Molecular docking analysis of flavonoid compounds with HIV-1 Reverse transcriptase for the identification of potential effective inhibitors

It is of interest to elucidate the binding mode analysis of 18 sulphonated flavones in the non nucleoside inhibitory binding pocket of the HIV-1 reverse transcriptase (PDB ID: 1RTD). We further compared them with the known Non Nucleosidic Reverse Transcriptase Inhibitors (NNRTI) drug molecules such as delaviridine, nevirapine and etravirine. Molecular docking studies of sulphonated flavones were performed in the binding pocket of reverse transcriptase using the PatchDock server. The flavones have different binding energies with RT and the atomic contact energy (ACE) value of sulfonated flavones range from-389 to-231 Kcal/mol while docking of the commercialized NNRTI showed the ACE value range from -486 to -224 Kcal/mol. This shows that most sulfonated flavones have ACE similar to the known NNRTI. Thus, seven compounds (FS-6, FS-7, FS-8, FS-9, FS-14, FS-15, FS-17) were reported as potent, selective, orally bio available, and nontoxic lead based on ADMET screening and effective binding analysis in the active site of the reverse transcriptase (PDB ID: 1RTD) for further consideration. We further document that compounds (FS-1, FS-10, FS-4 and FS-12) have unfavorable binding features to be considered as leads.


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©Biomedical Informatics (2019) and the complexity of the constituents fixed on the basic backbone [3, 4]. So far many studies were carried out for the synthesis of various functionalized flavonoids; these studies were also focused to improving their aqueous solubility and thus their bioavailability. In addition, previous research showed that the mechanism of action of flavones are related to their ability to scavenge free radicals but also to complexing essential metals for the catalytic activity of enzymes [5,6]. Furthermore, the pharmaceutical efficiency of the sulfonated compounds depends on their ability to bind specifically to target protein.
To better understand the mechanisms of interactions between sulphated macromolecules and amino acids Ojada et al have studied these interactions using X-ray crystallography [7].
Flavonoids are becoming the focus of medical research, they were used in many therapeutic application, such as anti-inflammatory, antimicrobial and anti-carcinogenic agents, others flavonoids were confirmed to be as inhibitors of numerous enzymes such as DNA synthetases and RNA polymerases, they have been also reported to present an excellent inhibition of human immunodeficiency virus (HIV-1) Protease (PR) and Reverse Transcriptase (RT) [8]. Previous studies have shown that some methoxylated flavones in position 3 have significant antiviral activities [9], citing for example the 4',7dihydroxy -3,5,6-timethoxyflavone tested in vitro have a significant antiviral activity against the poliovirus type 1 and rhinavirus type 15 [10], on the other hand, studies have shown that flavonoids carrying numerous hydroxyl groups, especially in position 5' and 4' (Apigenin, Myricetine...), exhibit very significant inhibitory activities for HIV1-RT with IC50 values less than 100 µg/mL [11], the baïcalein (5,6,7-trihydroxyflavone) tested also on the HIV-1 RT exhibited a 90% inhibition of the enzyme at a concentration of 2µg/ml [12]. Katsuhiko et al found that polyhydroxylated flavones have significant inhibitory activities against HIV1-RT, it was inhibited by 100%, 90% and 70% in the presence of 2 µg/mL of quercetin, myricetin, quercetagetin and baicalein, respectively [13]. The xanthohumol, a flavonoid extracted from Humulus lupulus, was tested also for its HIV-1 RT inhibitory activity by the Yong-Tang Zheng et al, the results showed a significant inhibitory potency at concentrations of approximately 24 µM [14].
The reverse transcriptase is a very interesting target for anti-HIV treatments, because it plays a central role in the viral replication and acts at the primary stage of infection whereas only two copies of gRNA are available. HIV-1 RT is known for its reverse transcription activity, nuclease activity (RNAse H) and DNA polymerase activity [15] HIV-1 RT is a multifunctional dimer formed of two subunits P51 and P66 with a molecular mass of 51-kDa and 66-kDa respectively. The P66 larger subunit is composed of 560 amino acids and includes both catalytic sites of the enzyme (RNAse H and DNA polymerase activity), on the other hand the P51 subunit it has no enzymatic activity but has an important structural role.  as Delaviridine, Nevirapine and Etravirine. The Docking study will gives a new insight into the investigation of molecular interaction between sulfonated compounds and allow us subsequently to select one or more best active flavones that will be the subject of an in vitro test in a future study.

