Investigations on interaction mechanism of chloroethyl nitrosourea derivatives with nucleic acid: In silico and spectroscopic STUDIES
THESIS SUBMITTED TO AcSIR FOR THE AWARD OF THE DEGREE OF
Doctor of Philosophy In Biological Sciences
By Shweta Agarwal Enrolment No: 10BB12A32008
Under the guidance of
Dr. Ranjana Mehrotra Chief Scientist & Head Quantum Phenomena & Applications
CSIR-NATIONAL PHYSICAL LABORATORY Dr. K.S. KRISHNAN MARG NEW DELHI-110012 INDIA
AUGUST 2015
ABSTRACT
Cancer is the second leading cause of death in the world after cardiovascular diseases.
In spite of good advancements in diagnosis and treatment, there is still a lack of better remedial options for cancer treatment. Cancer develops, when normal cells (in a particular part of the body) begin to divide in an uncontrolled manner. Generally, transformation of a normal cell to cancerous cell is associated with DNA mutation and damage. At present, there are several regimes in use to combat cancer and majority of them involve the utilization of chemotherapy (the use of chemicals to destroy cancer cells). Despite inexorably significant role of chemotherapy in cancer cure and control, cytotoxic mechanisms of several chemotherapeutic agents are not well characterized. Many of these anticancer chemotherapeutic drugs target nucleic acids and auxiliary processes (such as replication, transcription and translation) in course of their cytotoxic action. Keeping this in view, researchers are constantly putting their efforts to elucidate the underlying anticancer mechanism of drugs at molecular level by investigating the interaction mode between nucleic acid and drugs.
The information gathered from the outcome of such investigations is helpful in the establishment of a correlation between drug's molecular structure and its cytotoxicity.
Furthermore, this knowledge would be instrumental in the detection of those structural modifications in a drug that could result in sequence/structure specific binding to their target (nucleic acid). This comprehension can be exploited in the rational designing and synthesis of new drugs, possessing better efficacy and reduced side effects, since non-specific binding restricts drug dose and regularity in cancer treatment. At last, it can be deduced that interaction studies are fundamental in unraveling the mystery of molecular recognition, in general, and nucleic acid binding, in particular.
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Chapter 1, the introductory chapter, depicts aims and objectives of the present work.
Chloroethyl nitrosoureas derivatives (CENUs) constitute one of the classes of alkylating antineoplastic agents, which possess immense importance and direct relevance in the treatment of brain tumors, lymphomas, malignant melanoma and various solid tumors. Antineoplastic action of CENUs is believed to involve the inhibition of DNA replication, RNA transcription and protein translation by means of alkylation of nitrogenous bases in DNA double helix. Despite of the availability of detailed structural/chemical knowledge about the alkylating agents in literature, there remain deficits in the understanding of CENUs-nucleic acid interaction in particular.
Therefore, it is of great significance to elucidate the peculiarities of CENUs-nucleic acid complexation to comprehend their underlying cytotoxic action mechanism. In the present work, we aim to investigate the nucleic acid binding properties of chloroethyl nitrosourea derivatives, namely; nimustine, lomustine and semustine using molecular modeling and various spectroscopic techniques. Lomustine and semustine structurally differ from each other by single methyl group. Therefore, it is an interesting aspect to perform comparative exploration of nucleic acid binding features of these two, which is also demonstrated in work, presented here.
