p75 Interacting Inhibitor by

Aug 24, 2017 - Through this project, we have finished the docking screening of the Maybridge database against the p75 binding site of HIV-1 integrase ...
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Discovery of a Novel HIV‑1 Integrase/p75 Interacting Inhibitor by Docking Screening, Biochemical Assay, and in Vitro Studies Yan Wang,†,⊥ Huang-Quan Lin,†,‡,⊥ Ping Wang,§,⊥ Jian-Shu Hu,†,⊥ Tsz-Ming Ip,† Liu-Meng Yang,§ Yong-Tang Zheng,*,§ and David Chi-Cheong Wan*,† †

School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China Shenzhen Research Institute, the Chinese University of Hong Kong, Shenzhen 518057, China § Key Laboratory of Animal Models and Human Diseases Mechanisms of Yunnan, Kunming Institute of Zoology, KIZ/CUHK Joint Laboratory of Bioresources and Molecular Research in Common Diseases, Chinese Academy of Sciences, Kunming, Yunnan 650223, China ‡

S Supporting Information *

ABSTRACT: Protein−protein interaction between lens epithelium-derived growth factor (LEDGF/p75) and HIV-1 integrase becomes an attractive target for anti-HIV drug development. The blockade of this interaction by small molecules could potentially inhibit HIV-1 replication. These small molecules are termed as LEDGINs; and several newly identified LEDGINs have been reported to significantly reduce HIV-1 replication. Through this project, we have finished the docking screening of the Maybridge database against the p75 binding site of HIV-1 integrase using both DOCK and Autodock Vina software. Finally, we have successfully identified a novel scaffold LEDGINs inhibitor DW-D-5. Its antiviral activities and anticatalytic activity of HIV-1 integrase are similar to other LEDGINs under development. We demonstrated that the combination of DW-D-5 and FDA approved anti-HIV drugs resulted in additive inhibitory effects on HIV-1 replication, indicating that DW-D-5 could be an important component of combination pills for clinic use in HIV treatment.

1. INTRODUCTION Lens epithelium-derived growth factor (LEDGF/p75) is a cofactor of HIV-1 integrase that binds to integrase to facilitate its nuclear translocation. The crucial role of the interaction between LEDGF/p75 and HIV-1 integrase in the early steps of HIV-1 replication has been widely evidenced by mutagenesis, RNA interference (RNAi), and gene knockout studies. Apart from genetic approaches, the blockade of this interaction by small molecules could potentially inhibit HIV-1 replication.1 These small molecules, termed as LEDGINs, become an attractive target for anti-HIV drug development and several newly identified LEDGINs have been reported to significantly reduce HIV-1 replication.2−5 However, current discovery of LEDGINs relied on pharmacophore modeling and fragment-based discovery as two dominant tools of virtual screening.2−5 The newly identified LEDGINs were summarized in Table 1. Here we reported to use Maybridge database against p75 binding site of HIV-1 integrase to identify novel LEDGINs for docking analysis. The Maybridge Database was included in our analysis because this database has been demonstrated to have higher possibility © 2017 American Chemical Society

to get genuine compounds from the vendors. Three chemicals have been identified as new LEDGINs according to our docking screening and in vitro validation. Two of these chemicals, the DW-D-5 and DW-D-6 exhibited good antiviral activities in HIV-1 replication study. The combination cocktail of drugs against different target proteins is widely used in anti-HIV-1 therapy. Therefore, it is critical to test whether newly identified LEDGINs are not antagonistic with other FDA approved anti-HIV-1 drugs.6 Additionally, LEDGINs that synergistically inhibit the viral load in HIV-1 infected patients in combination with FDA-approved anti-HIV-1 drugs will be preferable for a first-line component of combination pills against HIV-1 in the future.

