Unveiling the Potential of Unfused Bichromophoric Naphthalimide to

║Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and. Biotechnology, University of .... can interfere with MT assembly...
0 downloads 11 Views 2MB Size
Subscriber access provided by Caltech Library

B: Biophysical Chemistry and Biomolecules

Unveiling the Potential of Unfused Bichromophoric Naphthalimide to Induce Cytotoxicity by Binding to Tubulin: Breaks Monotony of Naphthalimides as Conventional Intercalators Ritika S Joshi, Dipanwita Das Mukherjee, Subhendu Chakrabarty, Ansie Martin, Manojkumar Jadhao, Gopal Chakrabarti, Angshuman Sarkar, and Sujit Kumar Ghosh J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10429 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Unveiling

the

Bichromophoric

Potential Naphthalimide

of

Unfused to

Induce

Cytotoxicity by Binding to Tubulin: Breaks Monotony of Naphthalimides as Conventional Intercalators Ritika Joshi,† Dipanwita Das Mukherjee,║ Subhendu Chakrabarty,║ Ansie Martin,ǂ Manojkumar Jadhao,† Gopal Chakrabarti,║ Angshuman Sarkarǂ and Sujit Kumar Ghosh†*



Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur,

Maharashtra 440010, India ║

Department of Biotechnology and Dr. B. C. Guha Centre for Genetic Engineering and

Biotechnology, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, WB 700 019, India ǂ

CMBL, Department of Biological Sciences, BITS-Pilani, K.K. Birla Goa Campus,

Zuarinagar, Goa 403726, India Corresponding Author Dr. Sujit Kumar Ghosh, Department of Chemistry, Visvesvaraya National Institute of Technology, Nagpur, Maharashtra, PIN 440010, India, e-mail: [email protected] [email protected] Phone no: + (91) 712 2801775, Fax: + (91) 712-2223230/2801357

1 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2 ACS Paragon Plus Environment

Page 2 of 51

Page 3 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

ABSTRACT: In the development of small molecule drug candidates, naphthalimide based compounds hold a very important position as potent anti-cancer agents with considerable safety in drug discoveries. Being synthetically and readily accessible, naphthalimide compounds with planar architecture have been developed mostly as DNA targeting intercalators. However, in this article we demonstrate for the first time, wherein an unfused naphthalimide-benzothiazole bichromophoric compound 2-(6-chlorobenzo[d] thiazol-2-yl)1H-benzo[de] isoquinoline-1,3(2H)-dione (CBIQD), designed and synthesized by our group, seems to expand the bioactivity of naphthalimide as anti-mitotic agents also. Preliminary studies demonstrate that CBIQD interferes with human lung cancer (A549) cell proliferation, growth and caused cellular morphological changes. However, the underlying mechanism of its anti-tumor action and primary cellular target in A549 cells remained sceptical. Confocal microscopy in A549 cells revealed disruption of interphase microtubule network and formation of aberrant multi-polar spindle. Consistent with microscopy results, UV-Vis, steady-state fluorescence and time-resolved fluorescence (TRF) studies demonstrates that CBIQD efficiently binds to tubulin ( of 2.03 x 105 M-1 ±1.88%), inhibits its polymerization and depolymerises preformed microtubules (MTs). Low doses of CBIQD have also shown specificity towards tubulin protein in presence of a non-specific protein like BSA as well as other cytoskeleton component, actin. The in vitro determination of binding site coupled with in silico studies suggests that CBIQD may prefer to occupy the colchicine binding site. Further, CBIQD perturbed tubulin conformation to some extent and protected ~1.4 cysteine residues towards chemical modification by 5, 5ʹ-dithiobis-2-nitrobenzoic acid (DTNB). We also suggest the possible mechanism underlying CBIQD induced cancer cell cytotoxicity: CBIQD when bound to tubulin, may prevent it to maintain a straight conformation; consequently the α- and β-heterodimers might be no longer available for MT growth. Thus, the consolidated spectroscopic research described herein explores the potential of CBIQD as

3 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a new paradigm in design and development of novel unfused or non-ring fused naphthalimide based anti-mitotic cancer therapeutics in medicinal chemistry research.

4 ACS Paragon Plus Environment

Page 4 of 51

Page 5 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

INTRODUCTION The important therapeutic strategies to tackle cancer1 include modulation of angiogenesis, interfering with DNA synthesis, transcription and translation, inducing DNA damage, inhibiting the function of mitotic spindle etc. Of these, the highly dynamic mitotic-spindle microtubules (MTs) and their building block, αβ tubulin, that have an important role in key cellular events represent the most successful target to be identified so far for the anticancer therapy.2–5 At least one reason that cancer cells are relatively sensitive to mitotic inhibitors compared with normal cells is that cancer cells divide more frequently than normal cells and therefore are more susceptible to mitotic poisons. MTs, composed of α- and β-tubulin heterodimers, are highly integrated cytoskeletal fibers and their functions are well orchestrated in cells. MTs are hollow tubes approximately 25 nm in diameter that radiate from microtubule-organizing center located at the centrosome in the cytoplasm of interphase cells. They provide and maintain cell shape, structure, are involved in cell movement, intracellular transport and cell division.2,6,7 The integrity of MT structures is essential for cells to go through various cell cycle checkpoints because without it, programmed cell death or apoptosis is triggered. A very important structure generated from MTs is the mitotic spindle, which is used by eukaryotic cells to segregate their chromosomes correctly during cell division and allow transferring of chromosomes to the daughter cells. So the compounds that can suppress spindle MT dynamics and intervene with cell division process or metaphase to anaphase transition point may serve as potentially fruitful avenue for future anti-mitotic drug development. Several structurally diverse compounds,8–13 that may bind to any one of the three well known characterized tubulin binding sites (colchicine site and vinblastine sites as well as taxol site), either destabilize or stabilize MTs by perturbing the mitotic spindle functions and inhibiting cell division at the interphase or metaphase to anaphase transition of mitosis. The first of these group which 5 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

destabilize the MTs include Vinka alkaloids, cryptophycins, colchicine, vinblastine that have distinct binding sites on tubulin,14–17 while the second group of compounds which stabilize the MTs include epothilones, taxol, discodermolide that share overlapping binding sites in tubulin.14,18 The clinical success of Vinka alkaloids, estamustine, and taxanes has prompted the search for new anticancer agents that target MTs and several MT poisons are currently undergoing clinical trials for cancer chemotherapy.14,15,18 The naphthalimide core scaffold, with an enormous intrinsic modularity,19–21 has generated immense interest amongst the researchers who straddle the fields of chemistry and biology. The rigor of their traditional status as robust fluorescent probes has been preserved in almost all the derivatives reported21–30 yet their biological activity has been strictly confined either mostly as traditional DNA intercalators19,31–40 or topoisomerase II inhibitors,41,42 as far as their anti-cancer activity is concerned. A plethora of naphthalimides (including heterocyclic ring fused derivatives) have been developed till date that have shown anti-cancer activity against leukemia (KBM-3, HL-60), human CX-1 colon, MCF, A549, HeLa cell lines by targeting DNA directly (intercalator or groove binders), down regulating the transcription of genes, or inhibiting the activity of topoisomerase II.19 Nevertheless, it is important to highlight that heterocyclic ring tailored unfused naphthalimides have not been explored till date, and that no naphthalimide is (serendipitously) discovered till date which can interfere with MT assembly-disassembly, to the best of our knowledge. Under these premises, we recently designed and synthesized a bichromophoric unfused naphthalimide-benzothiazole conjugate (CBIQD) which fluoresces across the entire polarity scale on account of its rehybridised intramolecular charge transfer state (RICT)24 and has even shown the ability to phosphor at RT.43 Further, CBIQD has shown its antiproliferative effect on A549 lung cancer cells by causing severe morphological changes: the strongest evidence indicating that tubulin might be the target for CBIQD in A549 cells.

6 ACS Paragon Plus Environment

Page 6 of 51

Page 7 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Moreover, the efficacy of single molecular construct like CBIQD, which may integrate the advantage of imaging and therapeutic functions to achieve the ultimate goal of simultaneous diagnosis and treatment without the use of co-staining, is the need of the hour. So, in this article, we explore for the first time the mode of interaction of an unfused bichromophoric naphthalimide CBIQD to tubulin. Our study represents a small molecule based approach to better understand the mechanistic details of tubulin-CBIQD interactions, and thereby provide a molecular link to the cellular events that proved lethal to A549 cells.

EXPERIMENTAL SECTION Materials. CBIQD (Scheme 1) used for the present study is designed and synthesized by our group and is reported elsewhere.24 Colchicine, 4′, 6-diamidino-2-phenyl-indole (DAPI), Vinblastine, Bis-ANS, 5, 5′-dithiobis-2-nitrobenzoic acid (DTNB) is procured from Sigma Aldrich (USA) and used as received. Tubulin protein (>99%, bovine), Guanosine 5′triphosphate sodium salt (GTP), General Tubulin Buffer (GTB) (lyophilized powder) is acquired from Cytoskeleton Inc., USA. GTB powder constituting appropriate amounts of 80 mM PIPES, 2 mM MgCl2, 1 mM EGTA is re-suspended in 100 ml of Millipore water to give 1X strength buffer and is further used for experiments. Primary antibodies to α-tubulin and TRITC-conjugated secondary antibodies are purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Genei respectively. Millipore water is used wherever required. All general laboratory chemicals are purchased either from Sigma-Aldrich (USA), Invitrogen (USA) or from Sisco Research Laboratories. Human broncho-alveolar carcinoma-derived (A549) cells are obtained from National Centre for Cell Sciences (NCCS) Pune, India. Instrumentation and Methodology. In Vitro culture of A549 Cells. A549 cells are cultured in vitro in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) at 7 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

37 °C with 5% CO2. For all experiments, cells are seeded at the density of 3 × 104 cells/ml in 6 well plates. Sample Preparation for Anti-Proliferative Activity. Highly concentrated stock solution of CBIQD in spectroscopic grade DMSO (Spectrochem, India) is prepared initially. When the cells became 70% confluent they are exposed to different dosages like 7, 14, 55, 137, and

274 µM of CBIQD. A control with no CBIQD exposure is also maintained

alongside to compare and contrast the pattern of growth and cell morphology. Cell Growth, Cell Morphology Analysis. Following CBIQD exposure at various dosages cell proliferation and growth is monitored by determining the cell number using neubauershemocytometer and cell morphology is analysed and documented using inverted optical microscope (Nikon Eclipse TS 100).44 The photographs are captured using a digital compact camera system attached to the microscope. Confocal Microscopy. CBIQD is treated with A549 cells grown as a monolayer on a glass coverslip for a period of 24h. Subsequently, the cells are washed with phosphate buffer saline (PBS), fixed with 2% formaldehyde for 30 min and permeabilized with 0.1% sodium citrate and 0.1% Triton X for 15min. 5% BSA is used to block nonspecific binding. The cellular MT is probed with the anti-α-tubulin primary antibody (1:200 dilution) followed by the TRITC-conjugated secondary antibody (1:100 dilution), and the nuclear DNA is counterstained with DAPI. The images are obtained with an Olympus LSM IX81 confocal microscope and analysed with Olympus Fluoview.45 Cell Viability Assay. The effect of CBIQD on the viability of A549 cells and WI-38 cells (normal lung fibroblast) is determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) assay. Cells are plated in 96-well culture plate (1 x 104 cells per well) and treated with various concentrations of CBIQD (0-50 µM) for both 24h. MTT solution (5 mg/ml) is prepared in PBS, filter sterilized, and 20 µl is added to each well. The

