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Discovery of a natural PI3Kδ inhibitor through virtual screening and biological assay study
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study Jun-Fang Guo a, Zhong-Qi Ning a, Xia Wu a, c, Yan-Jiang Qiao b, *, Xing Wang a, c, ** a
School of Traditional Chinese Medicine, Capital Medical University, Fengtai District, Beijing, 100069, PR China Key Laboratory of TCM-information Engineer of State Administration of TCM, School of Chinese Materia Medica, Beijing University of Chinese Medicine, No.11, North San huan Road, Chaoyang District, Beijing, 100029, PR China c Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Capital Medical University, Fengtai District, Beijing, 100069, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2018 Accepted 3 December 2018 Available online xxx
Phosphoinositide-3-kinase-d (PI3Kd) is a key regulator in the process of IgE mediated mast cell degranulation, which directly induces allergic diseases, such as asthma. This study is aimed at discovery of natural PI3Kd inhibitors from Chinese medicine and evaluating their anti-mast cell degranulation activity. A combined virtual screening based on 3D pharmacophore model and molecular docking was used to screen for bioactive ingredients directly targeting PI3Kd. Then, an in vitro kinase inhibition assay was conducted to evaluate the PI3Kd inhibitory activity of the virtual screening hits. Subsequently, a bhexosaminidase release assay was performed to verify the anti-mast cell degranulation activity of the active compounds. Finally, ginkgoneolic acid was identified as a PI3Kd inhibitor (IC50 ¼ 2.49 mM) and exhibited anti-mast cell degranulation activity in vitro (IC50 ¼ 2.40 mM). Docking studies showed that Glu826, Val827 and Val828 were key amino acid residues for PI3Kd inhibitory activity. Ginkgoneolic acid may be a potential lead compound for developing effective and safe PI3Kd-inhibiting drugs. © 2018 Published by Elsevier Inc.
Keywords: Ginkgoneolic acid Phosphoinositide-3-kinase-d Virtual screening Mast cell degranulation
1. Introduction Phosphoinositide 3-kinase (PI3K), a class of lipid kinases that catalyses the phosphorylation of the 3-hydroxyl group of one or more phospholipid inositol rings of phosphatidylinositol [1], plays a pivotal role in signal transduction activation, histamine release and leukotrienes and prostaglandins synthesis in various immune cells, such as mast cells and B cells [2]. PI3K was reported to participate in multifarious physiological and pathological processes in the airway involved in inflammatory diseases. For example, experiments showed that PI3K inhibitors could modulate the production of T cytokines and reduce inflammation in vivo [3]. According to structural and functional characteristics, class I PI3K consists of p110a, p110b, p110d and p110g [4,5]. The expression and function of PI3Kd are predominantly confined to immune cells, and its inhibitors have captured much attention as a potential
* Corresponding author. ** Corresponding author. School of Traditional Chinese Medicine, Capital Medical University, Fengtai District, Beijing, 100069, PR China. E-mail addresses: [email protected] (Y.-J. Qiao), [email protected] (X. Wang).
therapeutic target for the treatment of inflammatory diseases. For instance, aerosol or systemic application of double selective PI3Kd/ g inhibitors can reduce inflammatory cell infiltration in inflammatory lung disease mice models, including neutrophil infiltration induced by lipopolysaccharide or ovalbumin [6,7]. Furthermore, selective PI3Kd inhibitors, such as LY 294002 and IC 87114, can reduce allergen-induced airway inflammation and airway hyperresponsiveness [8] and similar results can be found in PI3Kd deficient mice in comparison models [9]. More recently, a study explored the efficacy of a new inhaled PI3Kd (GSK2269557) in clinical chronic obstructive pulmonary disease (COPD) patients, and the results showed that levels of interleukin-8 and interleukin6 were reduced [10], which indicated the anti-inflammatory effects of targeting PI3Kd in chronic lung diseases. Consequently, there would be great therapeutic benefits to targeting PI3Kd for several allergic diseases [11]. Owing to various adverse reactions of the known PI3Kd inhibitors, discovery of novel PI3Kd inhibitors with the safety profile and improved efficacy is expected [12]. Meanwhile, natural products from TCM with unique skeleton and bioactivity could provide an important resource for lead compounds and candidates in drug discovery. This study aimed to identify PI3Kd inhibitors from an in-
https://doi.org/10.1016/j.bbrc.2018.12.009 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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house Chinese medicine database. Finally, ginkgoneolic acid was found to be a PI3Kd inhibitor through a kinase activity assay. Additionally, cellular experimental methods showed that ginkgoneolic acid had anti mast cell degranulation activity in vitro. The findings of this study provided a reference for drug discovery targeting of PI3Kd, and it would also be conducive to elucidate the mechanism of pharmacological activity of ginkgoneolic acid. 2. Materials and methods 2.1. Common feature-based 3D pharmacophore modelling All of the computational virtual screening was executed on a Dell Red Hat Linux workstation in the Discovery Studio 2.5 (DS2.5, Accelrys, Inc. San Diego, CA, USA) and SYBYL 1.3 (Tripos Inc., St. Louis, MO, USA), the default parameters were applied to build the pharmacophores (also called “hypotheses”), except those especially referred to. The hypotheses were generated using the HIPHOP protocol in DS2.5 as previously described [13]. The pharmacophore model refers to the ‘pharmacodynamic characteristic elements’ and their spatial arrangement that represent the important features of active molecules. The pharmacophore model in this study was obtained from 54 known PI3Kd inhibitors that were reported to have IC50 values in the range of 0.1 nMe10 mM found in the published literature [14e17]. The molecules were sketched or downloaded and saved in Mol format. After adding the hydrogen atoms and assigning the GasteigerMarsili charges, the structures of these molecules were verified and a modified Tripos force field was used to refine the conformers in SYBYL 1.3. The training set, which was used to establish the common-feature pharmacophore model, contained six PI3Kd inhibitors (Fig. 4) selected according to the diversity of molecular structures. The remaining 48 PI3Kd inhibitors served as a test set to evaluate the established model. Then, a set of 10 hypotheses was built utilizing the Common Feature Pharmacophore Generation tool implemented in DS2.5. To assess the reliability of the hypotheses, a total of 304 compounds including 48 known PI3Kd inhibitors and 256 non-PI3Kd inhibitors were used as a test set. The forecasting performance of the model was evaluated by four indicators proposed in the previous publication [18]: A%, Y%, N and comprehensive appraisal index (CAI). Ht refers to the hit number of molecules based on hypotheses, Ha refers to the number of active compounds, N refers to the ability to discriminate active PI3Kd inhibitors from non-PI3Kd inhibitors, and CAI is a comprehensive evaluation indicator [19]. A% ¼ Ha / A 100
(1)
Y% ¼ Ha / Ht 100
(2)