2. MATERIALS AND METHODS 2.1. Molecular Docking Screening. Molecular docking screening was performed using the three-dimensional crystal structures of substrate free HIV-1-IN/p75 (2B4J) obtained Received: June 30, 2017 Published: August 24, 2017 2336

DOI: 10.1021/acs.jcim.7b00402 J. Chem. Inf. Model. 2017, 57, 2336−2343

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Journal of Chemical Information and Modeling Table 1. Other LEDGINs under Development chemical D77 CHIBA-3003 CHIBA-3053 N-(cyclohexylmethyl)-2,3-dihydroxy-5(piperidin-1-ylsulfonyl) benzamide N-bis(4-methoxyphenyl)methylbenzamide Carbidopa Eprosartan Atorvastatin cmpd 6 of 2-(quinolin-3-yl)acetic acids CX14442 compound 11e of of N-aryl-naphthylamines Kuwanon-L Lovastatin 5-((p-tolylamino)methyl)quinolin-8-ol NPD170

biological activity inhibits inhibits inhibits inhibits

HIV-1IIIB replication by EC50 value of 23.8 μg/mL in MT-4 cell (5.03 μg/mL for C8166 cells)7 the HIV-1 IN−LEDGF/p75 interaction with an IC50 value of 35 μM2 the HIV-1 IN−LEDGF/p75 interaction with an IC50 value 3.5 μM11 the IN−LEDGF/p75 interaction with an IC50 value of 8 μM12

shows an IC50 of 8.1 μM in the AlphaScreen based LEDGF/p75−IN interaction assay and has moderate antiviral activity, with an EC50 of 29 μM4 inhibits the IN−LEDGF/p75 interaction with an IC50 value of 6.54 μM13 inhibits the IN−LEDGF/p75 interaction with an IC50 value of 35.85 μM13 inhibits the IN-LEDGF/p75 interaction with an IC50 value of 8.9 μM13 inhibits the IN−LEDGF/p75 interaction with an IC50 value of 1.37 μM; antiviral activity of compound 6 has been tested by MT-4 cell protection assay with EC50 2.35 μM1 is the first LEDGIN reported to display antiviral activity in the low nanomolar range, with an EC50 of 69 nM and a selectivity index of 13916 inhibits the IN−LEDGF/p75 interaction with an IC50 value of 2.5 μM; antiviral activity has been tested by MT-4 cell protection assay with an EC50 of 1.07 μM14 inhibits the IN−LEDGF/p75 interaction with an IC50 value of 22 μM; antiviral activity of Kuwanon-L has been tested by replication with an EC50 of 1.9 μM15 antiviral activity of Lovastatin has been tested by MT-4 assay with EC50 6.95 μM16 antiviral activity of 5-((p-tolylamino)methyl)quinolin-8-ol has been tested by MT-4 assay with an EC50 of 15.41 μM5 inhibits the IN−LEDGF/p75 interaction with an IC50 value of 0.25 μM; antiviral activity of NPD170 has been tested by CPE in C8166 cells with an EC50 of 1.81 μM17

cells were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature. The cells were rinsed in PBS, postfixed and made permeable in 0.5% Triton X-100 at room temperature for 30 min. After rinsing with PBS, the cells were incubated in 3% BSA for 1 h, washed by PBS, and incubated with anti-integrase1 antibody (Santa Cruz Biotechnology) diluted with PBS containing 1% BSA for 16 h at 4 °C. Then cells were washed 30 min each time for three times by PBS at room temperature and then incubated with FITC-conjugated antibody (Santa Cruz Biotechnology) diluted with PBS containing 1% BSA for 1 h at room temperature. Then cells were washed as above and stained with PI (1 μg/mL) for 20 min. Photos of stained cells were taken under a confocal fluorescence microscope. 2.4. Western Blot of Inhibitory Activity on Translocation of HIV-1 Integrase into the Nucleus. The transfection procedure was as described above. For Western blot analysis, the cells were treated with chemicals for 24 h and washed with PBS. Then the cells were dislodged and pelleted by centrifugation and resuspended in the cell lysis buffer (10 mM HEPES; pH 7.5, 10 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.5% Nonidet P-40 and 0.5 mM PMSF) along with the protease inhibitor cocktail (Sigma). After incubation on ice for 15−20 min with intermittent mixing, samples were vortexed to disrupt cell membranes and then centrifuged at 12 000g at 4 °C for 10 min. The supernatant was discarded and the pelleted nuclei were washed twice with the cell lysis buffer and resuspended in the nuclear extraction buffer (20 mM HEPES (pH 7.5), 400 mM NaCl, 1 mM EDTA, 1 mM DTT, 1 mM PMSF) containing protease inhibitor cocktail and incubated on ice for 30 min. Nuclear extracts were collected by centrifugation at 12 000g for 15 min at 4 °C. Protein concentrations of the nuclear extracts were determined using Bradford’s reagent (Bio-Rad, USA). The extracts were immediately analyzed by Western blot. Proteins in the total cell lysate were separated by 10% SDS polyacrylamide gel electrophoresis and were electro-transferred to a polyvinylidene difluoride membrane (Immobilon-P membrane; Millipore, Bedford, MA, US). After blocking in a solution of 5% bovine serum albumin for 1 h, the