8 ACS Paragon Plus Environment

Page 8 of 51

Page 9 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

purple precipitate formed is dissolved in 100 µl Triton-X and the absorbance is measured on an ELISA reader (MultiskanEX, Lab systems, Helsinki, Finland) at a test wavelength of 570 nm and a reference wavelength of 650 nm. Data is analyzed by plotting percent of viable cells against [CBIQD], considering control cells as 100% viable. Steady-state UV-Vis Absorption and Fluorescence Measurements: JASCO V-630 Spectrophotometer, equipped with a matched pair of quartz cuvettes (1.0 cm path length) and a Peltier temperature control system, is used to collect absorption spectra. The fluorescence spectra are collected by JASCO FP-8300 Spectrofluorometer equipped with 1.0 cm path length 9FL Semi-Micro fluorescence cuvette. The excitation and emission slit widths are set at 2.5 nm. The fluorescence intensities are corrected for inner filter effect at the excitation and emission wavelengths of protein, using the following equation:46  =   

 +   2

where,  and  are the corrected and observed fluorescence intensities, respectively, and  and  are the absorbance of CBIQD at the excitation and emission wavelengths, respectively. Time Resolved Fluorescence Measurements: Time-resolved fluorescence decay measurements are carried out by the method of time-correlated single-photon counting (TCSPC) technique using a PTI Pico Master TSCPC Spectrofluorometer with a PMH-100-4 detector. A 280 nm LED is used as an excitation source and the lifetime is recorded on a 150 ns scale. The FelixGX software is used for fluorescence data collection and analysis. The goodness of fit is judged by χ2 values and Durbin-Watson parameters and visual observations of fitted line, residuals, and autocorrelation function. For all lifetime measurements, the fluorescence decay curves are analyzed by single, bi and tri-exponential iterative fitting program such as:47    = ∑  exp −τ⁄τ$  9 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

where,  is a pre-exponential factor representing fractional contribution to the time-resolved decay of the component with a lifetime & , mean lifetimes &' for bi-exponential decays of fluorescence are calculated from decay times and pre-exponential factors using the following equation: &' =

() *) + (, *, () + (,

Inhibition of Tubulin Polymerization by CBIQD: Different concentrations of CBIQD

(0-25.48 µM) are mixed with bovine tubulin protein (1 µM), purchased from

Cytoskeleton Inc., in GTB (containing 80 mM PIPES, 2 mM MgCl2, 1 mM EGTA) in the presence of

1 mM GTP and the polymerization reaction is initiated by incubating the

sample at 37 ºC. The rate and extent of polymerization reaction is monitored by measuring absorbance at 350 nm48 for 40 min using JASCO V-630 Spectrophotometer. A well-known DNA intercalator, DAPI, is used as a fluorescent probe for tubulin and MTs.49 DAPI whose binding site is different from that of Colchicine, Vinblastine, or Taxol, does not interfere with MT dynamics but the relative fluorescence intensity of MT bound DAPI is significantly higher than tubulin bound DAPI.49 Thus, if CBIQD induces MT depolymerization, it would reduce the fluorescence intensity of MT-DAPI complex. Under similar conditions as described above, tubulin (1 µM) is polymerized along with 1 µM DAPI. The fluorescence response of the polymerized MT-DAPI complex is monitored to see the effect of different concentrations of CBIQD when added to this system. The excitation and emission wavelengths are 390 nm and 450 nm respectively. Tubulin-CBIQD Binding Measurements by Fluorometric Titration. Quenching of the Protein Fluorescence. Excitation of aqueous protein solution at 280 nm, leads to emission from both tryptophan (Trp) and tyrosine (Tyr) residues, but most of the fluorescence comes from Trp residues because of efficient resonance energy transfer from Tyr to Trp. So, the binding affinity of CBIQD to tubulin is assessed by monitoring the 10 ACS Paragon Plus Environment

Page 10 of 51

Page 11 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

alteration in the intrinsic fluorescence of tubulin at an excitation wavelength 280 nm. To explore the quenching phenomenon, fluorescence quenching data is subjected to SternVolmer (SV) analysis using the following well-known SV equation:46 = 1 +  / 012 = 1 + 34 &- 012  34 =

 / &-

where, - and  are the steady-state fluorescence intensities in the absence and presence of quencher, respectively,  / is the SV quenching constant, and 012 is quencher (CBIQD) concentration, &- is the average lifetime of protein in the absence of quencher. The binding constant and the number of binding sites is determined according to the double-logarithmic equation as follows:46 - −   5 6 =  + 012  where, F0 and F are the fluorescence intensities in the absence and presence of CBIQD, respectively, n is the binding stoichiometry and  is the binding constant. According to the above equation, a plot of log [(F0-F)/F] versus log [Q] will produce a straight line whose slope is equal to " ". Determination of Binding Site. To identify the CBIQD binding site inside the tubulin scaffold, the competitive binding with the standard site markers colchicine and vinblastine is experimented by measuring the change in the fluorescence of tubulin-CBIQD complex. CBIQD exhibits intense emission in aqueous medium on account of its RICT state24 and so this emission can be probed to decipher its binding location in tubulin in the presence of increasing concentrations of site markers (colchicine and vinblastine). The extent of CBIQD emission quenching in the presence of site markers may give a direct measure of its binding location in tubulin protein. For this, tubulin (1 µM) is incubated with CBIQD (1 µM) for 30 min at 37 ºC. Then different concentrations of colchicine (0-20 µM) are added to the tubulin11 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CBIQD complex reaction mixture and emission spectra are recorded following 5 min incubation at each aliquot of colchicine addition. The excitation and emission wavelengths are 344 nm and 397 nm respectively. The above procedure is also adopted for studying competition between CBIQD and vinblastine to occupy the same binding site on tubulin. The concentration of vinblastine used is 0-80 µM. Titration of Tubulin Sulfhydryl Groups. The sulfhydryl specific reagent, frequently called Ellman’s Reagent or DTNB complexes with thiol groups in tubulin to form a mixed disulfide of the protein and one mole of 2-nitro-5-thiobenzoate (TNB2-) per mole of protein sulfhydryl group. The rate and extent of sulfhydryl group modification can be monitored by measuring the absorbance changes at 412 nm.50,51 Tubulin (1 µM) is incubated with 5 µM CBIQD at 4 ºC for 15 min, and then 70 µM of DTNB is added. The number of sulfhydryl groups modified after 50 min of reaction is determined by using a molar extinction coefficient of 12000 for TNB2- at 412 nm. The linear rate of sulfhydryl group modifications is obtained by plotting  08⁄8 − 9 2 versus time, where 8 is the absorbance of TNB2- at saturation and 9 is the absorbance at different times of the reaction. In silico Studies. The computational molecular docking studies are carried out using Linux centos 6 operating system, 64 bit Intel ®core™ i5-2500 CPU @ 3.30 GHz, 4 GB RAM using Schrödinger suite 2014 by Maestro 9.9 (Maestro, version 9.9, Schrödinger, LLC, New York, NY, 2014). For Glide docking, CBIQD is prepared by Ligprep52 using OPLS (Optimized potential for liquid simulations) 2005 force field to rectify molecular geometries, retain specific chirality and to get least energy conformations required for docking. The tubulin protein (PDB ID: 3UT5) is first prepared using Protein Preparation Wizard workflow included in the Maestro suite.53 The protein is preprocessed to add hydrogen, all the missing atoms and loops are filled using prime followed by optimization of the H-bond especially for Asp, Glu, His, hydroxyl containing residues and then minimization is carried out with

12 ACS Paragon Plus Environment

Page 12 of 51

Page 13 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

OPLS2005 force field,54,55 with the aim of relaxing the atomic coordinates until geometric convergence (RMSD of 0.3 Å) is achieved. The position of the inhibitor in the corresponding crystal structure is used to define the binding site. It is here where the receptor grid is generated using the receptor grid generator workflow in the region which is defined as 5 Å around the co-crystallized ligand (Colchicine). Water molecules in the corresponding structures are treated as rigid molecules. Docking is performed using GLIDE’s extra precision (XP) mode56,57 where only the ligand is flexible. The conformer of ligand which is found to be best complementarily and least penalty scores with minimum binding energy against the receptors is picked up for further analysis. This energy based scoring function includes terms accounting for short range van der Waals, electrostatic interactions, Hbonding and penalty scoring function. Additionally, Induced Fit Docking58,59 (IFD) is also performed and the resulting top poses are used to sample the protein plasticity using the prime program in Schrödinger suite. Further, molecular dynamics (MD) simulation is performed using Desmond60,61 that involves three steps which are system building, minimization followed by NPT ensemble molecular dynamics. The TIP3P solvation model is set with orthorhombic box shape where box volume is calculated and minimized to least possible number. The ions are neutralized by adding Na+ salt with OPLS 2005 force field. The solvated ligand receptor complex is further minimized with maximum iterations of 2000 and convergence threshold of 1.0 kcal mol-1 Å-1. The minimized solvated ligand receptor complex is processed for 15 ns simulation period, recording interval of 1.2 ps, trajectory of 4.8 NPT ensemble method with Nose–Hoover chain, relaxation time 1.0 ps, temperature 298 K and pressure 1.01325 bar with facility to relax model before simulation with RESPA integrator by time step bonded 2.0 fs. Coulombic short range method cut off is with 9 Å and long range method of smooth particle mesh Ewald with Ewald tolerance of 10-9.

13 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 51

Scheme 1: Structure of CBIQD

RESULTS Effects of CBIQD on A549 Human Lung Cancer Cell Lines.

Our quest for

developing therapeutic anticancer agent, CBIQD (Scheme 1), will stay unreciprocated unless initial but indispensable anti-proliferative activity on cancer cell lines provides us a positive intimation for the same. By determining the cytotoxicity levels, the proposed unfused naphthalimide candidate can hinder the proliferation of target cells either by obstructing the function of the genetic material or by causing severe alterations in the cellular morphology. From the published literature, it is documented that sulfur containing heterocyclic naphthalimide derivatives mostly exhibit their anticancer activity on A549 (human lung cancer), HeLa (human cervical), MCF7 (human breast cancer), P388 (human colorectal adenocarcinoma) and HT29 (human melanoma) cell lines.19 Based on the literature survey as well as the availability of cell lines for in vitro studies with us, we have performed the antiproliferative activity on A549 and HeLa cell lines. CBIQD has shown promising cell growth inhibitory activity as well as retardation effects on A549 as well as in HeLa cell lines (SI: Figure S1). The anti-proliferative activity of CBIQD studied in a time as well as dose dependent manner on A549 cells cultured in vitro is discussed herewith in details which suggests that the cell numbers are reduced when treated with 7 µM of CBIQD (Figure 1A) in 24 h when 14 ACS Paragon Plus Environment

Page 15 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

compared to that in control. Although the cell numbers are further reduced on gradual increase in CBIQD concentration from 14 µM to 274 µM but the effect is more pronounced in 7 µM treatments in 24 h. At higher CBIQD concentration of 137 µM pronounced cell death is seen and the cell number reduces to 3 x 104 cells after 48 h exposure. Drastic changes in cell morphology are observed on treatment of 14 µM CBIQD in 24 h (Figure 1B), where cells started rounding up and retracting from the surface. This morphological change may suggest the cell death through cytoskeleton disturbances, which play a prominent role in cell cycle, morphogenesis and migration. Thus, it will be interesting to decode the detailed mechanism of such cell death and the involvement of CBIQD in such kind of phenomena. Effect of CBIQD on Normal Lung Fibroblast (WI-38) Cells. Upon comparison of the cytotoxic effect of CBIQD on normal lung fibroblast WI-38 cells with that on lung carcinoma A549 cells by performing MTT assay, it is demonstrated that cytotoxicity is much lower in WI-38 cells (Figure 1C) compared to A549 cells after 24 h treatment with CBIQD. In A549 cells, 3 µM CBIQD caused significant cell death (around 40%) and 50 µM CBIQD caused 80% cell death with calculated IC50 dose around 6 µM CBIQD in 24 h, while in WI38 cells, 20% cell death is observed even after treatment with 50 µM CBIQD (Figure 1C).

