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. 2022 Nov 14;5(12):1292–1304. doi: 10.1021/acsptsci.2c00172

Noscapine–Amino Acid Conjugates Suppress the Progression of Cancer Cells

Amardeep Awasthi †,*, Neeraj Kumar ∥, Abhijeet Mishra ⊥, Rangnath Ravi #, Anu Dalal ∇, Saurav Shankar †, Ramesh Chandra †,‡,§,*
PMCID: PMC9745893  PMID: 36524011

Abstract

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Lung cancer is the leading cause of cancer deaths globally; 1 in 16 people are diagnosed with lung cancer in their lifetime. Microtubules, a critical cytoskeletal assembly, have an essential role in cell division. Interference with the microtubule assembly leads to genetic instability during mitosis and cancer cell death. Currently, available antimitotic drugs such as vincas and taxanes are limited due to side effects such as alopecia, myelosuppression, and drug resistance. Noscapine, an opium alkaloid, is a tubulin-binding agent and can alter the microtubule assembly, causing cancer cell death. Amino acids are fundamental building blocks for protein synthesis, making them essential for the biosynthesis of cancer cells. However, the ability of amino acids in drug transportation has yet to be exploited in developing noscapine analogues as a potential drug candidate for cancer. Hence, in the present study, we have explored the ninth position of noscapine by introducing a hydroxymethylene group using the Blanc reaction and further coupled it with a series of amino acids to construct five target conjugates in good yields. The synthesized amino acid conjugate molecules were biologically evaluated against the A549 lung cancer cell line, among which the noscapine–tryptophan conjugate showed IC50 = 32 μM, as compared to noscapine alone (IC50 = 73 μM). Morphological changes in cancer cells, cell cycle arrest in the G1 phase, and ethidium bromide/acridine orange staining indicated promising anticancer properties. Molecular docking confirmed strong binding to tubulin, with a score of −41.47 kJ/mol with all 3D coordinates and significant involvement of molecular forces, including the hydrogen bonds and hydrophobic interactions. Molecular dynamics simulations demonstrated a stable binding of noscapine–tryptophan conjugate for a prolonged time (100 ns) with the involvement of free energy through the reaction coordinates analyses, solving the bioavailability of parent noscapine to the body.

Keywords: Noscapine, Amino acids, Lung cancer, Molecular dynamics simulation, Molecular docking, In vitro analyses

Cancer is the leading cause of human mortality, with an estimated 10 million people dying from it globally in 2020.1 Over the past decades, researchers have made unprecedented efforts to develop therapeutics against cancer. Amino acids are fundamental building blocks for protein synthesis, making them essential for the biosynthesis of cancer cells.2 Therefore, tumor cells always have an increased demand for amino acids to grow and proliferate rapidly inside the tissue. Amino acid transporters are responsible for facilitating the availability of amino acids to cancer cells, making them an essential component for tumor-targeting therapy.3 Traditional targeting therapies block the intake of nutrients and starve the cells to death.4 However, these traditional therapies lack selectivity, and to counter this, modern tumor-targeting therapies such as boron neutron capture therapy (BNCT)5 and positron emission tomography (PET)6 can avoid undesirable off-target side effects by the diverse distribution of transporters. Some of the commercially available drugs present in the market, such as levodopa (Stalevo), baclofen (Zentiva), and valaciclovir (Valtrex), utilize the transport function of amino acids.7

The prodrug approach can utilize the differential distribution of amino acid transporters by mimicking the amino acid substrates. These prodrugs can achieve high selectivity in drug delivery and lower the drug’s surface distribution into cancer cells. For instance, LAT1 (large amino acids transporter8)-derived prodrugs showed a higher affinity for branched and aromatic amino acids (Leu and Phe) and were reported to be effective in targeting brain parenchyma.9 Aspartate-modified doxorubicin (Asp-DOX) is another LAT1-derived prodrug that was reported to target HepG-2 cancer cell lines and inhibit cell growth actively.10 Some examples of amino acid-derived drugs are shown in Figure 1.

Figure 1.

