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Identification of a novel inhibitor for the Bromodomain (BRD4) through a receptor-based drug discovery approach

Recently, the demands on the drug discovery process have increased drastically because of the need to apprehend a novel target which might be both pertinent to cause disease and chemically tractable. The emergence of bioinformatics and computer strategies have given room to analyse conditions at the molecular level. The present work was to perform a molecular docking analysis and ADMET study of different δ-carboline derivatives with bromodomain (BRD4) receptor using receptor-based drug discovery approach. Based on the literature, 60 compounds were designed and subjected to molecular docking for the inhibition of brd4 receptor. The results showed that Compound 34 received the highest binding affinity with BRD4 receptor. Hence eight compounds were selected based on docked pose determined using AutoDock/Vina with the minimal energy of above -5.1. Then ADMET study was carried, in that, all the eight compounds had middle to high BBB permeability. During metabolism, all compounds except compounds 37, 42 and 47 showed no inhibition of CYP2C99 in the liver. Analysis of drug-likeness profile showed almost all compounds eligible in CMC rule, violation rule of CMC, MDDR rule with the value of 1 and violations of WDI showed 0 value. Such findings strongly implied that derivatives of δ-carboline could serve as lead molecules to inhibit BRD4, and this could lead to the future development of the right candidate for cancer research.


INTRODUCTION
Colorectal cancer (CRC) was a low incidence a few centuries ago, but is now predominant cancer and represents about 10% in the developed countries. The increase of CRC in developed countries can be attributed to modern nutritional practices and enhanced risk factors. Many treatments have been designed to provide the patient with extra opportunities for primary and metastatic CRC, which includes laparoscopic surgery, radiotherapy, neoadjuvant and palliative treatments. These treatments, however, had restricted effects on cure rates and long-term survival of the patients (Kuipers et al., 2015;Tang et al., 2017). Drug resistance was practically acknowledged at the outset in CRC treatment. The signi icance of drug resistance was demonstrated by intensive studies in causes of low sensitivity medications such as luoropyrimidines, irinotecan and oxaliplatin (Panczyk, 2014). Indoles deriva-tives are the most versatile compounds used for the synthesis of most of the FDA-approved drugs, ranked 9 th in the Top 25 most frequently approved nitrogen heterocycles in the FDA drugs. In recent decades there is an emphasis on the synthesis of indole derivatives. This emphasis is due to the endless architectural possibilities of the polycyclic structure by adding various fused heterocyclic scaffolds to achieve promising novel heterocycles. In many rational designs, the lexibility of the Indole framework can be observed in a broad spectrum of biological goals from topoisomerase inhibitors to G2/M inhibitors (Martins et al., 2015). Bromodomains (BRDs), which regulate gene transcription (Bason et al., 2011;Das et al., 2014) on histone at chromatin-modifying enzymes (Galdeano and Ciulli, 2016;Pérez-Salvia and Esteller, 2017). The protein acts as a gatekeeper for both the mitotic and the cell cycle (Conery et al., 2016;Zhan et al., 2015).
Contrary to most epigenetic modi iers, during mitosis BRD4 stays attached to chromosomes.
It uniquely connects to the late mitosis and early G1 by binding to acetylated H3 and H4 bookmark genes. For cell cycle progression, BRD4 is also essential and leads to G1 Arrest (Doroshow et al., 2017). Carbolines (pyridoindoles) are a heterocyclic group of compounds with a broad spectrum of biological activity (Lin et al., 2016;Kumar et al., 2008). The least researched four classes of compounds relative to their α, β, and γ analogues are the δ -carbolines (5H-pyrido[ 3,2-b]indole). This class of heterocycles has not yet discovered effective medicines. However, several compounds of this carboline class were relatively diverse in terms of biological activity. A large group has had powerful antimuscarinic, antihyperglycemic, antimalarial, antiplasmodial, antifungal, anticryptococcal, antiviral and antitumor activity. Crytolepine and its analogues have been recently revealed to be cytotoxic to melanoma cells and lung cancer cells. This research, therefore, discusses the identi ication of a novel δ-carboline derivatives to inhibit BRD4 protein by receptorbased drug discovery strategy, and ADMET to comprehend receptor (BRD4 proteins) binding patterns of δ-carboline to acquire more selective and potent medicines for the new possible CRC therapy.

Preparation of lead compounds
60 (Compounds 1-60) structurally diverse of δ-Carbolines side chains were selected for construction of a virtual library of the lead compound. The chemical structure of δ-Carbolines side chains was prepared by ChemBioDraw Ultra Version 12.0. The conformational energies of the inhibitors have been reduced. Minimalized structures were then subjected to docking experiments using Autodock/Vina.