Chapter 2 deals with the instrumentation and methodology used in the subsequent chapters of the thesis. Stock solution of nucleic acid (DNA and tRNA) were prepared in tris-HCl buffer (pH-7.4, 10 mM) and kept at 8 oC for 24 hours. The solutions were stirred at regular intervals to make sure the formation of homogenous nucleic acid solutions. The final concentration of DNA and tRNA stock solutions were measured spectrophotometrically using molar extinction coefficient of 6600 cm-1 M-1 and 9250 cm-1 M-1 respectively and calculated as 42 mM. Drug’s solutions of varying concentration were prepared by a series of dilutions of stock solution and mixed separately with stock DNA and tRNA solutions of constant concentration to attain different drug/DNA and drug/tRNA molar ratios. Vibrational [Fourier transform
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infrared spectroscopy (FTIR), surface enhanced Raman spectroscopy (SERS)], circular dichroism (CD) and absorption spectroscopic investigations were performed on free nucleic acid and CENUs-nucleic acid complexes at different molar ratios. In silico studies on drug-nucleic acid complexes were also carried out using AutoDock
4.2 software to get insight into the interaction site.
Molecular docking and spectroscopic investigations on the binding properties of chloroethyl nitrosourea derivative; nimustine, lomustine and semustine with DNA duplex are detailed in Chapter 3. The spectral outcomes on nimustine-DNA adducts show that nimustine is a major groove-directed alkylating agent. Further analysis illustrates that interaction of nimustine occurs via guanine (C6=O6) and thymine
(C4=O4) residues reactive sites located in DNA major groove. CD spectral results suggest the formation of an intermediate form of DNA during the transition from B- to C-form at local level after the formation of nimustine-DNA complexes, although globally, DNA remains in native B-form. Spectroscopic observations on lomustine-
DNA complexes suggest that lomustine interacts via guanine (N7) and cytosine (C4) base residues along with slight binding to sugar-phosphate backbone of DNA duplex.
Furthermore, formation of an intermediate stage (B-A-form) of DNA double helix after its interaction with lomustine was also noticed. In case of semustine-DNA complexation, initial interaction of semustine with thymine followed by dG-dC DNA cross-link formation is suggested, which further authenticates molecular modeling prediction. CD spectroscopic data indicates the formation of an intermediary form of
DNA during the transition from B- to C-form locally after semustine-DNA complexation. Furthermore, DNA binding mechanism of semustine has been compared with that of lomustine. This suggests that lomustine has more prominent effect than semustine with respect to their interaction with DNA double helix. These findings may add further understanding about the cytotoxic action mechanism of chloroethyl nitrosourea derivatives at molecular level.
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Study of nature and mode of interaction of chloroethyl nitrosourea derivative; nimustine, lomustine and semustine with transfer RNA (tRNA) using various biophysical and spectroscopic techniques such as attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), Fourier transform infrared difference spectroscopy, circular dichroism (CD) spectroscopy and UV-visible spectroscopy is illustrated in Chapter 4. In addition, molecular docking simulations were performed to predict the preferred orientation of CENUs binding to tRNA molecule, which further help in predicting the strength and site of interaction between the drug and tRNA moiety. Parameters involved in the complexation of CENUs with tRNA including binding affinities were studied in detail. FTIR spectral outcomes suggest that CENUs interact via guanine and cytosine residues of tRNA in addition to slight binding with its sugar-phosphate backbone, authenticating molecular modeling prediction. However, in case of nimustine-tRNA and semustine-tRNA complexes, primarily binding with uracil residue was observed as indicated by FTIR spectral analysis. This augments the possibility of groove-directed alkylation as their anticancer action mechanism. CD spectroscopic data signifies no conformational change in native A-form of tRNA after its complexation with CENUs.
Furthermore, UV-visible studies affirm weak type of binding of CENUs with tRNA.
These findings may further contribute in the development of RNA targeting chemotherapeutic agents.
Chapter 5 includes conclusions of the experimental observations and analysis along with the concluding remarks and future perspectives. Mechanistic understanding of cytotoxicity, exerted by the drugs, is vital to comprehend the molecular basis of their action and subsequent to improve their binding specificity & efficacy. In this perspective, explication of structural-conformational aspects of binding phenomenon with correlation of structure-function relationship becomes significant. Thereby, much attention has been paid towards the investigations of DNA and RNA recognizing agents that particularly target their structural components and can be developed as therapeutics.
