In vitro bioassessment of novel δ- carboline derivatives as an antiproliferative agent

Several carboline derivatives are anticancer agents and studied for antiproliferative action against various cancer cells. Based on the preliminary analysis using insilico strategies, we have selected eight compounds for the study. All compounds have been synthesised and characterised for their purity and chemical composition. Antiproliferative activity was assessed by insilico Carcinogenicity assay, Cytotoxicity analysis by sulphorhodamine B, Antiproliferation assay and DNA damage analysis. The cytotoxic effects of the CH5, CH17, CH29, CH34, CH37, CH39, CH42 and CH47 on Vero, HeLa, A549, BRL3A, HCT116, and MCF7 were determined using the SRB assay. CH5, CH34, CH37 and CH42 was the most potent cytotoxic towards HCT116 cells with CTC50 value of 62.1±0.19, 47.1±0.41, 78.5±1.26 and 32.1±1.11μg/ml respectively. The assay revealed a noticeable reduction in cell number for CH5 and CH37 tested except CH34 and CH42. CH5 and CH37 observed cytotoxic effects were found to destroy the cells according to time, and cell viability decreased with that time length. To learn their role in cell death, CH5 and CH37 were therefore taken up for a further screening. This study suggested that CH5 and CH37 had a separatemechanismof action to kill and that in the cell line. Such results will provide enrichment of scienti ic knowledge on the molecular mechanism and target therapies of CH5 and CH37, thereby potentially helpful for patients with Colon cancer.


INTRODUCTION
The protein bromodomain (BRD4) can serve as an effector of histone acetylation because it is capable of detecting acetylated residues in histone tails. Over the last decade, it has been shown that inhibitors that block the interaction of BRD4 with acetylated residues have therapeutic potential (Muller et al., 2011). BRD4 protein inhibitors have been identi ied as critical antiproliferative agents in many cancer cells, and most of the compounds are in clinical trials. These inhibitors mechanism of action is to block the expression of oncogenes associated with enhancers with a very high level of histone acetylation known as superenhancers (SE) and thus prevent oncogene-driven cancer cell propagation (Garcia-Carpizo et al., 2019;Wu et al., 2017;Xiang et al., 2018;Zhang et al., 2019). Based on the previous study using insilico methods, eight compounds belonging to carboline derivatives were selected and utilised against different cancer cells to see that these compounds could stop cell proliferation. It has been estimated that half of all therapeutic agent consists of tetracyclic heteroaromatic ring compounds. There are various biologically active molecules containing different heteroatoms such as nitrogen and oxygen that have often drawn the attention of chemists over the year, primarily due to their biological signi icance. Carbolines are a group of heterocyclic compounds with a wide range of biological activity. δ-Carbolines are the less-known group of compounds compared to their analogues of α, β and γ. Effective drugs have not yet been found among this class of heterocycles.
Nevertheless, for several compounds of this heterocycle class, a vast spectrum of biological activity has been identi ied. A broad group of analogue δ-Carbolines has demonstrated high antimuscarinic, antihyperglycemic, antimalarial, antiplasmodial, antifungal, anticryptococcal, antiviral, and antitumor activity. Recently, cryptolepine and its analogues have been identi ied as cytotoxic to B16 melanoma cells and M109 Madison lung cancer cells (Queiroz et al., 2006).

Chemical and Reagents
All of the glassware's were dried on the oven before use. All the major chemicals had been acquired from Sigma Aldrich Co., Ltd. All solvents used in the study have been of acceptable quality and have been used as such without further puri ication. African green monkey Kidney cell (Vero), Human cervical cell line (HeLa), lungs cancer cell line (A549), Liver Cell line (BRL3A), human-colon-cancer cell line (HCT116) and breast cancer cell line (MCF7) were collected from National Centre for Cell Science, Pune, India.

Synthesis and characterisation
The compounds which were used in this entire study were identi ied through insilico receptor-based drug discovery. BRD4 was used as a receptor in the previous study. This BRD4 is known for his histone acetylation and Post Transcriptional modi ication in the cell. More than 50 compounds were taken in the prior research against BRD4 protein. Based on the indings, we selected eight compounds Table 1 for antiproliferation activity. These selected compounds were then synthesised, puri ied and characterised by IR, NMR and Mass their structure based on the various spectra. The characterised compounds have now been used in an in vitro proliferation study.

Carcinogenicity activity of compounds
The synthesised compounds have been used for the production of carcinogenicity prediction models using CarcinoPred-EL (Zhang et al., 2017). The synthesised eight compounds were used as a data set to construct and validate predictive models. The purpose of the study was to establish various molecular ingerprints and machine learning methods to predict the carcinogenicity of multiple compounds. That also describes the structural characteristics of the carcinogenic effects. Five-fold cross-validation assessed the ef iciency of predictive model's, which is commonly used in insilico methods. The models are used to detect possible carcinogens in the early phases of drug development-a free carcinogenicity prediction online tool (CarcinoPred-EL) used for this purpose.

Cytotoxicity study of compounds on the cancer cell line
The cytotoxicity study was conducted on eight cancer cells, such as Vero, HeLa, A549, BRL3A, HCT116, and MCF7 for all eight compounds using an SRB assay method. The monolayer of various cultures of cancer cells was trypsinised, and the number of cells was balanced to 1x10 5 cells/ml using a 10 % NBCS medium. Added 100 µl (approximately 10,000 cells) of diluted suspension of cells into 96 microtitre plate. The partial layer was formed after 24 hours, then the supernatant was washed, and the cells in the microtitre plates were added with 100 µl of different compound concentrations, then incubated at 37 • C for 72 h. After 72 h of incubation, 25 µl containing 50% of TCA was added gently to the wells to form a layer over the dilutions of the synthesised compounds to create a total concentration of 10 % and incubated for 1h at 4 • C. Then the plates were washed with distilled water, then airdried and stained for 30 min with MTT dye. The remaining dye was removed by washing with a 1 % acetic acid further air-dried. Then wells were added with 100 µl of 10 mM tris base to solubilise the dye. The absorbance was measured at a wavelength of     (Bannister and Kouzarides, 2011).

