- Open Access
Binding interaction of a gamma-aminobutyric acid derivative with serum albumin: an insight by fluorescence and molecular modeling analysis
- Uttam Pal†1,
- Sumit Kumar Pramanik†2,
- Baisali Bhattacharya1,
- Biswadip Banerji2 and
- Nakul C. Maiti1Email author
© The Author(s) 2016
- Received: 7 January 2016
- Accepted: 4 July 2016
- Published: 19 July 2016
gamma-Aminobutyric acid (GABA) is a naturally occurring inhibitory neurotransmitter and some of its derivatives showed potential to act as neuroprotective agents. With the aim of developing potential leads for anti-Alzheimer’s drugs, in this study we synthesized a novel GABA derivative, methyl 4-(4-((2-(tert-butoxy)-2-oxoethyl)(4-methoxyphenyl)amino)benzamido)butanoate by a unique method of Buchwald–Hartwig cross coupling synthesis; with some modification the yield was significant (97 %) and spectroscopic analysis confirmed that the compound was highly pure (98.8 % by HPLC). The druglikeness properties such as logP, logS, and polar surface area were 3.87, −4.86 and 94.17 Å2 respectively and it satisfied the Lipinski’s rule of five. We examined the binding behavior of the molecule to human serum albumin (HSA) and bovine serum albumin (BSA) which are known as universal drug carrier proteins. The molecule binds to the proteins with low micromolar efficiency and the calculated binding constants were 3.85 and 2.75 micromolar for BSA and HSA, respectively. Temperature dependent study using van’t Hoff equation established that the binding was thermodynamically favorable and the changes in the Gibb’s free energy, ΔG for the binding process was negative. However, the binding of the molecule to HSA was enthalpy driven and the change of enthalpy (ΔH) was −10.63 kJ/mol, whereas, the binding to BSA was entropy driven and the change in entropy ΔS was 222 J/mol. The molecular docking analysis showed that the binding sites of the molecule lie in the groove between domain I and domain III of BSA, whereas it is within the domain I in case of HSA, which also supported the different thermodynamic nature of binding with HSA and BSA. Molecular dynamics analysis suggested that the binding was stable with time and provided further details of the binding interaction. Molecular dynamics study also highlighted the effect of this ligand binding on the serum albumin structure.
- Serum albumin
- Molecular docking
- Molecular dynamics
gamma-Aminobutyric acid (GABA) plays an important role as an inhibitory neurotransmitter of the central nervous system (Gajcy et al. 2010). Impaired secretion of GABA is associated with several important neurological disorders such as Parkinson’s (Kleppner and Tobin 2001) and Alzheimer’s disease (Jo et al. 2014) and other psychiatric disorders (Nutt and Malizia 2001). Amyloid-β (Aβ) is an intrinsically disordered protein and therefore, it does not have an ordered native structure under physiological condition (Lu et al. 2013). However, the structure of Aβ evolves or gets stabilized as it forms higher order aggregates (Lu et al. 2013) such as oligomers and thread like elongated fibril with cross beta sheet structure. Therefore, the binding sites on Aβ also evolve with the process of aggregation. Drugs that bind to amyloid beta at different stages of aggregation have been developed to arrest the further growth of oligomers (Padayachee and Whiteley 2011; Huy et al. 2013; Richard et al. 2013).
New GABA derivatives can be considered as potential drugs in the treatment of neurodegenerative disorders (Gajcy et al. 2010). The efficacy of GABA can be highly potentiated by benzodiazopines (Nutt and Malizia 2001). Recently there has been increasing interest in synthesizing new GABA derivatives, thus, these compounds could be the potential lead molecules in the development of anti-Alzheimer’s drugs to target Aβ peptide or its assembly structures (Jiang et al. 2013).
To realize the interaction pattern of the newly synthesized GABA derivative, methyl 4-(4-((2-(tert-butoxy)-2-oxoethyl)(4-methoxyphenyl)amino)benzamido)butanoate, in hydrophobic protein cavities we explored both the quantitative and qualitative aspects of the interaction and incorporation of the compound into the binding pockets of serum albumin using fluorescence, molecular docking and molecular dynamics analysis. This compound is very similar to a series of molecules which were previously tested by Dr. Banerji’s group for their anti-Alzheimer’s activity (Sanphui et al. 2013).
The incorporation and investigation of the molecule inside the hydrophobic protein environment was monitored following the changes in the intrinsic tryptophan fluorescence of the protein molecule. Intrinsic protein fluorescence originating from tryptophan and tyrosine residues provides ample information about the local environment, the changes in protein conformation and the interaction of a protein with a drug molecule (Möller and Denicola 2002). Perturbation in fluorescence intensity also provides significant insight into the interaction pattern of the molecules. Binding parameters were measured from fluorescence quenching in the presence of the compound and related thermodynamic parameters were obtained by measuring the effect of temperature on binding constant.