N ¼ (Ha D) / (Ht A)
(3)
CAI ¼ N A%
(4)
Based on the evaluation outcomes, the pharmacophore with the highest CAI value was selected to screen against an in-house chemical database containing 424 natural compounds, which was previously created using default parameters. First, all of the compounds were drawn and saved in Mol2 format, and then the molecules were converted into 3D structures using CONCORD method in SYBYL 1.3. Next, the structures were replenished and the energy was optimized with Tripos force field. The UNITY module in SYBYL 1.3 was used to establish the chemical database for virtual screening. The Search 3D Database tool implemented in DS2.5 was applied
to filter the database; each of the hits was assigned a fit value. The higher the fit value that is obtained, the higher the degree of matching between the hit and the 3D hypothesis. 2.2. Structure-based molecular docking simulation Further identification of virtual screening based on drug targets was conducted to predict the binding pattern between molecules and PI3Kd using Surflex-Docking in SYBYL 1.3 [20,21]. The crystal structure of PI3Kd (PDB ID: 5DXU. Resolution: 2.64 Å), that was resolved by the X-ray diffraction method and was used as a docking protein model. First, to identify the active sites of the target protein, the preparation of the protein for the docking experiments was performed: the co-crystallized water and small molecules of the protein were deleted and hydrogens were added. Second, the protein was assigned a force field using Gasteiger-Marsili charges, and the energy was optimized for 1000 iterations with the default parameters in SYBYL 1.3. Co-crystallized ligand (5H5, C19H22N6O3), a known PI3Kd inhibitor, was extracted and docked with the protein model. The amino acid residues within the range of 0.5 Å around the original ligand 5H5 were defined as a docking pocket, which turned into the ideal active site template combined with compounds. CH4, C]O and NeH were used as probes to represent hydrophobic groups, hydrogen bond donors and hydrogen bond acceptors, respectively. All of the molecules in the database were, in turn, docked with the ligand binding domain of PI3Kd that brought about a hit list with total scores for every molecule. The higher the score of the molecule in the list, the stronger the binding force is between the ligand and the protein. 2.3. Determination of the inhibitory activity targeting PI3Kd Based on availability, thirty compounds from the abovementioned hit set were purchased to evaluate their PI3Kd-inhibitory activity that was detected by the ADP-Glo™ Kinase Assay kit (Promega, Madison, WI, USA). The following experiment, including two steps of PI3Kd reaction and ADP detection, was carried out. First, 4.0 mL of PI3Kd (Abcam, USA, Cat: ab125633) solution (16.6 ng/ mL in kinase buffer [50.0 mM HEPES, 3.0 mM MgCl2, 50.0 mM NaCl, 0.025 mg/mL BSA, 625.0 mM DTT (pH 7.5)]) and 2.0 mL of compound solution (95.0 mM in kinase buffer containing 5% DMSO) were mixed uniformity and incubated for 15 min at 37 C. Second, 4.0 mL of substrate/ATP mixture (containing 0.125 mg/mL substrate and 250 mM ATP in kinase buffer) was added to initiate the PI3Kd reaction for 1 h at 37 C. Finally, a chemiluminescent detection assay was performed to measure the amount of ADP after a PI3Kd reaction according to the instructions of the manufacturer. The specific steps are as follows: 5.0 mL of ADP-Glo™ reagent was added to 5.0 mL of PI3Kd enzymatic reaction solution to stop the reaction and consume the remaining ATP, leaving only the ADP. The mixture was incubated in white 384-well plates with flat bottoms and nonbinding (Corning #3824, NY) for 40 min at 37 C. Then, ADP was converted into ATP by adding 10.0 mL of kinase detection reagent, and ATP was detected by luciferase and fluorescein. Finally, the mixture was incubated for 1 h at 37 C and the chemiluminescent value was calculated using a SpectraMax® iD3. The results were expressed as mean ± SEM from at least three replicates. L-a-phosphatidylinositol-3-phosphate sodium salt (Abcam, USA, Cat: ab145220) served as the PI3Kd substrate in the trial run. All compounds to be tested were acquired from the State Food and Drug Administration (Beijing, China). The positive inhibitor, AS605240 (Abcam, USA, Cat: ab120928), was commercially available and served as the reference compound for PI3Kd activity assay. In addition, the kinase buffer contained 5% DMSO was used as the
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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negative control. A positive and a negative control group must be added for each experiment. The value of the luminescence signal was expressed in relative fluorescence units (RFU) with no absolute values. The data obtained in this study was analysed with GraphPad Prism v. 5.00 (GraphPad Software Inc., San Diego, CA, USA). 2.4. IgE-mediated mast cells degranulation assay RBL-2H3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). EMEM was purchased from GIBCO/ Life Technologies Inc (Rockville, MD, USA). DMEM was purchased from Gibco BRL (Grand Island, NY, USA). Bovine serum albumin (BSA), penicillin, streptomycin, mouse anti-dinitrophenol (DNP) monoclonal IgE and foetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The RBL-2H3 mast cells were suspended in EMEM medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 mg/mL of streptomycin and then they were cultured in 96-well plate (Corning, NY, USA) keeping a density of 2 104 cells/well and incubated in a cell incubator containing 5% CO2 at 37 C for 24 h. After treatment with DNP-IgE (1 mg/mL, dissolved in 0.1 M phosphate buffer, pH 7.4) for 12 h of sensitization, the final concentration was 100 ng/mL. The cells were washed twice with PBS buffer (NaCl 8 g/L, KCl 0.20 g/L, Na2HPO4 1.44 g/L, KH2PO4 0.24 g/L, pH 7.4), and they were incubated for 1 h at 37 C after adding 80 mL of DMEM medium and 20 mL of compound solution. DNP-BSA (5 mg/mL, Merck, 324101-100 MG) was subsequently added into the wells to stimulate RBL-2H3 cells; the final concentration of DNP-BSA was 100 ng/mL and was incubated at 37 C for 30 min. Cells were followed transferred to a 0 C ice bath for 10 min. Finally, 50 mL of supernatant was mixed with 50 mL of p-nitrophenyl-N-acetyl-b-O-glucosamine solution (4 mM, dissolved in citric acid/sodium citrate buffer, pH 4.5). After incubation at 37 C for 1.5 h, 200 mL of sodium carbonate buffer was added to terminate the reaction (pH 10.5). The content of p-nitrophenol in supernatant was measured by a spectrophotometer at 405 nm. For the rest, the cells from the bottom of the wells in the 0 C bath were lysed with 100 mL of 0.1% Triton (Sigma, T8787), then 50 mL of the lysate was mixed with 50 mL of p-nitrophenyl-N-acetyl-b-Oglucosamine solution and incubated at 37 C for 1.5 h. The reaction was terminated by adding 200 mL of sodium carbonate buffer. Similarly, the absorbance (A) of the reaction solution was measured at 405 nm. The release rate of b-hexosaminidase (R) and the inhibition rate were calculated according to the following formula. A0, A1, A2: the absorbance of supernatant in the blank cells, the supernatant in stimulated cells and the lysate in stimulated cells, respectively; R0, R1, R2: the release rate of b-hexosaminidase in the blank group, the DNP-BSA group and the compound group, respectively.