Figure 1

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Amino acid-derived prodrugs utilizing differential distributions of amino acid transporters and their roles in various types of cancer.11−15

Inspired by these previous studies, we have synthesized a series of noscapine–amino acid conjugates as potential candidates for lung cancer therapies in the present study. Evaluation of the noscapine molecule (Figure 2) over the years has shown it to be promising as a potential anticancer drug candidate. Noscapine, one of the major constituents present in Papaver somniferum, or opium (∼10% of the total composition), was first isolated in 1817,16 and it has been used as an antitussive drug on the market for over 50 years. Its antitussive property was first discovered in 1930, and later, in 1958, noscapine was reported to inhibit cancer cell proliferation and established itself as an anticancer molecule.17,18 Noscapine exists in four optically isomeric forms, of which its (−)-α isomer (5′S,3′R) is considered the most biologically active. Noscapine binds stoichiometrically to each αβ-tubulin dimer and modifies the tubulin conformation, leading to cell apoptosis at the mitotic phase of the cancer cell progression. Unlike the other microtubule-targeting drugs such as vincas and taxanes, noscapine does not significantly alter the reversible monomer reaction to polymer and shows less cytotoxicity to normal healthy cells.19

Figure 2.

Figure 2

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Chemical structure of noscapine.

Over the past two decades, many research groups have developed several analogues of noscapine by incorporating several moieties in the molecule. In recent reports, 9-((perfluorophenyl)methylene)aminonoscapine (9-PAN) (IC50 = 20 ± 0.3 μM), N-propargylnoscapine (NPN) (IC50 = 1.35 ± 0.2 μM), and 1,3-diynyl derivatives of noscapine (IC50 = 6.23 μM) have shown excellent results against various breast cancer cell lines by arresting the cells at the G2/M phase of the cell cycle.20−22 1,3-Benzodioxole-modified noscapinoids also showed a wide range of activity against various cancer lines, particularly MCF-7, with IC50 = 0.6 ± 0.17 μM.23 Also, 9-ethynyl noscapinoids induced G2/M arrest and apoptosis by disrupting tubulin polymerization in cervical cancer.24 However, in all the synthesized derivatives of noscapine, the transporting ability of amino acids in drug delivery has not been explored to develop noscapine as a drug candidate. Our laboratory has continuously explored the noscapine molecule against various cancer for the past 20 years. Through this research article, we attempt to establish noscapine conjugated with amino acids as a potential anticancer agent against lung cancer.

Notably, in silico analysis has been widely employed to study the drug binding with the tubulin protein. Molecular docking assays are reported to assess the binding poses of the drug and groove of the target protein molecules.25 Molecular modeling approaches have played a vital role in the virtual screening of the extensive library of drugs and further assessment to elucidate the structure-assisted relationship and mechanism of action.26−29 Also, many studies have used molecular dynamics (MD) simulations to assess drug interactions with target proteins, employing the force fields with different pressure and volume states. MD simulation-generated trajectories have shown enormous potential to determine the root-mean-square deviation (RMSD), RMSF, radius of gyration (Rg), and many other parameters to evaluate the stable binding and conformations of drugs with target proteins.30−33

Altogether, we have synthesized novel noscapine–amino acid conjugates and further evaluated them with in silico and in vitro assays, which have shown significant outcomes for the noscapine–tryptophan conjugate as a potential anticancer drug targeting tubulin.

Results and Discussion

Chemistry

Noscapine, a 3-substituted phthalide-derived molecule, consists of a 1,3-benzodioxazole ring as a pharmacophore.34 Chemical modification of the noscapine molecule is slightly difficult due to the phthalide ring, which makes the molecule highly labile to give side products. The 9′-substituted noscapinoids have been reported to be more effective than the 7′-substituted noscapinoids. In this article, we explore the ninth position of noscapine by introducing hydroxymethylene as a group in molecule 2 using the Blanc reaction and then linking 2 with various amino acids by ester coupling to give the target conjugates. The generated −CH2OH group in molecule 2 and molecule 4a–e has an sp3 carbon, providing flexibility for incoming amino acids to rotate and bind with the target protein effectively. The synthetic route for the conjugates is depicted in Scheme 1.

