simulation. NOS binds HSA with binding affinity of −7.1 kcal mol−1 which shows strong binding of NOS with HSA81. This result also corroborateswith our experimental binding result (−5.8 kcal mol−1). Docking result give an idea about principle binding sites of NOS on HSA at subdomain IIA (Sudlow’s sites I) with a lesser extent at subdomain IIIA (Sudlow’s sites II).Figure8 shows clearly, NOS specifically interact with LYS195, TRP214, ARG218, GLN221, ASN295 at subdomain IIA and it also interact with residue of subdomain IIIA such as PRO339, ASP340, VAL343, LYS444, PRO447, CYS448, ASP451, TYR452 and VAL455. The subdomain IIA of HSA has binding sites occupied by electrostatic interaction with NOS within 5 Å range. The NOS also formed tow hydrogen bond with ARG218 and ASN295 residues of HSA, with distance 2.9 and 3.4 Å, respectively (Figure 8 B).


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The docking results suggested that the electrostatic interactions were the main forces for the binding between NOS and HSA are electrostatic interactions, whereas hydrogen bonds also plays minor contribution82.

The docking results are in accordance with above discussed

thermodynamic results. 3.8.2 Molecular Dynamic Simulation A molecular dynamic simulation (MD) is useful computational tool which provide a deep insight of protein-ligand binding with respect to time. HSA is an important transport protein in blood, therefore the rigidity, stability and structural alteration of HSA in presence of drug play a major role in drug metabolism. Thus, at this point we focused on MD simulations of HSA with NOS. The results obtained from the various methods from MD simulations like root mean square deviations (RMSD), radius of gyration (Rg), secondary structures, solvent accessible surface area (SASA) and root mean square fluctuations (RMSF) have been discussed for HSA and HSA-NOS complex. The rigidity and stability of pure HSA and HSA-NOS complex was evaluated by the RMSD plot of pure HSA and HSA-NOS complexagainst the 20000 ps simulation time scale, shown in figure 9A. From the figure, the RMSD value of pure HSA and HSA-NOS complex rapidly increases from starting to 4000 ps, after this system were achieved the equilibrium phase and oscillated around an average RMSD value till end of simulation, which represent systems were stable throughout simulation. The RMSD value from trajectory of pure HSA and HSANOS complex was determine where data points fluctuated as 0.39 ± 0.03 and 0.47 ± 0.03 nm, respectively. This increscent of RMSD value of HSA-NOS complex indicate the conformational change in HSA secondary structure on the binding of NOS 83. These results signify that rigidity and stability of HSA might be decrease with NOS.



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The protein reliability of HSA on binding of NOS was analysed by plotting Rg values against 20000 ps time scale. From Figure 9B, the Rg values of the pure HSA and HSA-NOS complex were gradually decrease with fluctuation about 10000 ps and after this both systems attain the equilibrium state till end of simulation. In the starting of simulation the Rg values of pure HSA and HSA-NOS complex were 2.64 nm, which is similar with other reported work. The experimental Rg value of HSA in aqueous medium reported was 2.74 ± 0.035 nm using neutron scattering technique84. MD simulations are similar to the value calculated by our experimental result. Rg value of pure HSA was initially fluctuated but after equilibration to be found at 2.54 ± 0.03 while Rg value of complex was 2.52 ± 0.02. At the end of simulation, a minute decrease was found in the Rg value of HSA upon binding with NOS. This result suggested that microenvironment of HSA was change on the binding of NOS, which lead to conformational changes in HSA structure. Previous studies by MD simulation have reported altered conformation of HSA after interaction with anticancer drug 85-86. The Rg and RMSD results are coherent to experimental CD result i.e. decrease of helical content of HSA and leads to change in the protein conformation. The SASA was performed to calculate the conformational change HSA binding of NOS against 20000 ps time scale. As shown in Figure 9C, initially the SASA values of both systems were steadily decreases till 5000 ps then system achieved the equilibrium phase and show low fluctuation until end of simulation. After attain equilibrium phase, SASA values of HSA was deceased from 165±3 to 154±3 nm2 upon the binding of NOS. The decrease suggests altered conformation in structure of HSA, and reduced pocket size with increased hydrobhobity around it 87. For further confirmation change in the secondary structure, we utilized do_dssp command to analyze the secondary structure of the HSA upon binding of NOS. Figure10 shows secondary