b-hexosaminidase release rate (%) ¼ (A1 - A0 / A1 þ A2 A0) 100
(5)
b-hexosaminidase inhibition rate (%) ¼ ((R1 - (R2 - R0)) / R1) 100
(6)
3
3. Results and discussion 3.1. Virtual screening for PI3Kd inhibitors In this work, a chemical dataset of 424 TCM sourced compounds were screened based on a 3D pharmacophore model and a molecular docking method. Primarily, during the process of HIPHOP pharmacophore generation, ten hypotheses based on PI3Kd inhibitors were produced and reordered by ranking score (Table 1), then the evaluation results of these models were presented in Table 2. Pharmacophore model_7 (Fig. 1A), having the highest N and CAI values, was chosen as the best hypothesis. One hydrophobic feature (HY, shown in blue), one hydrogen bond donor (HBD, shown in red) and one hydrogen bond accepter (HBA, shown
Table 1 Ten pharmacophore results produced by the HIPHOP. Model
Features
Rank
Direct Hit
Partial Hit
Max Fit
01 02 03 04 05 06 07 08 09 10
HDA HDA HDA HDA HDA HDA HDA HDA HDA HDA
41.58 40.85 39.93 39.33 38.49 38.15 37.92 37.53 37.51 37.28
111111 111111 111111 111111 111111 111111 111111 111111 111111 111111
000000 000000 000000 000000 000000 000000 000000 000000 000000 000000
3 3 3 3 3 3 3 3 3 3
Table 2 Evaluation results for the 3D pharmacophore models. Model
Ht
Ha
A (%)
Y (%)
N
CAI
Model_01 Model_02 Model_03 Model_04 Model_05 Model_06 Model_07 Model_08 Model_09 Model_10
165 164 170 175 174 188 187 181 189 190
39 40 41 39 45 42 47 45 44 44
72.22 74.07 75.93 72.22 83.33 77.78 87.04 83.33 81.48 81.48
23.64 24.39 24.12 22.29 25.86 22.34 25.13 24.86 23.28 23.16
1.36 1.40 1.38 1.28 1.48 1.28 1.44 1.43 1.34 1.33
0.98 1.04 1.05 0.92 1.24 1.00 1.26 1.19 1.09 1.08
2.5. Statistical analysis All data were presented as mean ± stand error of the mean (SEM). One-way analysis of variance (ANOVA) was used to analyse the statistical significance with GraphPad Prism 5 among the measurement groups. Values of *P < 0.05, **P < 0.01 or ***P < 0.001 were considered to be significant.
Fig. 1. 3D pharmacophore model_7 borne from PI3Kd inhibitors. (A) The optimal pharmacophore model involves three features: a hydrophobic feature (H, blue), a hydrogen bond donor (HBD, red) and a hydrogen bond accepter (HBA, green). Numbers represent the distances between every two pharmacodynamic characteristic elements; (B) The overlapping image between a known PI3Kd inhibitor (CHEMBL3805180) and pharmacophore model_7. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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Fig. 2. Binding mode between ginkgoneolic acid and PI3Kd protein. (A) 2D diagram illustrating the interactions. (B) 3D diagram illustrating the interactions. The hydrogen bonds were marked by red dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
in green) were considered to be the three characteristics of the model_7. A positive molecule (CHEMBL3805180) could match all of the characteristics of the hypothesis (Fig. 1B). Based on the model_7, a hit list of 182 compounds with FitValue >2.00 were exported by ligand-based virtual screening (part of the results were expressed in Table S1). Afterwards, the 182 compounds were further screened using the molecular docking method. Before developing the molecular docking, the reliability of the method was verified by extracting the original assembly ligand 5H5 (C19H22N6O3) and docking with the ligand binding domain of PI3Kd. The RMSD between the re-docked and co-crystallized conformation of ligand 5H5 was calculated as 1.79 Å. Furthermore, this finding showed that 5H5 could bind to PI3Kd via H-bond interaction with Glu826, Val827and Val828 and indicated that the Surflex-Docking procedure could optimally reproduce the co-crystal bioactive conformation of the PI3Kd inhibitor. Finally, all of the compounds were docked into the active sites of PI3Kd, and in turn, a hit list of 37 compounds was screened out with docking scores >7.50 (Table S2). Further investigation of the 37 compounds, possessing high fit values in pharmacophore matching and docking scores, was used for testing its PI3Kd inhibitory activity. The detailed results of the spatial arrangement between ginkgoneolic acid in the PI3Kd active site was demonstrated in Fig. 2, which showed that Tyr813, Ile825, Glu826, Val827, Val828, Met900, and Ile910 were key amino acid residues binding to ginkgoneolic acid, which is consistent with previous findings [22]. Moreover, the hydroxy group and the carboxyl group at the benzene ring are key structural features that affect biological activity. 3.2. Kinase inhibition assay These compounds were then assessed for their PI3Kd-inhibitory activity. The ADP-Glo™ Kinase Assay kit could quantify the activity by the amount of luminescent ADP, as described in the manufacturer's instructions. After preliminary screening, only the ginkgoneolic acid that exhibited at least 50% PI3Kd inhibition was qualified for further evaluation. The compound was subjected to the same enzymatic test again in a range of concentrations to accurately establish the inhibitory activity (IC50). Further dose responses were analysed and the results showed that the IC50 of ginkgoneolic acid
and (5Z)-5-(quinoxalin-6-ylmethylidene)-1, 3-thiazolidine-2, 4dione (AS-605240, a positive inhibitor) were 2.49 mM and 1.58 mM, respectively (Fig. 3A and B). 3.3. Mast cell degranulation inhibition experiment In this study, the effect of ginkgoneolic acid on mast cell degranulation was investigated in an IgE/BSA-induced model. As expressed in Fig. 3C, release rates of b-hexosaminidase from RBL2H3 cells were measured. Ginkgoneolic acid can significantly inhibit the release of b-hexosaminidase by DNP-BSA stimulation in mast cells, compared with the positive drug chloroquine. The inhibition rate of the chloroquine group (80.78%), the ginkgoneolic acid of 20 mM (74.65%) and the ginkgoneolic acid of 6.7 mM (92.09%) were less than the degranulation of the DNP-BSA group. The results made it clear that ginkgoneolic acid could attenuate the mast cell degranulation induced by antigen-specific IgE/BSA. Further doseeffect relationship analysis was shown in Fig. 3D, and the IC50 value of ginkgoneolic acid on mast cell degranulation was 2.40 mM. Ginkgoneolic acid, an alkyl phenolic acids, was commonly found in Ginkgo biloba and had been used for the treatment of allergic inflammation, such as allergic rhinitis and asthma in TCM [23,24]. A recent study had found that the extract of Ginkgo biloba had an inhibitory effect in a skin allergic reaction model [25]. Ginkgo biloba has been effective in treating various diseases, such as asthma [23], dementia, Alzheimer's disease and tinnitus [26]. However, the molecular mechanisms have not been thoroughly elucidated to date. This study reported that ginkgoneolic acid could decrease the release of inflammatory factors in vitro by inhibiting PI3Kd, providing a possible molecular mechanism for the antiallergic response of Ginkgo biloba. This study demonstrated that ginkgoneolic acid was an inhibitor of PI3Kd and showed inhibition of IgE-derived mast cell degranulation. These results provided a potential pharmaceutical source for anti-allergic therapeutics targeting PI3Kd and provided conditions for research and discovery of natural drug inhibitors. The interactions between PI3Kd and its inhibitors at the molecular level were carried out to discover its natural inhibitors. To the best of our knowledge, the virtual screening method in the study is beneficial to save time in discovering new bioactive compounds. Furthermore, the combined ligand- and structure-based approaches could
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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Fig. 3. Results of the biological experiments. (A) Efficiency curves of ginkgoneolic acid inhibiting PI3Kd based on enzymatic reaction. (B) Efficiency curves of AS-605240 based on enzymatic reaction (C) The release of b-hexosaminidase was measured after pretreatment with DNP-BSA, chloroquine and ginkgoneolic acid. (D) Dose-effect relationship of ginkgoneolic acid concentrations on the inhibition of mast cell degranulation.
Fig. 4. Training set compounds used for 3D pharmacophore generation.
show features of the PI3Kd active site by molecular simulation. This method provided a practical strategy for searching natural and efficient PI3Kd inhibitors and was supported with biological experiments.
Funding This research carried out in the paper was supported by the National Natural Science Foundation of China (No. 81603311) and
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Fiveeyear Plan (No. CIT&TCD201804084). Acknowledgments The authors would like to thank Yu-Hong Xiang at the Capital Normal University for supporting SYBYL-based computing services. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.009. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2018.12.009. References [1] P.T. Hawkins, L.R. Stephens, PI3K signalling in inflammation, Biochim. Biophys. Acta 1851 (2015) 882e898. [2] E.J. Yoo, C.A. Ojiaku, K. Sunder, J. Reynold, A. Panettieri, Phosphoinositide 3kinase in asthma: novel roles and therapeutic approaches, Am. J. Respir. Cell Mol. Biol. 56 (2016) 700e707. [3] E.E. Way, G. Trevejo-Nunez, L.P. Kane, B.H. Steiner, K.D. Puri, J.K. Kolls, K. Chen, Dose-dependent suppression of cytokine production from T cells by a novel phosphoinositide 3-kinase delta inhibitor, Sci. Rep. 6 (2016) 30384. [4] K. Okkenhaug, Signaling by the phosphoinositide 3-kinase family in immune cells, Annu. Rev. Immunol. 31 (2013) 675e704. [5] B. Vanhaesebroeck, J. Guillermet-Guibert, M. Graupera, B. Bilanges, The emerging mechanisms of isoform-specific PI3K signalling, Nat. Rev. Mol. Cell Biol. 11 (2010) 329e341. [6] J. Doukas, L. Eide, K. Stebbins, A. Racanelli-Layton, L. Dellamary, M. Martin, E. Dneprovskaia, G. Noronha, R. Soll, W. Wrasidlo, L.M. Acevedo, D.A. Cheresh, Aerosolized phosphoinositide 3-kinase gamma/delta inhibitor TG100-115 [3[2,4-diamino-6-(3-hydroxyphenyl)pteridin-7-yl]phenol] as a therapeutic candidate for asthma and chronic obstructive pulmonary disease, J. Pharmacol. Exp. Therapeut. 328 (2009) 758e765. [7] D.G. Winkler, K.L. Faia, J.P. DiNitto, J.A. Ali, K.F. White, E.E. Brophy, M.M. Pink, J.L. Proctor, J. Lussier, C.M. Martin, J.G. Hoyt, B. Tillotson, E.L. Murphy, A.R. Lim, B.D. Thomas, J.R. Macdougall, P. Ren, Y. Liu, L.S. Li, K.A. Jessen, C.C. Fritz, J.L. Dunbar, J.R. Porter, C. Rommel, V.J. Palombella, P.S. Changelian, J.L. Kutok, PI3K-delta and PI3K-gamma inhibition by IPI-145 abrogates immune responses and suppresses activity in autoimmune and inflammatory disease models, Chem. Biol. 20 (2013) 1364e1374. [8] K.S. Lee, H.K. Lee, J.S. Hayflick, Y.C. Lee, K.D. Puri, Inhibition of phosphoinositide 3-kinase delta attenuates allergic airway inflammation and hyperresponsiveness in murine asthma model, Faseb. J. 20 (2006) 455e465. [9] S.J. Park, K.S. Lee, S.R. Kim, K.H. Min, H. Moon, M.H. Lee, C.R. Chung, H.J. Han, K.D. Puri, Y.C. Lee, Phosphoinositide 3-kinase delta inhibitor suppresses interleukin-17 expression in a murine asthma model, Eur. Respir. J. 36 (2010) 1448e1459. [10] A. Cahn, J.N. Hamblin, M. Begg, R. Wilson, L. Dunsire, S. Sriskantharajah, M. Montembault, C.N. Leemereise, L. Galinanes-Garcia, H. Watz, A.M. Kirsten, R. Fuhr, E.M. Hessel, Safety, pharmacokinetics and dose-response characteristics of GSK2269557, an inhaled PI3Kdelta inhibitor under development for
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Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study Jun-Fang Guo a, Zhong-Qi Ning a, Xia Wu a, c, Yan-Jiang Qiao b, *, Xing Wang a, c, ** a
School of Traditional Chinese Medicine, Capital Medical University, Fengtai District, Beijing, 100069, PR China Key Laboratory of TCM-information Engineer of State Administration of TCM, School of Chinese Materia Medica, Beijing University of Chinese Medicine, No.11, North San huan Road, Chaoyang District, Beijing, 100029, PR China c Beijing Key Laboratory of Traditional Chinese Medicine Collateral Disease Theory Research, Capital Medical University, Fengtai District, Beijing, 100069, PR China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 November 2018 Accepted 3 December 2018 Available online xxx
Phosphoinositide-3-kinase-d (PI3Kd) is a key regulator in the process of IgE mediated mast cell degranulation, which directly induces allergic diseases, such as asthma. This study is aimed at discovery of natural PI3Kd inhibitors from Chinese medicine and evaluating their anti-mast cell degranulation activity. A combined virtual screening based on 3D pharmacophore model and molecular docking was used to screen for bioactive ingredients directly targeting PI3Kd. Then, an in vitro kinase inhibition assay was conducted to evaluate the PI3Kd inhibitory activity of the virtual screening hits. Subsequently, a bhexosaminidase release assay was performed to verify the anti-mast cell degranulation activity of the active compounds. Finally, ginkgoneolic acid was identified as a PI3Kd inhibitor (IC50 ¼ 2.49 mM) and exhibited anti-mast cell degranulation activity in vitro (IC50 ¼ 2.40 mM). Docking studies showed that Glu826, Val827 and Val828 were key amino acid residues for PI3Kd inhibitory activity. Ginkgoneolic acid may be a potential lead compound for developing effective and safe PI3Kd-inhibiting drugs. © 2018 Published by Elsevier Inc.
Keywords: Ginkgoneolic acid Phosphoinositide-3-kinase-d Virtual screening Mast cell degranulation
1. Introduction Phosphoinositide 3-kinase (PI3K), a class of lipid kinases that catalyses the phosphorylation of the 3-hydroxyl group of one or more phospholipid inositol rings of phosphatidylinositol [1], plays a pivotal role in signal transduction activation, histamine release and leukotrienes and prostaglandins synthesis in various immune cells, such as mast cells and B cells [2]. PI3K was reported to participate in multifarious physiological and pathological processes in the airway involved in inflammatory diseases. For example, experiments showed that PI3K inhibitors could modulate the production of T cytokines and reduce inflammation in vivo [3]. According to structural and functional characteristics, class I PI3K consists of p110a, p110b, p110d and p110g [4,5]. The expression and function of PI3Kd are predominantly confined to immune cells, and its inhibitors have captured much attention as a potential
* Corresponding author. ** Corresponding author. School of Traditional Chinese Medicine, Capital Medical University, Fengtai District, Beijing, 100069, PR China. E-mail addresses: [email protected] (Y.-J. Qiao), [email protected] (X. Wang).
therapeutic target for the treatment of inflammatory diseases. For instance, aerosol or systemic application of double selective PI3Kd/ g inhibitors can reduce inflammatory cell infiltration in inflammatory lung disease mice models, including neutrophil infiltration induced by lipopolysaccharide or ovalbumin [6,7]. Furthermore, selective PI3Kd inhibitors, such as LY 294002 and IC 87114, can reduce allergen-induced airway inflammation and airway hyperresponsiveness [8] and similar results can be found in PI3Kd deficient mice in comparison models [9]. More recently, a study explored the efficacy of a new inhaled PI3Kd (GSK2269557) in clinical chronic obstructive pulmonary disease (COPD) patients, and the results showed that levels of interleukin-8 and interleukin6 were reduced [10], which indicated the anti-inflammatory effects of targeting PI3Kd in chronic lung diseases. Consequently, there would be great therapeutic benefits to targeting PI3Kd for several allergic diseases [11]. Owing to various adverse reactions of the known PI3Kd inhibitors, discovery of novel PI3Kd inhibitors with the safety profile and improved efficacy is expected [12]. Meanwhile, natural products from TCM with unique skeleton and bioactivity could provide an important resource for lead compounds and candidates in drug discovery. This study aimed to identify PI3Kd inhibitors from an in-
https://doi.org/10.1016/j.bbrc.2018.12.009 0006-291X/© 2018 Published by Elsevier Inc.
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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house Chinese medicine database. Finally, ginkgoneolic acid was found to be a PI3Kd inhibitor through a kinase activity assay. Additionally, cellular experimental methods showed that ginkgoneolic acid had anti mast cell degranulation activity in vitro. The findings of this study provided a reference for drug discovery targeting of PI3Kd, and it would also be conducive to elucidate the mechanism of pharmacological activity of ginkgoneolic acid. 2. Materials and methods 2.1. Common feature-based 3D pharmacophore modelling All of the computational virtual screening was executed on a Dell Red Hat Linux workstation in the Discovery Studio 2.5 (DS2.5, Accelrys, Inc. San Diego, CA, USA) and SYBYL 1.3 (Tripos Inc., St. Louis, MO, USA), the default parameters were applied to build the pharmacophores (also called “hypotheses”), except those especially referred to. The hypotheses were generated using the HIPHOP protocol in DS2.5 as previously described [13]. The pharmacophore model refers to the ‘pharmacodynamic characteristic elements’ and their spatial arrangement that represent the important features of active molecules. The pharmacophore model in this study was obtained from 54 known PI3Kd inhibitors that were reported to have IC50 values in the range of 0.1 nMe10 mM found in the published literature [14e17]. The molecules were sketched or downloaded and saved in Mol format. After adding the hydrogen atoms and assigning the GasteigerMarsili charges, the structures of these molecules were verified and a modified Tripos force field was used to refine the conformers in SYBYL 1.3. The training set, which was used to establish the common-feature pharmacophore model, contained six PI3Kd inhibitors (Fig. 4) selected according to the diversity of molecular structures. The remaining 48 PI3Kd inhibitors served as a test set to evaluate the established model. Then, a set of 10 hypotheses was built utilizing the Common Feature Pharmacophore Generation tool implemented in DS2.5. To assess the reliability of the hypotheses, a total of 304 compounds including 48 known PI3Kd inhibitors and 256 non-PI3Kd inhibitors were used as a test set. The forecasting performance of the model was evaluated by four indicators proposed in the previous publication [18]: A%, Y%, N and comprehensive appraisal index (CAI). Ht refers to the hit number of molecules based on hypotheses, Ha refers to the number of active compounds, N refers to the ability to discriminate active PI3Kd inhibitors from non-PI3Kd inhibitors, and CAI is a comprehensive evaluation indicator [19]. A% ¼ Ha / A 100
(1)
Y% ¼ Ha / Ht 100
(2)
N ¼ (Ha D) / (Ht A)
(3)
CAI ¼ N A%
(4)
Based on the evaluation outcomes, the pharmacophore with the highest CAI value was selected to screen against an in-house chemical database containing 424 natural compounds, which was previously created using default parameters. First, all of the compounds were drawn and saved in Mol2 format, and then the molecules were converted into 3D structures using CONCORD method in SYBYL 1.3. Next, the structures were replenished and the energy was optimized with Tripos force field. The UNITY module in SYBYL 1.3 was used to establish the chemical database for virtual screening. The Search 3D Database tool implemented in DS2.5 was applied
to filter the database; each of the hits was assigned a fit value. The higher the fit value that is obtained, the higher the degree of matching between the hit and the 3D hypothesis. 2.2. Structure-based molecular docking simulation Further identification of virtual screening based on drug targets was conducted to predict the binding pattern between molecules and PI3Kd using Surflex-Docking in SYBYL 1.3 [20,21]. The crystal structure of PI3Kd (PDB ID: 5DXU. Resolution: 2.64 Å), that was resolved by the X-ray diffraction method and was used as a docking protein model. First, to identify the active sites of the target protein, the preparation of the protein for the docking experiments was performed: the co-crystallized water and small molecules of the protein were deleted and hydrogens were added. Second, the protein was assigned a force field using Gasteiger-Marsili charges, and the energy was optimized for 1000 iterations with the default parameters in SYBYL 1.3. Co-crystallized ligand (5H5, C19H22N6O3), a known PI3Kd inhibitor, was extracted and docked with the protein model. The amino acid residues within the range of 0.5 Å around the original ligand 5H5 were defined as a docking pocket, which turned into the ideal active site template combined with compounds. CH4, C]O and NeH were used as probes to represent hydrophobic groups, hydrogen bond donors and hydrogen bond acceptors, respectively. All of the molecules in the database were, in turn, docked with the ligand binding domain of PI3Kd that brought about a hit list with total scores for every molecule. The higher the score of the molecule in the list, the stronger the binding force is between the ligand and the protein. 2.3. Determination of the inhibitory activity targeting PI3Kd Based on availability, thirty compounds from the abovementioned hit set were purchased to evaluate their PI3Kd-inhibitory activity that was detected by the ADP-Glo™ Kinase Assay kit (Promega, Madison, WI, USA). The following experiment, including two steps of PI3Kd reaction and ADP detection, was carried out. First, 4.0 mL of PI3Kd (Abcam, USA, Cat: ab125633) solution (16.6 ng/ mL in kinase buffer [50.0 mM HEPES, 3.0 mM MgCl2, 50.0 mM NaCl, 0.025 mg/mL BSA, 625.0 mM DTT (pH 7.5)]) and 2.0 mL of compound solution (95.0 mM in kinase buffer containing 5% DMSO) were mixed uniformity and incubated for 15 min at 37 C. Second, 4.0 mL of substrate/ATP mixture (containing 0.125 mg/mL substrate and 250 mM ATP in kinase buffer) was added to initiate the PI3Kd reaction for 1 h at 37 C. Finally, a chemiluminescent detection assay was performed to measure the amount of ADP after a PI3Kd reaction according to the instructions of the manufacturer. The specific steps are as follows: 5.0 mL of ADP-Glo™ reagent was added to 5.0 mL of PI3Kd enzymatic reaction solution to stop the reaction and consume the remaining ATP, leaving only the ADP. The mixture was incubated in white 384-well plates with flat bottoms and nonbinding (Corning #3824, NY) for 40 min at 37 C. Then, ADP was converted into ATP by adding 10.0 mL of kinase detection reagent, and ATP was detected by luciferase and fluorescein. Finally, the mixture was incubated for 1 h at 37 C and the chemiluminescent value was calculated using a SpectraMax® iD3. The results were expressed as mean ± SEM from at least three replicates. L-a-phosphatidylinositol-3-phosphate sodium salt (Abcam, USA, Cat: ab145220) served as the PI3Kd substrate in the trial run. All compounds to be tested were acquired from the State Food and Drug Administration (Beijing, China). The positive inhibitor, AS605240 (Abcam, USA, Cat: ab120928), was commercially available and served as the reference compound for PI3Kd activity assay. In addition, the kinase buffer contained 5% DMSO was used as the
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negative control. A positive and a negative control group must be added for each experiment. The value of the luminescence signal was expressed in relative fluorescence units (RFU) with no absolute values. The data obtained in this study was analysed with GraphPad Prism v. 5.00 (GraphPad Software Inc., San Diego, CA, USA). 2.4. IgE-mediated mast cells degranulation assay RBL-2H3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). EMEM was purchased from GIBCO/ Life Technologies Inc (Rockville, MD, USA). DMEM was purchased from Gibco BRL (Grand Island, NY, USA). Bovine serum albumin (BSA), penicillin, streptomycin, mouse anti-dinitrophenol (DNP) monoclonal IgE and foetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The RBL-2H3 mast cells were suspended in EMEM medium supplemented with 10% FBS, 100 U/mL of penicillin and 100 mg/mL of streptomycin and then they were cultured in 96-well plate (Corning, NY, USA) keeping a density of 2 104 cells/well and incubated in a cell incubator containing 5% CO2 at 37 C for 24 h. After treatment with DNP-IgE (1 mg/mL, dissolved in 0.1 M phosphate buffer, pH 7.4) for 12 h of sensitization, the final concentration was 100 ng/mL. The cells were washed twice with PBS buffer (NaCl 8 g/L, KCl 0.20 g/L, Na2HPO4 1.44 g/L, KH2PO4 0.24 g/L, pH 7.4), and they were incubated for 1 h at 37 C after adding 80 mL of DMEM medium and 20 mL of compound solution. DNP-BSA (5 mg/mL, Merck, 324101-100 MG) was subsequently added into the wells to stimulate RBL-2H3 cells; the final concentration of DNP-BSA was 100 ng/mL and was incubated at 37 C for 30 min. Cells were followed transferred to a 0 C ice bath for 10 min. Finally, 50 mL of supernatant was mixed with 50 mL of p-nitrophenyl-N-acetyl-b-O-glucosamine solution (4 mM, dissolved in citric acid/sodium citrate buffer, pH 4.5). After incubation at 37 C for 1.5 h, 200 mL of sodium carbonate buffer was added to terminate the reaction (pH 10.5). The content of p-nitrophenol in supernatant was measured by a spectrophotometer at 405 nm. For the rest, the cells from the bottom of the wells in the 0 C bath were lysed with 100 mL of 0.1% Triton (Sigma, T8787), then 50 mL of the lysate was mixed with 50 mL of p-nitrophenyl-N-acetyl-b-Oglucosamine solution and incubated at 37 C for 1.5 h. The reaction was terminated by adding 200 mL of sodium carbonate buffer. Similarly, the absorbance (A) of the reaction solution was measured at 405 nm. The release rate of b-hexosaminidase (R) and the inhibition rate were calculated according to the following formula. A0, A1, A2: the absorbance of supernatant in the blank cells, the supernatant in stimulated cells and the lysate in stimulated cells, respectively; R0, R1, R2: the release rate of b-hexosaminidase in the blank group, the DNP-BSA group and the compound group, respectively.
b-hexosaminidase release rate (%) ¼ (A1 - A0 / A1 þ A2 A0) 100
(5)
b-hexosaminidase inhibition rate (%) ¼ ((R1 - (R2 - R0)) / R1) 100
(6)
3
3. Results and discussion 3.1. Virtual screening for PI3Kd inhibitors In this work, a chemical dataset of 424 TCM sourced compounds were screened based on a 3D pharmacophore model and a molecular docking method. Primarily, during the process of HIPHOP pharmacophore generation, ten hypotheses based on PI3Kd inhibitors were produced and reordered by ranking score (Table 1), then the evaluation results of these models were presented in Table 2. Pharmacophore model_7 (Fig. 1A), having the highest N and CAI values, was chosen as the best hypothesis. One hydrophobic feature (HY, shown in blue), one hydrogen bond donor (HBD, shown in red) and one hydrogen bond accepter (HBA, shown
Table 1 Ten pharmacophore results produced by the HIPHOP. Model
Features
Rank
Direct Hit
Partial Hit
Max Fit
01 02 03 04 05 06 07 08 09 10
HDA HDA HDA HDA HDA HDA HDA HDA HDA HDA
41.58 40.85 39.93 39.33 38.49 38.15 37.92 37.53 37.51 37.28
111111 111111 111111 111111 111111 111111 111111 111111 111111 111111
000000 000000 000000 000000 000000 000000 000000 000000 000000 000000
3 3 3 3 3 3 3 3 3 3
Table 2 Evaluation results for the 3D pharmacophore models. Model
Ht
Ha
A (%)
Y (%)
N
CAI
Model_01 Model_02 Model_03 Model_04 Model_05 Model_06 Model_07 Model_08 Model_09 Model_10
165 164 170 175 174 188 187 181 189 190
39 40 41 39 45 42 47 45 44 44
72.22 74.07 75.93 72.22 83.33 77.78 87.04 83.33 81.48 81.48
23.64 24.39 24.12 22.29 25.86 22.34 25.13 24.86 23.28 23.16
1.36 1.40 1.38 1.28 1.48 1.28 1.44 1.43 1.34 1.33
0.98 1.04 1.05 0.92 1.24 1.00 1.26 1.19 1.09 1.08
2.5. Statistical analysis All data were presented as mean ± stand error of the mean (SEM). One-way analysis of variance (ANOVA) was used to analyse the statistical significance with GraphPad Prism 5 among the measurement groups. Values of *P < 0.05, **P < 0.01 or ***P < 0.001 were considered to be significant.