from the Protein Data Bank. The receptor was pretreated by the Chimera software to get the standard three-dimensional structural information for docking. The ligands were approached from the Maybridge Database and transformed to the proper format for docking through the Openbabel. The software DOCK 6 and AutoDock Vina v.1.0.2 were used for all dockings in this study. The docking parameters for DOCK 6 and AutoDock Vina were set to their default values. The grid box was 20 Å × 20 Å × 20 Å, encompassing the surface position where the HIV-1-IN and p75 have interaction. For the docking results of AutoDock Vina, the binding modes were clustered through the root-mean square deviation (RMSD) among the Cartesian coordinates of the ligand atoms. The docking results were ranked by the binding free energy from the default score function in AutoDock Vina. The binding modes with lowest binding free energy and the most cluster members were chosen for the optimum docking conformation. For the results of DOCK, the binding modes were selected by the free energy based on the DOCK3.5 score function. The binding results were illustrated by PyMOL Molecular Graphics System Version 1.3 (Schrödinger, LLC). 2.2. Plasmid. Prokaryotic protein expression vector pRSET (Thermo Fisher Scientific) containing full-length HIV-1 integrase and pcDNA3.1 (Thermo Fisher Scientific) vector containing HIV-1 integrase were constructed in our laboratory. 2.3. Image Assay of Inhibitory Activity on Translocation of HIV-1 Integrase into the Nucleus. HeLa cells cultured and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 μg/mL G418, and 1% of streptomycin−penicillin (Invitrogen) at 37 °C in a 5% CO2 incubator. HeLa cells (5 × 106) were seeded onto 35 mm confocal dishes (BD) in DMEM containing 10% FBS for 24 h prior transfection. The plasmid HIV-1-IN-pcDNA3.1 was transfected into HeLa cells using Lipofectamine 2000 transfection reagent (Invitrogen) in accordance with the manufacturer’s instruction. Then, the medium was removed 4 h after transfection. Fresh medium containing test chemicals were added. After 24-h incubation, 2337