15 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. (A) Effect of different dosages of CBIQD on A549 cell viability after 24 h and 48 h of exposure time period (B) Changes in human A549 cell morphology after exposing the cells to different concentration of CBIQD.

16 ACS Paragon Plus Environment

Page 16 of 51

Page 17 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 1. (C) MTT assay showing effect of CBIQD on viability of both A549 cells and WI-38 cells after treatment with different doses of CBIQD (0-50 µM) for 24 h. Data represents mean ±SD.

Perturbation of Cellular Microtubules by CBIQD Treated A549 Cells.

As

CBIQD inhibited the cell growth and number and caused cellular morphological alterations in cultured A549 cells in vitro, it may be comprehended that the normal function of tubulin/ MTs, which is an important constituent of the cytoskeleton and plays a crucial role in cell division process, may be hampered. So, we further monitored the direct effect of CBIQD on the integrity of the mitotic spindle and cellular MT network of A549 cells by confocal microscopy. In the control cells treated with 0.1% DMSO, the interphase MTs illustrate a well-organized, intact and radial architecture (Figure 2A), however upon CBIQD treatment, a dose dependent disruption and disorganization of the interphase MT network is observed (Figure 2A). At low doses (3.5 µM), CBIQD started MT disruption at periphery, making the cells contracted. Near IC50 dose, i.e. 7 µM, MT network is more disrupted and cells become more round shaped, while a higher dose (15 µM) drastically distorts MT structure. The similar type of disorganization of interphase MT structure is observed upon treatment with 50 nM colchicine (well- known MT depolymerizing agent) (Figure 2A). On the other hand, the spindle MTs show normal bipolar organization enabling proper metaphase to anaphase chromosomal transition in the control cells (Figure 2B). At low doses of CBIQD (3.5 µM), the disruption of mitotic spindle is apparent. However, depolymerization of the spindle MTs is initiated at 7 µM CBIQD while at 15 µM, formation of aberrant multi-polar spindle is observed. Moreover, at high concentration of 25 µM, complete loss of spindle MTs is apparent (Figure 2B). To confirm MT loss, the change in the intensity of Rhodamine (tagged with secondary antibody) fluorescence by Olympus Fluoview

17 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ver. 4.2a is monitored. Treatment with CBIQD results in a gradual decrease in fluorescence intensity in a dose dependent manner as in case of colchicine treated positive control (SI: Figure S2). Thus, the above results are critical in contributing considerably to the more efficient evaluation of CBIQD’s direct effect on MT network, and hints that CBIQD might be interfering with the MT-tubulin dynamics in some way. So, at this juncture, it is highly probable that MT or tubulin is the appropriate target responsible for the anti-proliferative activity exhibited by CBIQD on A549 cells. Hence, it is worthwhile to investigate the interaction of the synthesized compound with tubulin and preformed MTs individually.

Figure 2. Dose dependent disruption of the (A) interphase microtubules (Scale bar = 20 µm) and (B) spindle microtubule (Scale bar = 10 µm) of cultured A549 lung cancer cells by

18 ACS Paragon Plus Environment

Page 18 of 51

Page 19 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

CBIQD upon 24h treatment. Colchicine (Col; 50 nM) is used as the positive control. Immunofluorescence images of the microtubules (red) and nucleus (blue) are obtained by an Olympus confocal microscope. Effect of CBIQD on Cell Cycle Progression of A549 Cells.

The cell cycle

progression of A549 cells is further examined by flow cyometric analysis, using BD Accuri flow cytometer, of the cellular content of DNA upon treatment with 3.5 µM and 7 µM CBIQD for 24 h. It is demonstrated that 3.5 µM and 7 µM CBIQD causes G0/G1 arrest, accumulating 61.6% and 63.7% cells, respectively, in that phase compared to 56% in untreated control cells (SI: Figure S3). The accumulation of cells in G0/G1 phase seem to occur at the expense of both S-phase and G2/M phase as cell population in S- and G2/M phases is reduced down in the treated cells compared to control cells. Although G2/M arrest is a hallmark of MT damage, several reports indicate where MT disintegration is associated with G0/G1 arrest. Interphase MT depolymerization causes activation of p53 through integrinErk signaling62 which activates G1 checkpoint leading to G0/G1 cell cycle arrest.63 Several MT inhibitors which are also inhibitors of checkpoint kinases block cell cycle at G0/G1 phase instead of G2/M phase.64 Moreover, some inhibitors which are reported to disrupt interphase MTs at low doses and spindle microtubule at higher doses, are found to arrest cell cycle at G1 phase at low doses.63,65 In our study we found that CBIQD depolymerizes interphase MTs at low doses, which could have activated G1 checkpoint or the compound might have other targets as well, inside cells, especially G1-checkpoint proteins, which may be investigated in future. Inhibition of Tubulin Polymerization (into microtubules) by CBIQD.

Since

CBIQD can disrupt the MT network in cultured A549 cells (as observed from microscopy studies), we wanted to examine whether it could effectively impede the self-assembly of tubulin into MTs, which is the most important property of tubulin. So tubulin is polymerized 19 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in the absence and presence of different CBIQD concentrations as described in experimental section. As depicted in Figure 3A, the bichromophoric compound produced a concentration dependent inhibition in the rate and extent of MT polymerization. A concentration of 23 µM CBIQD decreased the extent of polymerization by ~30%, while 32 µM CBIQD inhibited polymerization by ~40%. However, the time taken by the unfused naphthalimide each instance increased with an increase in [CBIQD]. Also, the half-maximal inhibition (IC50) of polymerization is calculated to be ~41 µM (Figure 3B).

Figure 3. Inhibition of microtubule assembly by CBIQD (A) Polymerization of tubulin (1µM) in assembly buffer is measured in the absence (■) and presence of 1 (●), 2 (▲), 7 (▼), 13 (◄), 26 (►), 40 (♦), 55 (□), 70 µM (○) CBIQD. Microtubule assembly is monitored by measuring absorbance at 350 nm with time. (B) Inhibition of tubulin polymerization as a function of [CBIQD]; time = 40 min. Data are the average of three independent experiments. 20 ACS Paragon Plus Environment

Page 20 of 51

Page 21 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Induction of Depolymerization of Preformed Microtubules by CBIQD. Since the synthesized unfused bichromophore could disrupt and disorganize the MT network of A549 cells, it becomes necessary to access whether it can disturb the tubulin-MT equilibrium dynamics or not. So, it is attempted to investigate the depolymerization action of CBIQD on preformed MTs by two different approaches. In the first approach, alteration in the optical density at 350 nm is monitored as a measure of polymer mass.48 Tubulin (1 µM) is polymerized in the presence of GTP as described in the experimental section, to this preformed MT suspensions, different concentrations of CBIQD are added. From Figure 4A it is apparent that concentration of 6.37 µM yielded ~59% diminution in the absorbance values of the polymer mass, while 25.48 µM downsized the absorbance values to ~69%. For all the concentrations of CBIQD used, the variation in the absorbance stabilized above 15 min. The pattern of decrease in O.D. values induced by CBIQD is also compared and contrasted against a positive control, colchicine. It is demonstrated that ~2.5 µM of CBIQD is able to cause nearly a same decrease in O.D. values as observed for 1 µM of colchicine, implying that ~2.5 µM of CBIQD and 1 µM of colchicine can cause the depolymerization of preformed MTs to the same extent, however, the depolymerization kinetics followed may be different in both the cases. To further confirm the depolymerizing effect of CBIQD on preformed MTs, second approach is employed wherein any decrease in the extrinsic fluorescence of MT-DAPI complex is monitored as a measure of MT depolymerization.49 It is observed that CBIQD quenches the fluorescence of MT-DAPI complex in a concentration dependent manner, suggesting that CBIQD induces MT depolymerization (Figure 4B). Control experiments using colchicine as a positive control and DMSO as a negative control are also performed to elucidate that the observed decrease in the emission intensity of MT-DAPI complex is not because of any artifacts but indeed is due to the depolymerization of preformed MTs (Figure

21 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

4B). It is observed that colchicine could effectively cause a diminution in MT-DAPI complex fluorescence, whereas the MT-DAPI complex fluorescence intensity remained steady without any detectable decrease. Hence, from the above two different experimental approaches it is indicated that CBIQD not only hinders association of free tubulin to form MTs but also depolymerizes preformed MT assembly. Therefore, it will be intriguing to monitor the binding association (if any) of CBIQD with tubulin heterodimer and to further decode the binding mechanism involved in the binding process.

Figure 4. Disruptive effects of CBIQD on preformed microtubules. (A) Depolymerization of preformed microtubules is monitored by recording absorbance at 350 nm in the absence (■) and presence of 1 µM colchicine (●), 1 (∆), 2 (▼), 4 (◄), 7 (►), 13 (○), 16 (□), 26 µM (▲) CBIQD. Tubulin is first polymerized in the assembly buffer for 30 min at 37 ºC and different concentrations of CBIQD are then added to the reaction mixtures. Data are the average of 22 ACS Paragon Plus Environment

Page 22 of 51

Page 23 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

three independent experiments. (B) Microtubule depolymerization is monitored by measuring the decrease in MT-DAPI fluorescence in the presence of different [CBIQD]. Tubulin (1 µM) is first polymerized in the assembly buffer with 1 µM DAPI for 30 min at 37 ºC. Then DMSO (■), 1 (●), 2 (▲), 4 (▼), 8 (□), 12 (►), 16 (∆), 26 µM (○) CBIQD and 1 µM colchicine (*) is added; λex = 390 nm; λem monitored = 450 nm. Data are the average of three independent experiments. Binding Association of CBIQD to Tubulin. The emission spectra of tubulin protein in the absence and presence of increasing concentration of CBIQD at 25 ºC is illustrated in Figure 5A. In the absence of CBIQD, the emission maximum of tubulin in GTB is centered at 327 nm. Successive addition of CBIQD results in the drop of fluorescence intensity at 327 nm along with a significant 7 nm blue shift. This hypsochromic shift in the emission maximum along with reduction in fluorescence intensity indicates that the interactions of tubulin with ligand change the microenvironment with probable increase in hydrophobicity in the vicinity of binding location. In Figure 5A, quenching is accompanied with simultaneous formation of a new band around 375 nm with a shoulder around 395 nm and the intensity rises with subsequent increase in [CBIQD]. This could be because of the fact that CBIQD also has an appreciable absorption near the excitation wavelength (280 nm) as well as in the range 340 nm24 where the protein emits. However, CBIQD does not show any considerable emission in the region of protein fluorescence.24 The new bands in the red region are out of scope of present discussion. The SV plot shows a slight upward curvature towards the y-axis at higher concentrations of the guest molecule (Figure 5B, inset), so the data points at lower ligand concentrations following linearity (Figure 5B), are used to determine SV quenching constant of 3.56 x 104 L.mol-1 which may suggests involvement of static quenching process only. Further, the binding constant (Kb) obtained for CBIQD-tubulin interaction from steadystate measurements, is found to be 2.03 x 105 M-1 (± 1.88%) implying a stronger binding. 23 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Additionally a straight line obtained by applying double-logarithmic equation yielded slope (n) = 1.15 (SI: Figure S4) which points towards a single binding site. Generally, the exact quenching mechanism operative in the binding process can be confirmed by lifetime measurements. The &' values of Trp residues in tubulin experienced only a slight decrease from 2.95 ns to 2.45 ns with increasing [CBIQD] (Table 1, Figure 6A). Moreover, the plot of &- ⁄&' versus [CBIQD] (Figure 6B) also follows linearity indicating involvement of only static quenching mechanism in the binding process. The minor alteration (decrease) in &' values (Table 1), signifies that the hydrophobicity might have been increased to a certain extent.

