key: cord-301349-m4nr3pqx
authors: Mirza, Muhammad Usman; Saadabadi, Atefeh; Vanmeert, Michiel; Salo-Ahen, Outi M.H.; Abdullah, Iskandar; Claes, Sandra; De Jonghe, Steven; Schols, Dominique; Ahmad, Sarfraz; Froeyen, Matheus
title: Discovery of HIV entry inhibitors via a hybrid CXCR4 and CCR5 receptor pharmacophore‐based virtual screening approach
date: 2020-09-02
journal: Eur J Pharm Sci
DOI: 10.1016/j.ejps.2020.105537
sha: 
doc_id: 301349
cord_uid: m4nr3pqx

Chemokine receptors are key regulators of cell migration in terms of immunity and inflammation. Among these, CCR5 and CXCR4 play pivotal roles in cancer metastasis and HIV-1 transmission and infection. They act as essential co-receptors for HIV and furnish a route to the cell entry. In particular, inhibition of either CCR5 or CXCR4 leads very often the virus to shift to a more virulent dual-tropic strain. Therefore, dual receptor inhibition might improve the therapeutic strategies against HIV. In this study, we aimed to discover selective CCR5, CXCR4, and dual CCR5/CXCR4 antagonists using both receptor- and ligand-based computational methods. We employed this approach to fully incorporate the interaction attributes of the binding pocket together with molecular dynamics (MD) simulations and binding free energy calculations. The best hits were evaluated for their anti-HIV-1 activity against CXCR4- and CCR5-specific NL4.3 and BaL strains. Moreover, the Ca(2+) mobilization assay was used to evaluate their antagonistic activity. From the 27 tested compounds, three were identified as inhibitors: compounds 27 (CCR5), 6 (CXCR4) and 3 (dual) with IC(50) values ranging from 10.64 to 64.56 μM. The binding mode analysis suggests that the active compounds form a salt bridge with the glutamates and π-stacking interactions with the aromatic side chains binding site residues of the respective co-receptor. The presented hierarchical virtual screening approach provides essential aspects in identifying potential antagonists in terms of selectivity against a specific co-receptor. The compounds having multiple heterocyclic nitrogen atoms proved to be relatively more specific towards CXCR4 inhibition as compared to CCR5. The identified compounds serve as a starting point for further development of HIV entry inhibitors through synthesis and quantitative structure-activity relationship studies.

The most effective treatment of patients infected with human immunodeficiency virus 1 (HIV-1) is currently highly active antiretroviral therapy (HAART), which utilizes a combination of several drugs targeting viral proteins such as reverse transcriptase, integrase and protease [1, 2] . An alternative approach relies on the multi-targeted inhibition of viral cell entry [3] , which consists of a series of structural events arbitrated by viral and cellular membrane proteins. The interaction between the HIV-1 glycoprotein gp120 and the cellular receptor CD4 is an important initial step in viral entry. It induces a conformational change in gp120 and mediates further interaction with either one of the two co-receptors, chemokine receptor 5 (CCR5) and CXC-chemokine receptor 4 (CXCR4) during viral entry into the host cells [4, 5] . Both chemokine receptors belong to the G-protein coupled receptor family and regulate HIV-1 cellular tropism. CCR5-tropic or R5 strains preferentially uses CCR5, whereas CXCR4-tropic or X4 strains preferentially use CXCR4. Dual-tropic strains use both co-receptors for their entry in the CD4 + target cells [5] [6] [7] .

The chemokine ligand 5 (CCL5) also known as RANTES (regulated upon activation, normal T cell expressed, and secreted) is the natural ligand for CCR5 [8] , whereas the CXCchemokine ligand 12 (CXCL12), also known as SDF-1 (stromal cell-derived factor 1) is the natural ligand for CXCR4 [9] . Inhibition of either CCR5 or CXCR4 leads HIV to shift to a more virulent dual-tropic strain. Therefore dual inhibition of both co-receptors is of paramount importance to improve the therapeutic strategies against HIV infection [10, 11] . Although numerous selective CCR5 and CXCR4 antagonists have been reported [12] [13] [14] [15] , only a few CCR5/CXCR4 dual antagonists have been identified so far [3] . CCR5 and CXCR4 share an overall similar architecture, and the overlapping regions of the binding pockets pave the way for the identification of dual co-receptor antagonists [16] .