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CHAPTER 5 Conclusions and Future Perspectives
““Research is to see what everybody else has seen, and to think, what nobody else has thought”
-Albert Szent-Gyorgi
Conclusions Mechanistic explication of cytotoxicity, exerted by chemotherapeutic agents, is essential to comprehend the molecular basis of their action and subsequent to improve their specificity & efficacy. In this perspective, understanding of structural- conformational aspects of binding phenomenon, correlation of structure-function relationship, thermodynamics properties, nitrogenous base sequence selectivity and linkage between ligand geometry become significant. Besides this, structural stability and conformational features of nucleic acid (DNA and RNA) has always been under an imperative focus owing to its vital role in cellular activities. Thereby, much attention has been paid towards the investigations of DNA and RNA recognizing agents, which particularly target their structural-conformational aspects of nucleic acid and subsequently, can be developed as therapeutics. In the present thesis, interaction mechanism of chloroethyl nitrosourea derivatives; nimustine, lomustine and semustine with nucleic acid (DNA and tRNA) has been investigated using various spectroscopic and molecular modeling techniques. The main conclusions drawn from this study are as follows:
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Mode of action of nimustine: . The docking results on nimustine-DNA complexes predict the major-groove-directed
binding of nimustine with DNA together with the formation of three hydrogen bonds
between nimustine and DNA nitrogenous bases; guanine and cytosine. While, docking
simulations on nimustine-tRNA adducts indicate the interaction of nimustine with C48
(cytosine), 5MC49 (methylated cytosine), U50 (uracil), 1MA58 (methylated adenine)
and U59 residues of tRNA. In addition, nimustine and tRNA complexation is
strengthen by the generation of two hydrogen bonds.
. The vibrational spectroscopic results authenticate docking simulations and
suggest that nimustine is a major groove-directed alkylating agent. Further
analysis illustrates that nimustine interaction occurs via guanine (C6=O6) and
thymine (C4=O4) reactive sites located in DNA major groove. Some degree of
external interaction with phosphate-sugar backbone has also been observed.
. CD spectral results suggest the formation of an intermediate form of DNA during
the transition from B- to C-form at local level after nimustine-DNA complex
formation, although globally DNA remains in native B-form.
. In case of nimustine-tRNA complexation, interaction occurs through guanine and
cytosine residues of tRNA in addition to slight binding with its sugar-phosphate
backbone. However, preliminary binding with uracil residue was also observed
as indicated by vibrational spectral analysis, which augments the possibility of
groove-directed alkylation as its anticancer action mechanism.
. No conformational change in native A-form of biomolecule after its
complexation with nimustine was observed.
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Mode of action of lomustine:
. Analysis of lowest energy docked model of lomustine with DNA reveals that
lomustine interacts with DNA through guanine and cytosine nitrogenous bases with
the formation of two hydrogen bonds between lomustine and guanine. Further,
docking simulations establish the contribution of hydrogen bonds (∆Ghbond) and van
der Waals forces (∆Gvdw) in the overall binding free energy of lomustine-DNA
adducts. In case of lomustine-tRNA interaction, nucleobase residues G51 (guanine),
U52 (uracil), G53, 5MU54 (methylated uracil), 1MA58 (methylated adenine) and
A62 of tRNA are involved in the direct binding with drug’s chloroethyl moiety. The
predicted binding free energy for lomustine-DNA complexation i.e. -6.36 kcal/mol is
found to be more than that of lomustine-tRNA interaction (-4.76 kcal/mol),
indicating more stability of lomustine-DNA adducts than lomustine-tRNA adducts.
. Spectroscopic observations indicate alkylating mode of action of lomustine.
Furthermore, vibrational spectroscopic (ATR-FTIR and SERS) results suggest
that lomustine interacts via guanine (N7, O6) and cytosine (C4) residues along
with minor binding to sugar-phosphate backbone of DNA.
