Molecular mechanistic vision on binding interaction of triptan drug, a serotonin (5-HT1) agonist with human serum albumin through multispectral and computational assessments European Journal of Chemistry 11 (2) (2020) 145-155 European Journal of Chemistry ISSN 2153-2249 (Print) / ISSN 2153-2257 (Online) – Copyright © 2020 The Authors – Atlanta Publishing House LLC – Printed in the USA. This work is published and licensed by Atlanta Publishing House LLC – CC BY NC – Some Rights Reserved. http://dx.doi.org/10.5155/eurjchem.11.2.145-155.1971 European Journal of Chemistry View Journal Online View Article Online Molecular mechanistic vision on binding interaction of triptan drug, a serotonin (5-HT1) agonist with human serum albumin through multispectral and computational assessments Manjushree Makegowda and Revanasiddappa Hosakere Doddarevanna * Department of Chemistry, University of Mysore, Manasagangothri, Mysuru 570006, Karnataka, India mmanjushreem@gmail.com (M.M.), hdrevanasiddappa@yahoo.com (R.H.D.) * Corresponding author at: Department of Chemistry, University of Mysore, Manasagangothri, Mysuru 570006, Karnataka, India. e-mail: hdrevanasiddappa@yahoo.com (R.H. Doddarevanna). 10.5155/eurjchem.11.2.145-155.1971 Received: 14 February 2020 Received in revised form: 24 March 2020 Accepted: 26 March 2020 Published online: 30 June 2020 Printed: 30 June 2020 The triptan drug such as eletriptan in combination with hydrochloride (ETP) is a 5-HT1 receptor agonist used to treat the migraine headache. Human serum albumin (HSA), the fundamental serum protein, executes various functions, that includes transporting and binding of many ligands. HSA binding interaction with ETP is elucidated from molecular docking in composite with fluorescence (emission, 3D and synchronous), UV-vis and FT-IR spectroscopy at 296, 304 and 312 K (pH = 7.40). ETP after interaction modified the HSA secondary structure and its micro-environments. Energy transfer and thermodynamic parameters were evaluated. Various quenching and binding constants were computed for formed ETP-HSA complex. The dominant interactive forces for ETP and HSA binding are hydrogen bonds join up with van der Waals extent possibly at site III (IB). The presence of Ca2+, Co2+, Na+, Mg2+ and Fe3+ ions significantly affected binding ability of ETP towards HSA. The essentialness of this investigation is beneficial in life sciences, medicinal chemistry, pharmaceutical industry and clinical medicine. Eletriptan Energy transfer Molecular docking Binding interaction Secondary structure Fluorescence quenching Cite this: Eur. J. Chem. 2020, 11(2), 145-155 Journal website: www.eurjchem.com 1. Introduction The severe outcome of cyclic headaches of moderate level, causes the migraine. Typically, about 15% world populations are affected by this disorder where it starts and becomes worst at puberty and middle age, respectively. Majorly, females are less affected than males. The main reasons for migraines are change in hormone level, genetic and environmental factors. Usually migraines occur in nature at one side of the head which involves the blood vessels and nerves of the brain with pulse to three days. This pain has common symptoms namely vomiting, nausea and sensitivity to smell or sound or light which leads to lose of physical activity. One of the best second generation triptan drugs for the treatment of migraine is eletriptan which is often used in combination with hydrochloride namely eletriptan hydrochloride (ETP) (Figure 1a). This drug was approved by U. S. Food and Drug Administration (FDA). ETP is a serotonin receptor agonist, particularly to family of 5-HT1 receptor agonist. The blood vessels sore around the brain is to be reduced during the migraine of head pain by ETP. ETP should not give to the patients having diseases like heart related problems and stroke. Moreover, it has common advert effects such as somnolence, nausea, hypertension, asthenia, dizziness and this side effect increases with dose [1-3]. Serum albumins are bind by drug in blood may strongly influence drugs distribution, metabolism, absorption, excretion