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Discovery of a novel phosphoinositide 3-kinase gamma (PI3Kγ) inhibitor against hematologic malignancies and theoretical studies on its PI3Kγ-specific binding mechanisms

Class IB phosphoinositide 3-kinase gamma (PI3Kγ) is vital for regulating intracellular signaling pathways and has become an attractive drug target for the treatment of malignant tumors. In the present study, one potent PI3Kγ inhibitor (JN-PK1) with a novel scaffold against hematologic malignancies was identified based on a series of biological experiments, and then the selective mechanism of PI3Kγ inhibition was explored by a systematic computational method. JN-PK1 shows an effective antiproliferative activity on several cancer cell lines, especially blood cancer cells. Cell-free enzymatic studies demonstrated that JN-PK1 specifically inhibits PI3Kγ at low micromolar concentrations without affecting other isoforms of PI3K. In the cellular context, JN-PK1 potently inhibits PI3K/Akt/mTOR signaling pathway in a time- and concentration-dependent manner, which leads to the apoptosis of cancer cells. Further, the specific binding mode of JN-PK1 with PI3Kγ was illustrated by molecular docking, and the selective inhibition mechanism of PI3Kγ by JN-PK1 was revealed by molecular dynamics simulation. Finally, some key residues of PI3Kγ required for specificity and activity were identified. Taken together, JN-PK1 may be developed as a promising therapeutic agent for the treatment of hematologic malignancies.


Introduction
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases which play critical roles in many cellular processes, such as cell survival, proliferation, differentiation, and motility. PI3Ks are mainly grouped into three classes (Class I, II, and III) according to their structure and lipid substrate specicity. Among these classes, Class I PI3Ks are the most studied to date because of their frequent dysregulation in various cancers. [1][2][3][4][5][6] Class I PI3Ks, which are further divided into Class IA (PI3Ka, PI3Kb, PI3Kd) and IB (PI3Kg), have the ability to phosphorylate PIP2 (phosphatidylinositol 4,5-bisphosphate), converting it to PIP3 (phosphatidylinositol 3,4,5-trisphosphate). 7 PIP3 recruits a series of downstream signaling proteins, such as Akt (protein kinase B), to the plasma membrane and activates them by directly binding to their pleckstrin homology (PH) domains. 8 Extensive research conducted over the past few years highlights the association of deregulated Class I PI3K signaling with various cancers and that depicts PI3Ks as novel therapeutic targets. Therefore, a large number of PI3K inhibitors have been developed and reported over the last couple decades. 9 Recently, more attention has been drawn to the development of PI3K isoform-selective inhibitors. Idelalisib (CAL-101), the rst marketed selective PI3Kd inhibitor, was successfully approved by the FDA in 2014 for the treatment of chronic lymphocytic leukemia and small lymphocytic lymphoma. 10 More interestingly, the expression of PI3Ka/b is ubiquitous, while PI3Kd/g are restricted expressed in hematopoietic cells. 11,12 Due to this, PI3Kd and PI3Kg may have more therapeutic potential for hematologic malignancies. [12][13][14][15][16][17] Recently, Duvelisib (IPI-145), a dual PI3Kd/g inhibitor, entered the market in 2018, 18 which will further promote the development of PI3Kg selective inhibitors.
The only member of class IB, PI3Kg, is mainly activated by GPCRs (G-protein-coupled receptors). PI3Kg is necessary for growth factors signaling, chemokine-stimulated chemotaxis, blocking stimulated T cell recruitment into tumors, and regulating innate immunity during inammation and tumorigenesis. [19][20][21][22] Additionally, the restriction of PI3Kg expression to the hematopoietic system may limit the toxicity of specic inhibitors compared to pan-PI3K inhibitors. In recent years, more and more small molecule inhibitors acting on PI3Kg have been discovered, some of which have entered clinical trials for hematologic malignancies or solid tumors, such as the specic PI3Kg inhibitors AS604850, 23 CAY10505, 24 AS252424, 25 CZC24832, 26 and some dual PI3Kd/g inhibitors IPI-549 (NCT03795610), 27 TG100-115, 28 RP6530 (NCT02017613). [29][30][31] However, the selective PI3Kg inhibitors are far less developed than other PI3K isoforms inhibitors, because PI3Kg shares high sequence similarity in the conserved ATP-kinase binding site with the Class IA isoforms, that directly leads to the great difficulty in the development of specic g isoform inhibitors. 32 Thus in this study, a high throughput virtual screening was preformed to screen the ChemBridge database, and as a result, 83 compounds were purchased and then submitted to bioassays. Finally, the novel PI3Kg selective inhibitor JN-PK1, 1,1 0 -(9-methyl-9H-carbazole-3,6-diyl)diethanone, was identied for the treatment of hematologic malignancies, which was conrmed using a range of biochemical and cellular studies. Cell-free assays exhibited that JN-PK1 selectively inhibits PI3Kg isoform. On a cellular level, JN-PK1 showed potent anti-proliferation activity against several hematologic cancer cell lines, and specically inhibited the PI3K signaling pathway. Meanwhile, a computational investigation on this inhibitor integrating molecular docking and molecular dynamics (MD) simulation was conducted, and lastly, the selective PI3Kg inhibition mechanism of this inhibitor was highlighted. All compounds were purchased from ChemBridge Corporation (San Diego, CA). PI-103 was from Selleck (Shang Hai, China). The Annexin V-FITC/PI Apoptosis Detection Kit was from BD Biosciences (Becton Dickinson, UAS). MTT or 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetra-sodium bromide was from Sigma (St Louis, MO). ADP-Glo Luminescent Assay was purchased from Promega (Madison, Wisconsin, USA). BCA Protein Assay Kit was purchased from Biosharp (China).