Figure 5. (A) Emission spectra illustrating quenching of intrinsic tubulin fluorescence with an increase in CBIQD concentration. Stock: [CBIQD] = [Tubulin] = 1mM; curves a → q: 24 ACS Paragon Plus Environment

Page 24 of 51

Page 25 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

[CBIQD] = 0, 2.12, 4.23, 6.33, 8.42, 10.50, 12.60, 14.60, 16.70, 18.80, 20.80, 22.80, 24.90, 26.90, 30.90 and 32.86 µM; λex = 280 nm; pH 7.0; T = 298 K (B) Inner filter effect corrected Stern-Volmer plots for the interaction of CBIQD and tubulin at lower [CBIQD]; inset: at all [CBIQD].

Figure 6. (A) Decay profile of representative tubulin-CBIQD systems at 25 ºC; curves a → f: [CBIQD] = 0, 2, 6, 13, 23, and 33 µM. (B) Plot of :; ⁄:< vs [CBIQD]. Table 1. Fluorescence Presence of CBIQD [CBIQD] τ1 (µM) (ns) 00.00 1.73 01.00 1.72 02.00 1.71 06.00 1.64 13.00 1.54 23.00 1.53

Decay Parameters of Tubulin in the τ2 (ns) 4.59 4.57 4.57 4.48 4.40 4.38

α1 (%) 57.44 58.06 59.08 59.10 60.16 64.06

α2 (%) 42.56 41.94 40.92 40.90 39.84 35.94

25 ACS Paragon Plus Environment

τf (ns) 2.95 2.92 2.88 2.80 2.68 2.55

χ2 1.14 1.08 1.05 1.03 1.16 1.03

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

25.00 1.51 4.36 64.11 35.89 2.53 1.12 33.00 1.50 4.29 66.74 33.26 2.45 1.08 Deciphering the Binding Site of CBIQD inside Tubulin. Colchicine and Vinblastine are well-characterized drugs that bind to tubulin, inhibit MT polymerization and suppress MT dynamic instability.66 Despite their similar actions, colchicine and vinblastine localizes themselves in different binding sites in tubulin. It has been demonstrated that structurally unrelated natural and synthetic compounds that inhibit MT polymerization bind either to colchicine or vinblastine binding site in tubulin.67–70 Similar to above two sitemarkers, our synthesized CBIQD also inhibits tubulin polymerization and induced depolymerization of preformed MTs as discussed earlier (Figure 3 & 4). Thus, we aimed to scrutinize the binding location of CBIQD i.e. whether it binds at the colchicine or vinblastine site. CBIQD is a highly fluorescent naphthalimide bichromophoric system due to formation of RICT state in aqueous medium24 and its fluorescence intensity remains unchanged with time on binding to tubulin (SI: Figure S5). So we probed the fluorescence of tubulin-CBIQD complex in the presence of increasing concentration of site markers independently (as described in experimental section). During the experimentation, the tubulin-CBIQD complex fluorescence is quenched to a greater extent (24.12%) upon subsequent addition of colchicine to the system (Figure 7A), whereas the diminution is to a much lesser extent (2.37%) when vinblastine is introduced into the tubulin-CBIQD system (Figure 7B). The total concentration of colchicine (21.29 µM) that brings about 24.12% quenching of CBIQD fluorescence, is much lesser than the total concentration of vinblastine in the system (53.01 µM) that hardly quenches CBIQD intrinsic fluorescence. Taking together, the presence of colchicine has some implications on CBIQD binding to tubulin and it can be suggested that colchicine strongly affected tubulin-CBIQD association while vinblastine could not. Thus, there is a competition that exists between colchicine and CBIQD to occupy the same binding site on tubulin, demonstrating that CBIQD binding site on tubulin overlaps with that of the

26 ACS Paragon Plus Environment

Page 26 of 51

Page 27 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

colchicine binding site. It is pertinent to mention here that the fluorescence band structure and shape of CBIQD in buffer when obtained at 37 ºC is different from the one obtained at 25 ºC. The reason for the observed variation in band structure might be because of the alteration in the emissive states of CBIQD on heating; nevertheless, no significant shift in fluorescence emission maximum is noted.

Figure 7. Colchicine inhibits the binding of CBIQD to tubulin, but vinblastine does not. Tubulin (1 µM) is incubated with CBIQD (1 µM) for 30 min and then different concentrations of colchicine or vinblastine is added to the mixture. CBIQD emission is monitored at 397 nm after an incubation of 15 min at every successive addition of the site marker; λex = 344 nm, T = 37 ºC. (A) Emission spectra of CBIQD-tubulin system in the presence of increasing [Colchicine]; curves a → p: [Colchicine] = 0, 1.10, 2.19, 3.28, 4.38, 27 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

6.54, 7.62, 8.69, 9.76, 10.83, 12.95, 15.06, 17.15, 19.23 and 21.29 µM. (B) Emission spectra of CBIQD-tubulin system in the presence of increasing [Vinblastine]; curves a → m: [Vinblastine] = 0, 3.87, 7.73, 11.57, 15.41, 19.23, 23.04, 26.83, 30.61, 34.38, 38.13, 45.59 and 53.01µM; λex = 344 nm, T = 37 ºC. In addition to the above method which requires monitoring CBIQD-tubulin complex fluorescence at 397 nm, attempts were also made to incubate tubulin with colchicine and vinblastine separately and then CBIQD is titrated into the two systems individually to concretize the binding site of CBIQD inside the tubulin scaffold. The first case with colchicine (as the site marker) requires to monitor the quenching of tubulin-colchicine complex fluorescence at 430 nm by keeping an excitation wavelength of 360 nm. However, when the system containing equimolar mixture of tubulin-colchicine complex together with CBIQD in buffer is excited at an excitation wavelength of 360 nm, CBIQD also gets excited due to its appreciable absorbance in 360 nm region.24 Additionally, since CBIQD is highly emissive in aqueous/buffer medium24 the high fluorescence from CBIQD overpowers or other way around it nearly tends to suppress the emission of tubulin-colchicine complex to be monitored in 430 nm region (figure not shown). Thus, under such unavoidable technical circumstances/ inconvenience as explained above, it is difficult to ascertain the binding site of CBIQD by monitoring the tubulin-colchicine complex fluorescence within acceptable systematic error. On the other hand, the site marker competitive experiment with vinblastine requires to monitor the quenching of tubulin-vinblastine complex fluorescence at 535 nm by keeping an excitation wavelength of 385 nm. In this case since, CBIQD has negligible absorbance around 385 nm; the emission spectrum of tubulin-vinblastine complex is found to be devoid of any contributions from CBIQD emission. It is observed that CBIQD did not affect the tubulin-vinblastine complex fluorescence (SI: Figure S6), indicating that CBIQD binding site is different from the vinblastine binding site. 28 ACS Paragon Plus Environment

Page 28 of 51

Page 29 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Conformational Modifications in Tubulin Due to CBIQD Binding. Any molecular recognition between host and guest is generally accompanied by conformational alterations in the former. So in order to investigate any conformational alteration that may occur in tubulin protein while accommodating CBIQD, the following two approaches are experimented. Inhibition of tubulin-bis-ANS Fluorescence by CBIQD. The hydrophobic molecule bis-8-anilinonaphthalene-1-sulfonate (bis-ANS) binds to tubulin and inhibits MT assembly.71 Tubulin contains one high-affinity binding site and multiple low-affinity binding sites for bisANS.71,72 It is known that the binding site of bis-ANS is distinct from the colchicine, vinblastine or podophyllotoxin binding site.71,72 The extreme environmental sensitivity of bisANS makes it a useful probe for examining the conformational states of the tubulin heterodimers.73 CBIQD (0-18 µM) is added to pre-formed bis-ANS tubulin complex, and bisANS fluorescence is used to monitor the effect of CBIQD on binding of bis-ANS to tubulin. CBIQD produced a concentration dependent quenching of tubulin-bis-ANS fluorescence (Figure 8). For example, 18 µM CBIQD reduced bis-ANS fluorescence by 35%. Since the binding domain of bis-ANS in tubulin heterodimer is different from that of CBIQD, the apparent depreciation of bis-ANS-tubulin complex fluorescence is probably because CBIQD binding to tubulin may induce a conformational change in tubulin leading to the reduction in bis-ANS binding.

29 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 8. Quenching of tubulin-bis-ANS complex fluorescence by CBIQD. Tubulin (1 µM) is incubated with Bis-ANS (1 µM) for 40 min to form tubulin-bis-ANS complex. Then 0-18 µM concentrations of CBIQD is added to the complex, and fluorescence measurements are recorded after 20 min of incubation at 25 ºC; λex = 430 nm, monitored λem = 490 nm. Kinetics of Chemical Modification of Tubulin Cysteine Groups by DTNB in Presence of CBIQD. The cysteine (Cys) residues in tubulin are actively involved in regulating ligand interactions and tubulin polymerization process both in vivo and in vitro. These residues contain sulfhydryl (-SH) groups that are sensitive reporters in determining the conformation of tubulin upon ligand binding.50,74 Modification of one or two Cys groups in tubulin can completely inhibit MT polymerization.50 Thus, the sulfhydryl specific reagent DTNB is used to determine the accessibility of Cys residues in tubulin towards modification in association with CBIQD binding. The reaction kinetics for Cys titration in tubulin with DTNB at 37 ̊C in the absence and presence of 4.25 µM CBIQD are shown in Figure 9A. There are 16.2 ± 0.3 sulfhydryl residues accessible per tubulin dimer in the absence of CBIQD and 14.8 ± 0.25 residues per tubulin dimer in the presence of CBIQD. This implies that CBIQD reduced the number of Cys residues accessible to DTNB for chemical modification. The difference in the number of modified Cys residues in the absence and presence of unfused naphthalimide is ~1.4 indicating that the binding induces a conformational change in tubulin in some way. Moreover, the pseudo-first-order plot (Figure 9B) showed that the average rate of Cys group modification remain unaffected by CBIQD.