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and incubated for 1 h at 37 °C. For measurement, OPD was used as substrate. By adding 2 M sulfuric acid 10 min, the OD was read with Elx800ELISA at 490 nm (reference wavelength was 630 nm). 2.7. HIV-1 CPE Inhibition Assay. In the presence or absence of various concentrations of samples, 4 × 104/well C8166 cells were infected with HIV-1 at a multiplicity of infection (MOI) of 0.07, and cultured in 96-well plates at 37 °C in 5% CO2 for 3 days. AZT was used as a positive control. At 3 days postinfection, the cytopathic effect (CPE) was measured by counting the number of syncytia (multinucleated giant cell) in each well of 96-well plates under an inverted microscope (100×). The inhibitory percentage of syncytia formation was calculated by the percentage of syncytia number in sample-treated culture compared to that in infected control culture. 50% effective concentration (EC50) was calculated. 2.8. MT-4 Protection Assay. MT-4 cells were suspended in culture medium at 4× 105 cells/mL and infected with HIV IIIB at a multiplicity of infection of 0.07. Immediately after viral infection, 100 μL of the cell suspension was placed in each well of a flat-bottomed microtiter tray containing various concentrations of the test compounds. The test compounds were dissolved in DMSO at 50 mM or higher. After 7 days of incubation at 37 °C, the cell viability was determined using the MTT method. Compounds were tested in parallel for cytotoxic effects in uninfected MT-4 cells. 2.9. HIV-1 Integrase Strand Transfer Assay. Full-length HIV-1 integrase constructed with a 6-histidine tag was expressed in Escherichia coli and purified following standard methods. The strand transfer reaction was performed in 96-well bottom polypropylene microplates (BD) containing 1 μL of compound or 25% DMSO. A 16 μL of IN mixture (20 mM HEPES pH 7.5, 10 mM MnCl2, 1 mM DTT, 0.50 μM His6-IN) was added. After a 15 min preincubation, 20 μL of substrate oligonucleotide mixture (0.20 μM Biotin-LTR preprocessed donor DNA, 0.20 μM Digoxigenin (DIG)−Target DNA) was added and the plate was incubated for 18 h at 37 °C. The reaction solution then was transferred to streptavidincoated white plates (Thermo Scientific Pierce). After 1 h incubation at room temperature with gentle shaking, integrase and unjoined DNA were removed by washing each well three times with 200 μL wash solution 1 (30 mM NaOH, 0.2 M NaCl, 1 mM Na2EDTA). A 100 μL portion of 2000-fold diluted AP-conjugated anti-DIG antibody (abcam) was added and the plate was incubated for 1 h at 37 °C. Unbound antibody was removed with wash solution 2 (PBS pH 7.4, 0.05% Tween-20, 0.1% bovine serum albumin), 100 μL of NPP ELISA substrate (Thermo Scientific Pierce) was added, and the OD was read with Elx800 ELISA at 380 nm. 2.10. AChE Assay. DW compounds dissolved in DMSO were tested for AChE inhibitory activity by the Ellman assay with minor modifications. A 10 μL of human recombinant AChE (prepared in-house) and 1 μL of drug (final conc. Ten μM) were added into 190 μL of PBS buffer (100 mM, pH 7.4) and incubated in a 96-well plate at 37 °C for 10 min. Then 25 μL of 12.5 mM ATCI and 25 μL of 10 mM DTNB were premixed and added into each well. After 10 min incubation with the substrate, the optical densities were measured in a 96-well plate reader at 412 nm. The optical density was inversely proportional to the inhibitory activity. By contrast, a blank control without the tested compound was also performed in parallel; the normal hydrolytic rate of the enzyme can be

Figure 1. Flowchart of screening procedures.

membrane was incubated overnight with anti-integrase-1 and Lamin B1 primary antibodies (Santa Cruz Biotechnology) followed by incubation with alkaline phosphatase-conjugated secondary antibodies for 1 h. Specific bands were detected with BCIP and NBT detection reagent (Sigma). 2.5. Cytotoxicity Test. MTT colorimetric assay was performed to determine the cytotoxicity of tested chemicals. Cells were seeded in 96-well plates at a density of 5 × 104 cells/ well and treated with tested chemicals at desired concentration at 37 °C for 24 h. The cells were then incubated with 20 μL MTT (5 mg/mL) for 4 h. The cells were eluted with DMSO and quantified with a spectrophotometer (Ultramark Microplate Reader, Bio-Rad) at a wavelength of 590 nm. 2.6. Inhibition of HIV-1 p24 Antigen Production in Acute Infection. HIV-1IIIB (HIV-1 laboratory-adapted strain) was used in acute infection assay. C8166 cell line and HIV-1IIIB were supplied by British Medical Research Council, AIDS Reagent Project. C8166 cells were infected with HIV-1IIIB (MOI = 0.07) at 37 °C in a 5% CO2 humidified incubator for 2 h. The culture medium was removed, and cells were washed for three times with PBS. Then, the cells (4 × 104 cells/well) were seeded with compounds and incubated at 37 °C in a 5% CO2 humidified incubator for 3 days. The cell culture supernatant were collected and mixed with 5% Triton X-100. HIV-1 p24 was assayed by ELISA. First, Anti-Fc antibody was embedded as 1 μg/well at 4 °C for overnight and wells were blocked for 2 h at 37 °C with 5% defatted milk powder. Second, a 100 μL mouse anti-p24 antibody was added to each well and incubated for 1 h at 37 °C. Third, 100 μL cell culture supernatant was added to each well and incubated for 2 h at 37 °C. Forthly, 100 μL rabbit anti-p24 antibody was added to each well and incubated for 1 h at 37 °C. Finally, 100 μL HRP-conjugated goat antirabbit IgG was added to each well 2338