Cell growth assays
Cell growth was assessed by MTT assay. In a 96-well plate, solid tumor cells (MKN-28, SGC7901, JF-305, Capan-1, Patu8988, SUM102, T47D, and MDA-MB-231) were plated at a concentration of 3 Â 10 3 cells in 100 mL medium, and suspension cells (OCI-My5, Jurkat, KMS11, HL-60, and u937) were plated with a concentration of 8 Â 10 3 cells in 100 mL medium. Cells were then treated with 5 mL of 10 mM compound. Aer 72 h, 10 mL of 5 mg mL À1 MTT was added to each well and cells were incubated at 37 C for 4 h. Subsequently, 100 mL MTT reagent was added to each well. On alternate days, the absorbance of each well was measured at 550 nm using a microplate reader. All data is shown as the percent viability of treated compared to nontreated cells.

PI3K kinase activity in cell-free assays
JN-PK1 activity against Class I PI3K kinases was tested using ADP-Glo Luminescent Assay and PI-103 33 was used as the positive control. Both compounds were tested thrice using 10 serial dilutions starting at 10 mM and subsequently diluted 2fold for each subsequent dilution with 100% DMSO, and reacted with substrate solution (PIP2 at 50 mM, ATP at 25 mM). The two assays were done in 384-well white plates with a total reaction volume of 10 mL per well (2.5 mL of compound, 2.5 mL of PI3Ka at 0.3 mg mL À1 , and 5 mL of substrate solution), or 15 mL (2.5 mL of compound, 2.5 mL of PI3Kd at 1 mg mL À1 or PI3Kb at 1 mg mL À1 or PI3Kg at 1.5 mg mL À1 , 5 mL of substrate solution, and 5 mL of ADP-Glo reagent). Aer incubating at room temperature for 1 h, the 10 mL Glo reagent assay was added to each well to stop the reaction. At the end, each plate was read for luminescence.