30 ACS Paragon Plus Environment

Page 30 of 51

Page 31 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 9. (A) Chemical modifications of cysteine residues of tubulin (1 µM) by DTNB in the absence (■) and presence of 4.25 µM CBIQD (●) are monitored at 412 nm as described in the experimental section. (B) Pseudo first-order plot of the sulfhydryl modification kinetics in the absence (■) and presence (●) of CBIQD. Data are the average of three independent experiments. In silico Prediction of CBIQD Binding Site on Tubulin. In silico studies provide an insight into the atomistic level details of CBIQD interacting with α, β-tubulin heterodimers, their binding affinity, binding pose, intermolecular interactions and vital features necessary for inhibition of microtubule assembly. The extra precision (XP) docking of CBIQD at the colchicine binding site provides a minimum energy pose of the ligand with its major portion buried in the β-subunit (Figure 10A); the glide score obtained is -5.617 kcal mol-1. The ligand-receptor complex is particularly stabilized by hydrophilic interactions: the 31 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

two carbonyl oxygen’s on the naphthalimide core form H-bonds each with Gln247 and Asn258 residues, while a weak halogen bonding interaction is also established between –Cl atom on the benzothiazole fragment with Val315 amino acid residue (Figure 11). Although CBIQD is not involved in a direct interaction with Cys241 residue, merely its presence in the vicinity of ligand in the binding pocket (Figure 10B) indicates the influence it might have on the depolymerization activity of CBIQD as shown from the experimental results also.

Figure 10. (A) Minimum energy conformation of CBIQD at the interface of α, β-tubulin, major portion buried in the β-subunit. PDB ID: 1UT5. (B) 2D-ligand interaction pattern of amino acid residues in the immediate vicinity of CBIQD at the binding site. Presence of Cys241 residue (encircled in pink) may influence the depolymerization activity of CBIQD.

32 ACS Paragon Plus Environment

Page 32 of 51

Page 33 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Figure 11. Various intermolecular interactions between CBIQD and the amino acid residues responsible for the stabilization of CBIQD-tubulin complex. Further, molecular dynamics simulation is implemented for a period of 15 ns to access the stability of tubulin-CBIQD complex especially when both are free to move under explicit solvation conditions, the properties are evaluated using RMSD and RMSF of the protein. The RMSD analysis (SI: Figure S7A), indicates that the simulation has equilibrated as the RMSD values of both protein and ligand stabilize around a fixed value. Further the fluctuations from the beginning towards the end of simulation fall within 1-3 Å for both protein and ligand indicating that the protein has undergone minor conformational changes during simulation. This conformational alteration induced in the protein ensemble may have an impact on the microtubule destabilizing effect of CBIQD as observed from the in vitro studies also. However, the ligand RMSD is found to be slightly larger than that of protein in the beginning, and then it is probable that the ligand might have been slightly diffused away from its initial binding area. The RMSF of all amino acid residues of tubulin in the complex (SI: Figure S7B) is examined to reveal the local changes along the protein chain. On this plot, peaks indicate areas of the protein that fluctuate the most during simulation. The results

33 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 51

indicates that all residues in the CBIQD binding site had a relatively certain degree of flexibility, illustrating slight conformational alterations undergone by tubulin to initiate and maintain optimum binding with the ligand. The B-factors of protein crystal structures reflect the fluctuation of atoms about their average positions and provide important information about protein dynamics. Further, the protein-ligand contacts when monitored throughout the simulation (SI: Figure S8, S9 & S10), suggests that Ala250, Lys254, Asn258, Lys352 are able to interact with CBIQD for about 10%, 27%, 86%, 17% time of the simulation run (SI: Figure S10) particularly Lys352 seems to make multiple contacts with the ligand over the entire time frame (SI: Figure S8 & S9). Since Lys352 and Lys254 are important for colchicine binding to tubulin, binding interaction of CBIQD at this site may interfere with colchicine binding that endorses the binding site experimental findings. Determination of Specificity of CBIQD towards Tubulin Influence of Bovine Serum Albumin (BSA) on CBIQD-Tubulin Binding. To examine the probable influence of transport protein like BSA on the binding efficiency and hence specificity of CBIQD towards bovine brain tubulin, competitive binding interaction is experimented in absence and presence of the other interacting partner (BSA/ tubulin). The binding constant (Kb) of CBIQD-BSA interaction is found to be 4.67 x 103 M-1 (SI: Figure S11), while CBIQD binds to tubulin with Kb = 2.03 x 105 M-1 (± 1.88%) as discussed earlier. Moreover, on gradual addition of BSA the fluorescence intensity of equimolar mixture of CBIQD-tubulin system is not altered (quenched) significantly

(Figure 12A), whereas, in

the reverse experiment, tubulin is able to efficiently decrease the emission intensity of an equimolar mixture of CBIQD-BSA system (Figure 12B). This competitive binding interaction is quantified in terms of binding constant (Kb) and documented in Table 2. Hence the weaker affinity as well as the lower binding constant [0.945 x 103 M-1 (±2.64%)] of

34 ACS Paragon Plus Environment

Page 35 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

CBIQD-BSA in presence of tubulin may indicate the specificity of this bichromophoric naphthalimide derivative towards tubulin protein.

Figure 12. (A) Variation in the emission spectra of an equimolar (1:1) mixture of CBIQD + Tubulin on gradual addition of BSA (B) Variation in the emission spectra of an equimolar (1:1) mixture of CBIQD + BSA on gradual addition of Tubulin; λex = 344 nm; λem monitored = 397 nm; T = 25 ºC Table 2. Binding Constants (Kb) of CBIQD with Tubulin and BSA in Absence and Presence of Each Other System In absence of BSA/ In presence of BSA/ Tubulin Tubulin 5 -1 Binding constant (Kb) 2.03 x 10 M (±1.88%) 1.39 x 104 M-1 (± 1.32%) of CBIQD-Tubulin Binding constant (Kb) 4.67 x 103 M-1 (±1.03%) 0.945 x 103 M-1 (± 2.64%) of CBIQD-BSA Influence of Actin Protein on CBIQD-Tubulin Binding. (A) By in vitro competitive experiment: To demonstrate the probable influence of another cytoskeletal filament such as actin on the binding efficiency and hence specificity of CBIQD towards tubulin, in vitro competitive experiments are performed. Here the emission wavelength monitored is 397 nm, which is the emission maximum of CBIQD in buffer obtained at an excitation wavelength of 344 nm. On gradual addition of actin protein the fluorescence intensity of equimolar mixture

35 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

of CBIQD-tubulin system almost remains unaffected (Figure 13A), whereas, in the reverse experiment, tubulin is able to efficiently decrease the emission intensity of an equimolar mixture of CBIQD-actin system (Figure 13B). This competitive binding interaction is quantified in terms of binding constant (Kb) and it is demonstrated a much weaker binding affinity (in the order of ~102) of CBIQD towards actin in the presence of tubulin when compared to the binding affinity of CBIQD towards tubulin in the presence of actin (in the order of ~104). Hence the weaker affinity as well as the lower Kb [0.764 x 102 M-1 (±1.07%)] of CBIQD-actin in presence of tubulin may indicate the specificity of this bichromophoric naphthalimide derivative towards tubulin protein.

Figure 13. (A) Variation in the emission spectra of an equimolar (1:1) mixture of CBIQD + Tubulin on gradual addition of Actin protein; (B) Variation in the emission spectra of an equimolar (1:1) mixture of CBIQD + Actin on gradual addition of Tubulin; λex = 344 nm; λem monitored = 397 nm; T = 25 ºC (B) By immunofluorescence assay: To check the status of other cytoskeleton protein, such as actin, A549 cells are treated with low dose of CBIQD (3.5 µM) and stained with anti-β-actin antibody to visualize the effect of actin. The actin structure of the treated cells (SI: Figure S12 E & F) remains almost same as that of untreated cells (Figure S12 A and B). Actin filaments are not that much distorted compared to cellular MT at this low concentration of 36 ACS Paragon Plus Environment

Page 36 of 51

Page 37 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

CBIQD (3.5 µM). The data of treatment with higher doses of CBIQD is not given, because at higher doses actin is also disrupted. It is quite usual since at higher doses of CBIQD total cell structure is distorted. Thus, it may be concluded that at low concentrations, CBIQD specifically targets MT over other cytoskeleton proteins.

DISCUSSION Most of naphthalimide based small molecules known till date; exert their anti-cancer activity, classically by intercalating into the DNA double helix base pairs19,50,75,76 or by inhibiting topoisomearse II activity.41 However, this study reports for the first time how a small, unfused naphthalimide bichromophoric compound, CBIQD emerged as an anti-mitotic agent (although serendipitously). Remarkably, CBIQD inhibits A549 cell proliferation, causes cellular shrinkage, alters cellular morphology and blocks the mitotic spindle function by perturbing the MT organization. It has been demonstrated that cellular shrinkage is related to depolymerization of the MT network in cells because MTs are important constituents of cells that impart shape, structure and integrity of cytoskeleton.77,78 Most importantly, MTs are important to mitosis in a number of ways: in the proper formation of mitotic spindle, in the process of attachment of chromosomes to spindle MTs, in chromosome congression to the metaphase equatorial plate, in the transition from metaphase to anaphase, and in anaphase movement to the spindle poles. During cell division, MTs in the cytoplasmic network depolymerize and the tubulin thus liberated is again polymerized to form the mitotic spindle.3,16 In the present study, we find CBIQD inhibits polymerization of tubulin in vitro, depolymerized preformed MTs and bound to tubulin at the colchicine binding site. Binding of CBIQD to tubulin induced slight conformational alterations in the protein as a consequence of diminution in the tubulin-bis-ANS fluorescence as well as reduced the average number of Cys residues toward chemical modification by DTNB. Taking together, it is reasonable to 37 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

believe that tubulin binding site for this bichromophoric naphthalimide may be an important site involved in regulation of MT polymerization dynamics in cells and, thus, an important drug-target site. By analyzing the binding of CBIQD to tubulin using fluorescence techniques, we identified a single high-affinity site for the compound with an apparent binding constant (Kb) of 2.03 x 105 M-1 (± 1.88%). The prominent quenching of the intrinsic tubulin emission (Figure 5A) by CBIQD is a clear demonstration of its strong binding to tubulin. This quenching which is accompanied with a significant 7 nm blue shift indicates slight structural deformations in the protein ensemble upon CBIQD binding, which may also result in an increase in the hydrophobicity in the binding region. Tubulin contains nearly eight Trp residues per dimer that are present in a viscous environment.79 As per certain reports, &' of Trp in tubulin protein and viscosity is often found to increase with red shift in emission wavelength when the excitation wavelength is kept constant.79 Our data in Table 1 suggest that &' of Trp fluorophore decreases upon increasing [CBIQD] implying that the average environment of these eight Trp residues might be more hydrophobic and less viscous accompanied with slight conformational alterations as indicated from the steady state measurements also. However, at this juncture, it is quite difficult to point out which Trp residue is affected the most since tubulin comprises of nearly eight Trp residues. MTs are the target of several exogenous small molecule based anti-proliferative agents, which mimic the function of endogenous regulatory proteins: these compounds either stabilize or destabilize MTs. Most of them bind to one of the three characterized tubulin sites: taxol site (MT stabilizing effect), colchicine and vinblastine sites (both have MT destabilizing effect). Since CBIQD depolymerized the preformed MTs and prevented the tubulin polymerization process, so it is highly probable that it may interfere with either colchicine or vinblastine binding site. Vinblastine did not inhibit the binding of CBIQD to tubulin,