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structure of the HSA-NOS complex generated by dssp program. The result provides the α-helix and other secondary structures of the HSA-NOS complex maintain rather stable during 20 ns MD simulation and the α-helix decreases with respect of pure HSA. This shows change in secondary structure upon binding of NOS. RMSF was outstanding tool to analyse the local protein mobility. RMSF value of pure HSA and HSA-NOS complex are plotted against the residue numbers over 20000 ps time scale in Figure11A. The atomic fluctuations profile show low fluctuation of HSA-NOS complex compared to pure HSA have same trend. It was clearly shown in Figure11A, HSA-NOS complex have lowest fluctuations in sub-domains IIIA, and IIA with respect of other sub-domains. To explore the atomic fluctuations at drug-binding sites in detail, we determined the RMSF value of each amino acid residue at the NOS binding site (Figure11B). The residues of both binding sites of HSA show binding with NOS but more rigidity and lower fluctuation were located to individual residues of HSA i.e. TRP214, ARG218, GLN221, ASN295at site IIA, which confirms sub-domains IIA is principle binding site for HSA-NOS binding88. Site IIA of HSA, the results show this site as main binding site for NOS. Above observations strongly support the results of previous discussed techniques for NOS specific binding in sub-domains IIA of HSA. 4. Conclusion The interaction between anticancer drug NOS and transport protein HSA have been done by various spectroscopic techniques, electrochemistry and computational method. The cytotoxicity results suggest that NOS have good potential in skin cancer and its IC50 value is 72 µM. The spectroscopic analyses suggested that the quenching process is a static in nature. The electrostatic interaction is the main binding forces between NOS-HSA complexes. The FRET results again suggested contribution of static quenching in binding mechanism. The cyclic



Page 30 of 46

voltammetry result confirmed the strong binding between HSA and NOS. The synchronous fluorescence, FTIR, 3D fluorescence, CD and MD result suggested conformational alteration in the native structure of HSA on binding with NOS at higher concentration. The esterase-like activity of HSA gets increase at low concentration and reduces at higher concentration of NOS. The molecular site marker, molecular docking and molecular dynamic simulation results show that principle binding site for NOS was site I (subdomain II A) of HSA. The study presents structural insights of HSA with anticancer drug NOS, it has applications in development of anticancer drugs for pharmaceutical and biomedical research. Supporting Information Time-Resolved Fluorescence, UV spectroscopy, Synchronous fluorescence and FTIR results. Notes The authors declare no competing financial interest. Acknowledgments Dr. Rajan Patel acknowledges the financial support from Science and Engineering Research Board (EEQ/2016/000339) New Delhi, India. Authors also thank DST for providing the FIST grant with Sanction Order No. (SR/FIST/LS-541/2012). Neha Maurya also thankful to the ICMR, New Delhi, India, for providing a research grant (file no. Gen.452/Estt./R.O./2018). The authors Dr. Amer M. Alanazi and Azmat Ali Khan would like to extend their sincere appreciation to the Deanship of Scientific Research at King Saud University for funding of this research through the Research Group Project No. RGP-212.

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Figure1 Cytotoxicity evaluation of NOS through the MTT assay after 48 hrs of treatment on A431.



Page 40 of 46

Figure2 Fluorescence quenching of HSA (5µM) at 340 nm in the absence and presence of NOS and the concentration of NOS were 0, 4.95, 9.80, 14.56, 19.23, 23.81, 28.30, 32.71, 37.04 41.28 and 45.45 µM at 298 K and pH 7.4 (inset: Fluorescence quenching of NOS (50µM)).

