Pharmacophore modeling and 3D-QSAR analysis of phosphoinositide 3-kinase p110α inhibitors

Pharmacophore modeling studies were undertaken for a series of compounds belonging several groups of phosphoinositide 3-kinase (PI3K) p110α inhibitors: 4-morpholino-2-phenylquinazolines derivatives, pyrido[3′,2′:4,5]furo-[3,2-d]pyrimidine derivatives, imidazo[1,2-a]pyridine derivatives, sulfonylhydrazone substituted imidazo[1,2-a]pyridines, and LY294002. A five-point pharmacophore with three hydrogen bond acceptors (A), one hydrophobic group (H), and one aromatic ring (R) as pharmacophore features was developed. The pharmacophore hypothesis yielded a statistically significant 3D-QSAR model, with a correlation coefficient of R ² = 0.95 for training set compounds. The model generated showed excellent predictive power, with a correlation coefficient of Q ² = 0.88 and r _pret² = 0.95 for a test set of 14 compounds. Furthermore, the structure–activity relationships of PI3K p110α inhibitors were elucidated and the activity differences between them discussed. Docking studies were also carried out wherein active and inactive compounds were docked into the active site of the PI3K p110α crystal structure to analyze PI3K p110α–inhibitor interactions. The results provide insights that will aid optimization of these classes of PI3K p110α inhibitors for better activity, and may prove helpful for further lead optimization and virtual screening.     

Dynamics of the phosphoinositide 3-kinase p110δ interaction with p85α and membranes reveals aspects of regulation distinct from p110α

Phosphoinositide 3-kinase δ is upregulated in lymphocytic leukemias. Because the p85-regulatory subunit binds to any class IA subunit, it was assumed there is a single universal p85-mediated regulatory mechanism; however, we find isozyme-specific inhibition by p85α. Using deuterium exchange mass spectrometry (DXMS), we mapped regulatory interactions of p110δ with p85α. Both nSH2 and cSH2 domains of p85α contribute to full inhibition of p110δ, the nSH2 by contacting the helical domain and the cSH2 via the C terminus of p110δ. The cSH2 inhibits p110β and p110δ, but not p110α, implying that p110α is uniquely poised for oncogenic mutations. Binding RTK phosphopeptides disengages the SH2 domains, resulting in exposure of the catalytic subunit. We find that phosphopeptides greatly increase the affinity of the heterodimer for PIP2-containing membranes measured by FRET. DXMS identified regions decreasing exposure at membranes and also regions gaining exposure, indicating loosening of interactions within the heterodimer at membranes.     

Phosphoinositide 3-kinase p110α negatively regulates thrombopoietin-mediated platelet activation and thrombus formation

Phosphoinositide 3-kinase (PI3K) plays an important role in platelet function and contributes to platelet hyperreactivity induced by elevated levels of circulating peptide hormones, including thrombopoietin (TPO). Previous work established an important role for the PI3K isoform; p110β in platelet function, however the role of p110α is still largely unexplored. Here we sought to investigate the role of p110α in TPO-mediated hyperactivity by using a conditional p110α knockout (KO) murine model in conjunction with platelet functional assays. We found that TPO-mediated enhancement of collagen-related peptide (CRP-XL)-induced platelet aggregation and adenosine triphosphate (ATP) secretion were significantly increased in p110α KO platelets. Furthermore, TPO-mediated enhancement of thrombus formation by p110α KO platelets was elevated over wild-type (WT) platelets, suggesting that p110α negatively regulates TPO-mediated priming of platelet function. The enhancements were not due to increased flow through the PI3K pathway as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) formation and phosphorylation of Akt and glycogen synthase kinase 3 (GSK3) were comparable between WT and p110α KO platelets. In contrast, extracellular responsive kinase (ERK) phosphorylation and thromboxane (TxA₂) formation were significantly enhanced in p110α KO platelets, both of which were blocked by the MEK inhibitor PD184352, whereas the p38 MAPK inhibitor VX-702 and p110α inhibitor PIK-75 had no effect. Acetylsalicylic acid (ASA) blocked the enhancement of thrombus formation by TPO in both WT and p110α KO mice. Together, these results demonstrate that p110α negatively regulates TPO-mediated enhancement of platelet function by restricting ERK phosphorylation and TxA₂ synthesis in a manner independent of its kinase activity.     