38 ACS Paragon Plus Environment

Page 38 of 51

Page 39 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

suggesting that CBIQD did not bind to the vinblastine domain in tubulin. However, colchicine inhibited CBIQD bound to tubulin in a concentration dependent manner (Figure 7A). These findings demonstrate that the binding pocket of the unfused bichromophore overlaps with that of colchicine. Further, the binding of CBIQD to tubulin decreased the fluorescence of tubulin-bis-ANS fluorescence and affected the accessibility of –SH groups of tubulin to DTNB, indicating that CBIQD binding may induce a slight conformational change in tubulin and that these Cys residues may not be directly involved in binding with CBIQD (Figure 10B). Nevertheless it is pertinent to mention that, tubulin comprises twenty Cys residues51 that are distributed throughout the protein, and it is quite possible that the residues affected by CBIQD are not located at the CBIQD binding site on tubulin but located elsewhere, and CBIQD induced conformational alteration prevented the chemical modification of these residues. Colchicine is known to protect 1.4 to 2 Cys residues from alkylating agents50,74 and causes a strong reduction in their reactivity indicating that although colchicine and CBIQD bind to the same site on tubulin, but they may modify the tubulin conformation in a different way. Since our studies indicate CBIQD as a first representative of an unfused naphthalimide derivative that exhibits its anti-cancer activity by binding to tubulin, it is hereby important to assess its binding interaction and specificity in the presence of another non-specific receptor i.e. BSA which also acts as a model transport protein for several exogenous and endogenous substances from the blood stream to the appropriate target site. As illustrated earlier, an increasing concentration of BSA could quench CBIQD-tubulin fluorescence to a lesser extent (Figure 12A) as compared to CBIQD-BSA fluorescence quenched by an increasing concentration of tubulin in the system (Figure 12B) when excited at 344 nm. Most importantly, the weaker binding of BSA to CBIQD in the presence of tubulin (Table 2) is a clear manifestation that the transport protein BSA is unable to hold the

39 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

CBIQD molecule which implies that CBIQD is more specific towards the target tubulin protein as compared to BSA. This information could be very useful for the effective delivery and specificity of the potential anti-mitotic candidate CBIQD towards the target tubulin protein in the presence of BSA. Mechanism of Inhibition of Tubulin Polymerization into Microtubules by CBIQD. There are several possible mechanisms through which CBIQD could inhibit tubulin polymerization and trigger MT disassembly. One possibility is that CBIQD can sequester tubulin dimers and the sequestration of tubulin reduces free tubulin concentration for microtubule assembly. Alternatively, CBIQD induces conformational change in MT that inhibits assembly. Other possibility may be based on the consequences of CBIQD binding at the colchicine binding domain, where it is buried into the β subunit at the interface with the α subunit of the same tubulin (Figure 10A). Nevertheless, there are minimal chances that α subunit may be positioned near a CBIQD-bound β subunit, because of the steric hindrance (s) of the naphthalimide molecule. Conversely, there might be no space available for CBIQD when β subunit is in a straight conformation due to the fact that in the core of MTs, tubulin is straight: α- and β- subunits are related by a translation along the protofilament axis. It is well known that the MT polymer is stabilized by specific longitudinal contacts between tubulin monomers within straight protofilaments and by lateral interactions between protofilaments. Since, CBIQD when bound to tubulin may prevent it to maintain a straight conformation or conversely stabilize it in a curved assembly, these tubulin heterodimers might be no longer available for MT growth as they cannot establish the required MT longitudinal contacts within the heterodimers. Consequently, lateral interactions may be prevented as partners remain too distant for the corresponding contacts to occur. Thus, the pool of uncomplexed tubulin is significantly lowered; MT growth is prevented or cannot be sustained, which leads

40 ACS Paragon Plus Environment

Page 40 of 51

Page 41 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

to disassembly. This suggests that MT dynamics requires the integrity of inter-subunit contacts within and between protofilaments to occur, which is probably disturbed by CBIQD.

CONCLUSION In contrast to the anticancer activity exhibited by other DNA targeted naphthalimides as reported in literature, the preliminary anti-proliferative effect exhibited by CBIQD, as well as the related confocal microscopy results reported herein, led us to believe that our synthesized naphthalimide derivative possibly interferes with normal tubulin-MT equilibrium dynamics that ultimately resulted in A549 cell cytotoxicity. This notion further got its justification from combined optical spectroscopic and molecular docking results, where CBIQD could effectively bind to tubulin, hinders its assembly and also could initiate depolymerization of preformed MTs. Thus, the synthesized molecule emerged as a depolymerizing agent which interacts with tubulin and exert effects on tubulin function (viz. affecting tubulin polymerization and alteration in conformation) that is crucial for its antitumor activity. This benzothiazole tethered unfused naphthalimide CBIQD contributes in the expansion of extant therapeutic tool box of naphthalimide derivatives as anti-mitotic agents. Thus, the exploration of the detailed mechanism of the antitumor activity of CBIQD can render this bichromophore as potentially useful candidate for future combination therapy with conventional nucleic-acidtargeted or novel cytoskeletal-directed agents. Although the idea of implementing CBIQD as a dual therapeutic and imaging agent is limited by its absorbance in the blue end of the visible region, but at present, it can be taken care of, by employing multiphoton excitation using an ultrafast pulsed laser system. However, our efforts are directed to tuning the structural framework to shift the absorption wavelength towards the therapeutic window without sacrificing its high emission quantum yield as well as tubulin targeted antitumor activity.

41 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ASSOCIATED CONTENT Supporting Information Available: Effect of different dosages of CBIQD on HeLa cell viability after 24 h of exposure (Figure S1), Bar diagram showing frequency of Rhodamine fluorescence of antibody stained A549 cells as obtained from confocal microscopic study and analyzed by Olympus Fluoview ver. 4.2a. Software. Treatment with CBIQD caused gradual decrease in fluorescence intensity in dose dependent manner as in case of colchicine treated positive control (Figure S2), Effect of CBIQD on cell cycle progression of A549 cells: Cell cycle distribution of A549 cells after treating the cells with 0 (A), 3.5 (B) and 7 µM (C) CBIQD for 24 h. Colchicine (10 nM) (D) is used as a positive control. Histogram showing cell counts against DNA content (FL-2A). Data shows one of the three individual experiments with similar results (Figure S3), Double-logarithmic plot to determine the binding constant (Kb) and number of binding sites (n) of CBIQD on tubulin at 25 ºC (Figure S4), Variation of fluorescence intensity of CBIQD (1µM) bound tubulin (1 µM) with time in buffer A; λex = 344 nm, λem = 397 nm (Figure S5), Stern-Volmer Plot for the variation in the fluorescence of vinblastine-tubulin complex upon addition of increasing [CBIQD]. λex = 385 nm; monitored λem = 535 nm; T = 25 ˚C (Figure S6), Tubulin and ligand (CBIQD) plots during 15 ns simulation (A) RMSD (B) Correlation of CBIQD bound protein RMSF with experimental X-ray B-factor indicating simulation results parallel the crystallographic data. Green represents protein backbone, maroon represents B factor and green vertical bars represent amino acid residues that interact with CBIQD (Figure S7), Tubulin protein interactions with the CBIQD monitored throughout the 15 ns simulation (Figure S8), A timeline representation of the interactions and contacts (H-bonds, hydrophobic, Ionic, Water bridges). The top panel shows total number of specific contacts the protein makes with ligand over the course of trajectory. The bottom panel shows residues that interact with CBIQD in each trajectory frame. Some residues make more than one specific contact with the ligand,

42 ACS Paragon Plus Environment

Page 42 of 51

Page 43 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

represented by a darker shade of orange according to the scale to the right of the plot (Figure S9), Normalized stacked bar chart illustrating the types of Tubulin-CBIQD interactions occurring during 15 ns and time fraction of each contact maintained over the course of trajectory (Figure S10), Double-logarithmic plot to determine the binding constant (Kb) and the number of binding sites of CBIQD with BSA at 298 K (Figure S11), Effect of CBIQD on actin filaments in A549 cells. Immunofluorescence images showing actin filaments of A549 cells and corresponding phase contrast pictures. (A-D) Untreated control cells stained with anti-β-actin antibody and Rhodamine tagged secondary antibody (A & B) and their respective phase contrast pictures (C & D). Cells treated with 3.5 µM CBIQD show no significant change in structure of actin filaments of A549 cells as evident from immunofluorescence pictures (E & F). Respective phase contrast images are also shown of the treated cells (G & H) (Figure S12). This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION Corresponding Author Dr. Sujit Kumar Ghosh, Department of Chemistry, Visvesvaraya National Institute of Technology,

Nagpur,

Maharashtra

440010,

India,

e-mail:

[email protected],

[email protected], Phone no: +(91) 712 2801775, Fax: +(91) 712-2223230/2801357

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT

43 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 44 of 51

RJ and DDM thanks CSIR, New Delhi, Government of India for the CSIR fellowship (09/1092(0001)/2012-EMR-I and 09/028(0838)/2011-EMR-I) respectively. AM is supported by BRNS fellowship (Project No. 2011/37B/25/BRNS). We would also like to thank respected anonymous reviewers for their critical comments and suggestions to enrich the quality of the manuscript.

REFERENCES (1)

Avendaño, C.; Menéndez, J. C. Medicinal Chemistry of Anticancer Drugs; Elsevier, 2008.

(2)

Jordan, M. A.; Wilson, L. Microtubules as a Target for Anticancer Drugs. Nat. Rev. Cancer 2004, 4, 253–265.

(3)

Wilson, L.; Jordan, M. A. Microtubule Dynamics: Taking Aim at a Moving Target. Chem. Biol. 1995, 2, 569–573.

(4)

Chan, K.-S.; Koh, C.-G.; Li, H.-Y. Mitosis-Targeted Anti-Cancer Therapies: Where They Stand. Cell Death Dis. 2012, 3, e411.

(5)

Gascoigne, K. E.; Taylor, S. S. How Do Anti-Mitotic Drugs Kill Cancer Cells? J. Cell Sci. 2009, 122, 2579–2585.

(6)

Nogales, E.; Whittaker, M.; Milligan, R. A.; Downing, K. H. High-Resolution Model of the Microtubule. Cell 1999, 96, 79–88.

(7)

Bhalla, K. N. Microtubule-Targeted Anticancer Agents and Apoptosis. Oncogene 2003, 22, 9075–9086.

(8)

McMicken, B.; Thomas, R. J.; Brancaleon, L. Partial Unfolding of Tubulin Heterodimers

Induced

by

Two-Photon

Excitation

of

Bound

Meso

-

Tetrakis(sulfonatophenyl)porphyrin. J. Phys. Chem. B 2016, 120, 3653–3665. (9)

Majumdar, S.; Ghosh Dastidar, S. Ligand Binding Swaps between Soft Internal Modes of Α,β-Tubulin and Alters Its Accessible Conformational Space. J. Phys. Chem. B 2017, 121, 118–128.