In addition, to find out the interaction of the drug molecule at atomic level, molecular docking and dynamics analysis were carried out. Molecular docking is a robust and efficient computational technique to understand the structure activity relationship of a drug-like molecule with a target protein (Jorgensen 2004; Morris and Lim-Wilby 2008; Meng et al. 2011). The binding sites of the GABA derivative on serum albumins, interacting residues and the type of interactions were probed by molecular docking analysis. Molecular dynamics study further ascertained the stability of binding and highlighted the specific interactions over a time period.
Bovine and human serum albumins were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Tris–HCl and Urea were also purchased from Sigma-Aldrich. All the samples were prepared in 20 mM Tris–HCl buffer of pH 7.0. Deionized and triple distilled water was used for preparing buffer solution that was passed through 0.22 µm pore size Millipore filters (Millipore India Pvt. Ltd., Bangalore, India).
All air and water sensitive reactions were carried out in oven dried glassware under nitrogen atmosphere using standard manifold techniques. All the chemicals were purchased from Acros organics and Sigma-Aldrich, and used without further purification unless otherwise stated. Compounds that are not described in the experimental part were synthesized according to the literature procedures. Solvents were freshly distilled by standard procedures prior to use. Flash chromatography was performed on silica gel (Merck, 100–200 mesh) with the indicated eluant. All 1H and 13C-NMR spectra were recorded on a Bruker 600 MHz spectrometer. For 1H NMR, tetramethylsilane (TMS) served as internal standard (δ = 0) and data are reported as follows: chemical shift, integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet) and coupling constant(s) in Hz. For 13C NMR, TMS (δ = 0) or CDCl3 (δ = 77.26) was used as internal standard and spectra were obtained with complete proton decoupling.
Procedure to synthesize the GABA derivative
Ground-state absorption spectra were recorded with a Shimadzu UV-2401PC Spectrometer. 1 cm path-length quartz cuvette was used and 250–450 nm wavelength range was scanned.
Fluorescence emission spectroscopy
The steady-state fluorescence emission and excitation spectra were recorded with a Cary Eclipse Fluorescence Spectrophotometer. The emission spectra of serum albumins were obtained by exciting the samples at the wavelength 295 nm. In all the cases, the excitation and emission slit widths were kept at 5 nm each. Protein fluorescence spectra as a function of ligand concentration was recorded by a simple titration method (Banerjee et al. 2012, 2013; Ray et al. 2012).
Determination of binding constants
Lipophilicity and solubility calculations
Lipophilicity in terms of calculated logP (clogP) and solubility in terms of calculated logS (clogS) were determined at Virtual Computational Chemistry Laboratory server (http://www.vcclab.org/lab/alogps/) (Tetko et al. 2005). Polar surface area was calculated with a 1.4 Å radius probe size.
Molecular docking experiments were performed using four different algorithms: AutoDock Vina (Trott and Olson 2010), AutoDock 4.2 (Morris et al. 2009), PatchDock/FireDock (Schneidman-Duhovny et al. 2005; Mashiach et al. 2008) and SwissDock (Grosdidier et al. 2011). BSA (PDB: 3V03) (Majorek et al. 2012) and HSA (PDB: 4L8U) (Bhattacharya et al. 2000) structural information was obtained from Protein Data Bank (Berman et al. 2000). Protein structures were chosen based on the validation report provided by wwPDB at the PDB website (Read et al. 2011; Gore et al. 2012). All the hetero atoms and water and multiple subunits were removed from the PDB structures and the missing side chain residues for BSA were modeled at PDB_hydro web server (Azuara et al. 2006). The ligand structures were drawn in Avogadro (Hanwell et al. 2012) and geometry optimized in vaccuo using the steepest descent followed by conjugate gradient algorithms in UFF forcefield as implemented in Avogadro.
AutoDockTools (Morris et al. 2009) was used to prepare the ligand and proteins For the docking in AutoDock 4.2 and AutoDock Vina. Polar hydrogen atoms and Gasteiger charges were added to the proteins and the ligand. All the rotatable bonds in the ligand were set free. No flexibility was added to the protein side chains. The whole protein was placed in the center of a simulation box. The box dimension was 87 × 66 × 80 cubic angstroms for BSA and 87 × 66 × 73 cubic angstroms for HSA. Grid point spacing of 0.775 Å was used for docking in AutoDock 4.2, while the grid point spacing for AutoDock Vina was 1 Å. Genetic algorithm was run (ga_run) 100 times to generate a statistically significant number of docked poses (Alam et al. 2012). All the other parameters were kept constant. AutoDock Vina results were rendered in PyMOL and AutoDock 4.2 results were rendered in MGLTools.