Scheme 1. Synthetic Methodology to Construct Noscapine–Amino Acid Conjugates.

Scheme 1

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Reaction conditions: (i) paraformaldehyde, HCl, rt, 1.5 h, 95%; (ii) 3a–e, EDC·HCl, HOBt, DIPEA, DMF, rt, 12 h, 70–86%.

First, the commercially available noscapine hydrochloride salt (α) 1 was treated with paraformaldehyde and HCl and stirred overnight at rt. After completion, the reaction mixture was quenched on crushed ice and made alkaline (pH = 9) by slowly adding sodium carbonate to give 2 in good yield (95%). Using the noscapine·HCl instead of free noscapine gave us a good yield in a shorter reaction time than previously reported procedures.35 Compound 2 was further treated with Boc-protected amino acids to give target conjugates 4a–e by utilizing 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBt) as coupling agents, N,N-diisopropylethylamine (DIPEA) as a base, and DMF as a solvent. The reaction was quenched with brine and washed with water. The mixture was extracted with DCM, and the solvent was removed under a vacuum. The crude compound was isolated by column chromatography in good yield using 35% ethyl acetate and hexane as eluent. All the synthesized compounds were stable at room temperature. Mechanistically, EDC reacts with the −COOH group of amino acids to form an active O-acylisourea intermediate, which is readily displaced by nucleophilic −OH present in molecule 2 to form an ester in 4a–e. The coupling reaction was also tried in DCM but produced moderate yields. In the synthesized library of noscapine–amino acid conjugates, Nos-Trp and Nos-Ala conjugates were synthesized in 85–88% yield. In addition, Nos-Val, Nos-Phe, and Nos-Leu were synthesized in 75–80% yield (Figure 3). However, attempts to synthesize noscapine conjugates with Isoleu and Met amino acids were unsuccessful. All the synthesized compounds were fully characterized by IR, 1H and 13C NMR, and mass (ESI and HRMS) spectrometry (Supporting Information).

Figure 3.

Figure 3

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Synthesized noscapine–amino acid derivatives.

Molecular Docking

Anticancer Target Protein Retrieval and In Silico Analyses

In our study, we have used tubulin as our target protein, which is reported to be the direct target of the parent compound noscapine. Tubulin protein has a significant role in cell cycle regulation, so its modulation through active-site interaction with the target drug can combat the cell cycle progression of cancer cells. In order to perform the molecular interaction analyses, tubulin protein was retrieved from the Protein Data Bank with PDB ID: 1TUB in .pdb format. The three-dimensional retrieved structure of tubulin protein was processed for molecular docking analyses. The tubulin protein structure availed from the Protein Data Bank was identified by electron crystallography, and it has two subunits of alpha helices, beta chains, and random coils of high resolutions. The 3D tubulin structure was energy minimized to obtain the most stable and confined structure via the Maestro workspace. The tubulin structure consisted of an alpha chain of 440 amino acids and beta chains size 442 amino acids with the involvement of coils, as shown in Figure 4.

Figure 4.

Figure 4

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Structural retrieval and assessment. (A) Three-dimensional secondary structure of tubulin protein, depicting the alpha helices, beta chains, and random coils in ribbon style. (B) Depiction of regulatory chain-A and chain-B of tubulin protein in two different colors in ribbon style. (C) Ramachandran plots of tubulin protein, showing that the residue exists in the acceptable region. (D) Depiction of tubulin structure with other reference native structures, showing the closeness and optimal quality similarity, residue atomic fluctuation plot of tubulin protein, and depiction of minimal deviations of residues of crystal structure.

Tubulin Structural Assessment

The 3D structure of the tubulin protein was investigated for its stereochemical parameters and analyzed through the Ramachandran plot. The Saves interface module generated the Ramachandran plot of tubulin to identify the number of residues lying in the allowed, disallowed, and outlier positions and dihedral angles. The outcome Ramachandran plot depicted that 88.2% of residues lie in the favored region, 5.3% in the allowed region, and only 5.3% in the disallowed region.