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Figure 2. Chemical structures of filtered compounds.

candidates from DOCK screening and Autodock Vina screening, respectively. According to druggable analysis, six chemicals from DOCK screening and eight chemicals from Autodock Vina screening were selected for in vitro validation. The structures of selected chemicals are presented in Figure 2. The docking scores of selected chemicals and benchmarks are summarized in Table 2. However, 4 out of 14 chemicals exhibited strong cytotoxicity; we therefore did not evaluate their anti-HIV-1 activities. 3.2. Three Chemicals Identified as Potential LEDGINs. The translocation of HIV-1 integrase assay was conducted by transfected pcDNA3.1/HIV-1 IN plasmid into HeLa cells and the HIV-1 IN nuclear distribution was probed by using both localization image analysis and Western blot. Figure 3A shows HIV-1 integrase staining with FITC, red nucleus staining with PI and merged picture obtained after treatment with the various

represented by the blank control. Each assay was performed in triplicate. The percentage inhibitory activities of the various compounds were calculated by comparison with the positive control and the blank control. The formula was shown as follows: percent of inhibitory activity of the compound = (1 − absorbance of sample/absorbance of blank control)/(1 − absorbance of positive control/absorbance of blank control) × 100%.

3. RESULTS 3.1. Total of 14 Chemicals Filtered as LEDGINs Using Docking Screening. The workflow of screening procedures was illustrated in Figure 1. In the primary docking screen, we docked ∼51 000 chemicals from the Maybridge database against wild type HIV-1 integrase and selected the top 50 2339

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Journal of Chemical Information and Modeling Table 2. Docking Scores of Filtered Compoundsa

p75-binding site of HIV-1 integrase. Additionally, these chemicals also formed hydrogen bonds to facilitate their binding affinities. For example, DW-D-1 formed hydrogen bond with Thr174, DW-D-5 formed hydrogen bond with Gln168, and DW-D-6 formed hydrogen bonds with Gln168 and Glu170, respectively. 3.3. DW-D-5 and DW-D-6 Showed HIV-1 p24 Production Inhibition in Acute Infection. DW-D-1, DW-D-5, and DW-D-6 showed HIV-1 IN nuclear translocation inhibition in both cell based imaging analysis and Western blot detection. The antiviral activity of these potential HIV-1-IN/ p75 PPI inhibitors was tested using p24 production assay. HIV-1 replication was tracked by production of soluble p24 that can be examined by quantification of p24 expression using ELISA assay. DW-D-5 and DW-D-6 dramatically reduced p24 production in HIV-1IIIB acutely infected C8166 cells with EC50 values of 2.03 and 74.34 μM, respectively (Table 3, Figure S1). In HIV-1IIIB acutely infected MT-4 cells, DW-D-5 and DW-D-6 dramatically reduced p24 production with EC50 values of 1.31 and 54.46 μM, respectively (Table 4, Figure S4). 3.4. DW-D-5 and DW-D-6 Inhibited HIV-1 Induced Syncytia Formation. To further evaluate the antiviral effects of DW-D-5 and DW-D-6, we tested whether these two chemicals protect C8166 cells from HIV induce cytopathic effects (CPE). The results showed that compound DW-D-5 and DW-D-6 could inhibit HIV-1IIIB induced CPE by EC50 value of 1.51 and 32.78 μM, respectively in C8166 cells (Table 3, Figure S2). However, neither DW-D-5 nor DW-D-6 exhibited good antiviral activities in MT-4 cell protection assay with EC50 of 45.85 and 72.64 μM, respectively (Table 4, Figure S5). The EC50 value of antiviral activities of D77 and BI-1001 are 3.99 and 5.80 μM, respectively.7,8 CX series compounds exhibit better antiviral activities with IC50 value at the nanomolar level.6 3.5. DW-D-5 and DW-D-6 Inhibited HIV-1 Integrase Catalytic Activity. Previous study demonstrated that p75 binds to the interface of an HIV-1 integrase dimer and promotes HIV-1 integrase tetramerization so that HIV-1 integrase could form functionally which is required for strand transfer integration. Therefore, LEDGINs might also inhibit HIV-1 integrase catalytic activity. We found that both DW-D-5 and DW-D-6 inhibited the HIV-1 integrase strand transfer activity as quantitated by ELISA assay with IC50 values of 0.85 and 0.14 μM respectively (Figure S7). Compound CX14442 also significantly inhibits HIV-1 integrase catalytic activity with IC50 values of 0.66 μM.6 Given that some DW compounds have reactive functionality, they may be nonspecific. However, none of the DW compounds inhibited acetylcholinesterase (AChE) activity (Figure S8), indicating that both DW-D-5 and DW-D-6 are specific against HIV integrase. Together, we concluded here that DW-D-5 and DW-D-6 showed good antiviral activities as leading compounds for antiHIV-1 drug development. Their antiviral activities are similar to other LEDGINs previously reported. 3.6. Combination of DW-D-5 and FDA Approved AntiHIV Drugs Resulted in an Additive Inhibitory Effect on HIV-1 Replication. The combination cocktail of drugs against different target proteins is widely used in anti-HIV therapy. Therefore, it is critical to test whether newly identified LEDGINs are not antagonistic with other FDA approved anti-HIV drugs.6 We selected three FDA approved drugs for combination effect test, including Saquinavir as the representative protease inhibitor, Raltegravir as the representative integrase inhibitor, and Zidovudine as the representative reverse transcriptase inhibitor.