Antiproliferation assay on the colorectal cell line
Cell proliferation assay was conducted on colorectal (HCT116) cell line for an all the four compounds using SRB assay method. The procedure described here was optimised in a 96-well model for the proliferation assay of compounds to adherent cells. The monolayer was made on tissue culture lask according to the MTT assay described above (Bannister and Kouzarides, 2011). This setup helps the lask to assess the ability of the selected compounds for antiproliferative activity. After incubation, cell monolayers were ixed with 10 % trichloroacetic acid and stained with SRB for 30 min; then, the excess dye was removed by repeatedly washing the cells with 1 % acetic acid. Ten mM Tris base was added to dissolve the dye and measured at 510 nm (Orellana and Kasinski, 2016;Vichai and Kirtikara, 2006).

Measurement of DNA damage
We selected four compounds by SRB assay for further cytotoxicity. The DNA damage caused by the compounds was assessed during this process. The cells were suspended in 10 mm Tris HCl and 10 mm EDTA in alkaline pH. The cells were incubated and treated with proteinase-K. The combination was incubated (37 • C/3h) in phenol: chloroform: isoamyl (25:24:1) for DNA extraction. The extracted DNA was treated with 20 mg/ml of DNase and RNase at 4 • C for one hour and precipitated with sodium acetate and ethanol (1:3). The Gel electrophoresis method was used to measure the DNA damage for the synthesised compounds. The mixture was prepared by adding 10 µg of DNA from selected cancer cells on a 2 % agarose gel containing ethidium bromide and visualised at 100 V for 45 min under the Gel Doc method (Thangam et al., 2014).

Carcinogenicity nature of the compounds
Carcinogenicity is one of the cell-killing property of the chemical compound. To know the carcinogenicity nature of the compound CarcinoPred-EL was used (Mady et al., 2018;Roman et al., 2019).
The results showed that all the compound are mutagenic in Amis test either by point frameshift mutation (Table 2 and Table 3), and none of the compounds showed positive mutagenicity on a carcinoma mouse model. Compounds CH5, CH34, CH37, and CH42 were shown positive mutation on carcino rat except for compound CH17, CH29, CH39, and CH47. Inhibition of these prolongs QT C intense along with the risk of cardiac arrhythmias all the compounds showed a medium risk of hERG inhibition (Table 2 and Table 3).

In vitro Cell Killing property of selected compounds
Based on the in silico study, eight compounds were selected for in vitro cell killing property against various cancer cell line such as Vero, HeLa, A549, BRL3A, HCT116, and MCF7 to know their cell-killing property. Cell-based assays, particularly those using human cancer cell lines, gives a fast and inexpensive way of assessing compounds in vitro activity.
To differentiate the cytostatic and cytotoxic effects of a compound SRB assay is the best of choice. It measures cell density based on the amount of cellular protein. Such tests are less sensitive to metabolic errors and measure the overall concentration of cells directly by the amounts of protein present (Aslantürk, 2018;Gordon et al., 2018). The cytotoxic effects of the CH5, CH17, CH29, CH34, CH37, CH39, CH42 and CH47 on Vero, HeLa, A549, BRL3A, HCT116, and MCF7 were determined using SRB assay Table 4. As displayed in Table 4

Inhibition of cancer cell proliferation using reduction assay
Based on the cell-killing property of the cell, four compounds were selected for the study. These four compounds were taken for further research. In vitro cell proliferation of CH34, CH37, CH39 and CH42 against HCT116 were assessed by MTT reduction assay. Some recent studies have shown that chemicals can inhibit cell proliferation, modulate the detention of the cell cycle and trigger apoptosis in cancer cells but not in healthy cells. The assay revealed a noticeable reduction in cell number for CH5 and CH37 tested except CH34 and CH42. CH5 and CH37 were observed to kill the cells in a timedependent manner. We also found that cell viability was indirectly proportional to time Figure 1. Hence, CH5 and CH37 were taken up for further screening to know their mechanism in cell death.

Measurement of DNA damage
Selected two compounds CH5 and CH37, were used against PBR322 Plasmid DNA to check their activity on Raw DNA sample. The results showed no fragmentation on Plasmid DNA after addition of the compounds. This study indicated that those two compounds had a different mechanism of action in killing and reduction of the cell line in invitro. Sev-eral scientists found that the compound that displayed anticancer activity did not show DNA damaging and apoptosis (Bernstein et al., 2013;Jackson and Bartek, 2009). So to know the mechanism, various anticancer assays were taken into consideration to reveal the mechanism of action of the two compounds.

CONCLUSION
Our study demonstrates the nature of carcinogenicity, properties of cell killing, antiproliferation activity, and DNA damage assessment of synthesised derivatives of δ-carboline. Among the eight compounds, CH5 and CH37 showed promising cellkilling properties and halted HCT116 cell line proliferation at a lower concentration. These indings can provide enrichment of scienti ic information about the molecular mechanism and target therapies of CH5 and CH37, which is therefore potentially bene icial to Colon cancer patients.

Funding Support
Nil

Con lict of Interest
The author declares that there is no con lict of interest.