Also, the tubulin structure was examined by the Verify3D module to confirm the conformation and quality of the crystal structure, with >80% of the amino acids possessing the 3D-1D profiles. Interestingly, we found that the retrieved tubulin structure resembles the native protein structures of the same size. Altogether, structure evaluation with various interfaces affirms the optimal quality of the tubulin structure for it to be considered for further molecular modeling analyses.

Molecular Binding Analysis of Noscapine Derivative with Specific Target Tubulin

Synthesized compounds were examined and screened against the specific anticancer target protein tubulin using molecular docking assays (Figure 5). Molecular docking analyses were executed by employing the Hex 8.0 CUDA software, which provided 100 binding poses of the drugs to the target protein, and the top-scored poses were further considered. The Hex gave parameters of shape and electrostatics for all derivatives, and the results were also compared with those of the parent compound noscapine. Interestingly, we found that 4e displayed a higher docking score of −41.49 kJ/mol than noscapine, −36.25 kJ/mol, with tubulin protein (see Table 1).

Figure 5.

Figure 5

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Molecular interaction analyses. (A) 3D surface view of binding of 4e (rainbow color) with the binding pocket of tubulin (gray color) in ribbon style. (B) Enlarged view of the interaction of the 4e cavity with the binding groove of tubulin and depiction of its strong binding to both chains involving molecular forces. (C) Depiction of molecular forces: hydrogen bonds and tubulin residues involved in hydrophobic interactions with 4e. (D) Native contact analysis of drug–protein structure, which shows the consistent involvement of molecular forces. (E) Reaction coordinate analysis of binding of 4e with tubulin, which shows the involvement of free energy to stabilize the interaction.

Table 1. Molecular Docking Results of Synthesized Noscapine–Amino Acid Derivatives.

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The docked complex of 4e and tubulin with the highest score was analyzed through the Protein–Ligand Interaction Profiler (PLIP) and LIGPLOT. As a result, we found the involvement of strong molecular forces from the drug toward the binding groove of tubulin protein. 4e has shown the closest binding by forming eight hydrogen bonds and potential hydrophobic interactions. In detailed analyses of the molecular interactions, we found the first hydrogen bond was formed between the Gln residue at the 11 position of chain-A and the N3 atom of the drug with a bond length of 2.74 Å. The second hydrogen bond was formed between the Leu amino acid at position 70 of chain-A and acceptor drug atom O3 of 4ewith bond length 2.92 Å. The third hydrogen bond was formed between the Glu residue at the 71 position of chain-A and the O3 atom of the drug with bond length 2.55 Å. The fourth hydrogen bond was formed between the Val residue at position 74 of chain-A and the O3 atom of the ligand with bond length 2.90 Å. The fifth hydrogen bond was formed between the Asn residue at position 101 of chain A and the O3 atom of the drug with bond length 3.17 Å. The sixth hydrogen bond was formed between the Val residue at the 177 position of chain-A and the N3 atom of the ligand with bond length 3.30 Å. The seventh bond was formed between the Asn amino acid at position 687 of chain-B and the O2 atom of the drug with bond length 2.26 Å. The eighth hydrogen bond was formed between the Lys residue at position 790 of chain-B and the O3 atom of 4e with bond length 3.22 Å. Moreover, solid hydrophobic interactions were found in the range of 3–4 Å between Gln at position 11 of chain-A, Leu at position 70 of chain-A, Glu at position 71 of chain-A, Gln at position 176 of chain-A, Tyr at position 210 of chain-A, Tyr at position 224 of chain-A, Leu at position 686 of chain-B, and Asp at position 767 of chain-B and target drug molecule 4e. In addition, we also evaluated the reaction coordinates of 4e binding with tubulin through the structure mode MD of the complex for 1 ns using the Amber program. Altogether, we found the stabilized binding of 4e with tubulin with consistent involvement of free energy and native contacts (non-covalent) along with the hydrogen bonds and hydrophobic interactions. These results signified that 4e has higher potential against the target protein tubulin and showed that it is crucial to perform further in vitro and in vivo analyses.

