Potent anticancer activity of cystine-based dipeptides and their interaction with serum albumins
© Banerji et al.; licensee Chemistry Central Ltd. 2013
Received: 19 January 2013
Accepted: 15 May 2013
Published: 24 May 2013
Cancer is a severe threat to the human society. In the scientific community worldwide cancer remains a big challenge as there are no remedies as of now. Cancer is quite complicated as it involves multiple signalling pathways and it may be caused by genetic disorders. Various natural products and synthetic molecules have been designed to prevent cell proliferation. Peptide-based anticancer drugs, however, are not explored properly. Though peptides have their inherent proteolytic instability, they could act as anticancer agents.
In this present communication a suitably protected cystine based dipeptide and its deprotected form have been synthesized. Potent anticancer activities were confirmed by MTT assay (a laboratory test and a standard colorimetric assay, which measures changes in colour, for measuring cellular proliferation and phase contrast images. The IC50 value, a measure of the effectiveness of a compound in inhibiting biological or biochemical function, of these compounds ranges in the sub-micromolar level. The binding interactions with serum albumins (HSA and BSA) were performed with all these molecules and all of them show very strong binding at sub-micromolar concentration.
This study suggested that the cystine-based dipeptides were potential anticancer agents. These peptides also showed very good binding with major carrier proteins of blood, the serum albumins. We are currently working on determining the detailed mechanism of anticancer activity of these molecules.
KeywordsPeptide Anticancer Serum albumin Spectroscopy Docking
Neura 2a (neuroblastoma cell line), Hek 293 (kidney cancer cell line) and Hep G2 (liver cancer cell line) were procured from the National Centre for Cell Sciences (NCCS, Pune, India) and were grown in Dulbecco’s modified Eagle medium antibiotics (penicillin/streptomycin and gentamicin). Cells were cultured at 37°C in 95% air and 5% CO2 humidified incubators. Hep G2 cells were seeded at a density of 105 well plated in 96 well plates. Cells were typically grown to 60–70% confluence, rinsed in phosphate-buffered saline (PBS) and placed into serum-free medium overnight prior to treatments. After overnight incubation, the Hep G2, HEK 293, and Neura 2a cells were treated with these compounds separately at the concentration of 1 μM, 10 μM and 20 μM, respectively. After 48 hours the medium was removed and a 50 μl of fresh medium was added along with 10 μl of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). MTT solution (5mg/ml) was slowly removed after 4 hours and the purple crystals with solubilization in 1.4 ml of DMSO. The absorbance was measured at test wavelength of 550 nm in Elisa Plate Reader [22, 23].
The steady-state fluorescence spectra were recorded with a Perkin Elmer LS-45 spectrofluorophotometer. Emission spectra were recorded with an excitation wavelength of 280 nm and emission range of 290–450 nm. Both the excitation and emission slit widths kept at 5 nm each. The intrinsic fluorescence of tryptophan residue(s) in the protein was measured in the presence and in the absence of the dipeptides. Most of the experiment was carried out at room temperature (25°C), Some temperature dependent studies were carried out using water bath.
The fluorescence of the protein was found to quench in the presence of the peptides. The quenching experiment was carried out simply by adding small aliquote (1–10 μL from 100 μM stock solution) of concentrated peptide solution to 1 mL solution containing an appropriate concentration of HSA/BSA (0.5 μM in 20 mM Tris–HCl buffer, pH 7.5) taken in 1 cm path length quartz cuvette. The optical density of the solution at the excitation wavelength was kept less than 0.05. Small error due to dilution upon addition of the peptide was neglected. The peptides showed negligible absorbance at the excitation wavelength (280 nm). Fluorescence intensities at 340 nm were recorded as a function of ligand concentration. To derive the binding parameters, obtained data were analyzed using modified Stern–Volmer equation [24–26].
Measurements of circular dichroism (CD)
The Far-UV CD spectra have been measured on a Jasco J-810 spectrometer using a 1.0 mm quartz cell under constant nitrogen flow condition and at room temperature. The CD spectra of HSA and BSA have been recorded in the absence and presence of these compounds within the wavelength range of 200–250 nm. The CD results have been represented in terms of ellipticity (θ).