docking score chemical name

Autodock Vina

DOCK

CC50 [μM]

Benchmark −6.3 −51.1 128.89b −6.0 −38.2 96.00b −5.1 −43.1 72.20b −6.3 −40.4 >100b Filtered Chemicals from DOCK Screening DW-D-1c −5.1 −67.5 >50 DW-D-2c −6.1 −59.7 1.92 DW-D-3c −6.4 −64.6 7.89 DW-D-4c −6.5 −61.4 10.92 DW-D-5c −5.4 −59.9 >50 DW-D-6c −6.5 −58.9 >50 Filtered Chemicals from Autodock Vina Screening DW-V-1c −8.0 −50.2 >50 DW-V-2c −7.2 −46.2 >50 DW-V-3c −7.0 −43.5 >50 DW-V-4c −7.3 −46.4 >50 DW-V-5c −7.6 −47.7 3.37 DW-V-6c −9.4 −49.3 >50 DW-V-7c −7.2 −43.4 >50 DW-V-8c −7.1 −47.9 >50 D77 CX14442 CX05045 BI-1001

a

Cytotoxic concentration 50% in HeLa cells (μM) was tested. CC50 values of >50 μM indicates that the respective inhibitors were not cytotoxic at the tested concentrations up to 50 μM. bCC50 of benchmarks value and the assay method have been described elsewhere. c Indicates the Maybridge code.

test chemicals. The merged picture shows merging of the green and red colors after treatment with DW-V-1, DW-V-2, DW-V-3, DW-V-4, DW-V-6, and DW-V-8, indicating the absence of an inhibitory action on nuclear translocation of HIV-1 integrase. The green fluorescence staining to HIV-1 integrase did not merge with the red nucleus staining with PI after treatment with DW-D-1, DW-D-5, DW-D-6, and DW-V-7, indicating an inhibitory action on nuclear translocation of HIV-1 integrase. Western blot results for HIV-1 integrase nuclear translocation assay indicated that DW-D-1, DW-D-5, and DW-D-6 could significantly reduce HIV-1 integrase nuclear distribution. However, DW-V-1, DW-V-2, DW-V-3, DW-V-4, DW-V-6, DW-V-7, and DW-V-8 did not inhibit HIV-1 integrase nuclear distribution (Figures 3B). A comparison with previous image data revealed that DW-D-1, DW-D-5, and DW-D-6 could be identified as potential LEDGINs. Interestingly, we noticed that all of newly identified LEDGINs were selected from DOCK screening. On the other hand, none of chemicals that were selected from Autodock Vina screening exhibited inhibitory action on nuclear translocation of HIV-1 integrase. We concluded here that DOCK provides better predictions on the identification of LEDGINs. Molecular docking analysis illustrates the favorable binding positions of these chemicals with lowest binding free energy in the p75-binding site of HIV-1 integrase. Figure 4 shows the interactions of LEDGINs (gray stick) to HIV-1 integrase (gray cartoon) with labeled amino residues. DOCK uses geometric algorithms to predict the binding modes of small molecules. In this case, DOCK uses spheres placed in the p75-binding site of HIV-1 integrase and performs bipartite matching between those spheres and the chemicals from the Maybridge database. Therefore, all of selected three chemicals perfectly occupy the 2340