and its transportation [4]. Bioavailability and circulating lifetimes of drug could be determined from drug binding affinity to protein. Weak interactions result in inadequate distribution and reduce the lifetimes while strong one diminishes the drug free fraction in plasma. There is necessary to establishing a balance between drugs’s free and bound type to obtain precise therapeutic doses, hence reasonable amounts could be supplied to targeted tissues and to reduce noxious side effects. Broadly utilized protein i.e. serum albumins in drug binding investigations is human serum albumin (HSA) (Figure 1b) where it assists to maintain neutralize toxins and intravascular colloid osmotic pressure [5]. The heart shaped HSA been constituted with three homologous domains [6]. ETP binding to HSA at 296, 304 and 312 K was investigated via various spectroscopic and docking methods. Effects of Ca2+, Co2+, Na+, Mg2+ and Fe3+ ions were also examined. On the basis of literature observe [7-15], this work has not been done. ABSTRACT RESEARCH ARTICLE KEYWORDS http://dx.doi.org/10.5155/eurjchem.11.2.145-155.1971 http://www.eurjchem.com/ http://dx.doi.org/10.5155/eurjchem.11.2.145-155.1971 mailto:mmanjushreem@gmail.com mailto:hdrevanasiddappa@yahoo.com mailto:hdrevanasiddappa@yahoo.com http://www.eurjchem.com/ https://crossmark.crossref.org/dialog/?doi=10.5155/eurjchem.11.2.145-155.1971&domain=pdf&date_stamp=2020-06-30 146 Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 (a) (b) Figure 1. (a) ETP and (b) HSA 3D structure. 2. Experimental 2.1. Reagents and stocks Human serum albumin (Lyophilized powder, fatty acid free, globulin free, ≥99%: agarose gel electrophoresis), calcium chloride hexahydrate (Ca2+, 98%), cobalt(II) chloride hexahydrate (Co2+, ≥97%), sodium chloride (Na+, ≥99%), magnesium sulfate heptahydrate (Mg2+; ≥97%), iron(III) oxalate hexahydrate (Fe3+), warfarin (analytical standard), digitoxin (≥92%) and ibuprofen (≥98%) were products of Sigma-Aldrich (USA). Analytical standards were employed for other exploited chemicals. Tris buffer was made from double- distilled water at pH = 7.40 where this was executed throughout every experiment by simultaneously corrected its background. Concentration of 1.0×10-4 mol/L for HSA and 1.0×10-3 mol/L for site probes, ETP and metal ions as stocks. 2.2. UV-vis absorption study Absorbance estimations (200-300 nm) at 304 K were executed on Life Sciences DU 730: UV-visible Spectrophoto- meter (Beckman Coulter, U.S.A) for HSA (2.70×10-6 mol/L) with increased ETP solution (0.0 to 4.50×10-6 mol/L about each increment of 0.45). 2.3. FT-IR spectral observations Free HSA (2.70×10-6 mol/L) and ETP-HSA system’s FT-IR spectra at 304 K were conducted from FT-IR spectrometer (Spectrum Two, Perkin Elmer, USA) in 1700-1500 cm-1 region where ETP concentration was 2.70×10-6 mol/L. ORIGINPRO 9.0 Software analyzed the curve fitting measurements to secondary structures. 2.4. Emission fluorescence Fluorescence estimations were implemented on fluore- scence spectrophotometer (F-4600, Hitachi, Japan) with 10 nm (excitation/emission) slit width. HSA was 2.70×10-6 mol/L with different ETP (0 to 4.50×10-6 mol/L) concentrations with λem = 290-420 nm and λex = 295 nm at 296, 304 and 312 K. Absorbance of ETP at emission (Aem) and excitation (Aex) wavelengths was note down to decline the effects from inner filter for entire fluorescence intensities of HSA (Fobs = observed and Fcor = corrected) [16]. 𝐹𝐹𝑐𝑐𝑐𝑐𝑐𝑐 = 𝐹𝐹𝑐𝑐𝑜𝑜𝑜𝑜 × 𝑒𝑒(𝐴𝐴𝑒𝑒𝑒𝑒+ 𝐴𝐴𝑒𝑒𝑒𝑒) 2⁄ (1) 2.5. Synchronous fluorescence These spectra were note down at 304 K in 210-320 nm for ETP-HSA system. Concentration of HSA persisted at 2.70×10-6 mol/L while ETP differed from 0 to 4.50×10-6 mol/L. 2.6. Energy transfer between ETP and HSA HSA (2.70×10-6 mol/L) emission and ETP absorption spectrum (2.70×10-6 mol/L) were recorded at 304 K in 290-420 nm. These two overlapping determine the efficient energy transfer. 2.7. Metal ions on ETP-HSA binding HSA (2.70×10-6 mol/L) fluorescence spectra with Ca2+, Co2+, Na+, Mg2+ and Fe3+ ions (2.70×10-6 mol/L) by increased ETP amounts from 0 to 4.50×10-6 mol/L at 304 K were recorded. 