The crystal structure of HSA and BSA were obtained from Protein Data Bank (PDB ID: 1E78 and 3V03 respectively). Structures of the synthesized compounds were drawn in Gauss View followed by geometry optimization in Gaussian 09 with DFT level of theory using B3LYP/6-31 + G(d,p) basis set. AutoDock 4 and MGLTools of The Scripps Research Institute were used to perform the docking calculations [27, 28]. Docking was performed following the previously published protocol [29–33]. The PyMOL molecular (http://pymol.org/) viewer and the MGLTools were used to render the output.
Results and discussion
In order to determine the biological efficacy of these newly synthesized compounds in vitro cell culture system has been used.
Cell viability was quantified by MTT, a yellow tetrazole assay, where the viable cells were determined by the reduction of the yellow MTT into purple formazan product. For this assay, the cells were plated in 96 well plates and grown in monolayer and then treated with these compounds of interest. The viability of cells by MTT assay was performed 48 hours post treatment as described before. Finally, the medium was removed and replenished with 80 μl of fresh medium along with 20 μl of MTT (5 mg/ml). After 4 hours, MTT solution was slowly removed and the purple crystals were solubilised in 100 μl of DMSO. The absorbance was measured by a plate reader at a wavelength of 550 nm. The absorbance obtained from treated cells were expressed as percentages of absorbance obtained from untreated cells and are reported as mean ± SEM (n =3).
Action of a drug molecule to a cell is initiated by drug receptor and many of the receptors have high specificity for a drug molecule and the chemical structure of a drug may significantly alter the cell's response to the drug molecule. Also the concentration of drug molecule to the receptor site directly affects the drug response. For example, amphetamine and methamphetamine act as powerful stimulus for nervous system and act via the same receptor. These two compounds differed slightly in their chemical structure; however, methamphetamine exerts more powerful action. There are small structural changes present in our synthesized dipeptides. NH2 groups in 1A and 1B are protected with carbamates, also the carboxylic acid moiety is as a methyl ester. The receptor that initiates the drug action of the dipeptides may show difference in action due to these structural changes. However, similar to many chemical reactions, drug action of the receptor also depends on the effective concentration of the drug molecule at the receptor site. Amount of drug that penetrates to the cell/receptor site again depends on structure of the drug molecule and their physical parameter such as hydrophobicity. One possible explanation is that 1A and 1B (cLogP: 4.01, see Additional file 1: Computation of partition coefficient (cLogP)) are more hydrophobic than 1C and 1D (cLogP: 1.75). So, the membrane permeability of these two are more than the other two. So, 1A and 1B can penetrate the cells better than that of 1C and 1D and could be sensed by the receptor more strongly apart from the structural specificity.
Cell viability tests were performed using cultured cells. However, in real systems, like cells in human body/other animals drugs need to be reached to the body/effected cells by blood. All the drug molecules that enter into the body via systemic circulation get exposed to the blood milieu. In blood, serum protein albumins (HSA, BSA) are the major carrier proteins. They bind to a wide variety of small molecules and fatty acids and carry of them to different parts of the body. Very good binding to these proteins means very good distribution of the drug all over the body i.e., increased bioavailability. Therefore, the binding behaviour of the synthesized peptides to HSA and BSA was carried out using the unique and intrinsic fluorescence from the tryptophan residues. The dipeptides showed very good binding with plasma carrier proteins of both bovine and human. Interaction site of the peptides to the protein was established via molecular docking analysis as discussed later.
Binding constant from fluorescence study
Stern-Volmer quenching constant (K) with HSA and BSA at temperature 298 K as obtained from equation 1
Stern-Volmer quenching constant (M-1)
18.37 × 105
7.95 × 105
4.32 × 105
3.52 × 105
9.56 × 105
4.88 × 105
10.97 × 105
5.90 × 105
Binding dissociation constants (K d ) with HSA and BSA at temperature 298 K
(SEM = standard error of mean; NA = not available) Compounds
Binding constant (Kd) ± SEM in μM
0.546 ± 0.05
1.257 ± NA
2.312 ± NA
2.840 ± NA
1.044 ± 0.08
2.051 ± NA
0.912 ± NA
1.703 ± 0.07
There is the same relationship between 1A and 1B that between 1C and 1D: diastereoisomers. However, all the four compounds showed similar binding efficiency (Tables 1 and 2). It indicated that both the conformations are equally significant in the attenuation of HSA/BSA fluorecence. Eftink et. al. and others clearly indicated how the quenching volume and the entry of the quencher to the hydrophobic protein pocket influence both the static and dynamic quenching [24–26]. As in this investigation no significant difference occurred in quenching efficiency (quenching / binding constant), the dipeptides had similar accessibility of the tryptophan residues in the proteins.
Circular dichroism (CD)
Thermodynamic parameter of binding as obtained from molecular docking simulation experiments
Binding free energy (kcal mol-1)
Domain I also binds to other drug molecules such as, 2,3,5-triiodobenzoic acid . Small molecules are also reported to bind in to a site in between the domain I and III of HSA . The major contributing forces involved in the binding of these compounds with serum albumins are hydrogen bonding (Additional file 1: Figure S4 and Additional file 1: Figure S5), hydrophobic interaction and van der Waals attraction.
In conclusion, in this work, we have synthesized four dipeptides made of cystine amino acid (both protected and unprotected form) and studied their interaction with BSA and HSA. Routine solution phase synthesis was employed to prepare these peptides. The cell viability of these compounds was quantified by MTT assay. They show anticancer activity in sub micro molar range. The phase contrast images show massive cell death. The interaction between these compounds with HSA and BSA was investigated by employing different spectroscopic techniques (fluorescence and CD spectroscopy). Fluorescence study indicates strong binding of these compounds with both BSA and HSA. The CD results revel that the secondary structure of BSA and HSA were very slight affected upon interaction with these compounds. The molecular modeling studies show that the binding of these compounds with BSA and HSA are thermodynamically favorable and no cluster formation occurs, which suggest that the bindings are nonspecific in nature. Although detail mechanistic studies of anticancer properties of these molecules are still going on, the initial results indicate DNA intercalation (Additional file 1: Figure S6) may be responsible for the cell death. Further studies in this aspect are going on in our laboratory and the results will be published in due course of time.
Biswadip Banerji achieved the following in his academic years: M.Sc. in Chemistry, University of Calcutta, Kolkata, India; Ph.D., Indian Institute of Technology, Kanpur, India; Postdoctoral Research Fellow from Oxford Centre for Molecular Science & Chemistry Research Laboratory, Oxford University, UK; and Postdoctoral Research Fellow from the School of Chemical and Life Sciences, Institute of Chemical & Engineering Sciences-Agency for Science, Technology and Research (ICES-A*STAR), Singapore. He was the Team Leader at Chembiotek, Kolkata, India. He is a Senior Scientist from the Indian Institute of Chemical Biology, Kolkata, India. His research area interests cover smart nanobiomaterials, peptide based drug designing, self assembly of biomaterials and natural product derived hybrid scaffolds and its application in therapeutics.
Sumit Kumar Pramanik obtained his B.Sc. in chemistry from Vidyasagar University, India. He earned his M.Sc. in applied chemistry from Bengal Engineering and Science University, Shibpur, India. He is a Ph.D. Student from the Chemistry Division, Indian Institute of Chemical Biology, Kolkata, India. His research area interests include nanobiomaterials and peptide based drug design and biophysical chemistry.
Uttam Pal earned his B.Sc. in Physiology from the Presidency College, Kolkata, India. He is a M.Sc. degree holder of Biophysics and Molecular Biology from University of Calcutta, Kolkata, India. He is a Ph.D. Student from the Structural Biology and Bioinformatics Division, Indian Institute of Chemical Biology, Kolkata, India. His research area covers structural biology and bioinformatics.
Nakul Chandra Maiti achieved M.Sc. in Chemistry, University of Calcutta, Kolkata, India; Ph.D. From Tata Institute of Fundamental Research, Mumbai, India; Postdoctoral JSPS visiting scientist, Institute for Molecular Science, Japan; Postdoctoral Senior Research Associate, Biochemistry, Case, Cleveland, Ohio, USA; Postdoctoral Research Associate/lecturer, California State University, Los Angeles, USA. He is a Senior Scientist from the Indian Institute of Chemical Biology, Kolkata, India. His research area interests cover structure based amyloid research, structural aspects and in-vitro behavior of natively unfolded proteins and peptides those are linked to human diseases, applications of NMR, fluorescence and Raman spectroscopy to biological systems, computational biochemistry and bioinformatics.
Sumit Kumar Pramanik thanks CSIR, India, Uttam Pal thanks INSPIRE Fellowship Programme, DST, India for financial support. We would also like to thank the miND project, CSIR for providing financial assistance towards this work. The authors would also like to thank the central instrumental fascilities of CSIR - IICB.
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