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Figure 3. (A) To identify the compounds that interfere with IN nuclear translocation, IN expression vector pcDNA3.1 was transfected into HeLa cells for immunofluorescence assay. FITC-conjugated anti-HIV-1 IN polyclonal antibody (Santa Cruz Biotechnology) was used to label IN in the immunofluorescence assay. The nucleus was stained by PI. IN was mainly localized in the cellular nucleus (control group). In the presence of the compounds DW-D-1, DW-D-5, DW-D-6, and DW-V-7, the FITC tagged IN accumulated in the peripheral area of the nucleus. The other compounds showed no influence on IN nuclear translocation. (B) Additionally, Western blot analysis was employed to measure nuclear protein amounts of IN in Hela cells transfected with IN. Numbers above bands correspond to the fold change compared with the control. Numbers less than 0.2 indicate that the corresponding chemicals inhibit IN nuclear translocation.

using both DOCK and Autodock Vina software. According to druggable analysis, six chemicals from DOCK screening and eight chemicals from Autodock Vina screening were selected for following in vitro test. After preliminary cytotoxicity test, three chemicals from DOCK screening and seven chemicals from Autodock Vina screening were selected for the inhibitory test on HIV-IN/p75 protein−protein interaction. Interestingly, three chemicals from DOCK screening significantly inhibited p75-induced integrase nuclear translocation, indicating that these three chemicals are potential LEDGINs. On the other hand, none of chemicals from Autodock Vina screening showed inhibitory effect on HIV-IN/p75 protein−protein interaction in cell-based assay. In this study, DOCK provided 100% positive hits but Autodock Vina provided 100% false positive hits in LEDGINs

The dosage of selected FDA approved drug is identical to their IC50 values according to previous study. MacSynergy II software was employed to evaluate the synergy and antagonism effects of the antiviral effects of two-drug combinations (Table 5). Unfortunately, DW-D-6 antagonized the inhibitory effects of these FDA approved anti-HIV drugs on HIV-1 replication (data not shown). The combination of DW-D-5 and FDA approved anti-HIV drugs resulted in additive inhibitory effects on HIV-1 replication. Furthermore, Raltegravir exhibited 35.34% inhibitory effect at 5 nM. When cotreating with 1 μM DW-D-5, Raltegravir exhibited 51.69% inhibitory effect at the same dose.

4. DISCUSSIONS In this project, we have finished the docking screening of Maybridge database against p75 binding site of HIV-1 integrase 2341

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Figure 4. DW-D-1, DW-D-5, and DW-D-6 are LEDGINs identified by molecular docking. It shows the interactions of LEDGINs (gray stick) to HIV-1 integrase (gray cartoon) with labeled amino residues.

Table 3. Antiviral Effects of DW-D Chemicals in C8166 Cellsa CPE

p24

compounds

CC50 [μM]

EC50 [μM]

TI

CC50 [μM]

EC50 [μM]

TI

AZT DW-D-5 DW-D-6

>100 >100 >100

0.01 1.51 32.78

>7000 >66.23 >3.05

>100 >100 >100

0.01 2.03 74.34

>11000 >45.05 >1.31

a

The therapeutic index (TI) is determined in cells as lethal dose of a drug for 50% of the population (CC50) divided by the minimum effective dose for 50% of the population (EC50).