Molecular Dynamics

Conformational Dynamics and Stability of Protein–Ligand Complex

The MD simulation studies were performed to understand better the stability and conformational dynamics of the tubulin and noscapine–tryptophan conjugate complex 4e. A 100 ns simulation was performed by taking the most stable binding conformation of the tubulin–noscapine–tryptophan conjugate complex.

In Figure 6a, the calculated root-mean-square deviations (RMSDs) of the tubulin backbone, the noscapine–tryptophan conjugate 4e, and their complex (tubulin–noscapine–tryptophan conjugate complex) are plotted according to the simulation time. From this RMSD trajectory plot, it can be noticed that the tubulin immediately reached a steady equilibrium in the initial 10 ns of simulation. The stable structure of tubulin throughout the simulation suggests that the noscapine–tryptophan conjugate does not employ any odd structural alternations in the protein conformation. Additionally, the noscapine–tryptophan conjugate RMSDs were computed (Figure 6b), and it was found that the noscapine–tryptophan conjugate is tightly bound, though it had some structural flexibility in the active-site pocket of protein for the initial 20 ns and rapidly attained equilibrium, depicting its tight binding with tubulin.

Figure 6.

Figure 6

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(a) Root-mean-square deviation (RMSD) of the tubulin backbone (C-α atoms) during 100 ns. (b) RMSD of the noscapine–tryptophan conjugate bound to the active site of tubulin during 100 ns simulation. (c) Time evolution plot of the radius of gyration (Rg) for all Cα-atoms of the tubulin–noscapine–tryptophan conjugate complex. (d) Solvent-accessible surface area (SASA) of the tubulin and noscapine–tryptophan conjugate complex.

The radius of gyration (Rg) is calculated as a time-dependent function to infer the impact of noscapine–tryptophan conjugate binding on the compactness and the structural integrity of the tubulin protein (Figure 6c). The stable Rg values throughout the simulation length depict the conservation.

In the prescribed time for simulation, the stable Rg values imply the conservation of the structural veracity of the protein and support a clear manifestation of a stable tubulin–noscapine–tryptophan conjugate complex. Also, since the solvent-accessible surface area (SASA) helps in understanding the role of solvent accessible to the protein, it is also computed to gain a better insight. It was found (Figure 6d) that the tubulin confirmation does not undergo a stark conformational change, indicating no prominent role of the solvents. Considering all the data, it can be seen that the binding of ligands imparted no conformational perturbation in the active site of tubulin and the tubulin–noscapine–tryptophan conjugate complex is stable.

In Vitro Studies

Noscapine–Tryptophan Inhibits the Growth and Survival of A549 Cancer Cells

To study the cytotoxic effects of noscapine and noscapine–tryptophan, A549 cells were treated with different concentrations for 24 h, and an MTT assay was performed (Figure 7). The result shows that treatment with 50 μM of noscapine–tryptophan decreases cell viability by 79% in A549 cells as compared to treatment with noscapine (31%). The IC50 of noscapine–tryptophan was calculated to be 32 μM, while for noscapine alone, the IC50 value was 73 μM. This clearly indicates that noscapine–tryptophan has better anticancer activity against A549 cells than noscapine alone. In previous reports, some noscapinoids showed good anticancer activity against A549 cell lines.36 However, these compounds cannot be synthesized at an industrial scale because of the excessive use of toxic chemicals such as acetic anhydride, sodium azide, and benzyl chloride and production in moderate yield. Also, the cytotoxicity of previously reported compounds against normal cells was slightly higher than that of 4e.

Figure 7.

Figure 7

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In vitro anticancer efficacy of noscapine alone and noscapine–tryptophan. Results are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control.

Effects of Noscapine–Tryptophan on Proliferation and Viability of A549 Cancer Cells

The most significant effect of 4e on monolayered cultured cells is the noticeable alteration in their structure and morphology. Under microscopic observation, treated cancer cells showed a changed morphological appearance compared to control cells (Figure 8). Morphological alterations caused by the noscapine–tryptophan action were considerable. The untreated A549 cells looked elliptical, were closely associated with each other, and were nurtured well. However, the A549 cells treated with noscapine–tryptophan had different morphologies, such as contracted, fragmented, round-shaped, detached from the base, and floated in their growth media.