Maraviroc received marketing approval by the Food and Drug Administration (FDA) as a very potent and selective CCR5 antagonist with a favourable HIV resistance profile. It inhibits RANTES binding to a recombinant CCR5 expressed in the human embryonic kidney (HEK-293) cell line with an IC 50 value of 5.2 nM [17] . Plerixafor (or AMD3100) is a selective CXCR4 antagonist that specifically inhibits SDF-1α binding to CXCR4 (IC 50 = 12.5 nM) [18] Its close analogue AMD3451 (a tetraazamacrocycle) was first reported as a dual CXCR4/CCR5 antagonist [11] . Alongside, tetraazamacrocycle-based transition metal complexes were shown to act as dual CXCR4/CCR5 antagonist and were endowed with excellent in vitro activity against HIV [19] . Other dual CXCR4/CCR5 antagonists include, peptide-based triazoles analogues [20] , diterpene derivatives (EC 50 0.02 and 0.09 µM against R5 and X4 HIV strains, respectively [21] ), and pyrazole derivatives (IC 50 3.8 and 0.8 µM against CCR5-and CXCR4-utilizing HIV-1 strains) [22] . An analogue of the antiparasitic drug suramin NF279 [23] , the natural penicillixanthone A [24] and a coumarin-based analogue GUT-70 [25] are also known as dual CCR5/CXCR4 antagonists (Figure 1 ). Modern structure-based virtual screening approaches are indispensable in the discovery of novel hits targeting specific proteins and is nowadays a crucial part in the hit-to-leadoptimization phase of drug discovery [26] [27] [28] . This approach resulted in the discovery of various potent antiviral compounds against viruses such as Ebola [29] , dengue [30] , zika [31, 32] , SARS-coronavirus [33, 34] and influenza [35] . The aim of this study was to discover selective CCR5, CXCR4 and dual CCR5/CXCR4 antagonists based on both receptor-and ligand-based virtual screening methods together with molecular dynamics (MD) simulations and binding free energy calculations. The most promising hits were evaluated for CXCR4 and CCR5 antagonism via a Ca 2+ mobilization assay using U87.CD4.CXCR4 and U87.CD4.CCR5-transfected cell lines. In addition, their anti-HIV-1 activity was evaluated in TZM-bl cells, using NL4.3 (CXCR4 tropic) and BaL (CCR5 tropic) HIV-1 strains.

Databases of commercially available compounds (MolPort and Interbioscreen databases, ~1.2 million compounds) were used for the virtual screening (VS) and prepared using the LigPrep module of Maestro v.11 (Schrödinger, LLC, New York) software. Possible protonation states were generated using the OPLS3 force field [36] at pH 7±2. At most, one stereoisomer was generated per ligand such that the specified chiralities were retained. For the pharmacophorebased screening, the database was converted to the Phase database format with the Generate Phase Database module of Maestro [37, 38] .

The VS was specifically performed on CCR5, based on the X-ray crystal structure of CCR5 complexed with maraviroc (PDB ID: 4MBS; A chain) [39] was retrieved from the Protein Data Bank (PDB; www.rcsb.org). The co-crystallized ligands, ions and water molecules were removed from the receptor and all hydrogen atoms, missing loops and side chains were added, and bond orders assigned using the Protein Preparation Wizard of Maestro [40] . The protonation states of histidine residues were inspected interactively; His88 and His231 were changed to HIE (protonated at Nɛ atom) and HIP (double protonated), respectively, to optimize the H-bonding network. Restrained minimization using the OPLS3 force field was first applied for hydrogen atoms only and then for heavy atoms until non-hydrogen atoms attained an average root-mean-square deviation (RMSD) of 0.3 Å. The top hits from this virtual screening campaign against CCR5 were comparatively docked with CXCR4 in order to discover dual acting CCR5/CXCR4 antagonists. Therefore, the X-ray crystal structure of CXCR4 complexed with an isothiourea derivative (IT1t) (PDB ID: 3ODU) [41] was prepared for docking, similarly as described above. In CXCR4, the protonation state of His113 and His281 were changed to HIE and HIP, respectively. 2.3 Receptor-based virtual screening and Prime/MM-GBSA binding free energy VS by molecular docking was conducted with the Glide module of Maestro [42] . The docking site was defined with the Receptor Grid Generation tool of Maestro. The enclosing cubic grid was centered at coordinates (150.03, 108. 25, 22.41) and ligand diameter midpoint box was set to 10 Å × 10 Å × 10 Å . The limitation length for ligands to be docked was set to 12 Å . A maximum of five poses per ligand were considered. The Glide high-throughput virtual screening (HTVS) mode was used for the initial docking of the large database after which the top 10% of the hits based on the docking score were chosen and then re-docked using the standard precision (SP) algorithm. Subsequently, 10% of the highest-ranked compounds from this step were redocked using the extra-precision (XP) mode and finally, the top 10% of the Glide XP score-ranked ligands were selected for the binding free energy calculations with the Prime/MM-GBSA module of Maestro [43] . The approximate energies were calculated using the VSGB 2.0 solvation model [44] and OPLS3 force field, first keeping all binding site residues fixed and then allowing the residues within 5 Å from the ligand to move. Flexible sampling was done by minimization of the complex.