PI3-kinase p110α mediates β1 integrin-induced Akt activation and membrane protrusion during cell attachment and initial spreading

Integrin-mediated cell adhesion activates several signaling effectors, including phosphatidylinositol 3-kinase (PI3K), a central mediator of cell motility and survival. To elucidate the molecular mechanisms of this important pathway the specific members of the PI3K family activated by different integrins have to be identified. Here, we studied the role of PI3K catalytic isoforms in β1 integrin-induced lamellipodium protrusion and activation of Akt in fibroblasts. Real-time total internal reflection fluorescence imaging of the membrane–substrate interface demonstrated that β1 integrin-mediated attachment induced rapid membrane spreading reaching essentially maximal contact area within 5–10 min. This process required actin polymerization and involved activation of PI3K. Isoform-selective pharmacological inhibition identified p110α as the PI3K catalytic isoform mediating both β1 integrin-induced cell spreading and Akt phosphorylation. A K756L mutation in the membrane-proximal part of the β1 integrin subunit, known to cause impaired Akt phosphorylation after integrin stimulation, induced slower cell spreading. The initial β1 integrin-regulated cell spreading as well as Akt phosphorylation were sensitive to the tyrosine kinase inhibitor PP2, but were not dependent on Src family kinases, FAK or EGF/PDGF receptor transactivation. Notably, cells expressing a Ras binding-deficient p110α mutant were severely defective in integrin-induced Akt phosphorylation, but exhibited identical membrane spreading kinetics as wild-type p110α cells.We conclude that p110α mediates β1 integrin-regulated activation of Akt and actin polymerization important for survival and lamellipodia dynamics. This could contribute to the tumorigenic properties of cells expressing constitutively active p110α.     

Both p110α and p110β isoforms of PI3K can modulate the impact of loss-of-function of the PTEN tumour suppressor

The PI3K (phosphoinositide 3-kinase) pathway is commonly activated in cancer as a consequence of inactivation of the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), a major negative regulator of PI3K signalling. In line with this important role of PTEN, mice that are heterozygous for a PTEN-null allele (PTEN+/? mice) spontaneously develop a variety of tumours in multiple organs. PTEN is a phosphatase with selectivity for PtdIns(3,4,5)P3, which is produced by the class I isoforms of PI3K (p110α, p110β, p110γ and p110δ). Previous studies indicated that PTEN-deficient cancer cell lines mainly depend on p110β, and that p110β, but not p110α, controls mouse prostate cancer development driven by PTEN loss. In the present study, we investigated whether the ubiquitously expressed p110α can also functionally interact with PTEN in cancer. Using genetic mouse models that mimic systemic administration of p110α- or p110β-selective inhibitors, we confirm that inactivation of p110β, but not p110α, inhibits prostate cancer development in PTEN+/? mice, but also find that p110α inactivation protects from glomerulonephritis, pheochromocytoma and thyroid cancer induced by PTEN loss. This indicates that p110α can modulate the impact of PTEN loss in disease and tumourigenesis. In primary and immortalized mouse fibroblast cell lines, both p110α and p110β controlled steady-state PtdIns(3,4,5)P3 levels and Akt signalling induced by heterozygous PTEN loss. In contrast, no correlation was found in primary mouse tissues between PtdIns(3,4,5)P3 levels, PI3K/PTEN genotype and cancer development. Taken together, our results from the present study show that inactivation of either p110α or p110β can counteract the impact of PTEN inactivation. The potential implications of these findings for PI3K-targeted therapy of cancer are discussed.     

Hot-spot mutations in p110α of phosphatidylinositol 3-kinase (PI3K): Differential interactions with the regulatory subunit p85 and with RAS

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is frequently upregulated in cancer. PIK3CA, the gene coding for the catalytic subunit p110α of PI3K, is mutated in about 12% of all human cancers. Most of these mutants are single amino acid substitutions that map to three positions (hot spots) in the helical or kinase domains of the enzyme. The mutant proteins show gain of enzymatic function, constitutively activate AKT signaling and induce oncogenic transformation in vitro and in animal model systems. We have shown previously that hot-spot mutations in the helical domain and kinase domain of the avian p110α have different requirements for interaction with the regulatory subunit p85 and with RAS-GTP. Here, we have carried out a genetic and biochemical analysis of these "hot-spot" mutations in human p110α. The present studies add support to the proposal that helical and kinase domain mutations in p110α trigger a gain of function by different molecular mechanisms. The gain of function induced by helical domain mutations requires interaction with RAS-GTP. In contrast, the kinase domain mutation is active in the absence of RAS-GTP binding, but depends on the interaction with p85.     