Figure 8.

Figure 8

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Noscapine–tryptophan-treated A549 lung cancer cells.

As per the MTT data, we further assessed the growth inhibitory potential of noscapine–tryptophan on human lung cancer cells (A549). It was noted that the application of noscapine–tryptophan (1, 5, 10, 25, and 50 μM) at 24 h, 48 h, and 72 h decreased the cell proliferation and caused cell death in a dose-dependent manner (Figure 9). After completion of treatment, significant induced cell death up to 5–8.8% (24 h), 16–17% (48 h), and 30–32% (72 h) and decreases in the total cell numbers of 30–37% (24 h), 50–52% (48 h), and 70–73% (72 h), respectively, were observed after treatment with noscapine–tryptophan conjugate (Figure 9). These results indicated that noscapine–tryptophan could restrict cell proliferation and induce cell death in A549 cells.

Figure 9.

Figure 9

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Effects of treatment by tryptophan–noscapine on cell growth and viability after (a) 24 h, (b) 48 h, and (c) 72 h using Trypan Blue assay. Results are representative of three independent experiments. **P < 0.01, ***P < 0.001 compared with control.

Noscapine–Tryptophan Induces Cell Cycle Arrest in A549 Cells

Initiation of cell cycle arrest phenomena is one of the critical steps of reduced cell proliferation by various anticancer drugs. We investigated the effects of noscapine–tryptophan on the cell cycle progression of lung cancer cells. When A549 cells were challenged with the IC50 concentration of noscapine–tryptophan, A549 cells growth was arrested in the G1 phase of the cell cycle (Figure 10).

Figure 10.

Figure 10

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Cell cycle analysis.

Noscapine–Tryptophan Induces Apoptosis in A549 Cancer Cells

To decide whether the restriction of cancer cell multiplication by noscapine–tryptophan was partly due to the induction of programmed cell death or apoptosis, we applied the acridine orange/ethidium bromide (AO/EtBr) staining technique to quantify the apoptotic cells in the treated sample. Figure 11 shows the apoptotic effects of noscapine–tryptophan in human lung cancer cells (A549). When compared with the control, 57% of the cell population in 32 μM (IC50) noscapine–tryptophan-treated A549 cells showed apoptosis after exposure for 24 h (Figure 11).

Figure 11.

Figure 11

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AO/EtBr staining of lung cancer cells to detect apoptosis induced by noscapine–tryptophan conjugate. Live cells appear uniformly green in color, while apoptotic cells and necrotic cells are characterized by yellow/orange and red staining, respectively, due to chromatin condensation and loss of membrane integrity.

ADMET Profile

Table 2 summarizes the absorption, distribution, metabolism, excretion, and toxicity (ADMET) profile of Nos-Trp conjugate 4e. Most of the parameters qualify the molecule to be an anticancer molecule. Lower log P and AMES score signify its low toxicity in the body, whereas a high MDCK score represents the permeability to the cells.

Table 2. ADMET Profile of Noscapine–Tryptophan Derivative (4e).
entry property value comment
1 SA score 4.44 SA score < 6 represents ease of synthesis
2 MCE-18 140.0 MSC-18 > 45 represents a medicinal evaluation of the molecule
3 Pfizer rule log P < 3;TPSA > 4 molecules with high log P and low TPSA are considered to be more toxic
4 MDCK permeability 2.6 × 10–5 high permeability
5 Pgp-substrate 0.007 probability of Pgp-substrate
6 human intestinal absorption (HIA) 0.013 probability of HIA+
7 F20% 0.013 probability of bioavailability
8 volume distribution (VD) 1.217 optimal
9 AMES toxicity 0.225 less toxic
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Materials and Methods

In Vitro Studies

Cell Culture and Treatment

Lung cancer cells (A549) cells were maintained as a monolayer in RPMI 1640 (Roswell Park Memorial Institute) media (Himedia Laboratories) blended with fetal bovine serum (10%) and an antibiotic cocktail of penicillin–streptomycin (1%). Cancer cells were grown at 37 °C in a 95% humidified incubator with 5% carbon dioxide gas and maintained by sub-culturing the cancer cells twice a week. All the cancer cells (A549) were treated with different concentrations of Nos-Trp 4e (5, 10, 25, 50, 75, and 100 μM) for 24 h; PBS buffer was used as a control.