2.8. Displacement of ETP by site selective assess ETP displacement was monitored using warfarin (for IIA), ibuprofen (for IIIA) and digitoxin (for IB) site probes. Presence of site probes (2.70×10-6 mol/L) with increased ETP (0 to 4.50×10-6 mol/L) concentration, HSA (2.70×10-6 mol/L) fluorescence spectra were summarized at 304 K. Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 147 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 Table 1. FT-IR spectral aspects at 304 K. System Amide I (cm-1) Amide II (cm-1) 1615-1637 1638-1648 1649-1660 1660-1680 1680-1692 1548 Free HSA 1635±0.40 1644±0.90 1658±0.38 1680±0.63 1687±0.72 1543±0.13 ETP-HSA 1627±0.31 1641±0.59 1657±0.44 1666±0.26 1682±0.43 1538±0.88 Table 2. Evaluated secondary structures (FT-IR) at 304 K. System Secondary structures (%) β-sheet Random coil α-helix β-turn β-antiparallel Free HSA 7.00±0.06 24.24±0.03 50.75±0.08 13.81±0.10 4.19±0.03 HSA-ETP 0.89±0.05 15.15±0.02 42.03±0.05 34.34±0.04 7.78±0.09 Figure 2. Spectrum of UV-vis absorption of HSA (2.70×10-6 mol/L) in addition of ETP (0 to 4.50×10-6 mol/L): curve L is ETP alone (2.70×10-6 mol/L) at 304 K. 2.9. Docking studies Docking analysis for binding mode between ETP and HSA was executed on Autodock Tools to three binding sites. Energy minimization for 3D structures of ETP (PubChem CID 121596618) and HSA (PDB ID: 1AO6) were achieved according to Marvin View and GROMOS96 force field. Lamarckian genetic algorithm with 60 grid maps was applied and binding visualization made on Discovery Studio software. 2.10. 3D fluorescence 3D fluorescence of HSA (2.70×10-6 mol/L) and ETP-HSA system at 304 K were measured. Concentration of ETP was kept at 81.0×10-6 mol/L with λem= 250-500 nm and λex = 200-380 nm. 3. Results and discussion 3.1. UV-vis measurements Complexation and structural modifications are exploited from UV-vis spectroscopy for protein-drug binding [17]. Responsible characteristic peaks are at 214 nm (n → π* transitions: amide cluster) and at 278 nm (π → π* transitions: Tyr and Trp residues) gathered for HSA over ETP interaction in UV-vis spectrum (Figure 2). Herein, these peaks suffer red shift that manifesting the altered secondary structure together with polarity in microenvironments (Tyr/Trp) of HSA. Accordingly, increased in absorbance as ETP increased concentration at each peaks of HSA dictates the formation of ETP-HSA complex. 3.2. FT-IR spectroscopy Further evidence of ETP-HSA interaction was affirmed by FT-IR out comes from characteristic amide bands where amide I (C=O stretch: 1700-1600 cm-1) and II (associated C-N stretch across N-H bend: 1600-1500 cm-1) have direct replicate of HSA secondary structure. Spectral variations noticed from Table 1 signify that ETP modified the HSA conformations (Figure 3). Curve-fitting operations with second derivative to infrared self- deconvolution were subjected to amide I (high compassion to weak conformational alterations) band to measure free HSA and its ETP complex secondary structures (Figure 4). Upon ETP interaction, a decrease of random coil, α-helix and β-sheets with increment in β-turns and antiparallel were noticed for HSA (Table 2). This is indicative of a biased unfolding of HSA in ETP [18]. 3.3. Fluorescence quenching Decreased remarkable fluorescence intensity (blue shift ~3 nm at 329 nm) was found for HSA by varying of ETP amounts evidenced from Figure 5 at 304 K. Decreased polarity, change and less hydrophilic of Trp (λex = 295 nm and λem = 340 nm) micro-environment might exhibited from this blue shift. This ensures that ETP bound to HSA with high affinity by efficiently quenched the HSA intrinsic fluorescence [19]. Quencher induced HSA fluorescence quenching possibly classified as static/dynamic. Dynamic: Faster diffusion causes the larger collision amounts at enhanced temperatures thereby increased quenching constants whereas static: weaken complex stability at higher temperatures leads to reduced quenching constants. To analyze the ETP-HSA system’s fluorescence quenching, in addition to Stern-Volmer Equation (2) a modified Equation (4) was exploited and are summarized in Table 3. 