For the best-ranked compounds, molecular property prediction was carried out with Maestro's QikProp module. Besides the Lipinski's rule of five (≤ 2 violations), favorable cell permeability (QPPCaco > 500 nm s -1 ; QPPMDCK > 500 nm s -1 ) and acceptable aqueous solubility (-6.5 < QPlogS < 0.5), a series of PAINS filters were applied to eliminate compounds with potential toxicophores.

As a second screening approach, pharmacophore modeling was carried out with the Phase module of Maestro [37, 38] against CCR5. After preparing the maraviroc-CCR5 crystal complex (PDB ID: 4MBS) with the Protein Preparation Wizard of Maestro, this structure was used for generating the pharmacophore hypothesis. The Nɛ nitrogen atom of the triazole ring and the amide NH of maraviroc form H-bonds with the hydroxyl groups of Tyr37 and Tyr251, respectively ( Figure 2 ). In addition, the positively charged nitrogen in the bicyclic ring engages in an ionic interaction with Glu283. Therefore, a H-bond acceptor (A), a H-bond donor (D) and a positive ionic (P) feature were selected as the pharmacophoric features for the hypothesis (Figure 2 ). This pharmacophore model was then used to screen through the commercial ligand database in search of matching ligands. The ligands were scored and ranked by the Phase Screen Score according to how well they superimpose on the features associated with the hypothesis. The previously calculated QikProp properties were then used to filter the best-ranked 3000 hit compounds as described above. The 500 ligands that passed the filter were then docked into the maraviroc binding site with Glide using the XP mode and the subsequent Prime/MM-GBSA binding free energy calculation according to the abovedescribed protocol. Phase generated pharmacophore model. Pharmacophoric features: red sphere for hydrogen-bond acceptor (A); light blue sphere for hydrogen-bond donor (D); blue sphere for the positive ionic (P) feature. The arrows are pointing in the direction of the lone pair. Ligand atom color code: carbongreen; nitrogenblue; oxygenred; fluorinelight green. All hydrogen atoms except for maraviroc's polar hydrogens have been omitted for clarity.

After a careful analysis of the best-ranked virtual hits, the receptor-bound docked poses of the most promising compounds were subjected to MD simulation to estimate the stability of the binding complexes. All simulations were performed with the AMBER 18 simulation package [45] using the same MD simulation protocol as described previously [46, 47] . Each solvated system was minimized stepwise, followed by the heating and equilibration stages. Finally, a production run of 20 ns was performed at 300 K and 1 bar pressure. The time step was set to 2 fs and the trajectory snapshots were saved every 2 ps and analysed using the CPPTRAJ program [48] of AMBER. Moreover, H-bond occupancy was also examined for best compounds over simulation period. The binding free energies (ΔG total ) of the most promising hit compounds were calculated using the MM-GBSA (molecular mechanics-generalized Born surface area) method, implemented in AMBER 18. The AMBER ff99SB molecular mechanics (MM) force field [49] was used to estimate energy contributions from the atomic coordinates of the ligand, receptor and the complex in a gaseous phase. The total binding free energy is calculated as a sum of the molecular mechanics binding energy (ΔE MM ) and solvation free energy (ΔG sol ) as given below: Where, ΔE MM is further divided into internal energy (ΔE int ), electrostatic energy (ΔE ele ), and van der Waals energy (ΔE vdw ), and the total solvation free energy (ΔG sol ) is contributed by the sum of polar (ΔG p ) and non-polar (ΔG np ) components. ΔG bind is the free energy of binding of the ligand evaluated after entropic calculations, which is (TΔS). The MM-GBSA approach has been successfully used in binding free energy calculations [50] of antiviral inhibitors [51, 52] .

The selected compounds were purchased from MolPort (Riga LV1011, Latvia) in 2 to 5 mg masses. The purity the compounds was greater than 95%.

The most promising compounds resulting from VS were evaluated for antiviral activity by a luciferase assay in TZM-bl cells infected with wild type HIV-1 strains NL4.3 (CXCR4-tropic strain, X4) and BaL (CCR5-tropic strain, R5). AMD3100 (specific CXCR4 antagonist) and maraviroc (specific CCR5 antagonist) were included as best-in-class positive controls. The cellular toxicity of the compounds was also evaluated in TZM-bl cells. The anti-HIV replication assays in TZM-bl cells have been described previously [53] .