Cell Growth and Viability Assays

A549 cells were spread at 1.0 × 105 in each culture (60 mm) and nurtured at 37 ± 1 °C overnight. Cancer cells were challenged with various doses of noscapine and Nos-Trp 4e (1, 5, 10, 25, and 50 μM) for 24 h, 48 h, and 72 h. After the chosen treatments, the A549 cells were isolated after trypsinization and washed two times with PBS buffer. Isolated cells were mixed with trypan blue dye and counted using a hemocytometer described earlier. We used trypan blue dye to determine the number of living and dead cells. The experiment was performed twice, and each set of experiments was carried out in triplicate.37

Flow Cytometry Analysis of Cell Cycle Progression

A549 cells were seeded the same as in the cell growth and viability assay and treated with noscapine–tryptophan. At the accomplishment of each action, the total number of cells was pooled and processed for cell cycle analysis as described earlier. Cells were suspended on treated Petri plates and centrifuged at 500g for 5 min. The pellet was collected, washed twice with PBS buffer, and suspended in total darkness in 1.0 mL of a saponin–propidium iodide (PI) cocktail solution that contained 0.3% saponin (w/v), 25 mg/mL of PI (w/v), 0.1 mM EDTA, and 10 mg/mL RNase A (w/v). All samples were incubated at 4 °C overnight in the dark for staining. The data was retrieved using CellQuest software in a FACS Calibur (Becton Dickinson, USA) for 10,000 events per sample and analyzed using Win MDI software after suitable gating to determine the cell percentage in each phase of the cell cycle.37

Quantification of Apoptotic Cells by Acridine Orange (AO)/Ethidium Bromide (EtBr) Staining

A549 lung cancer cells were plated at a density of 2.0 × 105 cells per 60 mm cell culture plate. Different noscapine–tryptophan concentrations (5, 10, 25, 50, 75, and 100 M) were combined with various concentrations of cells in the growing medium. Cells were collected by trypsinization after the treatment period of 24 h. 25 μL of each cell suspension was isolated into a 1.5 mL microcentrifuge tube kept on ice. 1 μL of a dye comprising of 100 μg/mL AO and 100 μg/mL EtBr was prepared in PBS buffer and mixed well with the cell suspension. 10 μL of the dye and cell mixture was visualized under a fluorescent microscope (Nikon Eclipse Ti-S; Nikon Corp., Tokyo, Japan), and pictures were taken at 200× using a blue filter for AO stain and a green filter for EtBr stain; later both images were merged for a final image as reported earlier.37

Statistical Analysis

All experiments were carried out in triplicate. Their mean ± SD data were observed for statistical significance based on one-way ANOVA software along with Dunnett’s post hoc test. The statistical mean average values were found *P < 0.05, **P < 0.01, and ***P < 0.001, matching those reported earlier.37