𝐹𝐹0 𝐹𝐹⁄ = 1 + 𝑘𝑘𝑞𝑞𝜏𝜏0[𝑄𝑄] = 1 + 𝐾𝐾𝑆𝑆𝑆𝑆[𝑄𝑄] (2) 𝑘𝑘𝑞𝑞 = 𝐾𝐾𝑆𝑆𝑆𝑆 𝜏𝜏0⁄ (3) 𝐹𝐹0 (𝐹𝐹0 − 𝐹𝐹)⁄ = (1 𝑓𝑓𝑎𝑎⁄ ) + (1 𝐾𝐾𝑎𝑎⁄ 𝑓𝑓𝑎𝑎[𝑄𝑄]) (4) 148 Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 (a) (b) Figure 3. FT-IR spectra at 304 K: (a) HSA alone and (b) ETP bounded to HSA. c(HSA) = c (ETP) = 2.70×10-6 mol/L. (a) (b) Figure 4. Curve fitting for (a) Free HSA and (b) ETP-HSA system at amide I band. where, F0 is fluorescence intensity without ETP, F is fluorescence intensity with ETP, kq is quenching rate constant, τ0 is HSA average life time without ETP (~2.7×10-9 s), KSV is Stern-Volmer quenching constant and [Q] is ETP concentration. Ka is effective static quenching constant and 𝑓𝑓𝑎𝑎 is fluorophore fraction. Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 149 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 Table 3. Quenching constants to ETP-HSA system. Temperature (K) KSV (L/mol)×104 kq (L/mol.s)×1013 Ka (L/mol)×105 296 20.9±0.05 7.74±0.03 4.32±0.10 304 19.0±0.09 7.03±0.02 3.69±0.01 312 17.4±0.04 6.44±0.02 0.24±0.08 Figure 5. HSA (2.70×10-6 mol/L) fluorescence emission spectrum in distinct amounts of ETP (0 to 4.50×10-6 mol/L), curve L is ETP alone (2.70×10-6 mol/L) at 304 K. (a) (b) Figure 6. Plots for ETP-HSA system: (a) Stern-Volmer and (b) Modified Stern-Volmer at 296, 304 and 312 K. KSV from slope of plot F0/F versus [Q] and kq at 296, 304 and 312 K diminished to higher temperatures (Table 3 and Figure 6a). Also, kq values from Equation (3) are more comparable to 2.0×1010 L/mol.s (limiting diffusion rate constant) which established that static quenching (predominant ETP-HSA complex formation) [20]. This result is well supported from evaluated Ka values (lowered at raised temperatures) of modified Stern-Volmer plot (Table 3 Figure 6b) [21,22]. 3.4. Binding characteristics Drugs exist in fringe flow with protein as unbound and bound which follow the guideline of reversible equilibrium and the mass action law. Typically, for any provided drug, bound- protein and free drug always there is equilibrium. This is defined by accompanying Equation (5), log [(𝐹𝐹0 − 𝐹𝐹) 𝐹𝐹⁄ ] = log 𝐾𝐾𝑜𝑜 + 𝑛𝑛 log[𝑄𝑄] (5) 150 Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 Table 4. Binding and thermodynamic specifics for ETP-HSA interaction. Temperature (K) Kb×105 (L/mol) n ΔG (kJ/mol) ΔH (kJ/mol) ΔS (J/mol.K) 296 13.46±0.03 1.351±0.06 -34.63±0.02 -153.8±0.07 -402.9±0.08 304 2.15±0.02 1.164±0.03 -31.41±0.10 312 0.58±0.07 1.023±0.04 -28.19±0.05 (a) (b) Figure 7. Plots for ETP-HSA system: (a) log[(𝐹𝐹0 − 𝐹𝐹) 𝐹𝐹⁄ ] versus log[𝑄𝑄] and (b) van’t Hoff at 296, 304 and 312 K. The intercept gives Kb (binding constant) and slope imparts n (number of binding sites) values from the plot log [(𝐹𝐹0 − 𝐹𝐹) 𝐹𝐹⁄ ] versus log[Q] (Table 4 and Figure 7a). In plasma, Kb determines the drug’s degree of circulation: Strong binding could diminish the free drug concentration, making it not accessible for the component of activity whereas weak binding prompt to poor distribution or short lifetime [23]. More affinity ETP-HSA binding (Kb of order 105) was reduced when decreasing in Kb was observed at raised temperatures. Assumed that this in vitro study is might be similar to in vivo study when going to perform [24,25]. Since, n=1.351, 1.164 and 1.023 at 296, 304 and 312, respectively, representing that the molecular population of ETP decreased in HSA binding interaction at higher temperatures. Obviously, n values were roughly 1 evincing that existence of one ETP to bind HSA. 3.5. Binding and thermodynamic attributes Responsible foremost interactive forces (electrostatic, van der Waals, hydrophobic and hydrogen bonds) in ligands to proteins binding could be manifesting from thermodynamic parameters specifically changes in enthalpy (ΔH), free energy (ΔG) and entropy (ΔS). Gathered binding data at 296, 304 and 312 K were utilized to explore the thermodynamic parameters from Equations (6) and (7) [26]. ln 𝐾𝐾𝑜𝑜 = (− ∆𝐻𝐻 𝑅𝑅𝑅𝑅⁄ ) + (∆𝑆𝑆 𝑅𝑅)⁄ (6) ∆𝐺𝐺 = ∆𝐻𝐻 − 𝑅𝑅∆𝑆𝑆 (7) Linear interrelation between ln Kb and 1/T determined the values of ΔS and ΔH in ETP-HSA system (Table 4 and Figure 7b). The evaluated values in minus ΔH, ΔG and ΔS suggested van der Waals forces with exothermic, spontaneity and hydrogen bonding, respectively, for ETP-HSA interaction. Furthermore, this ETP-HSA binding proceeded by entropy driven (ΔH <ΔS). 