Virtual screening and docking
The virtual library comprising of 60 compounds were used as lead and PDB ID: 4NUD as the target for this docking study. The 3D structures of all the compounds and co-crystal of BRD4 were then docked using AutoDock/Vina (Morris et al., 2009;Seeliger and de Groot, 2010). For AutoDock docking, more speci ic preparations have been made using AutoDock Tools (ADT). For each residue, the structure of the receptor was built with gasteiger loads attached to each atom. The active site has been determined using the grid boxes used in ADT (x, y, z axes were 102×94×124~0.375 Å). The indings below 1.0 Å in RMSD were clustered together, and the binding energy was matched with the receptor structure for further study.

ADMET studies
PreADMET online tool (PreADMET, 2020) used to assess the drug-likeness of all the selected compounds. PreADMET measures the drug-like value of substances based on the Lipinski rule of ive. 2D structures were built using ChemBioDraw (Daina et al., 2014;Waseem et al., 2017). Mol ile data has been applied to predict drug properties.

Binding nature of BRD4
Based on the literature survey, BRD4 receptor was selected for the study. This BRD4 was known for controlling the growth and metastasis of cancer. BRD4 proteins conduct regulatory transcription under normal conditions, though the transcription of several oncogenes (Fu et al., 2015). AutoDock/Vina was used to predict binding af inity in molecular docking studies of δ-Carbolines with receptors as well as to distinguish potent lead compounds widely used. The docking revealed the top ten positions for each ligand (Figure 1). Among all the studied lead compounds, compound 5, 17, 29, 34, 37, 39, 42, 47 were found to be the best    with minimal energy (Figure 2). The highest binding af inity with minimal energy was used for the comparison (Table 1). The highest binding af inity was obtained for compound 34 followed by compound 42 > compound 37 and 39 > compound 47 > compound 5 > compound 17 > compound 29 with af inity kcal/mol of -6.2, -6.1, -6.0, -6.0, -5.9, -5.6 and -5.1., and all molecules exhibited the good binding shown in Figure 2. The interactions between the compounds and the amino acids of Glu151, Tyr137, Lys141, Pro142, Asn140, Val147, Tyr98, Ile101, Pro104, Pro46, Pro45, Lys102, Pro45, Val90, Ala89, Asn44, Tyr98, Pro45, Ala89, Val90, Asn44, Pro45, Tyr98, Asn93, Ala89, Tyr98, Lys99, Tyr98, Pro104, Pro45, Glu49, Pro45, Pro46, Val147, Pro142, asn140, Lys141, Tyr137, Trp81, and Glu78 which is speci ically involved in the catalytic enzyme mechanism. Hydrogen bonding and hydrophobic interactions stabilised the ligand complex. All of the top poses created by the tool exhibited binding to the amino acids in the pocket. The top pose with the lowest binding af inities and high docking scores is typical in most docking programs. The indole side chain displayed robust hydrophobic and hydrophilic interactions with non-polar and polar residues indicated an improvement in binding af inity (Table 1). By understanding the results, we selected eight compounds based on pose generated by Vina and generated the best results with the minimal energy of above -5.1. Hydrogen, hydrophobic and ion inter-actions with BRD4 consequently stabilise the structure. Usually, a particular pose with low binding energy has a strong af inity for a long time and is thus the strongest docked conformation.

ADMET prediction
Pre ADMET predicts that all eight compounds are medium to high BBB based on the Cbrain/ Cblood ratio (Table 2). P-glycoprotein is located on the surface of endothelial cells of BBB that selectively permits the drugs. The selected compounds are tightly bound to plasma proteins and have GI absorption of cacao-2 celline and preferred for the oral dosage form. No compound inhibits the CYP2C19, CYP2C9, CYP2D6, CYP3A4 and all the others are except CH 37, CH 42 and CH 47 that showed inhibition of CYP2C99 in the liver during metabolism. CH42 and CH47 are strong substrates for CYP3A4 metabolism, and all other compounds showed weak for the metabolism of CYP3A4.

Drug likeness pro ile of the selected compounds
All the eight compounds were taken for the druglikeness pro ile study. This pro ile checks the compounds pharmacological and toxicity properties. Lipinski's rule showed drug suitability for all compounds with the number of hydrogen bond < 5, hydrogen bond acceptor < 10, MW below 500, ClogP with below 5. Compound CH 15, CH17, CH29 and CH47 showed <<0.1µm is suitable for its binding af inity nature except for Compounds CH34, CH37, CH39, and CH47. Almost all the compounds quali ied in CMC rule, CMC rule of violation (0), MDDR rule with structure with the value of 1 and WDI violations showed 0 value (Table 3).

CONCLUSION
The above work demonstrated that the compounds 5,17,29,34,37,39,42 and 47 belonging to δ-Carbolines derivatives would work against BRD4 by blocking their binding site. Therefore, in MYC transcription, these molecules play an essential role in interfering with signal transduction of chromatin by inhibiting the acetyl-lysine domains involved in transcriptional initiation and elongation.