Computational Details

Molecular Docking Analysis

The advancements in the development of computational algorithms, applications, and software have made it easy to screen a large number of chemical compounds against their target-specific receptors for any number of diseases.38−40 We employed such a detailed computational analysis to screen the synthesized compounds and evaluate their binding mechanism with the target receptor tubulin. Tubulin protein has a significant role in cell cycle regulations leading to cancerous states.41,42 For screening the compounds, molecular docking assays were performed using the Hex 8.0 module.43 A Hex 8.0 Unix-based environment was used to execute the docking simulation run. Prior to docking, drug files were drawn using the ChemDraw software in 3D conformer coordinates, and drawn files were energy minimized and optimized. After that, the target receptor file of the tubulin protein was availed from the Protein Data Bank. The retrieved tubulin protein was also processed and its energy was minimized for molecular docking. In our studies, we have used the 3D coordinates conformations of the target protein to get the best binding cavity and drug pose, allowing the target drug to interact with all regulatory regions/cavities of the tubulin protein. After the molecular docking run, drug poses were analyzed using Chimera, and the best binding pose with a high molecular docking score was further evaluated with MD analysis. Additionally, we studied the reaction coordinates of the drug binding interface with tubulin, which defined the involved free energy as work profiling of the drug while binding to tubulin. The responsible molecular forces for a binding lead drug with tubulin were identified using the LIGPLOT and the PLIP ligand interaction profiler, which helped us define the binding pocket of tubulin involved with the interaction drug.

Molecular Dynamics Simulations

MD simulations were carried out to understand the stability of tubulin and Nos-Trp conjugate 4e. GROMACS 5.1.4 software with the GROMOS96 43a1 force field was used to perform the MD simulations.44 Using the PRODRG server, the topology file of the Nos-Trp conjugate was created.45 The tubulin–noscapine–tryptophan conjugate complex was solvated in a cubic box having a 10 nm edge length. The system was then fed with single-point charge (SPC) water molecules. An adequate number of ions was added to maintain the system’s electroneutrality.

Further, the system was minimized to remove the short contacts and atom overlaps.46 A cutoff radius of 0.9 nm was applied for both the van der Waals and Coulombic interactions. The particle Ewald mesh method was used to describe the long-range electrostatic interactions. A two-step equilibration was performed. At first, the solvent and ion molecules were allowed to relax, keeping the coordinates of the tubulin–noscapine–tryptophan conjugate complex confined at their respective positions. In the second step, the restraints from the tubulin–noscapine–tryptophan conjugate complex were gradually reduced, and the whole system was equilibrated in the NPT ensemble. All the bonds involving hydrogen atoms were restrained by exploiting the LINCS algorithm.47 A Berendsen thermostat48 was employed to control the system’s temperature at 300 K, while a Parrinello–Rahman barostat49 was used to keep the pressure constant at 1 bar. The long production simulations commenced with configurations gathered from the previous equilibration steps. All the systems were simulated for 100 ns in the NPT ensemble, and trajectory frames were saved at every 2 ps interval.

Conclusion

We have designed a series of noscapine–amino acid conjugates and established the noscapine–tryptophan amino acid conjugate as a potential anticancer drug molecule against A549 lung cancer cell lines. The molecule can follow the prodrug approach mechanistically for its delivery to the target tubulin protein. The data obtained from molecular docking and molecular dynamics simulations reveal strong binding of the noscapine–tryptophan amino acid conjugate with tubulin protein. The binding of ligands imparted no conformational perturbation in the protein’s active site, and the noscapine–tryptophan amino acid–protein complex is stable; thereby it is anticipated that the ligand is a potential anticancer drug molecule.

Acknowledgments

The authors are thankful to CSIR (02(0265)/16/EMR-II), DST-SERB (EMR/2016/002976), DST (PURSE) for funding to carry out research and to USIC, University of Delhi, for providing instrumentation facilities. A.A. gratefully acknowledge DST-INSPIRE (IF170256) for the award of the Senior Research Fellowship. N.K. particularly thanks CSIR for the Research Associate fellowship.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00172.

  • Experimental procedures for all the compounds with1H NMR, 13C NMR, mass spectra and IR data given in the supplementry information (PDF)

Author Contributions

A.A. and R.C. designed the studies. A.A. and S.S. synthesized the molecules. R.R. and A.M. carried out in vitro studies. N.K., A.D., and A.A. performed the in silico experiments. A.A., N.K., A.M., and R.C. wrote the manuscript.

The authors declare no competing financial interest.

Special Issue

Published as part of the ACS Pharmacology & Translational Science virtual special issue “New Drug Modalities in Medicinal Chemistry, Pharmacology, and Translational Science”.

Supplementary Material

pt2c00172_si_001.pdf (1.1MB, pdf)

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