Engineering of an isolated p110α subunit of PI3Kα permits crystallization and provides a platform for structure-based drug design

PI3Kα remains an attractive target for the development of anticancer targeted therapy. A number of p110α crystal structures in complex with the nSH2-iSH2 fragment of p85 regulatory subunit have been reported, including a few small molecule co-crystal structures, but the utilization of this crystal form is limited by low diffraction resolution and a crystal packing artifact that partially blocks the ATP binding site. Taking advantage of recent data on the functional characterization of the lipid binding properties of p110α, we designed a set of novel constructs allowing production of isolated stable p110α subunit missing the Adapter Binding Domain and lacking or featuring a modified C-terminal lipid binding motif. While this protein is not catalytically competent to phosphorylate its substrate PIP2, it retains ligand binding properties as indicated by direct binding studies with a pan-PI3Kα inhibitor. Additionally, we determined apo and PF-04691502 bound crystal structures of the p110α (105-1048) subunit at 2.65 and 2.85 Å, respectively. Comparison of isolated p110α(105-1048) with the p110α/p85 complex reveals a high degree of structural similarity, which validates suitability of this catalytically inactive p110α for iterative SBDD. Importantly, this crystal form of p110α readily accommodates the binding of noncovalent inhibitor by means of a fully accessible ATP site. The strategy presented here can be also applied to structural studies of other members of PI3KIA family.     

Estradiol increases cell growth in human astrocytoma cell lines through ERα activation and its interaction with SRC-1 and SRC-3 coactivators

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERβ, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERβ agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.     

miR-497-5p inhibits tumor cell growth and invasion by targeting SOX5 in non–small-cell lung cancer

MicroRNAs plays an important role in the ccurrence and development of non–small-cell lung cancer (NSCLC). miR-497-5p has been reported to function as a tumor suppressor in various cancers. However, the role of miR-497-5p in NSCLC remains poorly understood. In this study, we aimed to investigate the biological role and potential molecular mechanism of miR-497-5p in NSCLC. Our results showed that the messenger RNA (mRNA) expression level of miR-497-5p was notably downregulated in human NSCLC tissues and cell lines. miR-497-5p overexpression remarkably inhibited NSCLC cell proliferation and increased cell apoptosis in A549 and H460 cells, whereas inhibition of miR-497-5p had an opposite effect. The ability of cell migration and invasion was inhibited by miR-497-5p overexpression but was increased by miR-497-5p inhibition. Moreover, our findings indicated that SOX5 was a direct target of miR-497-5p. The protein and mRNA expression levels of SOX5 in A549 cells were remarkably inhibited by miR-497-5p overexpression but was upregulated by miR-497-5p inhibition. Furthermore, SOX5 overexpression notably reversed the effect of miR-497-5p mimic on NSCLC cell proliferation, cell apoptosis, cell migration, and invasion. Taken together, these results indicated that miR-497-5p overexpression inhibited NSCLC cell proliferation, migration and invasion, and induced cell apoptosis through inhibiting SOX5 gene expression. It was conceivable that miR-497-5p might serve as a potential molecular target for NSCLC treatment.     

Addition of N-terminal peptide sequences activates the oncogenic and signaling potentials of the catalytic subunit p110α of phosphoinositide-3-kinase

Addition of short (6 to 16 amino acids) peptide sequences to the N-terminus of p110α induces a gain of function. Such sequences include the common Flag, His, and VSV tags as well as random sequences. An N-terminal myristylation signal generally believed to activate p110α by providing a constitutive membrane address is also activating, if myristylation is mutationally abolished. The gain of function seen with N-terminally tagged (NTT) p110α constructs extends to signaling, oncogenic transformation and stimulation of cell growth. The activating effect of N-terminal tags requires a functional Ras-binding domain in p110α. Mutations in that domain (T208D and K227A) abolish the gains of function in oncogenicity and signaling. The dominant negative mutant of Ras, RasN17, interferes with transformation induced by NTT p110α. In contrast, binding to p85 activity is not required for cellular transformation and enhanced signaling by NTT p110α.     