(10)

Ginsburg, A.; Shemesh, A.; Millgram, A.; Dharan, R.; Levi-Kalisman, Y.; Ringel, I.; Raviv, U. Structure of Dynamic, Taxol-Stabilized, and GMPPCP-Stabilized Microtubule. J. Phys. Chem. B 2017, 121, 8427–8436.

(11)

Chakraborti, S.; Das, L.; Kapoor, N.; Das, A.; Dwivedi, V.; Poddar, A.; Chakraborti, 44 ACS Paragon Plus Environment

Page 45 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

G.; Janik, M.; Basu, G.; Panda, D.; et al. Curcumin Recognizes a Unique Binding Site of Tubulin. J. Med. Chem. 2011, 54, 6183–6196. (12)

Prinz, H.; Ridder, A.-K.; Vogel, K.; Böhm, K. J.; Ivanov, I.; Ghasemi, J. B.; Aghaee, E.; Müller, K. N-Heterocyclic (4-Phenylpiperazin-1-Yl)methanones Derived from Phenoxazine and Phenothiazine as Highly Potent Inhibitors of Tubulin Polymerization. J. Med. Chem. 2017, 60, 749–766.

(13)

Peng, J.; Risinger, A. L.; Fest, G. A.; Jackson, E. M.; Helms, G.; Polin, L. A.; Mooberry, S. L. Identification and Biological Activities of New Taccalonolide Microtubule Stabilizers. J. Med. Chem. 2011, 54, 6117–6124.

(14)

Jordan, M. Mechanism of Action of Antitumor Drugs That Interact with Microtubules and Tubulin. Curr. Med. Chem. Agents 2012, 2, 1–17.

(15)

Correia, J.; Lobert, S. Physiochemical Aspects of Tubulin-Interacting Antimitotic Drugs. Curr. Pharm. Des. 2001, 7, 1213–1228.

(16)

Wilson, L.; Panda, D.; Ann Jordan, M. Modulation of Microtubule Dynamics by Drugs. A Paradigm for the Actions of Cellular Regulators. Cell Struct. Funct. 1999, 24, 329–335.

(17)

Hamel, E. Antimitotic Natural Products and Their Interactions with Tubulin. Med. Res. Rev. 1996, 16, 207–231.

(18)

Giannakakou, P.; Gussio, R.; Nogales, E.; Downing, K. H.; Zaharevitz, D.; Bollbuck, B.; Poy, G.; Sackett, D.; Nicolaou, K. C.; Fojo, T. A Common Pharmacophore for Epothilone and Taxanes: Molecular Basis for Drug Resistance Conferred by Tubulin Mutations in Human Cancer Cells. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 2904– 2909.

(19)

Banerjee, S.; Veale, E. B.; Phelan, C. M.; Murphy, S. A.; Tocci, G. M.; Gillespie, L. J.; Frimannsson, D. O.; Kelly, J. M.; Gunnlaugsson, T. Recent Advances in the Development of 1,8-Naphthalimide Based DNA Targeting Binders, Anticancer and Fluorescent Cellular Imaging Agents. Chem. Soc. Rev. 2013, 42, 1601-1618.

(20)

Mati, S. S.; Roy, S. S.; Chall, S.; Bhattacharya, S.; Bhattacharya, S. C. Unveiling the Groove

Binding

Mechanism

of

a

Biocompatible

Naphthalimide-Based

Organoselenocyanate with Calf Thymus DNA: An “Ex Vivo” Fluorescence Imaging Application Appended by Biophysical Experiments and Molecular Docking Simulations. J. Phys. Chem. B 2013, 117, 14655–14665. (21)

Calatrava-Pérez, E.; Bright, S. A.; Achermann, S.; Moylan, C.; Senge, M. O.; Veale, E. B.; Williams, D. C.; Gunnlaugsson, T.; Scanlan, E. M.; Monneret, C.; et al. 45 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Glycosidase Activated Release of Fluorescent 1,8-Naphthalimide Probes for Tumor Cell Imaging from Glycosylated “Pro-Probes.” Chem. Commun. 2016, 52, 13086– 13089. (22)

Lee, M. H.; Kim, J. Y.; Han, J. H.; Bhuniya, S.; Sessler, J. L.; Kang, C.; Kim, J. S. Direct Fluorescence Monitoring of the Delivery and Cellular Uptake of a CancerTargeted RGD Peptide-Appended Naphthalimide Theragnostic Prodrug. J. Am. Chem. Soc. 2012, 134, 12668–12674.

(23)

Zhang, L.; Lei, K.; Zhang, J.; Song, W.; Zheng, Y.; Tan, S.; Gao, Y.; Xu, Y.; Liu, J.; Qian, X. One Small Molecule as a Theranostic Agent: Naphthalimide Dye for Subcellular Fluorescence Localization and Photodynamic Therapy In Vivo. MedChemComm 2016, 7, 1171–1175.

(24)

Joshi, R.; Meitei, O. R.; Kumar, H.; Jadhao, M.; Ghosh, S. K. Design, Synthesis, and Proticity Inclined Conformational Modulation in a Highly Fluorescent Bichromophoric Naphthalimide Derivative: Hint Directed from RICT Perspective. J. Phys. Chem. A 2016, 120, 1000–1011.

(25)

Izawa, H.; Nishino, S.; Sumita, M.; Akamatsu, M.; Morihashi, K.; Ifuku, S.; Morimoto, M.; Saimoto, H.; Lu, Z. Y. A Novel 1,8-Naphthalimide Derivative with an Open Space for an Anion: Unique Fluorescence Behaviour Depending on the Binding Anion’s Electrophilic Properties. Chem. Commun. 2015, 51, 8596–8599.

(26)

Shanmugaraju, S.; Dabadie, C.; Byrne, K.; Savyasachi, A. J.; Umadevi, D.; Schmitt, W.; Kitchen, J. A.; Gunnlaugsson, T.; Su, Z.-M. A Supramolecular Tröger’s Base Derived Coordination Zinc Polymer for Fluorescent Sensing of PhenolicNitroaromatic Explosives in Water. Chem. Sci. 2017, 8, 1535–1546.

(27)

Sidhu, J. S.; Singh, A.; Garg, N.; Singh, N. Carbon Dot Based, Naphthalimide Coupled FRET Pair for Highly Selective Ratiometric Detection of Thioredoxin Reductase and Cancer Screening. ACS Appl. Mater. Interfaces 2017, 9, 25847–25856.

(28)

Xu, Z.; Yoon, J.; Spring, D. R.; Slawin, A. M. Z.; Spring, D. R.; Chan, W.; Kim, J. S.; Kim, K.-M.; Yoon, J.; Yoon, J. A Selective and Ratiometric Cu2+ Fluorescent Probe Based on Naphthalimide Excimer–Monomer Switching. Chem. Commun. 2010, 46, 2563-2565.

(29)

Zhong, C.; Mu, T.; Wang, L.; Fu, E.; Qin, J.; Dossena, A.; Galaverna, G.; Duchateau, A.; Marchelli, R. Unexpected Fluorescent Behavior of a 4-Amino-1,8-Naphthalimide Derived β-Cyclodextrin: Conformation Analysis and Sensing Properties. Chem. Commun. 2009, 44, 4091-4093. 46 ACS Paragon Plus Environment

Page 46 of 51

Page 47 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

(30)

Elmes, R. B. P.; Erby, M.; Bright, S. A.; Williams, D. C.; Gunnlaugsson, T.; Moran, N.; Keyes, T. E.; Grills, D. C.; George, M. W.; Matousek, P.; et al. Photophysical and Biological Investigation of Novel Luminescent Ru(ii)-Polypyridyl-1,8-Naphthalimide Tröger’s Bases as Cellular Imaging Agents. Chem. Commun. 2012, 48, 2588-2590.

(31)

Gudeika, D.; Grazulevicius, J. V.; Volyniuk, D.; Butkute, R.; Juska, G.; Miasojedovas, A.; Gruodis, A.; Jursenas, S. Structure-Properties Relationship of the Derivatives of Carbazole and 1,8-Naphthalimide: Effects of the Substitution and the Linking Topology. Dye. Pigment. 2015, 114, 239–252.

(32)

Ji, L.; Yang, S.; Li, S.; Liu, S.; Tang, S.; Liu, Z.; Meng, X.; Yu, S. A Novel Triazolonaphthalimide Induces Apoptosis and Inhibits Tumor Growth by Targeting DNA and DNA-Associated Processes. Oncotarget 2017, 8, 37394–37408.

(33)

Ingrassia, L.; Lefranc, F.; Kiss, R.; Mijatovic, T. Naphthalimides and Azonafides as Promising Anti-Cancer Agents. Curr. Med. Chem. 2009, 16, 1192–1213.

(34)

Verma, M.; Luxami, V.; Paul, K. Synthesis, In Vitro Evaluation and Molecular Modelling of Naphthalimide Analogue as Anticancer Agents. Eur. J. Med. Chem. 2013, 68, 352–360.

(35)

Dai, F.; Li, Q.; Wang, Y.; Ge, C.; Feng, C.; Xie, S.; He, H.; Xu, X.; Wang, C. Design, Synthesis,

and

Biological

Evaluation

of

Mitochondria-Targeted

Flavone-

Naphthalimide-Polyamine Conjugates with Antimetastatic Activity. J. Med. Chem. 2017, 60, 2071–2083. (36)

Zhu, H.; Miao, Z.-H.; Huang, M.; Feng, J.-M.; Zhang, Z.-X.; Lu, J.-J.; Cai, Y.-J.; Tong, L.-J.; Xu, Y.-F.; Qian, X.-H.; et al. Naphthalimides Induce G(2) Arrest Through The ATM-Activated Chk2-Executed Pathway in HCT116 Cells. Neoplasia 2009, 11, 1226–1234.

(37)

Meyer, A.; Oehninger, L.; Geldmacher, Y.; Alborzinia, H.; Wölfl, S.; Sheldrick, W. S.; Ott, I. Gold(I) N-Heterocyclic Carbene Complexes with Naphthalimide Ligands as Combined Thioredoxin Reductase Inhibitors and DNA Intercalators. ChemMedChem 2014, 9, 1794–1800.

(38)

Li, S.; Xu, S.; Tang, Y.; Ding, S.; Zhang, J.; Wang, S.; Zhou, G.; Zhou, C.; Li, X. Synthesis, Anticancer Activity and DNA-Binding Properties of Novel 4-Pyrazolyl-1,8Naphthalimide Derivatives. Bioorg. Med. Chem. Lett. 2014, 24, 586–590.

(39)

Luo, Y.-L.; Baathulaa, K.; Kannekanti, V. K.; Zhou, C.-H.; Cai, G.-X. Novel Benzimidazole Derived Naphthalimide Triazoles: Synthesis, Antimicrobial Activity and Interactions with Calf Thymus DNA. Sci. China Chem. 2015, 58, 483–494. 47 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(40)

Lv, M.; Xu, H. Overview of Naphthalimide Analogs as Anticancer Agents. Curr. Med. Chem. 2009, 16, 4797–4813.