The ability of the compounds to inhibit the Ca 2+ flux induced by CXCL12 in U87.CD4.CXCR4 cells and by RANTES/CCL5 in U87.CD4.CCR5 cells was investigated via calcium mobilization assays. Intracellular calcium fluxes were measured with the FLIPR Tetra system as described previously [54] . The IC 50 (i.e., the compound concentration that inhibits the CXCL12-induced CXCR4 signaling or CCL5-induced CCR5 signaling by 50%) was calculated for each compound. The well-known antagonists, AMD3100 (CXCR4) and Maraviroc (CCR5) were used as a positive control.

To identify novel CCR5 antagonists, two parallel virtual screening campaigns were carried out using both receptor-and a ligand-based (pharmacophore) approaches. A total of ~1.2 million compounds were pre-filtered using a set of selected criteria: the Lipinski Rule of Five (≤ 2 violations), predicted permeability in Caco-2 and MDCK cells (> 500 nm/s) [55] , and the predicted IC 50 value for the blockage of human ether-a-go-go related gene potassium ion (HERG K + ) channel (QPlogHERG value set to -5). HERG K + inhibition is a well-known safety issue encountered in the medicinal chemistry optimization of various chemokine receptor antagonists, including maraviroc [56] . As a result, the database was reduced to ~30%, which then underwent the subsequent Glide docking protocol and the compounds were ranked based on XP docking and Prime/MM-GBSA scores. In the pharmacophorebased screening, the top compounds were selected from the pool of the best-ranked 3000 compounds predefined by the pharmacophore hypothesis (as explained in methods). Collectively, 137 compounds were selected and interactively visualized for molecular interactions. Receptor-based virtual hits included the compounds with the lowest (best) binding affinity values and a good interaction profile with the binding site residues of CCR5. Whereas, the main criteria in the pharmacophore-based screening included the potential to establish strong H-bond interaction (salt bridge) with Glu283, π-π stacking interaction with Trp86, and hydrophobic interactions with Tyr108, Ile198, and Tyr198. Furthermore, the visual inspection process contributed to the identification of compounds that revealed significant molecular interactions with the critical binding pocket residues of CCR5 and helped to exclude compounds that showed unrealistic docking conformations.

Among the top hits, 77 were found to follow the set selection criteria and showed strong interactions throughout the 20-ns MD simulations with favourable binding free energies. Although some of these compounds exhibited significantly different binding conformations, all top hits established an extensive interaction network with CCR5. These compounds were further investigated by MM-GBSA calculations and H-bond analysis. After a careful post-MD inspection, 43 compounds were selected based on the following criteria; (1) overall backbone stability of the protein/ligand complex, (2) electrostatic (ΔE ele ) and van der Waals (ΔE vdw ) interaction energy, (3) H-bonds occupancy, and (4) binding pocket residual contribution towards ligands. All these 43 hits were comparatively docked at CXCR4 receptor to investigate their potential for dual CCR5/CXCR4 antagonists [3] . As described earlier, both CCR5 and CXCR4 share marked similarities in their overall structures and in their active site geometry. These similarities include the presence of seven transmembrane domains with a high number of proline residues, a C-terminal threonine and serine-rich cytoplasmic region, four extracellular loops with a high number of cysteine residues and an N-terminal extracellular domain. The regions of the binding pockets contain critically important conserved residues which might allow CCR5 and CXCR4 to host the same ligand. Based on these evidences, we specifically sorted out potential hits and 27 structurally diverse compounds were purchased and tested for their anti-HIV activity (Figure S1) 

The Ca 2+ mobilization assay allowed to evaluate the antagonistic activity (i.e., the potency to inhibit the RANTES/CCL5 and SDF-1/CXCL12-induced calcium mobilization) of the selected compounds. Both RANTES (natural CCR5 ligand) and CXCL12 (natural CXCR4 ligand) produced a robust transient increase of cytosolic Ca 2+ flux in U87.CD4.CCR5 and U87.CD4.CXCR4 cells, respectively. Among the tested compounds, compounds 3, 6, and 27 showed antagonistic activity and dose-dependently inhibited the intracellular-induced Ca 2+ flux ( Table 1 ). The structures of these compounds are shown in 

The values of CC 50 , EC 50 and IC 50 are in micromolar if not otherwise stated. The results represent the mean of four readings.