Table 4. Antiviral Effects of DW-D Chemicals in MT-4 Cells MT-4 protection compounds

CC50 [μM]

EC50 [μM]

AZT DW-D-5 DW-D-6

9.40 14.78 >100

3.81 45.85 72.64

Table 5. Impact of Combinations of DW-D-5 and FDA Approved Anti-HIV-1 Drugs on HIV-1 Replication by Use of the Bliss Independent Modela

p24 TI

CC50 [μM]

EC50 [μM]

TI

2.47 0.32 >1.38

ND 66.58 92.78

ND 1.31 54.46

ND 50.82 1.70

mean vol (μM2%)b

a

The therapeutic index (TI) is determined in cells as lethal dose of a drug for 50% of the population (CC50) divided by the minimum effective dose for 50% of the population (EC50).

drug combination

synergy

antagonism

antiviral effect

DW-D-5 + Saquinavir DW-D-5 + Raltegravir DW-D-5 + Zidovudine

2.88 5.2 1.72

−4.64 −0.6 −6.16

additive additive additive

a

Antiviral activity of our inhibitors was tested by HIV-1 p24 production assay. Saquinavir (30 nM) was selected as the representative protease inhibitor; Raltegravir (5 nM) was selected as the representative HIV-1 integrase inhibitor; Zidovudine (10 nM) was selected as the representative reverse transcriptase inhibitor. b MacSynergy II software was employed to evaluate the synergy and antagonism effects of the antiviral effects of two-drug combinations. The positive and negative values were individually summed to give the mean synergy and antagonism volumes. Volumes of synergy or antagonism greater than ±50 μM2% can be considered significant and may be important in vivo.

virtual screening. The shape of ligand and receptor plays a crucial role in both matching procedure and scoring function in DOCK. However, Autodock Vina does not employ shape in either searching function or scoring function. We believed here shape is one of the most important criteria to identify small molecules that occupies p75-binding site of HIV-1 integrase. In the searching algorithm, DOCK has a different prefilter compared with Autodock Vina. The shape of ligands is estimated before the docking process and orienting ligands based on their shape is used as a filter before calculating scores. For details of this step, anchor-and-grow method in DOCK gives us the outlines of the ligands. Then it orients the shape of the ligand-fragments into the docking site and gets rid of the chemicals that do not fit the pocket based on their shape.

This part could be the reason that DOCK hits better candidates than Autodock Vina.9,10 DW-D-5 is the most potent LEDGINs from our screening, exhibiting antiviral activity with an IC50 value of 2.05 μM in HIV-1 replication assay. It also inhibited the HIV-1 integrase strand transfer activity with IC50 values of 850 nM. Its antiviral 2342

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Article

Journal of Chemical Information and Modeling

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activities and anticatalytic activity of integrase are similar to other LEDGINs under development. DW-D-5 provides a novel scaffold for LEDGINs development; we believe that the after the modification of DW-D-5 using QSAR approach, the derivatives of DW-D-5 should provide better antiviral activities.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.7b00402. IC50 curves, EC50 curves, CC50 curves, and other biochemical data were illustrated in Figures S1−8 (PDF) Complete docking score list and corresponding interaction residues analysis (XLSX)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86-871-519-5684. Fax: +86-871-519-5684. Postal address: Kunming Institute of Zoology, the Chinese Academy of Sciences, No. 32 Jiaochang Donglu, Kunming 650223, P.R. China (Y.-T.Z.). *E-mail: [email protected]. Tel.: 852-39436252. Fax: 852-39420991. Postal address: 326A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, P.R. China (D.C.-C.W.). ORCID

David Chi-Cheong Wan: 0000-0001-7356-6855 Author Contributions ⊥

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported partially by HMRF grants (ref no: 12110462) to D.C.-C.W. and the National Natural Science Foundation of China (81102483) to L.M.Y.

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REFERENCES

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DOI: 10.1021/acs.jcim.7b00402 J. Chem. Inf. Model. 2017, 57, 2336−2343