. Formation of an intermediate conformation (B to A-form) of DNA double helix
after its interaction with lomustine was demonstrated by CD spectroscopic
observations.
. Binding effect on the structure of RNA was limited to its interaction with guanine
and cytosine nitrogenous bases in combination with no conformational change in
native A-form of biomolecule (tRNA) after its complexation with lomustine.
. Results, presented here, can be helpful to delineate the anticancer action
mechanism of lomustine at molecular level.
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Mode of action of semustine: . Vibrational spectroscopic investigation concludes that semustine induces major
groove-directed DNA alkylation and produces G-C interstrand cross-links of DNA.
Further, spectral outcomes suggest primarily binding of semustine with thymine
residues followed by G-C cross-link formation, which validate molecular modeling
prediction. In addition, the degree of cross-linking augments with the incubation
time in a drug concentration dependent manner, which at the end achieves a stable
form. Moreover, minor external binding of semustine with phosphate-sugar
backbone of DNA is also indicated by ATR-FTIR spectral results.
. CD spectroscopic data indicates the formation of an intermediary form of DNA
during the transition from B- to C-form locally after semustine-DNA
complexation, although overall DNA remains in native B-form.
. DNA binding mechanism of semustine has been compared with that of
lomustine. This suggests that lomustine has more prominent effect than
semustine with respect to their interaction with DNA double helix.
. In case of semustine-tRNA complexation, interaction occurs through guanine and
cytosine residues of tRNA in addition to preliminary binding with uracil residues.
Besides this, a slight external binding with its sugar-phosphate backbone has also
been observed, which augments the possibility of groove-directed alkylation as
its anticancer action mechanism.
. Biomolecule (tRNA) remains in its native conformation (A-form) after formation
of semustine-RNA complex.
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Future Prospects Outcomes of present investigations address the concern of exploring molecular basis for the anticancer action mechanism of chloroethyl nitrosourea derivatives. The marked difference in their (chloroethyl nitrosourea derivatives) activities in spite of similarity into their molecular structure and cellular target can provide us evidences on utilizing the information for rational development of new chemotherapeutic agents.
In particular, here, we have observed that nucleic acid binding pattern of chloroethyl nitrosourea derivatives; lomustine and semustine differ from each other. This might be due to the distinction in their carbon skeletons, which differ by single methyl group. Outcomes presented in this thesis can not only help in finding the basis behind diverse actions of chloroethyl nitrosourea derivatives inside a cell; like their role in inhibition of DNA replication, DNA repair, RNA transcription and protein translation, also possibly aid in developing analogues for structure-function relationship studies and to find potentially more active and less toxic chemotherapeutic derivatives.
Further, the study can be extended to chemically modify and/or substitute methyl group of semustine to some other reactive groups in cyclohexyl moiety of chloroethyl nitrosourea derivatives. This may enhance the binding of drug with nucleic acid and hence might result in better specificity and efficiency.
Spectroscopic techniques and methodologies used in the present work can be extended to study sequence specific binding of chloroethyl nitrosourea derivatives.
This can be achieved by the construction of sequence specific oligonucleotides stretches and analyzing their binding properties with chloroethyl nitrosourea derivatives. Further, spectroscopic investigations can be extended to explore drugs interaction with modified DNA (methylayed, alkylated and depurinated etc.) in an attempt to observe how modifications in DNA bases affect chloroethyl nitrosourea derivatives binding. Attempts could be made to use DNA from in vivo sources (such
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as from cancerous cell) for similar investigations. Alternatively, drugs treated tumor cells could be used for extraction of chloroethyl nitrosourea derivatives-DNA/tRNA complexes and such complexes could be studied further along similar lines of investigations for verifications of all results, documented here. In future, we wish to collaborate with chemists for incorporating the key modifications into those chemical groups and side chains of CENUs, which can be potential cause for differential mode of action of these chloroethyl nitrosourea derivatives.
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