The ability of the compounds to inhibit the cytopathogenic effect induced by NL4.3 (CXCR4-tropic strain, X4) and BaL (CCR5-tropic strain, R5) HIV-1 strains was further evaluated in TZM-bl cells ( 

The calcium mobilization clearly indicated that compounds 3, 6, and 27 bind to the host coreceptors. Although compound 27 showed an IC 50 value in the low micromolar range as CCR5 antagonist, no antiviral activity was observed even at the highest tested concentration of 100 μM. The predicted binding modes of compounds 3 (CCR5/CXCR4 antagonist), 6 (CXCR4), and 27 (CCR5) were investigated to obtain an atomic understanding inside the binding pocket. For all three compounds, the MD simulation time was further extended from 20 to 50 ns to enhance the investigations on the stability of the complexes and the compounds' binding energy contributions.

The statistical correlation was also evaluated between the average Amber/MM-GBSA total binding free energy (ΔG total ) at the co-receptors and the experimental IC 50 values from Ca 2+ mobilization assay ( 

The stability analysis of the top-ranked compounds against CCR5 is diagrammatically illustrated in (Figure 4, S2-S3) . Overall, all 27 tested compounds occupied the bottom of the CCR5 binding cavity defined by the residues of the transmembrane (TM) helices (1, 2, 3, 4, 5 and 7), and these binding poses were supported by the co-crystallized conformation of maraviroc ( Figure 4A) . Moreover, the protonated nitrogen of the compounds identified through the pharmacophore-based hypothesis was found in the close vicinity of the negatively charged Glu283, and most of these nitrogen atoms engaged in salt bridge interactions. All compounds exhibited excellent binding energies against CCR5 as indicated via the energy plot, where compounds 3, 6, and 27 ranked among the top, albeit no inhibition of Ca 2+ flux evoked by RANTES was observed for 6 ( Figure 4B ). The stability of these compounds inside the deep binding cavity of the corresponding co-receptors revealed convergence in RMSD values ( Figure S2) . Compound 27 reached an equilibrium value of ~1.5 Å (relative to the equilibrated position of the ligand) somewhat late than the others, at around 20 ns, due to its flexible fatty acid side-chain ( Figure S2) . Compound 6 adopted a favourable conformation inside the CXCR4 binding pocket at around 13 ns and the dual antagonist 3 displayed consistently stable binding at both co-receptors already from the early steps of the simulation (~ 2-3 ns) (Figures S2) . The stable RMSD value in the last ~ 30 ns was evident from the stable molecular interactions governed by the ligand inside the respective binding site of the co-receptors. Furthermore, the TM helices of CXCR4 and CCR5 also exhibited significant stability as observed from the root-mean-square fluctuations (RMSF < 1 Å), although TM5, which is connected to TM4 by the largest extracellular loop ECL2, showed somewhat larger fluctuations (around 2 Å) ( Figure 4A and S3). The binding cavity of both co-receptors is mostly hydrophobic due to the presence of a relatively large number of nonpolar amino acids. The analysis of the binding interactions demonstrated that the stability of the compounds inside the binding pocket of CXCR4 and CCR5 is mostly governed by π-stacking and H-bond interactions ( Figure 5A-D) . For further in-depth understanding of interactions, H-bond occupancy was also recorded (distance ≤ 3 Å; angle ≥120°) throughout the 50 ns simulations ( Figure 5E ). Per-residue decomposition was also performed for the key residues involved binding the ligands.

In 27/CCR5 complex, the interactions were mainly governed by multiple H-bond interactions with the binding site residues. Compound 27 contains a short flexible fatty acid side-chain (9hydroxynonanoic acid) linked to monic acid by an ester linkage. As evident from the molecular interactions, compound 27 established a network of H-bonds over a period of 50 ns and adopted a more favourable conformation inside the binding cavity ( Figure 5A) chain (Figure 5A) . The per-residue decomposition analysis revealed favourable binding free energy contributions (< -1.5 kcal/mol) with Trp86, Tyr108, Phe109, Tyr148, whereas the interaction with Glu283 revealed a positive value due to the desolvation effect.

In the CXCR4/6 complex, the phenyl rings on either side of the molecule were fluctuating during the simulation, with especially the terminal phenyl moiety connected to the aliphatic carbon side chain, whereas the central piperazine ring remained stable in its position. As shown in Figure 5B , the terminal side-chain benzene established a strong π-π stacking with Trp94 and revealed the most favourable energy decomposition value of -4.6 kcal/mol as compared to the other π-π stacking interactions (with Tyr108 and Phe109). This π-π stacking interaction was found completely missing with Trp86 in the 6/CCR5 complex because the molecule was flipped inside the binding cavity of CCR5 (Figure S4) , which might explain the lack of activity against CCR5. Moreover, the oxygen atom of the side-chain carboxylic acid of Glu288 formed a strong and well populated (82.7%) salt-bridge (average distance, 2.47 Å) with the protonated nitrogen atom of the piperazine ring of compound 6, which could stabilize the central piperazine ring. Glu288 also showed a favourable binding free energy contribution of -3.37 kcal/mol.