(41)

Chen, Z.; Liang, X.; Zhang, H.; Xie, H.; Liu, J.; Xu, Y.; Zhu, W.; Wang, Y.; Wang, X.; Tan, S.; et al. A New Class of Naphthalimide-Based Antitumor Agents That Inhibit Topoisomerase II and Induce Lysosomal Membrane Permeabilization and Apoptosis. J. Med. Chem. 2010, 53, 2589–2600.

(42)

Bailly, C. Contemporary Challenges in the Design of Topoisomerase II Inhibitors for Cancer Chemotherapy. Chem. Rev. 2012, 112, 3611–3640.

(43)

Joshi, R.; Meitei, O. R.; Jadhao, M.; Kumar, H.; Ghosh, S. K. Conformation Controlled Turn on–Turn off Phosphorescence in a Metal-Free Biluminophore: Thriving the Paradox That Exists for Organic Compounds. Phys. Chem. Chem. Phys. 2016, 18, 27910–27920.

(44)

Celisa Santimano, M.; Martin, A.; Kowshik, M.; Sarkar, A. Zinc Oxide Nanoparticles Cause Morphological Changes in Human A549 Cell Line Through Alteration in the Expression Pattern of Small GTPases at mRNA Level. J. Bionanoscience 2013, 7, 300–306.

(45)

Das Mukherjee, D.; Kumar, N. M.; Tantak, M. P.; Das, A.; Ganguli, A.; Datta, S.; Kumar, D.; Chakrabarti, G. Development of Novel Bis(indolyl)-hydrazide–Hydrazone Derivatives as Potent Microtubule-Targeting Cytotoxic Agents against A549 Lung Cancer Cells. Biochemistry 2016, 55, 3020–3035.

(46)

Lakowicz, J. R. Principles of Fluorescence Spectroscopy, Third.; Springer, 2006.

(47)

Becker, W. Advanced Time-Correlated Single Photon Counting Techniques; Castleman, A. W., Toennies, J. P., Zinth, W., Eds.; Springer Series in Chemical Physics; Springer Berlin Heidelberg: Berlin, Heidelberg, 2005; Vol. 81.

(48)

Gaskin, F.; Cantor, C. R.; Shelanski, M. L. Turbidimetric Studies of the In Vitro Assembly and Disassembly of Porcine Neurotubules. J. Mol. Biol. 1974, 89, 737–755.

(49)

Bonne, D.; Heuséle, C.; Simon, C.; Pantaloni, D. 4’,6-Diamidino-2-Phenylindole, a Fluorescent Probe for Tubulin and Microtubules. J. Biol. Chem. 1985, 260, 2819– 2825.

(50)

Luduena, R. F.; Roach, M. C. Tubulin Sulfhydryl Groups as Probes and Targets for Antimitotic and Antimicrotubule Agents. Pharmacol. Ther. 1991, 49, 133–152.

(51)

Chaudhuri, A. R.; Khan, I. A.; Ludueña, R. F. Detection of Disulfide Bonds in Bovine Brain Tubulin and Their Role in Protein Folding and Microtubule Assembly in Vitro: A Novel Disulfide Detection Approach. Biochemistry 2001, 40, 8834–8841. 48 ACS Paragon Plus Environment

Page 48 of 51

Page 49 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

(52)

Schrodinger LLC. LigPrep, Version 2.5. New York, NY 2011.

(53)

Schrodinger LLC. Maestro, Version 9.2. New York, NY 2011.

(54)

Jorgensen, W. L.; Maxwell, D. S.; Tirado-Rives, J. Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids. J. Am. Chem. Soc. 1996, 118, 11225–11236.

(55)

Shivakumar, D.; Harder, E.; Damm, W.; Friesner, R. A.; Sherman, W. Improving the Prediction of Absolute Solvation Free Energies Using the Next Generation OPLS Force Field. J. Chem. Theory Comput. 2012, 8, 2553–2558.

(56)

Halgren, T. A.; Murphy, R. B.; Friesner, R. A.; Beard, H. S.; Frye, L. L.; W. Thomas Pollard, A.; Banks, J. L. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. J. Med. Chem. 2004, 47, 1739– 1749.

(57)

Friesner, R. A.; Murphy, R. B.; Repasky, M. P.; Frye, L. L.; Greenwood, J. R.; Halgren, T. A.; Paul C. Sanschagrin, A.; Mainz, D. T. Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein−Ligand Complexes. J. Med. Chem. 2006, 49, 6177–6196.

(58)

Sherman, W.; Day, T.; Jacobson, M. P.; Friesner, R. A.; Farid, R. Novel Procedure for Modeling Ligand/receptor Induced Fit Effects. J. Med. Chem. 2006, 49, 534–553.

(59)

Sherman, W.; Beard, H. S.; Farid, R. Use of an Induced Fit Receptor Structure in Virtual Screening. Chem. Biol. Drug Des. 2006, 67, 83–84.

(60)

Shivakumar, D.; Williams, J.; Wu, Y.; Damm, W.; Shelley, J.; Sherman, W. Prediction of Absolute Solvation Free Energies Using Molecular Dynamics Free Energy Perturbation and the OPLS Force Field. J. Chem. Theory Comput. 2010, 6, 1509–1519.

(61)

Guo, Z.; Mohanty, U.; Noehre, J.; Sawyer, T. K.; Sherman, W.; Krilov, G. Probing the α-Helical Structural Stability of Stapled p53 Peptides: Molecular Dynamics Simulations and Analysis. Chem. Biol. Drug Des. 2010, 75, 348–359.

(62)

Sablina, A. A.; Chumakov, P. M.; Levine, A. J.; Kopnin, B. P. p53 Activation in Response to Microtubule Disruption Is Mediated by Integrin-Erk Signaling. Oncogene 2001, 20, 899–909.

(63)

Sablina, A. A.; Agapova, L. S.; Chumakov, P. M.; Kopnin, B. P. p53 Does Not Control the Spindle Assembly Cell Cycle Checkpoint but Mediates G1 Arrest in Response to Disruption of Microtubule System. Cell Biol. Int. 1999, 23, 323–334.

(64)

Tanabe, K. Microtubule Depolymerization by Kinase Inhibitors: Unexpected Findings of Dual Inhibitors. Int. J. Mol. Sci. 2017, 18, 2508. 49 ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(65)

Zhang, C.; Yang, N.; Yang, C.; Ding, H.; Luo, C.; Zhang, Y.; Wu, M.; Zhang, X.; Shen, X.; Jiang, H.; et al. S9, a Novel Anticancer Agent, Exerts Its Anti-Proliferative Activity by Interfering with Both PI3K-Akt-mTOR Signaling and Microtubule Cytoskeleton. PLoS One 2009, 4, e4881.

(66)

Gigant, B.; Cormier, A.; Dorléans, A.; Ravelli, R. B. G.; Knossow, M. MicrotubuleDestabilizing Agents: Structural and Mechanistic Insights from the Interaction of Colchicine and Vinblastine with Tubulin; Springer Berlin Heidelberg, 2008; pp 259– 278.

(67)

Ganguli, A.; Choudhury, D.; Chakrabarti, G.; John, R.; Dasgupta, A. K.; Pradeep, T.; Chakrabarti, G.; Skinnider, L. F.; Choi, N. W. 2,4-Dichlorophenoxyacetic Acid Induced Toxicity in Lung Cells by Disruption of the Tubulin-Microtubule Network. Toxicol. Res. 2014, 3, 118-130.

(68)

Panda, D.; Ananthnarayan, V.; Larson, G.; Shih, C.; Jordan, M. A. and; Wilson, L. Interaction of the Antitumor Compound Cryptophycin-52 with Tubulin. Biochemistry 2000, 39, 14121-14127.

(69)

Gupta, K. K.; Bharne, S. S.; Rathinasamy, K.; Naik, N. R.; Panda, D. Dietary Antioxidant Curcumin Inhibits Microtubule Assembly through Tubulin Binding. FEBS J. 2006, 273, 5320–5332.

(70)

Gupta, K.; Panda, D. Perturbation of Microtubule Polymerization by Quercetin through Tubulin Binding: A Novel Mechanism of Its Antiproliferative Activity. Biochemistry 2002, 41, 13029-13038.

(71)

Horowitz, P.; Prasad, V.; Luduena, R. F. Bis (1,8-Anilinonaphthalenesulfonate). A Novel and Potent Inhibitor of Microtubule Assembly. J. Biol. Chem. 1984, 259, 14647–14650.

(72)

Ward, L. D.; Timasheff, S. N. Cooperative Multiple Binding of BisANS and Daunomycin to Tubulin. Biochemistry 1994, 33, 11891–11899.

(73)

Mazumdar, M.; Parrack, P. K.; Mukhopadhyay, K.; Bhattacharyya, B. Bis-ANS as a Specific Inhibitor for Microtubule-Associated Protein-Induced Assembly of Tubulin. Biochemistry 1992, 31, 6470–6474.

(74)

Roychowdhury, M.; Sarkar, N.; Manna, T.; Bhattacharyya, S.; Sarkar, T.; Basusarkar, P.; Roy, S.; Bhattacharyya, B. Sulfhydryls of Tubulin. A Probe to Detect Conformational Changes of Tubulin. Eur. J. Biochem. 2000, 267, 3469–3476.

(75)

Banerjee, S.; Kitchen, J. A.; Gunnlaugsson, T.; Kelly, J. M.; Berriman, M.; Hyman, R. W.; Carlton, J. M.; Pain, A.; Nelson, K. E.; Bowman, S.; et al. Synthesis and 50 ACS Paragon Plus Environment

Page 50 of 51

Page 51 of 51 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Photophysical Evaluation of a Pyridinium 4-Amino-1,8-Naphthalimide Derivative That Upon Intercalation Displays Preference for AT-Rich Double-Stranded DNA. Org. Biomol. Chem. 2012, 10, 3033–3043. (76)

Banerjee, S.; Kitchen, J. A.; Bright, S. A.; O’Brien, J. E.; Williams, D. C.; Kelly, J. M.; Gunnlaugsson, T.; Giorgi, C.; Gratteri, P.; Ingraín, A. M.; et al. Synthesis, Spectroscopic and Biological Studies of a Fluorescent Pt(ii) (Terpy) Based 1,8Naphthalimide Conjugate as a DNA Targeting Agent. Chem. Commun 2013, 49, 85228524.

(77)

Choudhury, D.; Xavier, P. L.; Chaudhari, K.; John, R.; Dasgupta, A. K.; Pradeep, T.; Chakrabarti, G. Unprecedented Inhibition of Tubulin Polymerization Directed by Gold Nanoparticles Inducing Cell Cycle Arrest and Apoptosis. Nanoscale 2013, 5, 44764489.

(78)

Mukherjee, S.; Acharya, B. R.; Bhattacharyya, B.; Chakrabarti, G. Genistein Arrests Cell Cycle Progression of A549 Cells at the G2/M Phase and Depolymerizes Interphase Microtubules through Binding to a Unique Site of Tubulin. Biochemistry 2010, 49, 1702–1712.

(79)

Guha, S.; Rawat, S. S.; Chattopadhyay, A.; Bhattacharyya, B. Tubulin Conformation and Dynamics: A Red Edge Excitation Shift Study. Biochemistry 1996, 35, 13426– 13433.

TOC GRAPHIC

51 ACS Paragon Plus Environment