3.6. Synchronous fluorescence estimations For Tyr/Trp (Δλ = 15/60 nm) synchronous fluorescence spectra in HSA when ETP varied concentration are designated in Figure 8. Emission maxima of Trp (Figure 8b) have red shift (~11 nm) which manifested that the modification in HSA conformation; increment in polarity by diminished hydrophobicity around Trp [27]. Simultaneously, Tyr emission maximum (Figure 8a) is reduced regularly, yet no significant wavelength change was detected. It recommends the ETP interaction with HSA doesn't influence the conformational Tyr micro-region. 3.7. Energy transfer assessments Apparently, a theory named Förster’s energy transfer relates energy transfer efficiency (E) and distance (r) between ETP and HSA is defined by Equation (8). 𝐸𝐸 = 𝑅𝑅0 6 (⁄ 𝑅𝑅0 6 + 𝑟𝑟6) = ( 𝐹𝐹0 − 𝐹𝐹 ) 𝐹𝐹0⁄ (8) The acquired E value is 0.067. Overlapping integral (J) for ETP and HSA is attributed as, 𝐽𝐽 = {∑[𝐹𝐹𝑑𝑑(𝜆𝜆)Ɛ𝑎𝑎(𝜆𝜆)𝜆𝜆4∆𝜆𝜆]} {∑[𝐹𝐹𝑑𝑑(𝜆𝜆)∆𝜆𝜆]⁄ } (9) Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 151 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 (a) (b) Figure 8. Synchronous fluorescence spectra of HSA (2.70×10-6 mol/L) after interaction with ETP (0.0 to 4.50×10-6 mol/L): (a) Tyr and (b) Trp at 304 K. Figure 9. Overlapped HSA (2.70×10-6 mol/L) fluorescence emission and ETP (2.70×10-6 mol/L) UV-vis absorption spectrum at 304 K. where, Fd(𝜆𝜆) and εa(𝜆𝜆) are fluorescence intensity of HSA and molar absorption coefficient of ETP at λ, respectively. J is obtained as 1.14×10-14 cm3.L/mol (from Figure 9). R0 (Critical distance) about excitation energy of 50% is transferred to ETP which is evaluated from Equation (10): 𝑅𝑅0 6 = 8.8 × 10−25𝐾𝐾2𝑁𝑁−4Ф𝐽𝐽 (10) In the current case, K2 (2/3), Φ (0.15) and N (1.36) are dipole spatial orientation element, HSA fluorescence quantum yield and medium’s refractive index, respectively. FoundR0 is 0.26 Å. From E and R0 values, calculated r is 0.41 Å. These consequences indicate that ETP to HSA binding reaction is via energy transfer, which offers to static quenching (R0 < r) [28]. 3.8. Metal ions on ETP binding to HSA Abundant elements like organic, inorganic and metal ions (trace and essential) in plasmatic fluid are well predictable in diverse biological processes. The accurate body functions are supervised by trace elements at chemical, biological and molecular forms. Several inorganic ligands and metal ions execute crucial part in medicinal diagnosis and therapy. Required Ca2+, Co2+, Na+, Mg2+ and Fe3+ ions portray greater affinity in relation to protein where assorted functions of metallo enzymes, organs and metabolic processes are played. Kb values of ETP-HSA system in presence of Ca2+, Co2+, Na+, Mg2+ and Fe3+ ions were evaluated from Equation (5) (Table 5 and Figure 10a). Co2+, Mg2+ and Fe3+ ions diminished Kb values of ETP-HSA system where these ions decrease the ETP binding with HSA and lower the ETP accumulation in blood for long period. Increased Kb values implied the formation of ETP-metal ion- HSA stable complexes across metal ion bridge for Ca2+ and Na+ ions. Hence, presence of Ca2+ and Na+ ions might boost the delivery exploit of ETP to the target spot by prolonging its storage time. 152 Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 Table 5. Binding constants to ETP-HSA system with metal ions and site probes at 304 K. System Kb (L/mol) * HSA-ETP 2.15±0.06×105 HAS-ETP-Ca2+ 3.56±0.07×105 HSA-ETP-Co2+ 1.02±0.01×104 HSA-ETP-Na+ 1.71±0.04×106 HSA-ETP-Mg2+ 2.44±0.06×103 HSA-ETP-Fe3+ 1.84±0.10×102 HAS-ETP-Warfarin 1.82±0.09×105 HAS-ETP-Ibuprofen 1.88±0.01×104 HAS-ETP-Digitoxin 1.46±0.03×105 * Kb is binding constant. (a) (b) Figure 10. Binding constants to ETP-HSA system (a) with (A-Co2+, B-Mg2+, C-Fe3+, D-Ca2+ and E-Na+) and (b) with (A-Ibuprofen, B-Warfarin and C-Digitoxin) at 304 K. Subsequently these aspects assist in regulating the ETP dose limits for successful treatment and in accomplishing the ideal therapeutic outcomes [29]. 