The dual CCR5/CXCR4 antagonist 3 contains a salicyl alcohol moiety, a chiral β-hydroxyl group, a secondary amine, and an ether-linked aryloxyalkyl sidechain. The nonpolar side chain facilitated binding with the hydrophobic binding cavity of the co-receptors. The protonated nitrogen of secondary amine in 3 retained similar spatial coordinates in both complexes and established well-populated salt-bridge (64.2 and 58.5%; average distance, 2.15 and 2.64 Å) with the glutamate residue of CCR5 (Glu283) and CXCR4 (Glu288) at respective sites ( Figure 5C-E) . The salt-bridge with glutamate provided an anchor point for a dual antagonist to extend in both directions. On one side, the aryloxyalkyl tail reached deep into the binding cavity of CCR5, where the terminal phenyl ring formed hydrophobic interactions with Tyr37, Tyr89, Gln280, Thr284 and Met287 ( Figure 5C) . Whereas, the phenyl ring formed a π-π interaction with Trp252 and established one H-bond with Tyr255 (46.4%; average distance, 2.55 Å) in CXCR4 ( Figure 5D ). On the other side, the two hydroxyl groups of the salicyl alcohol moiety established H-bonds with Thr195 of CCR5 (35.1%; average distance, 2.23 Å), although no stable H-bond was observed by this group in CXCR4, except an additional stacking interaction with Trp94. Among the interacting residues, Tyr108 (CCR5) and Tyr116 (CXCR4) showed favourable interactions, while Glu283 (CCR5) and Glu288 (CXCR4) revealed a slightly negative value.

The EC 50 values of the different compounds against CCR5 and CXCR4 were correlated quantitatively with the Amber/MM-GBSA binding energy values derived from the MD simulations in order to assess the performance of the calculations. For each MD simulated coreceptor complex with the bound ligand, a total of 5000 snapshots (every 4 ps) were generated. The ΔG total values were calculated at 10 different time scales (0-2 to 0-20 ns) and compared with the experimental EC 50 values. Table 3 tabulates average ΔG total values for CCR5 at different time scales (0-2 to 0-20 ns) of MD simulation along with the pEC 50 (-log of EC 50 ) values. The quantitative correlation between ΔG total and experimental pEC 50 values at the shortest time scales (0-2 ns and 0-4 ns) was rather poor (R 2 = 0.347 and 0.285), albeit a gradual increase in correlation was observed with larger time scales (> 0-12 ns and above). The initial poor correlation was due to the event of a single conformation of the proteinligand complex, which did not incorporate the flexibility in the beginning of the simulation. This underlying phenomenon was also evident from the correlations obtained from the docking scores (Glide XP) alone, which showed poor R 2 value (0.353) ( Figure S5) .

Afterwards, improved correlations were observed with a longer time scales of 0-18 (R 2 = 0.622) and 0-20 ns (R 2 = 0.631) (Table 3, Figure 6 ). The overall trend of ligand RMSD trajectories of most complexes (except for few) showed stability after ~8 ns (8-20 ns), which is rationalized through the electrostatic and vdW interaction energies. Consequently, the plot of ΔE MM components (ΔE elec and ΔE vdW ) with respect to MD simulations at various time scales (8-10 to 8-20 ns) were correlated separately with experimental pIC 50 values, as shown in Figure 6 . 

CCR5 and CXCR4 are both important co-receptors involved in the HIV-1 entry process [3, 4, 11, 57] . The CCR5 binding cavity is much deeper and open, while the entrance to the active site of CXCR4 is relatively covered having a strong negative charge on the surface as compared to CCR5 [39] . A highly negative surface charge density across the binding site makes CXCR4 atypical among the chemokine receptors and this difference is evident form the structural attributes of the CXCR4-specific inhibitors. Despite the substantial difference in the surface electrostatic potential, many compounds have been reported against both coreceptors [11, 19, 22, 58, 59] .