Methodology: Preparation of the protein structure
The three dimensional structure of HIV-1 reverse transcriptase (PDBID: 1RTD) was retrieved from protein data bank (http://www.pdb.org) at 3.2A˚ RMSD resolution. The protein molecule were prepared mainly using VEGA ZZ software [22], the preparation process involves several steps: Adding hydrogen bonds, deleting water molecules and co-crystallized DNA -primer complex, than the active site residues within a range of 3.5 Å were selected and saved in PDB format for energy minimization. Before energy minimization, the atom constraints was applied the protein backbone and the Magnesium Ion were fixed to avoid any modifications in the experimental structure. After, the active site residues were minimized using NAMD with a 50000 steps conjugate gradients minimization. After refinement of the structure for correct formal charge the active site residues were saved as a PDB file, which will be used for the docking study.

Preparation and optimization of ligands structures
A total of 18 sulphonated flavones ( Table 4) were drawn using ACD/ChemSketch version 12 [23]. The 2D structures of sulphonated flavones were generated and the SMILE notations of the compounds were generated as well as the MOL2 format files, to convert the generated MOL2 files to PDB format we used the OPEN BABEL software [24] and for 3D structure refinement we used GlycoBioChem PRODRG2 Server [25], this operation allows us to select the lowest energy conformer for each compound.

Protein-ligand docking
In silico docking studies were performed using PatchDock server [26,27], which uses the local geometric characteristics of proteins by limiting the search to certain areas of the molecular surface. For this, in a first step, the molecular surface undergoes a segmentation process using a segmentation algorithm for detection of geometric patches (concave, convex and flat surface pieces). These patches are then connected to form a three-dimensional graph, which includes convex, concave and flat patches then the surface patches of the protein are matched to surface patches of the ligand molecules. The complexes obtained are then reclassified by a score function depending on the geometric fit and atomic desolvation energy.

Drug likeness and ADMET prediction
Drug-likeness was calculated with OSIRIS Property Explorer [28] (http://www.organic-chemistry.org), which predicts for pharmaco kinetic descriptors including drug likeness, drug score, mutagenicity, irritancy, and reproductive effect. The in silico ADMET profiling of a drug molecule which constitutes the pharmacokinetic profile and its possible effects on health such as blood/brain barrier penetration, HIA (Human Intestinal Absorption), Caco-2 cell permeability and aqueous solubility. In this study, we have used the ACD/Labs I-lab 2.0 web-based application (https://ilab.acdlabs.com/iLab2/).    Table 4: Chemical structures, IUPAC names and SMILES notation of the eighteen flavonoid compounds used in this study Compound ID

Molecular Modeling
In order to gain insight into the interaction modes of the studied flavones, the frontier orbitals HOMO and LUMO were used to measures the electron donor character and acceptor, this energy is represented in the (Figure 3), The molecular orbital shows that the contribution of HOMO is especially located on the core B comprising the sulfonyle group (Sulfonate) in position 5' which is responsible for electron transfer between molecules and target key residue in the HIV1-RT. The predominant acceptor character of the flavones presented in our study relative to the donor character explains their ability to interact with the amine function of target key residues of the HIV1-RT. In the same context, the study conducted by Gaydou et al [33][34][35] has allowed synthesizing tens of sulfonated flavones some of which, their structures were resolved by X-ray diffraction, It was also demonstrated that these sulfonated flavones have the ability to form a complexes with amino acids such as the paratoluidine, as well as a complexation of nucleic acids [34,36]. The flavonoids which form a complexes with amino compounds in an aqueous medium reflect of their potential to interact with the proteins, this is due to the sulfonic acid group of flavonoids who is bioisostere of the carboxylic function which plays a predominant role in the interaction between this group and any target amino acid of the protein, thus, the reactivity pattern that occur with flavones compounds is due to the labile proton of the acid function which binds to the amine function of amino acids. This finding corroborates with the LUMO and /or HOMO states attributed to the sulfonic acid group in the studied compounds.

Molecular docking studies
In silico, docking was carried to improve our knowledge of the interactions patterns between the reverse transcriptase (PDBID: 1RTD) and the studied sulfonated flavones, the docking results are summarized in the Table 1 (Figure 1). That case seems obvious, because for the flavones, the methoxy in position 3 'are oriented out of the plane of the cycle, and the Electron density is important at this oxygen, this promotes the establishment of hydrogen bonds. The frontier orbitals HOMO and LUMO measured for this flavone (Figure 2) has allowed us to have an additional argument to explain the location of the resulting interactions. The HOMO confirms the high electron density in the oxygen of the methoxy in 3 'which is favorable to form a hydrogen bond with the Lys103 with an ACE value of -224 Kcal/mol. . In our study FS-9, FS-17, FS-3 (Figure 3) was found to be forming a hydrogen bond interactions with the Tyr 188 and the Lys 103 with an ACE values of -231, -377, -323 Kcal/mol respectively, this compound can be considered as new drugs that are selectively act on HIV1-RT taking into account drug-resistance mutations in the cited residues. In addition the interaction of the other sulfonated flavones with the key residues of the p66 subunit of HIV-1 RT (PDBID: 1RTD) showed different ACE binding energies and occupy the active site unlike that of the commercialized NNRTI (Figure 3).