Immunoblotting analysis
2.5.1 Protein preparation. OCI-My5 or HL-60 were dispersed into 6-well plates with 1 Â 10 6 cells per well and then treated with JN-PK1 at the following concentrations: 0, 2.5, 5, 10 and 20 mM. Aer 24 h, the cells of each well were collected, washed with 4 C PBS three times, incubated with 80 mL RIPA lysis buffer for 20 min on ice, and then centrifuged at 14 800 rpm for 20 min at 4 C. The supernatant was collected, and the total protein concentration was quantied using a BCA Protein Assay Kit. Loading buffer (5Â) was added to all protein samples isolated for Western blot analysis and then boiled at 100 C for 5 min.
2.5.2 Western blot assay. The inhibition of the PI3K/Akt signal pathway by JN-PK1 was detected by Western blot. The samples were loaded onto a 10% acrylamide gel for SDS-PAGE and run for 30 min at 80 V for the stacking gel, and 60 min at 100 V for the separation gel. The protein was transferred onto a membrane at 100 V for 70 min, which was then blocked in 5% skim milk diluted in 1 Â TBST for 2 h. The membrane was incubated with relevant primary antibody overnight at 4 C, and then washed with 1 Â TBST four times, 10 min each and then incubated in secondary antibody for 1 h at room temperature on a shaker. Aer washing four times, the membrane was imaged with the hypersensitive chemiluminescence detection kit (Proteintech).

Flow cytometry assay
Using Annexin V-FITC/PI detection kits (Beyotime Company), OCI-My5 and HL-60 cells treated with JN-PK1 for 24 h were detected on a ow cytometer, and the apoptosis rates were measured according to the manufacturer's instructions.

Docking studies
The 3D structure of JN-PK1 was sketched and minimized using maestro in Schrödinger. Then JN-PK1 was prepared by Ligprep module with the OPLS-2005 force eld, where generated 10 low energy ring conformations per ligand. The crystal structures of four PI3K isoforms (a, b, d, g) with high resolution were downloaded from protein data bank (PDB code: 4JPS, 34 2Y3A, 35 4XEO, 36 4ANW 36 ), and prepared as the initial receptors for the docking studies using Protein Preparation Wizard in Schrödinger: removing all crystal water molecules, adding hydrogen atoms, assigning partial charges, terminating the minimization if the RMSD reached the value of 0.3Å with the OPLS-2005 force eld. The grid les of each protein were generated with the ligands as centroids using Glide Grid Generation module.
Then JN-PK1 was respectively docked into each grid le of four isoforms using Glide Docking module (XP mode), with writing out 10 poses per ligand while other parameters as defaults. Aer completing each docking calculation, the best docked structures of JN-PK1 in each receptor-ligand system were chosen to the next analysis.

MD simulations
The best docked JN-PK1 structures with each PI3K isoforms (a, b, d, g) were used as the starting structures for MD simulations with AMBER14. 37 The AMBER ff03 33 force led was used for the proteins, the general AMBER force led (GAFF) 39 for the inhibitors and the semi-empirical AM1 method was employed for the optimization of ligands. 40 Then the restrained electrostatic potential (RESP) technique was used to t the electrostatic potential at the HF/6-31G* level 41 and the atomic partial charges of ligands were obtained.
Each system was rstly neutralized with sodium ions and then immersed in an octagonal box of TIP3P water molecules which extended 10Å from solute atoms. The long-range electrostatic interaction was handled by the Particle Mesh Ewald (PME) scheme, 42 and the non-bonded cutoff for the van der Waals interaction was set to 10Å. For relaxing any steric conicts probably generated during MD simulations, each system was rstly adopted three-stage minimizations: 1000 cycles of minimizations for constraining the backbone carbons (50 kcalÅ À2 ), 1000 cycles of minimizations with a weaker harmonic potential for constraining (10 kcalÅ À2 ) were conducted, 5000 cycles of minimizations containing 1000 cycles of steepest descend and 4000 cycles of conjugate gradient without any constrain were nally performed. The MD simulations were run over 50 ps in the NVT ensemble with the gradually rising temperature from 0 to 300 K, which were nally performed 30 000 ps. The time step was set to 2 fs, and all bonds including hydrogen atoms were constrained with the SHAKE algorithm.