Addition of N-terminal peptide sequences activates the oncogenic and signaling potentials of the catalytic subunit p110α of phosphoinositide-3-kinase

Addition of short (6 to 16 amino acids) peptide sequences to the N-terminus of p110α induces a gain of function. Such sequences include the common Flag, His and VSV tags as well as random sequences. An N-terminal myristylation signal generally believed to activate p110α by providing a constitutive membrane address is also activating, even if myristylation is mutationally abolished. The gain of function seen with N-terminally tagged (NTT) p110α constructs extends to signaling, oncogenic transformation and stimulation of cell growth. The activating effect of N-terminal tags requires a functional Rasbinding domain in p110α. Mutations in the RBD domain (T208D and K227A) abolish the gains of function in oncogenicity and signaling. The dominant negative mutant of Ras, RasN17, interferes with transformation induced by NTT p110α. In contrast, binding to p85 activity is not required for cellular transformation and enhanced signaling by NTT p110α.Key words: N-terminal tags, p110a, oncogenicity, Ras binding domain, p85 binding mutations     

Multiple roles for the p85α isoform in the regulation and function of PI3K signalling and receptor trafficking

The p85α protein is best known as the regulatory subunit of class 1A PI3Ks (phosphoinositide 3-kinases) through its interaction, stabilization and repression of p110-PI3K catalytic subunits. PI3Ks play multiple roles in the regulation of cell survival, signalling, proliferation, migration and vesicle trafficking. The present review will focus on p85α, with special emphasis on its important roles in the regulation of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and Rab5 functions. The phosphatidylinositol-3-phosphatase PTEN directly counteracts PI3K signalling through dephosphorylation of PI3K lipid products. Thus the balance of p85α-p110 and p85α-PTEN complexes determines the signalling output of the PI3K/PTEN pathway, and under conditions of reduced p85α levels, the p85α-PTEN complex is selectively reduced, promoting PI3K signalling. Rab5 GTPases are important during the endocytosis, intracellular trafficking and degradation of activated receptor complexes. The p85α protein helps switch off Rab5, and if defective in this p85α function, results in sustained activated receptor tyrosine kinase signalling and cell transformation through disrupted receptor trafficking. The central role for p85α in the regulation of PTEN and Rab5 has widened the scope of p85α functions to include integration of PI3K activation (p110-mediated), deactivation (PTEN-mediated) and receptor trafficking/signalling (Rab5-mediated) functions, all with key roles in maintaining cellular homoeostasis.     

The phosphoinositide 3'-kinase p110δ modulates contractile protein production and IL-6 release in human airway smooth muscle

Transforming growth factor (TGF) β1 increases pro‐inflammatory cytokines and contractile protein expression by human airway smooth muscle (ASM) cells, which could augment airway inflammation and hyperresponsiveness. Phosphoinositide 3′ kinase (PI3K) is one of the signaling pathways implicated in TGFβ1 stimulation, and may be altered in asthmatic airways. This study compared the expression of PI3K isoforms by ASM cells from donors with asthma (A), chronic obstructive pulmonary disease (COPD), or neither disease (NA), and investigated the role of PI3K isoforms in the production of TGFβ1 induced pro‐inflammatory cytokine and contractile proteins in ASM cells. A cells expressed higher basal levels of p110δ mRNA compared to NA and COPD cells; however COPD cells produced more p110δ protein. TGFβ1 increased 110δ mRNA expression to the same extent in the three groups. Neither the p110δ inhibitor IC87114 (1, 10, 30 µM), the p110β inhibitor TGX221 (0.1, 1, 10 µM) nor the PI3K pan inhibitor LY294002 (3, 10 µM) had any effect on basal IL‐6, calponin or smooth muscle α‐actin (α‐SMA) expression. However, TGFβ1 increased calponin and α‐SMA expression was inhibited by IC87114 and LY294002 in all three groups. IC87114, TGX221, and LY294002 reduced TGFβ1 induced IL‐6 release in a dose related manner in all groups of ASM cells. PI3K p110δ is important for TGFβ1 induced production of the contractile proteins calponin and α‐SMA and the proinflammatory cytokine IL‐6 in ASM cells, and may therefore be relevant as a potential therapeutic target to treat both inflammation and airway remodeling. J. Cell. Physiol. 227: 3044–3052, 2012. © 2011 Wiley Periodicals, Inc.     

Targeted inhibition of phosphatidyl inositol-3-kinase p110β, but not p110α, enhances apoptosis and sensitivity to paclitaxel in chemoresistant ovarian cancers