3.9. ETP selective site binding Drug competitive nature affects the bound and unbound drug concentration in HSA to bind compatible site [30]. The estimated Kb values from Equation (5) for each site markers on ETP-HSA system’s binding affinity is supplied in Table 5 and Figure 10b. The subsequent altered Kb value for ibuprofen in contrast to other two from Table 5 intimates that ETP effectively competed with ibuprofen for site II (IIIA) by relocating ibuprofen from its posture. 3.10. Molecular docking examinations Docking speculates the best fit inclination of ETP where it goes and proceeds to bind HSA to frame stable complex. Cluster analysis for individual binding sites were done using 2.0 Å RMSD tolerance to 80 docking runs (Figure 11 and Table 6) with lowest average binding energy received to site II [31]. This is energetically finest favorable ETP conformation immediately bound to HSA. Less Evdw+HB+desol value contrasted with EElec (Table 7) associated to hydrogen bonds bearing van der Waals comprised for HSA and ETP binding at site II (IIIA). 3.11. Three-dimensional fluorescence HSA conformational modulations upon ETP binding is thoroughly investigated from 3D fluorescence. Figure 12 depicts the 3D fluorescence spectra of bound ETP and free HSA which demonstrating 4 peaks namely ‘a’ portraying first-order (λem = λex), ‘b’ illustrating second-order (λem = 2λex) Rayleigh scattering, ‘1’ designating aromatic amino acids (Tyr/Trp: n → π* transition) and ‘2’ denoting polypeptide sequence (π → π* transition) [32]. On binding with ETP, substantial reduction in peak intensities was noticed for HSA (Table 8) signifying the alterations in polypeptide framework and Trp/Tyr micro- environment. 4. Conclusions ETP binding characteristics with HSA was schematically investigated from spectroscopic tools. ETP subsequently quenched the HSA across static with ETP-HSA complex formation. Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 153 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 Table 6. Docking results to ETP-HSA system at distinct sites Binding site Interactions Amino acid-ETP atom Type Distance (Å) Site I (IIA) van der Waals - SER287 - VAL241 - ILE264 - HIS247 - GLN196 - TRP214 - LEU219 - LEU238 Conventional Hydrogen Bond 2.19 ARG257:HE-O1 1.96 ARG257:HH22-O2 Carbon Hydrogen Bond 3.59 GLU292:OE1-C7 Pi-Cation 3.72 ARG222:NH2-aromatic ring 3 3.38 ARG222:NH1-aromatic ring 2 4.06 LYS199: NZ-aromatic ring 2 2.71 LYS199: NZ-aromatic ring 3 Pi-sigma 3.80 ILE290:CG2-aromatic ring 1 Alkyl 5.50 LYS199:(CB-CG-CD)-ring Pi-Alkyl 5.04 ALA291:CB-aromatic ring 2 5.23 ALA291:CB-aromatic ring 1 5.07 ARG257:(CB-CG)-aromatic ring 1 4.27 ALA261:(CB)-aromatic ring 1 4.78 LEU260:(CB-CG-CD2)-aromatic ring 1 5.25 ARG218:(CB-CG)-aromatic ring 1 5.47 ARG218:(CB-CG)-aromatic ring 2 Site II (IIIA) van der Waals - GLU492 - SER489 - LEU387 - ASN391 - GLN390 - PHE403 - LEU407 - LYS413 Conventional Hydrogen Bond 1.89 LYS414:HZ2-O1 1.69 LYS414:HZ1-O2 1.64 ARG410:HE-O2 2.69 ARG410:HH21-O2 Unfavorable Acceptor-Acceptor 2.90 LEU491:O-O1 Pi-Alkyl 4.83 ARG410:aromatic ring 2-(CG-CB) 4.49 ARG410:aromatic ring 3-(CG-CB) 5.11 ALA406:CB - aromatic ring 3 5.32 LEU394:(CD1-CG-CD2)-aromatic ring 3 Alkyl 5.43 VAL409:(CG2-CB-CG1)-ring Pi-Cation 3.30 ARG410:NH1-aromatic ring 3 3.38 ARG410:NH1-aromatic ring 1 Site III (IB) van der Waals - LYS137 - GLU141 - ARG117 - PRO118 - MET123 - TYR161 - VAL116 - HIS146 - LYS190 Conventional Hydrogen Bond 2.26 TYR138: OH-H15 Pi-Sigma 3.55 LEU115:CD2-aromatic ring 1 3.55 ILE142:CD1-aromatic ring 3 Pi-Pi T-shaped 5.13 TYR138:aromatic ring-aromatic ring 1 4.14 LEU185:C-aromatic ring 3 Amide-Pi stacked 5.33 LEU182:(CD1-CG-CD2)-aromatic ring 2 Alkyl 5.44 LEU115:(CD1-CB-CG)-ring 4.88 ARG145:(CG-CB)-ring 4.74 ILE142:(CG1-CB-CG2)-ring Pi-Alkyl 4.06 ARG186:(CG-CG)-aromatic ring 3 3.72 ARG186: (CB-CG)-aromatic ring 2 Table 7. Diverse energies to ETP-HSA system from Lamarckian Genetic Algorithm *. Rank Run 𝚫𝚫G (kJ/mol) Einter-mol (kJ/mol) Evdw+HB+desol (kJ/mol) EElec (kJ/mol) 1 8 -37.95 -45.44 -43.81 -1.67 2 78 -36.44 -43.92 -43.30 -0.67 3 16 -36.19 -43.68 -42.68 -1.00 4 69 -35.77 -43.26 -42.59 -0.67 5 41 -32.30 -39.79 -38.70 -1.09 * ΔG is the binding free energy, Einter-mol is the intermolecular interaction energy; sum of Vander Waals energy, hydrogen bonding energy, desolvation free energy and electrostatic energy, Evdw+HB+desol is the sum of Vander Waals energy, hydrogen bonding energy and desolvation free energy, EElec is the electrostatic energy. 