Figure 3 Binding isotherm of the fraction of protein binds to Noscapine (α) vs [NOS] for NOSHSA interaction.


Page 41 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(A)

(B)

Figure4 Effect of site-specific probe to drug-HSA system, (A) indomethacin (B) ibuprofen

Figure5(A) CV spectra of HSA (5 µM) in absence and in presence of NOS (0, 4.95, 9.80, 14.56, 19.23, 23.81, 28.30, 32.71, 37.04 41.28 and 45.45 µM) at pH 7.4) (B)Linear relationship between reciprocal of the current drop vs the reciprocal of the NOS concentration.



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Figure6(A) Far-UVCD spectra of HSA in presence and absence of different concentration of NOS (B) Near-UV-CD spectra of HSA in presence of different concentration of NOS.

Figure7(A)Relative esterase activity (%) of HSA and HSA:NOS complex (B) Michaelis-Menten plot of HSA (5µM) in varying concentration of NOS (A) Lineweaver Burk plots of HSA (5µM) in varying concentration of NOS. (A)

(B)

Figure8(A)Surrounding amino acid residue of HSA within 5 Åfrom docked NOS (B) 2D structure of interaction of HSA with NOS


Page 43 of 46 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 9(A) RMSD (nm) value of HSA and HSA -NOS complex. (B) Rg (nm) value of HSA and HSA-NOS complex. (C) SASA values of HSA and HSA -NOS complex.

Figure10 Variation of the secondary structures versus time for HSA-NOS complex



Page 44 of 46

Figure 11 (A) RMSF values of HSA and HSA-NOS complex (B) The RMSF values of HSA and HSA-NOS complex at binding site.

Table1Fluorescence decay of HSA at different concentrations of NOS τ1 (ns)

τ2 (ns)

α1

α2

‹τ› (ns)

χ2

0.00

1.61

5.69

31.61

68.39

5.22

1.65

4.95

2.00

5.98

39.41

60.59

5.27

1.30

9.80

1.93

5.85

40.66

59.34

5.13

1.19

14.56

1.87

5.72

40.02

59.98

5.04

1.28

19.23

1.78

5.58

39.45

60.55

4.92

1.24

23.81

1.74

5.55

40.82

59.18

4.88

1.23

28.30

1.69

5.44

40.55

59.45

4.79

1.41

32.71

1.66

5.42

42.07

57.93

4.73

1.35

37.04

1.63

5.39

43.32

56.68

4.69

1.34

41.28

1.60

5.30

43.15

56.85

4.61

1.23

45.45

1.61

5.35

45.42

54.58

4.60

1.36

NOS(µM)


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Table 2The binding constant (Kb) and binding site (n) of HSA and NOS interaction at three temperatures Temp (K)

Kb(L mol-1)

n

R2

298

1.77 x 104±0.022

0.8604±0.016

0.9966

308

1.54 x 104±0.027

0.8533±0.019

0.9951

318

1.44 x 104±0.022

0.8632±0.016

0.9966

∆H 0 (kJ M-1)

∆S0 (J M-1 K-1)

-15.96±0.062 27.60±0.206

∆G0 (kJ M-1) -24.19 -24.33 -24.47

Table 3 Michaelis-Menten kinetic parameters of HSA in the presence of NOS concentrations HSA:NOS

RA(%)

Km(μM)

Vmax(μMs-1)

kcat(s-1)

kcat/ Km(μM-1 s-1)

1:0 1:1 1:2 1:5

100 137 86 66

170.4±3.1 138.0±2.6 204.0±2.8 230.9±1.7

17.4 x10-2±0.16 18.4 x10-2±0.18 16.1 x10-2±0.21 15.0 x10-2±0.12

34.8 x10-3 36.8 x10-3 32.1 x10-3 30.0 x10-3

20.4 x10-5 26.7 x10-5 15.8 x10-5 13.0 x10-5



Graphical Abstract


Page 46 of 46

In Vitro Cytotoxicity and Interaction of Noscapine with Human Serum

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