The phosphatidylinositol 3-kinase (PI3K) pathway is one of the critical signaling cascades playing important roles in the chemoresistance of human cancer cells, including ovarian cancer. In this study, we investigated the potential of targeting the PI3K p110β-isoform as a novel approach to overcome the chemoresistance in ovarian cancer. The effects on apoptosis, cell viability, proliferation and migration in chemoresistant ovarian cancer cell were determined following targeted p110β inhibition by small interfering RNA (siRNA). Seven paclitaxel (PTX)-resistant sublines (SKpacs and A2780pac) were produced from SKOV3 and A2780 ovarian cancer cell lines. We, first, evaluated the expression of PI3K p110 isoforms in chemosensitive and chemoresistant ovarian cancer cell lines and patient specimens, and found that p110β-isoform was significantly overexpressed both in a panel of ovarian cancer samples, and in PTX-resistant sublines compared with their parent cell lines. RNA interference-mediated p110β silencing augmented PTX-mediated apoptosis (31.15 ± 13.88 %) and reduced cell viability (67 %) in PTX-resistant cells, whereas targeting p110α did not show a significant change in cell viability and apoptosis. In addition, p110β silencing impaired cell proliferation (60 %) in PTX-resistant SKpac cells. We also found the combined treatment group with p110β siRNA and PTX showed a significant inhibition of tumor growth of SKpac cells compared to the PTX-only treated group in a xenograft nude mouse model. Thus, the siRNA-mediated silencing of PI3K p110β resensitizes PTX-resistant ovarian cancer cells, and may be a useful therapeutic strategy for PTX-resistant ovarian cancers.     

Modulation of YY1 and p53 expression by transforming growth factor-β3 in prostate cell lines

Transforming growth factor-β (TGF-β) is the prototype of a family of secreted polypeptide growth factors. These cytokines play very important roles during development, as well as in normal physiological and disease processes, by regulating a wide array of cellular processes, such as cell growth, differentiation, migration, apoptosis, and extracellular matrix production. TGF-β utilizes a multitude of intracellular signalling pathways in addition to Smads with actions that are dependent on circumstances, including dose, target cell type, and context. The aims of this research were (i) to verify the effects of dose-dependent TGF-β3 treatment on YY1 and p53 expression, in BPH-1 cell line, human benign prostate hyperplasia, and two prostate cancer cell lines, LNCaP, which is androgen-sensitive, and DU-145, which is androgen-non responsive, (ii) establish a correlation between p53 and YY1 and (iii) determine the expression of a number of important intracellular signalling pathways in TGF-β3-treated prostate cell lines. The expression of YY1, p53, PI3K, AKT, pAKT, PTEN, Bcl-2, Bax, and iNOS was evaluated through Western blot analysis on BPH-1, LNCaP, and DU-145 cultures treated with 10 and 50 ng/ml of TGF-β3 for 24 h. The production of nitric oxide (NO) was determined by Griess reagent and cell viability through MTT assay. The results of this research demonstrated profound differences in the responses of the BPH-1, LNCaP, and DU-145 cell lines to TGF-β3 stimulation. We believe that the findings could be important because of the clinical relevance that they may assume and the therapeutic implications for TGF-β treatment of prostate cancer.     

Activation of Myocardial Phosphoinositide-3-Kinase p110α Ameliorates Cardiac Dysfunction and Improves Survival in Polymicrobial Sepsis

Phosphoinositide-3-kinase (PI3K)/Akt dependent signaling has been shown to improve outcome in sepsis/septic shock. There is also ample evidence that PI3K/Akt dependent signaling plays a crucial role in maintaining normal cardiac function. We hypothesized that PI3K/Akt signaling may ameliorate septic shock by attenuating sepsis-induced cardiac dysfunction. Cardiac function and survival were evaluated in transgenic mice with cardiac myocyte specific expression of constitutively active PI3K isoform, p110α (caPI3K Tg). caPI3K Tg and wild type (WT) mice were subjected to cecal ligation/puncture (CLP) induced sepsis. Wild type CLP mice showed dramatic cardiac dysfunction at 6 hrs. Septic cardiomyopathy was significantly attenuated in caPI3K CLP mice. The time to 100% mortality was 46 hrs in WT CLP mice. In contrast, 80% of the caPI3K mice survived at 46 hrs after CLP (p<0.01) and 50% survived >30 days (p<0.01). Cardiac caPI3K expression prevented expression of an inflammatory phenotype in CLP sepsis. Organ neutrophil infiltration and lung apoptosis were also effectively inhibited by cardiac PI3k p110α expression. Cardiac high mobility group box–1 (HMGB-1) translocation was also inhibited by caPI3K p110α expression. We conclude that cardiac specific activation of PI3k/Akt dependent signaling can significantly modify the morbidity and mortality associated with sepsis. Our data also indicate that myocardial function/dysfunction plays a prominent role in the pathogenesis of sepsis and that maintenance of cardiac function during sepsis is essential. Finally, these data suggest that modulation of the PI3K/p110α signaling pathway may be beneficial in the prevention and/or management of septic cardiomyopathy and septic shock.