154 Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 Figure 11. Numerous binding sites depicts to ETP docked with HSA. (a) (b) Figure 12. 3D fluorescence spectra: (a) HSA only and (b) ETP-HSA system. Makegowda and Doddarevanna / European Journal of Chemistry 11 (2) (2020) 145-155 155 2020 – European Journal of Chemistry – CC BY NC – DOI: 10.5155/eurjchem.11.2.145-155.1971 Table 8. At 304 K, 3D fluorescence aspects: free HSA and ETP-HSA system. Peak position (λex/λem, nm/nm) Peak Intensity Free HSA HSA-ETP 270/270 → 370/370 a 683.4 → 1141 741.7 → 1006 250/500 b 269 199.6 280/340 1 779.3 278.2 230/340 2 417.3 154.2 The achieved results supported the Trp micro-environment alterations and HSA secondary structure modifications. Hydrogen bonds grouping with van der Waals are validated from thermodynamic constituents to stabilize ETP-HSA system through spontaneously. Best configuration of HSA showed site II for ETP binding. The gained data from present examination can aid in exploring the pharmacodynamics and pharmaco- kinetics specificities of those significant serotonin agonist drugs. Acknowledgements Manjushree Makegowda thanks to University Grants Commission of Basic Scientific Research (UGC-BSR) for awarding the Senior Research Fellowship (SRF) under meritorious students and Institute of Excellence (IOE), Vijnana Bhavan, Mysuru for instrumental assistances. Disclosure statement Conflict of interests: The authors declare that they have no conflict of interest. Author contributions: All authors contributed equally to this work. Ethical approval: All ethical guidelines have been adhered. Sample availability: Samples of the compounds are available from the author. ORCID Manjushree Makegowda http://orcid.org/0000-0003-0298-4333 Revanasiddappa Hosakere Doddarevanna http://orcid.org/0000-0003-0127-3599 References [1]. Migraine, Wikipedia, https://en.wikipedia.org/wiki/Migraine, 2020 (Accessed 11 February 2020). [2]. Eletriptan-hydrochloride, NPS Medicinewise, https://www.nps.org.au/australian-prescriber/articles/eletriptan- hydrochloride, 2020 (Accessed 11 February 2020). [3]. Eletriptan, Wikipedia, https://en.wikipedia.org/wiki/Eletriptan, 2020 (Accessed 11 February 2020). [4]. Kremer, J. M.; Wilting, J.; Janssen, L. H. Pharmacol. Rev. 1988, 40, 1-47. [5]. Ghuman, J.; Zunszain, P. A.; Petitpas, I.; Bhattacharya, A. A.; Otagiri, M.; Curry, S. J. Mol. Biol. 2005, 353, 38-52. [6]. Fasano, M.; Curry, S.; Terreno, E.; Galliano, M.; Fanali, G.; Narciso, P.; Notari, S.; Ascenzi, P. IUBMB Life 2005, 57, 787-796. [7]. Zolfagharzadeh, M.; Pirouzi, M.; Asoodeh, A.; Saberi, M. R.; Chamani, J. J. Biomol. Struct. Dyn. 2013, 32, 1936-1952. [8]. Kamshad, M.; Shah Talab, M.; Beigoli, S.; Sharifi, R. A.; Chamani, J. J. Biomol. Struct. Dyn. 2018, 37, 2030-2040. [9]. Manjushree, M.; Revanasiddappa, H. D. Spectrochim. Acta A 2019, 209, 264-273. [10]. Sanei, H.; Asoodeh, A.; Hamedakbari-Tusi, S.; Chamani, J. J. Solution Chem. 2011, 40, 1905-1931. [11]. Ariga, G. G.; Naik, P. N.; Nandibewoor, S. T.; Chimatadar. S. A. J. Biomol. Struct. Dyn. 2016, 35, 3161-3175. [12]. Manjushree, M.; Revanasiddappa, H. D. Bioinorg. Chem. Appl. 2018, 6954951, 1-13. [13]. Mokaberi, P.; Reyhani, V.; Amiri-Tehranizadeh, Z.; Saberi, M. R.; Beigoli, S.; Samandar, F.; Chamani, J. New J. Chem. 2019, 43, 8132- 8145. [14]. Sharif-Barfeh, Z.; Beigoli, S.; Marouzi, S.; Rad, A. S.; Asoodeh, A.; Chamani, J. J. Solution Chem. 2017, 46, 488-504. [15]. Manjushree, M.; Revanasiddappa, H. D. Chem. Phys. 2020, 530, 110593 [16]. Shakibapour, N.; Dehghani Sani, F.; Beigoli, S.; Sadeghian, H.; Chamani, J. J. Biomol. Struct. Dyn. 2018, 37, 359-371. [17]. Yue, Y.; Sun, Y.; Dong, Q.; Liu, R.; Yan, X.; Zhang, Y.; Liu, J. Luminescence 2016, 31, 671-681. [18]. Yue, Y.; Liu, J.; Liu, R.; Sun, Y.; Li, X.; Fan, J. Food Chem. Toxicol. 