Fig. 1. 3D pharmacophore model_7 borne from PI3Kd inhibitors. (A) The optimal pharmacophore model involves three features: a hydrophobic feature (H, blue), a hydrogen bond donor (HBD, red) and a hydrogen bond accepter (HBA, green). Numbers represent the distances between every two pharmacodynamic characteristic elements; (B) The overlapping image between a known PI3Kd inhibitor (CHEMBL3805180) and pharmacophore model_7. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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Fig. 2. Binding mode between ginkgoneolic acid and PI3Kd protein. (A) 2D diagram illustrating the interactions. (B) 3D diagram illustrating the interactions. The hydrogen bonds were marked by red dashed lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
in green) were considered to be the three characteristics of the model_7. A positive molecule (CHEMBL3805180) could match all of the characteristics of the hypothesis (Fig. 1B). Based on the model_7, a hit list of 182 compounds with FitValue >2.00 were exported by ligand-based virtual screening (part of the results were expressed in Table S1). Afterwards, the 182 compounds were further screened using the molecular docking method. Before developing the molecular docking, the reliability of the method was verified by extracting the original assembly ligand 5H5 (C19H22N6O3) and docking with the ligand binding domain of PI3Kd. The RMSD between the re-docked and co-crystallized conformation of ligand 5H5 was calculated as 1.79 Å. Furthermore, this finding showed that 5H5 could bind to PI3Kd via H-bond interaction with Glu826, Val827and Val828 and indicated that the Surflex-Docking procedure could optimally reproduce the co-crystal bioactive conformation of the PI3Kd inhibitor. Finally, all of the compounds were docked into the active sites of PI3Kd, and in turn, a hit list of 37 compounds was screened out with docking scores >7.50 (Table S2). Further investigation of the 37 compounds, possessing high fit values in pharmacophore matching and docking scores, was used for testing its PI3Kd inhibitory activity. The detailed results of the spatial arrangement between ginkgoneolic acid in the PI3Kd active site was demonstrated in Fig. 2, which showed that Tyr813, Ile825, Glu826, Val827, Val828, Met900, and Ile910 were key amino acid residues binding to ginkgoneolic acid, which is consistent with previous findings [22]. Moreover, the hydroxy group and the carboxyl group at the benzene ring are key structural features that affect biological activity. 3.2. Kinase inhibition assay These compounds were then assessed for their PI3Kd-inhibitory activity. The ADP-Glo™ Kinase Assay kit could quantify the activity by the amount of luminescent ADP, as described in the manufacturer's instructions. After preliminary screening, only the ginkgoneolic acid that exhibited at least 50% PI3Kd inhibition was qualified for further evaluation. The compound was subjected to the same enzymatic test again in a range of concentrations to accurately establish the inhibitory activity (IC50). Further dose responses were analysed and the results showed that the IC50 of ginkgoneolic acid
and (5Z)-5-(quinoxalin-6-ylmethylidene)-1, 3-thiazolidine-2, 4dione (AS-605240, a positive inhibitor) were 2.49 mM and 1.58 mM, respectively (Fig. 3A and B). 3.3. Mast cell degranulation inhibition experiment In this study, the effect of ginkgoneolic acid on mast cell degranulation was investigated in an IgE/BSA-induced model. As expressed in Fig. 3C, release rates of b-hexosaminidase from RBL2H3 cells were measured. Ginkgoneolic acid can significantly inhibit the release of b-hexosaminidase by DNP-BSA stimulation in mast cells, compared with the positive drug chloroquine. The inhibition rate of the chloroquine group (80.78%), the ginkgoneolic acid of 20 mM (74.65%) and the ginkgoneolic acid of 6.7 mM (92.09%) were less than the degranulation of the DNP-BSA group. The results made it clear that ginkgoneolic acid could attenuate the mast cell degranulation induced by antigen-specific IgE/BSA. Further doseeffect relationship analysis was shown in Fig. 3D, and the IC50 value of ginkgoneolic acid on mast cell degranulation was 2.40 mM. Ginkgoneolic acid, an alkyl phenolic acids, was commonly found in Ginkgo biloba and had been used for the treatment of allergic inflammation, such as allergic rhinitis and asthma in TCM [23,24]. A recent study had found that the extract of Ginkgo biloba had an inhibitory effect in a skin allergic reaction model [25]. Ginkgo biloba has been effective in treating various diseases, such as asthma [23], dementia, Alzheimer's disease and tinnitus [26]. However, the molecular mechanisms have not been thoroughly elucidated to date. This study reported that ginkgoneolic acid could decrease the release of inflammatory factors in vitro by inhibiting PI3Kd, providing a possible molecular mechanism for the antiallergic response of Ginkgo biloba. This study demonstrated that ginkgoneolic acid was an inhibitor of PI3Kd and showed inhibition of IgE-derived mast cell degranulation. These results provided a potential pharmaceutical source for anti-allergic therapeutics targeting PI3Kd and provided conditions for research and discovery of natural drug inhibitors. The interactions between PI3Kd and its inhibitors at the molecular level were carried out to discover its natural inhibitors. To the best of our knowledge, the virtual screening method in the study is beneficial to save time in discovering new bioactive compounds. Furthermore, the combined ligand- and structure-based approaches could
Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009
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Fig. 3. Results of the biological experiments. (A) Efficiency curves of ginkgoneolic acid inhibiting PI3Kd based on enzymatic reaction. (B) Efficiency curves of AS-605240 based on enzymatic reaction (C) The release of b-hexosaminidase was measured after pretreatment with DNP-BSA, chloroquine and ginkgoneolic acid. (D) Dose-effect relationship of ginkgoneolic acid concentrations on the inhibition of mast cell degranulation.
Fig. 4. Training set compounds used for 3D pharmacophore generation.
show features of the PI3Kd active site by molecular simulation. This method provided a practical strategy for searching natural and efficient PI3Kd inhibitors and was supported with biological experiments.
Funding This research carried out in the paper was supported by the National Natural Science Foundation of China (No. 81603311) and
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Please cite this article as: J.-F. Guo et al., Discovery of a natural PI3Kd inhibitor through virtual screening and biological assay study, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2018.12.009