In silico ADMET prediction
Among the criteria that define a good drug, we find its absorption, distribution, metabolism, excretion and toxicity (ADMET), this descriptors and pharmaceutically relevant properties helps to evaluate biologically activity molecules and eliminate biologically inactivate one according to Lipinski rule of Five [40]. In our study we have analyzed pharmacokinetics descriptors (ADME) of sulfonated flavones using admet SAR toolbox [41] and the toxicity assessment for lethal dosages and probability of health effects using ACD/ I-Lab 2.0 (guest) and OSIRIS Property Explorer [28]. The results from OSIRIS Property Explorer shown that all the studied flavones were found to be in accordance of Lipinski's Rule of Five (Ro5) except for tow flavones, who does not respect the Lipinski Ro5 because the number of hydrogen bond acceptors (HBA) is greater than 10, the concerned flavones are: FS-1 and FS-10 ( Table 5), the HBA was 11 and 12 respectively, in addition two others flavones which present mutagenic effect were observed FS-4 and FS-12. However the studied compounds represent good solubility, bioavailability and have no reproductive, no irritant and no tumorigenic effect. Further pharmacokinetic properties were used to select the best drug candidate ( Table 2) among which were PlogBB (blood/brain), logHIA (intestinal barrier), Pcacoc (cell permeability), logpGI (substrate/non-Inhibitor), PlogS (aqueous solubility) and Logpapp (cell permeability), all sulfonated flavones showed significant values for the properties analyzed within acceptable range in comparison with the commercialized NNRTI ( Table 2) Table 2), while for predicted cell permeability (PCaco-2), all tested flavones shows to be in the acceptable range ,which reflects a good permeability across intestinal barrier and help in good transport of drug metabolic compounds.

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©Biomedical Informatics (2019) The logPGI Drug-drug interactions (DDIs) within tissue that transforms xenobiotics of vigorous reduction drug absorption and released more bile (liver) and urine (kidney) [42]. The acceptable range of −5 (poor) to +1 (good) and substrate inhibitor from 0 to 1 in which the commercialized NNRTI and tested flavones shows good activity with human intestinal absorption and metabolism. For the aqueous solubility (PlogS) of sulfonated flavones came within acceptable arrange which reflect their good solubility. The toxicity of sulfonated flavones was assessed based on lethal dosage (LD50) and functional probability effect on different tissues in rat model. The LD50 mouse and probability of health effects were predicted using ACD/I-Lab 2.0 (guest). The toxicity of selected flavones was summarized in the ( Table 3). The lower the dose the more toxic the compound, from our results ( Table 3) we found that tested flavones have less reliability on oral, subcutaneous, intra peritoneal, and intravenous when compared to reference molecule, the reliability index lie between 0.47-0. 12 borderline which reflect that some tested flavones (e.g., FS-1, FS-3, FS-11, FS-14, FS-16) were within the border line effect on reliability index and others were observed note reliable (Table 3) The toxicity and health effects predicted for blood, cardiovascular system, gastrointestinal system, kidney, liver, and lungs within the therapeutic dose range revealed that the sulfonated flavones FS-6, FS-8, FS-9, FS-18, FS-15, FS-17, FS-14 are less toxic on blood, cardiovascular system, gastrointestinal system, kidney, liver, and lungs, respectively and no side effect was observed in the tested dosages.

Conclusion:
Flavones are a subclass of natural compounds belonging to the class of flavonoids. They are widely studied for their ability to inhibit several enzymes such HIV-1 reverse transcriptase (RT), protease (PR), and integrase (IN). It is of interest to select such compounds as lead molecules with optimal binding features for RT (PDBID: 1RTD at the sites of Tyr 188, Lys102, Lys103 and Tryp 239) using molecular docking and ADMET/SAR screening. Thus, we report seven compounds (FS-6, FS-7, FS-8, FS-9, FS-14, FS-15, FS-17) as potent, selective, orally bioavailable, and non toxic leads based on the ADMET screening and effective binding analysis in the active site of the reverse transcriptase (PDBID: 1RTD) for further consideration.