2014, 71, 244-253. [19]. Bourassa, P.; Dubeau, S.; Maharvi, G. M.; Fauq, A. H.; Thomas, T. J.; Tajmir-Riahi, H. A. Biochimie. 2011, 93, 1089-1101. [20]. Abdelhameed, A. S.; Alam, P.; Khan, R. H. J. Biomol. Struct. Dyn. 2016, 159, 199-208. [21]. Sohrabi, T.; Hosseinzadeh, M.; Beigoli, S.; Saberi, M. R.; Chamani, J. J. Mol. Liq. 2018, 256, 127-138. [22]. Chamani, J.; Vahedian-Movahed, H.; Saberi, M. R. J. Pharmaceut. Biomed. 2011, 55, 114-124. [23]. Feroz, S. R.; Mohamad, S. B.; Bujang, N.; Malek, S. N.; Tayyab, S. J. Agric. Food Chem. 2012, 60, 5899-5908. [24]. Dehghani Sani, F.; Shakibapour, N.; Beigoli, S.; Sadeghian, H.; Hosainzadeh, M.; Chamani, J. J. Lumin. 2018, 203, 599-608. [25]. Moosavi-Movahedi, A.; Chamani, J.; Gharanfoli, M.; Hakimelahi, G. Thermochim. Acta 2004, 409, 137-144. [26]. Abdelhameed, A. S.; Alanazi, A. M.; Bakheit, A. H.; Darwish, H. W.; Ghabbour, H. A.; Darwish, I. A. Spectrochim. Acta A 2017, 171, 174-182. [27]. Jirgensons, B. J. Biol. Chem. 1965, 240, 1064-1071. [28]. Weiss, S. Science 1999, 283(5408), 1676-1683. [29]. Roy, A. S.; Tripathy, D. R.; Chatterjee, A.; Dasgupta, S. Spectrochim. Acta A 2013, 102, 393-402. [30]. Jacobsen, J.; Brodersen, R. J. Biol. Chem. 1983, 258, 6319-6326. [31]. Sharifi-Rad, A.; Mehrzad, J.; Darroudi, M.; Saberi, M. R.; Chamani, J. J. Biomol. Struct. Dyn. 2020, 1-27. [32]. Guo, X. J.; Sun, X. D.; Xu, S. K. J. Mol. Struct. 2009, 931, 55-59. Copyright © 2020 by Authors. This work is published and licensed by Atlanta Publishing House LLC, Atlanta, GA, USA. The full terms of this license are available at http://www.eurjchem.com/index.php/eurjchem/pages/view/terms and incorporate the Creative Commons Attribution-Non Commercial (CC BY NC) (International, v4.0) License (http://creativecommons.org/licenses/by-nc/4.0). By accessing the work, you hereby accept the Terms. This is an open access article distributed under the terms and conditions of the CC BY NC License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited without any further permission from Atlanta Publishing House LLC (European Journal of Chemistry). No use, distribution or reproduction is permitted which does not comply with these terms. Permissions for commercial use of this work beyond the scope of the License (http://www.eurjchem.com/index.php/eurjchem/pages/view/terms) are administered by Atlanta Publishing House LLC (European Journal of Chemistry). http://orcid.org/0000-0003-0298-4333 http://orcid.org/0000-0003-0127-3599 https://en.wikipedia.org/wiki/Migraine https://www.nps.org.au/australian-prescriber/articles/eletriptan-hydrochloride https://www.nps.org.au/australian-prescriber/articles/eletriptan-hydrochloride https://en.wikipedia.org/wiki/Eletriptan http://www.eurjchem.com/index.php/eurjchem/pages/view/terms http://creativecommons.org/licenses/by-nc/4.0 http://www.eurjchem.com/index.php/eurjchem/pages/view/terms 1. Introduction 2. Experimental 2.1. Reagents and stocks 2.2. UV-vis absorption study 2.3. FT-IR spectral observations 2.4. Emission fluorescence 2.5. Synchronous fluorescence 2.6. Energy transfer between ETP and HSA 2.7. Metal ions on ETP-HSA binding 2.8. Displacement of ETP by site selective assess 2.9. Docking studies 2.10. 3D fluorescence 3. Results and discussion 3.1. UV-vis measurements 3.2. FT-IR spectroscopy 3.3. Fluorescence quenching 3.4. Binding characteristics 3.5. Binding and thermodynamic attributes 3.6. Synchronous fluorescence estimations 3.7. Energy transfer assessments 3.8. Metal ions on ETP binding to HSA 3.9. ETP selective site binding 3.10. Molecular docking examinations 4. Conclusions Acknowledgements Disclosure statement ORCID References PrintField10: PrintField11: PrintField12: PrintField13: PrintField14: PrintField15: PrintField16: PrintField17: PrintField18: PrintField19: PrintField110: PrintField20: PrintField21: PrintField22: PrintField23: PrintField24: PrintField25: PrintField26: PrintField27: PrintField28: PrintField29: PrintField210: