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Molecular Recognition of tRNA with 1-Naphthyl Acetyl Spermine, Spermine and Spermidine: A Thermodynamic, Biophysical and Molecular Docking Investigative Approach Ayesha Kabir, Devawati Dutta, Chhabinath Mandal, and Gopinatha Suresh Kumar J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 3, 2016
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Molecular Recognition of tRNA with 1-Naphthyl Acetyl Spermine, Spermine and Spermidine: A Thermodynamic, Biophysical and Molecular Docking Investigative Approach Ayesha Kabir1, Devawati Dutta2, Chhabinath Mandal3 and Gopinatha Suresh Kumar1* 1
Biophysical Chemistry Laboratory, Organic and Medicinal Chemistry Division, CSIR- Indian
Institute of Chemical Biology, Kolkata 700 032, India 2
Cancer Biology and Inflammatory Disorder Division, CSIR-Indian Institute of Chemical
Biology, Kolkata 700 032, India 3
National Institute of Pharmaceutical and Educational Research, Kolkata 700 032, India
Correspondence to: Prof. (Dr.) G. Suresh Kumar Chief Scientist, Organic and Medicinal Chemistry Division CSIR-Indian Institute of Chemical Biology 4, Raja S. C. Mullick Road, Kolkata 700032, INDIA Phone: +91 33 2472 4049, Fax: +91 33 2473 0284 / 5197 e-mail:
[email protected] 1
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ABSTRACT: The role of tRNA in protein translational machinery and the influence of polyamines on the interaction of acylated and deacylated tRNA to ribosomes, make polyaminetRNA interaction conspicuous. We studied the interaction of two biogenic polyamines spermine and spermidine with tRNAPhe, and compared the results with the analog, 1-naphthyl acetyl spermine. The binding affinity of spermine was comparable to 1-naphthyl acetyl spermine; both were higher than spermidine. The interactions led to significant thermal stabilization of the tRNAPhe and an increase in the enthalpy of transition. All the interactions were exothermic in nature and displayed prominent enthalpy-entropy compensation behavior. The entropy driven nature of the interaction, the structural perturbations observed and docking results proved that the polyamines were bound in the groove of the anticodon arm of tRNAPhe. The amine groups of polyamines were involved in extensive electrostatic, H-bonding and van der Waals interactions with tRNAPhe. The naphthyl group of 1-naphthyl acetyl spermine made additional stacking interaction with G24 and G26 of tRNAPhe, which was absent in others. The results demonstrate that 1-napthyl acetyl spermine can target the same binding sites as the biogenic polyamines without substituting for the functions played by them, which may lead to exhibition of selective anti-cancer cytotoxicity.
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INTRODUCTION RNA plays central role in transcription, translation, protein functions, enzymatic processes, gene regulation and also RNA interference.1-6 Therefore, RNA binding small molecules may serve as leads for RNA based antiviral and anticancer agents.7-11 Aminoglycoside antibiotics are one group of RNA binding molecules that interact at the functional centers of the 16S rRNA.12-14 Transfer RNAs (tRNA) are one of the smallest RNA molecules; they function as cardinal decoding adaptors in the synthesis of proteins.15-16 tRNA has a well characterized structure with a sequence of about 74-95 bases. The cloverleaf secondary structure of tRNA consists of 4 constant arms and an extra arm in the longer tRNAs. The molecule folds back on itself forming an L-shaped tertiary structure consisting of two segments of double helix.17 Even though tRNA is mainly recalled for its central role in translation, they also engage in a number of nontranslational activities in bacteria, eukaryotes and archaea.17-18 They play pivotal roles in stress signalling, adaptive translation and complex human diseases.18 Additionally, the tRNA structure represents a unique site for recognition of small molecules and such binding interactions have been documented through extensive studies. 19-22 Polyamines, on the other hand, regulate gene expression and cellular growth and activities through its interaction with nucleic acids and proteins.23-26 Cancerous cells often display formidable concentration of polyamines whereas a waning in their concentration may lead to cellular growth suppression followed by apoptosis.27-31 For these reasons understanding polyamine action and metabolism has been crucial for antiproliferative and anti-cancer drug development.32 Minor structural changes in the biogenic polyamines have yielded analogs with paramount biological relevance in recent times.32-35 The selective polyamine transporter of the cell may be utilized by a potent polyamine analog to enter the cell and tussle with the targets of
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the natural polyamines.32-35 Considering the fact that the analog does not replace the biogenic polyamines in proliferative function, it may display selective anti-cancer cytotoxicity.32-35 The bis(ethyl) polyamines were the most notable analogs which were ever synthesized.34-35 Various reports have shown the potency of a number of other polyamine analogs against cancerous growth in numerous cell lines.34-40 Reports have shown that polyamines and its analogs interact with and stabilize tRNA.41-46 Interaction of natural polyamines to tRNA is pertinent for optimal translational preciseness and efficiency.46 Polyamines have been reported to attune protein synthesis and stimulate the binding interactions of acylated and deacylated tRNA to ribosomes.47-48
Fig. 1. (A) Secondary and tertiary structures of tRNA, (B) chemical structure of spermine (SPM), 1-naphthyl acetyl spermine (NASPM) and spermidine (SPD). Thus, the vital and expansive nature of tRNAs make them crucial targets of drugs. For these reasons, it will be interesting to validate the interaction of polyamines and their analog with tRNA. Our work on the comparative binding of biogenic polyamines and spermine analog, 1naphthylacetyl spermine (NASPM) with RNA and DNA polynucleotides have given us
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interesting results where we observed different specificity behavior of the analog NASPM over natural polyamines.49,50 The aim of the present work is to widen our knowledge related to the comparative binding of polyamines and analogs with tRNAPhe. The tRNAPhe binding potency of NASPM is not known yet. NASPM (Figure 1) is a synthetic analog of the Joro spider toxin which has been reported to display long-lasting anticonvulsant effect on previously kindled rats.51,52 Thus, biophysical and calorimetric studies were performed to elucidate the structural, thermodynamic, and conformational aspects of the binding interaction. Additionally, molecular docking studies were carried out to evaluate strength and mode of the interactions involved in the binding. MATERIAL AND METHODS Materials. tRNAPhe and the polyamines, spermine hydrochloride (SPM), 1-naphthyl acetyl spermine trihydrochloride (NASPM), spermidine hydrochloride (SPD), and ethidium bromide were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). tRNAPhe solution was prepared by dissolving the tRNAPhe in buffer followed by continuous gentle stirring at 4°C overnight. The concentration of tRNAPhe was determined by absorbance using the molar absorption coefficient (Ɛ) of 6900 (M-1 cm-1) at 260 nm formulated in terms of nucleotide phosphates.19-22 The tRNAPhe solution was free from any kind of protein contamination as indicated from the absorbance ratio at 260/280 nm. Each day the polyamines were weighed and dissolved to obtain the polyamine solutions. The experiments were done in 20 mM [Na+] in Citrate-Phosphate (CP) buffer, pH 7.0, containing 10 mM Na2HPO4 and the desired pH was attained by using citric acid. 0.22 µM millipore filters (Millipore, India Pvt. Ltd, Bangalore, India) were used to filter the buffer solutions. Different amounts of NaCl were added to the
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buffer solution to obtain buffers of different salt concentration required for the salt dependent studies. Experimental Methods. Differential scanning calorimetry (DSC) is a dependable methodology used to measure the heat energy uptake of a sample during controlled increase (or decrease) in temperature. The measurements were performed with a Microcal VP DSC unit (MicroCal, LLC., Northampton, MA, USA, now Malvern Instruments Ltd., Malvern, UK).53 In a set of continuous DSC scans, the cells were loaded with the buffer and equilibrated at 25 oC for 15 min. It was then scanned from 25 to 120 oC, at approximately 30 psi pressure, at a scan rate of 60 oC/hour. The buffer scans were repeated till the base line was reproducible (noise specification