MM/GBSA free energy calculation and decomposition analysis
Each system was calculated the binding free energy (DG bind ) using the MM/GBSA method according to the following eqn (1). 43,44 All components extracted from the last 10 ns stable MD trajectories were calculated with the MM_PBSA program of AMBER 14.

JN-PK1 selectively inhibits PI3Kg in a cell-free system and effectively inhibits the PI3K signaling pathway
The above results indicate that JN-PK1 effectively inhibits the proliferation of hematologic tumor cells. To determine whether this effect on proliferation is caused by the inhibition of PI3Kg, the inhibitory potency of JN-PK1 on PI3K enzymes was measured in a cell-free system. The results of the JN-PK1 treatment on four isoforms (PI3Ka, PI3Kb, PI3Kd and PI3Kg) are summarized in Table 1. Compared to PI-103, JN-PK1 showed lower activity on PI3K kinases, but signicantly and specically inhibited PI3Kg compared to the other isoforms at a lower concentration of 7.5 mM, while there was no signicant inhibitory effect on Class IA PI3Ks even at high concentrations. This suggests that JN-PK1 is a selective inhibitor of PI3Kg.
To further study whether JN-PK1 can be used to suppress the PI3K/Akt signaling pathway to treat hematologic malignancies, immunoblotting was used to detect the relative expression of proteins in the leukemia cell line HL-60 and MM cell line OCI-My5. It is well established in the literature that PI3K/Akt/mTOR (mammalian target of rapamycin) signaling plays signicant roles in regulating survival and proliferation of hematologic cancer cells, and PI3Kg is frequently overexpressed in different blood cancer cells. 47 Therefore, the phosphorylation levels of  Akt (p-Akt Thr308 and Ser473 ), the key downstream target of PI3K/ Akt signaling, were detected aer the treatment of JN-PK1. As illustrated in Fig. 2A, the phosphorylation of Akt at Thr308 was rst downregulated by JN-PK1, and that of Ser473 site was subsequently inhibited, suggesting that the total phosphorylation of Akt was suppressed by JN-PK1 with the inhibition of PI3Kg. And then, the phosphorylation level of mTOR (p-mTOR Ser2448 ), another key downstream protein, was measured.   Fig. 2A shows that the phosphorylation of mTOR was also suppressed upon inhibition of PI3Kg by JN-PK1 treatment. On the other hand, we found that the phosphorylation of these three proteins was notably decreased at a JN-PK1 concentration of 10 mM, but p-Akt Thr308 and p-mTOR Ser2448 decreased at a concentration as low as 5 mM. These results indicate that JN-PK1 remarkably suppresses Akt and mTOR phosphorylation in a concentration-dependent manner, and has no effect on total Akt and mTOR expression. Next, a time-course study revealed that JN-PK1 inhibits p-Akt Ser473 and especially p-Akt Thr308 within 6 h aer treatment, shown in Fig. 2B. Similarly, p-mTOR Ser2448 was associatively suppressed aer JN-PK1 treatment. Therefore, JN-PK1 inhibits the PI3K pathway in a timedependent manner. Additionally, as shown in Fig. 2A and B, JN-PK1 suppressed p-Akt and p-mTOR more effectively in HL-60 cells than OCI-My5 cells in a time-and concentrationdependent manner, suggesting that JN-PK1 would more effectively inhibit PI3K/Akt signaling in leukemia compared to other cancer cell lines.