Docking was also carried out at two different web servers: SwissDock and PatchDock/FireDock. SwissDock results were rendered in UCSF Chimera (Pettersen et al. 2004). PatchDock does not consider ligand flexibility, therefore, best poses of the ligand obtained by AutoDock 4.2, Vina and SwissDock were used as input ligand orientation for docking with PatchDock. 10 Best PatchDock results were further refined by FireDock web interface. FireDock results were rendered in PyMOL.
Molecular dynamics (MD) analysis was carried out in Schrodinger Maestro Molecular Modeling environment (academic release 2015-4). 12 ns dynamics were carried out for the protein ligand complexes and for the proteins as well, in SPC water environment using Desmond (Bowers et al. 2006) molecular dynamics program implemented in Schrodinger Maestro. The proteins or the complexes were placed in the center of the simulation box with periodic boundary conditions. The periodic boundary box dimensions are given in the supporting information (Additional file 1: Table S1). The whole systems were charge neutralized using sodium ions. MD was run in OPLS 2005 force field (Banks et al. 2005). Five step relaxation protocol was used starting with Brownian dynamics for 100 ps with restraints on solute heavy atoms at NVT (with T = 10 K) followed by 12 ps of dynamics with restraints at NVT (T = 10 K) and then at NPT (T = 10 K) using Berendsen method. Then the temperature was raised to 300 K for 12 ps followed by 24 ps relaxation step without restraints on the solute heavy atoms. The production MD was run at NPT with T = 300 K for 12,000 ps. The molecular dynamics output was rendered in Schrodinger Maestro Suite.
Absorbance and fluorescence of the GABA derivative
Molecular structure of compound 5 is shown in Scheme 1. Additional file 1: Figure S1 shows the absorption spectrum of the GABA derivative. Due to the presence of conjugate systems, it showed absorption in UV region (below 300 nm). However, the absorbance was very weak. The compound is non-fluorescent in nature.
Interaction with albumins
The Kd and Ka values for the binding of the GABA derivative to serum albumins as determined by the fluorescence quenching experiments at room temperature
3.85 × 10−6
2.60 × 105
2.75 × 10−6
3.64 × 105
Thermodynamics of serum albumin binding
Thermodynamics of the GABA derivative binding to serum albumins
ΔG° (kJ mol−1) at 25 °C
ΔH° (kJ mol−1)
ΔS° (J mol−1 K−1)
Drug like properties of the GABA derivative
Molecular properties of the compound
3.87 ± 0.53
Polar surface area
Lipinski’s rule of five
Molecular modeling provides insight into the interaction with serum albumins
Theoretical binding free energies as obtained by molecular docking experiments using four different algorithms
AutoDock 4.2 (kJ mol−1)a
AutoDock Vina (kJ mol−1)
PatchDock/FireDock (kJ mol−1)
SwissDock (kJ mol−1)
−14.37 ± 0.36
−17.34 ± 0.37
Dynamics of compound 5 binding with serum albumins
Figure 8, on the other hand, highlights the residue-wise fluctuations in the backbone and the side chains of BSA and HSA. The ligand contact sites are also highlighted. Figure 8 suggests that the backbone fluctuation slightly decreased near the ligand binding site, both, in BSA and in HSA. Decrease in the backbone fluctuations near the ligand indicated that the binding site attained a stable conformation. A video of the dynamics of BSA-compound 5 complex is shown in Additional file 2: Video S1. Additional file 3: Video S2 shows the dynamics of HSA-compound 5 in water.
We have reported here, the synthesis and physicochemical properties of a new derivative of the naturally occurring inhibitory neurotransmitter GABA and its interactions with the drug carrier protein in blood, the serum albumin. Binding of this molecule with two orthologs of serum albumins, human and bovine, were compared. The compound shows drug like properties and binds to the human and bovine serum albumins with the binding constants in low micromolar concentration range. Thermodynamics analysis showed that the binding of compound 5 with HSA was enthalpy driven, whereas binding with BSA was driven by entropy. Molecular docking studies by various different algorithms further showed that the compound binds to the groove between domain I and domain III of BSA and within the domain I in case of HSA. Molecular dynamics analysis showed that the compound forms stable complexes with the serum albumins. Binding of the compound with BSA was stabilized mainly by hydrophobic and ionic interactions, whereas, interactions with HSA was maintained predominantly through hydrogen bonding.
Conceived and designed the experiments: UP, SKP and NCM. Performed the experiments: UP, SKP and Baisali Bhattacharya. Analyzed the data: UP, SKP, Baisali Bhattacharya, Biswadip Banerji and NCM. Contributed reagents/materials/analysis tools: Biswadip Banerji and NCM. Wrote the paper: UP, SKP, Baisali Bhattacharya, Biswadip Banerji and NCM. All authors read and approved the final manuscript.
Uttam Pal thanks INSPIRE Fellowship Programme, Department of Science and Technology, Government of India, India for financial support. Sumit Kumar Pramanik thanks CSIR-Indian Institute of Chemical Biology for financial support.
The authors declare that they have no competing interests.
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