Comprehensive virtual screening experiments were carried out to investigate the multiple topographies of the CCR5 binding pocket using both receptor-and ligand-based approaches. The top hits from the CCR5 virtual screening campaign were afterwards docked in the binding cavity of CXCR4 to investigate the dual antagonism of these compounds. 27 Compounds were selected for biological evaluation, from which three displayed promising activity. Compound 3 is salmeterol, a β 2 -adrenergic receptor agonist acting as a dual CXCR4/CCR5 antagonists. Compound 6 is the antihistamine drug cinnarizine with CXCR4 antagonistic potency and compound 27 is the antibiotic mupirocin displaying potent CCR5 antagonism. To date, no antiviral activity of these compounds has been reported, except for moderate activity of salmeterol against the dengue virus [60] . Although, the pharmacological properties of these compounds have already been explained in the previous studies, very briefly, salmeterol is indicated to improve airflow in chronic obstructive pulmonary disease, and asthma. It falls under the category of long acting β-2 adrenergic receptor inhibitors with an elimination half-life of 5.5 hr when administered via pulmonary route. This long-acting feature of salmeterol is considered associated with its ability to bind with both active and exo-sites of the receptor. A blood maximum concentration of salmeterol is achieved within 15 min of pulmonary administration [61] . CYP3A4 is considered predominantly involved in the metabolism of salmeterol through α-hydroxylation. Some frequently known adverse effects of salmeterol may include hyperlactatemia, metabolic acidosis, depression, anxiety, hypophosphatemia, and hypokalemia [62] . Although, a 6-month long clinical trial of salmeterol have proved satisfactory in context of safety profile [63] . In context of these findings and keeping in view the serious side-effects of antiretroviral therapy, it could be most likely stated that further development and repurposing salmeterol for HIV treatment could serve a promising approach.

Cinnarizine is an antihistaminic compound used for motion sickness and vestibular disorders and is also considered as a nootropic agent [64, 65] . It is a long-known calcium channel blocker with slight potency towards certain dopamine receptors [66] . After oral administration, a maximum plasma concentration approaches in nearly 3 hours with elimination half-life of 3 to 4 hours [67] . Its six metabolites are known, which are produces via different of CYPs [68] . Although, cinnarizine has long been used in clinical practice with considerable tolerance but concerns has been raised for its association with drug-induced acute and chronic parkinsonism, linked to its ability to interact with dopamine receptors [69] .

On the other hand, long-term clinical trials up to 4-month have demonstrated reasonable safety [70] suggesting its beneficial application for further development as an anti-HIV agent.

Mupirocin, also known as pseudomonic acid A, is a natural produced by Pseudomonas fluorescens that can inhibit bacterial isoleucyl-tRNA synthetase [71] . It is found effective against many gram-positive bacteria and some gram-negative bacteria. The presence of epoxide residue in mupirocin makes it prone to extensive metabolism quickly converting it into inactive monic acid, which makes its applications limited to topical antibacterial ointment [71] . Its elimination half-life ranges between 20 and 40 minutes after an intravenous administration [72] . Due to this short half-life and lack of detailed pharmacological data from parenteral or oral administration requires extensive further experimentation for the use of mupirocin against HIV.

Compounds 3, 6 and 27 showed highly significant Amber/MM-GBSA (calculated) binding energy values during screening. Through molecular modelling studies, the compounds were found well-fitted inside the binding site of both co-receptors and remained stable throughout the MD simulation period. During MD simulations, the compounds remained in a close proximity to a negatively charged glutamate residue in the respective co-receptors. Molecular interactions of compounds 3 and 6 were in line with the pharmacophore hypothesis and the co-crystalized complexes of CCR5/maraviroc [39] and CXCR4/IT1t [41] , respectively. The protonated nitrogen atoms of the tropane group of maraviroc engaged in a salt-bridge interaction (2.78 Å) with Glu283 of CCR5, while the protonated nitrogen of the imidazothiazole moiety (N1) of IT1t showes a salt-bridge interaction (2.8 Å) with Glu288 of CXCR4. Likewise, the piperazine nitrogen of compound 6 and the secondary amine of compound 3 established a consistent salt-bridge interaction over a period of 50 ns with the respective glutamates. The importance of the glutamate residue deep inside the active site in the stabilization of the complexes has been reported previously [73] [74] [75] [76] [77] [78] [79] and, therefore, Glu288 of CXCR4 and Glu283 of CCR5 are important residues for structure-based drug discovery of CXCR4 and CCR5 antagonists. In addition, both compounds engaged in significant nonpolar interactions with the corresponding hydrophobic residues of the respective co-receptors, and these interactions are supported with previous mutagenesis and modelling studies [39, 41, 80, 81] . Compound 27, the most promising compound after receptor-based screening (IC 50 = 10.64 µM), showed a significant H-bond interaction profile with the residues lining the binding pocket of CCR5, as reported also for maraviroc [39, 80] .