JN-PK1 induces apoptosis of hematologic tumor cells
JN-PK1 effectively suppressed the proliferation of hematologic cancer cells. To further investigate whether JN-PK1 could induce apoptosis in hematologic cancer cells by inhibiting the PI3K/Akt pathway, Western blots were performed to detect relative levels of two apoptosis biomarkers, PARP (poly ADPribose polymerase) and Caspase-3, with increasing incubation time and concentration. As illustrated in Fig. 3A and B, the cleavage levels of PARP and caspase-3 were obviously increased following increased incubation time and concentration of JN-PK1 treatment, notably at a JN-PK1 concentration of 10 mM.
To further observe the number of apoptotic hematologic cancer cells induced by JN-PK1, Annexin V-FITC/PI double staining was performed on OCI-My5 and HL-60 cells treated with varying concentrations of JN-PK1 for 24 h. The percentage of apoptotic cells induced by JN-PK1 was increased in a concentrationdependent manner, and over half of the cells were at a late stage of apoptosis aer treatment with 10 mM (Fig. 3C and D). These results suggest that JN-PK1 efficiently inhibits the survival and proliferation of MM and leukemia cells.

Theoretical studies on the mechanism of selective inhibition of PI3Kg by JN-PK1
In order to elucidate the mechanism of selective inhibition of PI3Kg by JN-PK1, a computational strategy combining molecular docking, MD simulation, free energy calculations and decomposition was employed to reveal the binding modes between JN-PK1 and the four PI3K isoform enzymes: PI3Ka, PI3Kb, PI3Kd and PI3Kg. The molecular docking protocol was rst performed to generate the appropriate binding poses of JN-PK1 to each PI3K isoform. The docking scores (showed in Table  1) were À8.282 for PI3Ka, À6.713 for PI3Kb, À6.531 for PI3Kd and À8.876 for PI3Kg. This suggests that JN-PK1 binding is most specic to PI3Kg, in agreement with our experimental results. And then, the interactions between JN-PK1 and the four PI3K isoforms were respectively illustrated through 2D presentations. As shown in Fig. S1B, † one of the acetyl groups of JN-PK1 formed hydrogen bonds (H-bonds) with Lys799, Ser775 and Met773 of PI3Kb, while two acetyl groups of JN-PK1 respectively formed H-bonds with Lys779 and Ser831 of PI3Kd (Fig. S1C †). The two carbazole phenyl-rings of JN-PK1 formed two arene-H interactions with Val850 and Ile932 of PI3Ka   (Fig. S1A †), and in PI3Kg system, JN-PK1 formed three arene-H interactions with Ile881, Ile963 and Ile879 (Fig. 4A), these interactions are essential for the binding of JN-PK1 to these proteins. Besides, as shown in Fig. 4A, an H-bond formed between one acetyl group of JN-PK1 and Lys833 of PI3Kg, and the steric hindrance that was created by the side chain of residue Lys833 may help JN-PK1 remain in the active pocket of PI3Kg (Fig. 4B). 36,48 This may explain the stronger binding affinity of JN-PK1 to PI3Kg despite the fewer interactions that were formed with PI3Kg than other isoforms. Therefore, the contributions from individual residues of each isoform to JN-PK1 binding need to be further investigated. Next, the four docked JN-PK1/PI3K complexes were used as the initial structures for the subsequent MD simulations. As shown in Fig. S2, † the RMSD (root-mean-square deviations) values of the binding pockets were quite stable in the last 10 ns, indicating that the complexes reached equilibrium within last 10 ns. Subsequently, 1000 snapshots extracted from the last 10 ns of each MD trajectory were used to calculate the binding free energies through the MM/PBSA method. As summarized in Table 2, the predicted binding free energies were À20.50 kcal mol À1 for PI3Ka/JN-PK1, À25.75 kcal mol À1 for PI3Kb/JN-PK1, À23.43 kcal mol À1 for PI3Kd/JN-PK1 and À27.40 kcal mol À1 for PI3Kg/JN-PK1. This suggests that JN-PK1 specically interacts with PI3Kg, in agreement with our experimental data and docking results.
In order to understand the g-specic binding modes of JN-PK1, the binding free energies of all systems were decomposed into the contributions from individual residues and the JN-PK1-residues interaction spectrums are illustrated in Fig. S3. † Furthermore, we calculated the free energy changes, DDG total ¼ DG total (PI3Kg) À DG total (PI3Ka/PI3Kb/PI3Kd), and the residues located in the ATP-binding pockets were selected to discussed in detail. 49 As shown in Fig. 5E, the negative values of The free energy changes DDG total which were defined as DG total (PI3Kg) À DG total (PI3Ka/PI3Kb/PI3Kd), with PI3Kg residues colored in red and PI3Ka/PI3Kb/PI3Kd residues colored in purple.
DDG total represent a favorable contribution for PI3Kg. In general, as shown in Fig. 5D, JN-PK1 binding with PI3Kg was sandwiched between residues Ile831 and Ile963, and encompassed by residues Ile881, Val882 and Trp812 above and residues Tyr867 and Ile879 below, which formed strong hydrophobic interactions and highly improved the binding affinity of JN-PK1. Compared to the binding mode of PI3Ka (Fig. 5A), JN-PK1 was packed more tightly with PI3Kg, especially residues Phe961, Ile831, Ile881 and Val882, shown in Fig. 5E. This tight binding prevented JN-PK1 from exiting the active pocket of PI3Kg. Compared to PI3Kd (Fig. 5C), JN-PK1 remained in closer proximity to the active pocket of PI3Kg through tighter binding with residues Ile831, Tyr867, Ile879, Ile881, Val882 and Phe961. Comparing the free energy change DDG total of Ile800 of PI3Ka and Ile777 of PI3Kd (Fig. 5E), the free energy DG total of Ile797 of PI3Kb was similar to that of Ile831 of PI3Kg combined with JN-PK1, which may explain the reason why JN-PK1 bound more tightly to PI3Kb (Fig. 5B) with better predicted binding free energies. Therefore, residue Ile831 of PI3Kg is important for the binding selectivity of JN-PK1 to PI3Kg. Moreover, comparing the free energy changes DDG total as showed in Fig. 5E, the backbone residue Val882 uniquely formed an Hbond between one acetyl group of JN-PK1 and PI3Kg, common for selective PI3Kg inhibitors. 48 Additionally, hydrophobic interactions formed by the hinge residue Ile881 and Trp812 may restrain the movement of Trp812 and further promote JN-PK1 stabilization in the active pocket of PI3Kg. 50 As a potent PI3Kg-selective inhibitor, JN-PK1 exhibited stronger affinity for PI3Kg (Tables 1 and 2), and more tightly connected with important residues Ile831, Ile881 and Val882 in the active pockets of PI3Kg compared to the other three PI3K isoforms (Fig. 5E).

Conclusions
In summary, using a series of studies at both cellular and computational models, the highly potent and selective PI3Kg inhibitor JN-PK1 was identied for treating hematologic malignancies. JN-PK1 exhibited high anti-proliferative activity on most cancer cell lines, especially hematologic cell lines. The selective inhibition on PI3Kg of JN-PK1 was conrmed by a cellfree enzymatic system, and then the PI3K/Akt signaling pathway was specically inhibited in a time-and concentrationdependent manner aer JN-PK1 treatment. Immunoblotting and ow cytometry analysis visually presented that JN-PK1 induced apoptosis of cancer cells in association with its potent PI3K inhibition. Furthermore, the molecular docking and MD simulation were used to deeply study the binding modes between JN-PK1 and the four PI3K isoforms, and the selective inhibition mechanism of JN-PK1 on PI3Kg was revealed with the highlight of some key residues of PI3Kg relating to selectivity. In general, JN-PK1 may be an effective treatment of hematologic malignancies as a promising PI3Kg inhibitor with a novel scaffold, and the theoretical studies on its g-specic inhibition mechanisms may provide some important guidelines for further study.