Compound 6 did not inhibit the calcium flux induced by RANTES in U87.CD4.CCR5 cells, despite the significant MM-GBSA binding energy with CCR5 (ΔG total = -56.48 kcal/mol). The difference in electrostatic potential of the entrance of the binding site may provide the reason to this discrepancy, which has long been recognized between R5 and X4 strains [82] , and is supported by numerous mutational studies [83] [84] [85] [86] . CCR5 bears a neutral to positive electrostatic surface at the binding site entrance, with a deep negatively charged binding pocket, which might hinder the cationic piperazine moiety of compound 6 [74] . CXCR4 has a strongly negative surface charge due to a highly negative N-terminus that can favourably accommodate compound 6 [87] . Likewise, compounds 15 and 21, having relatively similar electronegative cyclic nitrogen atoms, showed selective anti-HIV activity against X4 HIV-1 strain (IC 50 of 41.13 and 64.33 µM, respectively). Furthermore, the binding pocket of the H 2histamine receptor (PDB ID: 6BQG), the primary target of cinnarizine [88] , has a similar, negatively charged surface ( Figure S6) , which is in agreement with the selective binding of compound 6 with CXCR4. In contrast, compound 27 possesses an overall negative charge [89] and is a potent CCR5 antagonist, lacking activity against CXCR4.

Per-residue decomposition analysis revealed binding free energy contributions by important binding site residues involved in electrostatic and vdW interactions. Among the residues that were found to stabilize the complex formation, glutamate residues produced positive decomposition values for compounds 3 and 27. This was due to the contribution of unfavourable solvation energy by blocking out a sufficient amount of solvent that might stabilize these negatively charged glutamates. However, due to the presence of long carbon chains in 3 and 27 reduced the solvent exposure of glutamate residues inside the binding cavity resulting in a more favourable value. Compound 6 showed a fairly negative energy value for glutamate interaction due to the presence of two protonated nitrogen atoms of the piperazine ring, which counteracted the desolvation effect. This more negative per-residue decomposition value of Glu288 in CXCR4 is supported by the evidence from Taylor et al., [59] . They changed the protonated piperidine ring [22] to a doubly protonated piperazine ring aiming at increasing the electrostatic interactions, hence counteracting the desolvation effect.

Although the identified compounds were less active against CCR5 and CXCR4 as compared to the positive controls maraviroc and AMD300, it should be underlined that these compounds are directly derived from virtual screening approaches without any further optimization. According to the literature, this is the first account of the activities of these compounds against CCR5 and CXCR4. Moreover, the structural pharmacophores that are present in compounds 2, 3, 6, and 27 were not observed in the previously reported CCR5 and CXCR4 antagonists. Pyrimidine, quinoline, tetrahydroquinoline, guanide, p-xylylenediamine, indole, and cyclic pentapeptide are commonly known pharmacophores as CXCR4 inhibitors [90] . The compounds 3, 6, and 27 possess relatively different structural architectures that do not know resemble with these moieties. This suggests that the discovery of 3, 6, and 27 as anit-HIV drugs could provide and new platform with a unique set of compounds that do not resemble with already known CXCR4 inhibitors. Among potent CCR5 inhibitors, maraviroc has 8-azabicyclo[3.2.1]octane and triazole residues, vicriviroc has piprazine, trifluoromethyl, and pyrimidine residues, aplaviroc contain 1,4,9triazaspiro [5.5] undecane and cenicriviroc is composed of biphenyl with fused tetrahydroazocine and imidazole [91] . The only similarity of the compounds reported in this study was found in the form of piprazine in 6, which resembles vicriviroc.

Additionally, it is worth mentioning that the high selectivity of compound 27 towards CCR5 could serve as a promising starting point for further optimization towards novel and more potent CCR5 antagonists. Moreover, it is notable that among the 27 screened compounds, 13 of them displayed antiviral activity against HIV-1 in low to medium micromolar range, which indicates an enrichment rate of approximately 48.14%. Hence, this systematic study focuses on an additional argument emphasizing the advantages of virtual screening for discovering new drug candidates.

The study outlines the identification of new selective CCR5 and CXCR4 antagonists, as well as the discovery of a dual CCR5/CXCR4 antagonist. The important pitfalls that must be addressed in virtual screening efforts against chemokine receptors is defining an effective scoring method using electrostatic surface potentials towards hit selection and prioritization. The pipeline of receptor-and ligand-based virtual screening, used in this study, proved as a promising tool to find antagonists for the selected targets. The results were validated through specific Ca 2+ GPCR signaling assays and via an anti-HIV-1 replication assay using CXCR4and CCR5-tropicNL4.3 and BaL strains. Quantitative structure activity relationship studies and synthesis of related derivatives could serve as a future direction for anti-HIV discovery. 

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