A mechanism for synergy with combined mTOR and PI3 kinase inhibitors

Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up-regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond.     

Novel imidazolopyrimidines as dual PI3-Kinase/mTOR inhibitors

This article describes the syntheses and SAR of a series of imidazolopyrimidine derivatives, which are evaluated as inhibitors of PI3-Kinase (PI3 K) and mTOR. These compounds were found to be ATP competitive with good tumor cell growth inhibition, and suppression of pathway specific biomakers such as phosphorylation of Akt at T308.     

G0S2 represses PI3K/mTOR signaling and increases sensitivity to PI3K/mTOR pathway inhibitors in breast cancer

G0/G1 switch gene 2 (G0S2) is a direct retinoic acid target implicated in cancer biology and therapy based on frequent methylation-mediated silencing in diverse solid tumors. We recently reported that low G0S2 expression in breast cancer, particularly estrogen receptor-positive (ER+) breast cancer, correlates with increased rates of recurrence, indicating that G0S2 plays a role in breast cancer progression. However, the function(s) and mechanism(s) of G0S2 tumor suppression remain unclear. In order to determine potential mechanisms of G0S2 anti-oncogenic activity, we performed genome-wide expression analysis that revealed an enrichment of gene signatures related to PI3K/mTOR pathway activation in G0S2 null cells as compared to G0S2 wild-type cells. G0S2 null cells also exhibited a dramatic decreased sensitivity to PI3K/mTOR pathway inhibitors. Conversely, restoring G0S2 expression in human ER+ breast cancer cells decreased basal mTOR signaling and sensitized the cells to pharmacologic mTOR pathway inhibitors. Notably, we provide evidence here that the increase in recurrence seen with low G0S2 expression is especially prominent in patients who have undergone antiestrogen therapy. Further, ER+ breast cancer cells with restored G0S2 expression had a relative increased sensitivity to tamoxifen. These findings reveal that in breast cancer G0S2 functions as a tumor suppressor in part by repressing PI3K/mTOR activity, and that G0S2 enhances therapeutic responses to PI3K/mTOR inhibitors. Recent studies implicate hyperactivation of PI3K/mTOR signaling as promoting resistance to antiestrogen therapies in ER+ breast cancer. Our data establishes G0S2 as opposing this form of antiestrogen resistance. This promotes further investigation of the role of G0S2 as an antineoplastic breast cancer target and a biomarker for recurrence and therapy response.     

Recent syntheses of PI3K/Akt/mTOR signaling pathway inhibitors

This review focuses on the syntheses of PI3K/Akt/mTOR inhibitors that have been reported outside of the patent literature in the last 5 years but is largely centered on synthetic work reported in 2011 and 2012. While focused on syntheses of inhibitors, some information on in vitro and in vivo testing of compounds is also included. Many of these reported compounds are reversible, competitive adenosine triphosphate (ATP) binding inhibitors, so given the structural similarities of many of these compounds to the adenine core, this review presents recent work on inhibitors based on where the synthetic chemistry was started, that is, inhibitor syntheses which started with purines/pyrimidines are followed by inhibitor syntheses which began with pyridines, pyrazines, azoles, and triazines then moves to inhibitors which bear no structural resemblance to adenine: liphagal, wortmannin and quercetin analogs. The review then finishes with a short section on recent syntheses of phosphotidyl inositol (PI) analogs since competitive PI binding inhibitors represent an alternative to the competitive ATP binding inhibitors which have received the most attention.     

Inhibition of autophagy sensitizes malignant pleural mesothelioma cells to dual PI3K/mTOR inhibitors

Malignant pleural mesothelioma (MPM) originates in most of the cases from chronic inflammation of the mesothelium due to exposure to asbestos fibers. Given the limited effect of chemotherapy, a big effort is being made to find new treatment options. The PI3K/mTOR pathway was reported to be upregulated in MPM. We tested the cell growth inhibition properties of two dual PI3K/mTOR inhibitors NVP-BEZ235 and GDC-0980 on 19 MPM cell lines. We could identify resistant and sensitive lines; however, there was no correlation to the downregulation of PI3K/mTOR activity markers. As a result of mTOR inhibition, both drugs efficiently induced long-term autophagy but not cell death. Autophagy blockade by chloroquine in combination with the dual PI3K/mTOR inhibitors significantly induced caspase-independent cell death involving RIP1 in the sensitive cell line SPC212. Cell death in the resistant cell line Mero-82 was less pronounced, and it was not induced via RIP1-dependent mechanism, suggesting the involvement of RIP1 downstream effectors. Cell death induction was confirmed in 3D systems. Based on these results, we identify autophagy as one of the main mechanisms of cell death resistance against dual PI3K/mTOR inhibitors in MPM. As PI3K/mTOR inhibitors are under investigation in clinical trials, these results may help interpreting their outcome and suggest ways for intervention.Malignant pleural mesothelioma (MPM) is sensitive to phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signaling inhibitors due to the activation of PI3K/mTOR signaling.^{1, 2} The activation may result from inactivation of INP4A phosphatase, which is downregulated in 44% of MPM (presented at IMIG2014), or alterations in PI3K signaling components, which are mutated in 9% of MPM,³ while receptor tyrosine kinase mutations/amplifications have not been identified in two recent high-throughput studies.^{4, 5}One of the tumor-suppressor genes frequently mutated in MPM is NF2 and NF2-null cells were shown to be sensitive to growth-inhibitory effects of rapamycin⁶ via mechanisms involving PI3K signaling-independent mTORC1 activation. However, the mTOR inhibitor, everolimus, showed no therapeutic benefit in unselected MPM patients.⁷ As mTORC1 inhibitors often lead to a feedback activation of PI3K activation in cancers,^{8, 9} we postulated that dual PI3K–mTOR inhibitors may yield greater therapeutic benefit. Furthermore, NF2 was also shown to inhibit PI3K activity by binding to PI3K enhancer-L (PIKE-L), which disrupts binding of PIKE-L to PI3K¹⁰ and loss of NF2 in schwannoma was shown to sensitize to PI3K inhibitors.¹¹In a screen on the dual PI3K/mTOR inhibitor NVP-BEZ235, within the Sanger Institute/MGH''s ‘Genomics of Drug Sensitivity'' screening panel,¹² CDKN2A deletion was shown to be associated with increased sensitivity. Because NF2 and CDKN2A are indeed the genes most frequently mutated in MPM, blocking PI3K/mTOR signaling might be a valid approach to circumvent the difficulty of applying targeted therapy in the absence of an identified oncogene. The rationale for targeting the PI3K/mTOR pathway is also supported by the association of increased activity with a worse clinical outcome.^{13, 14}NVP-BEZ235^(ref15) and GDC-0980^(ref16) are small-molecule inhibitors of class I PI3K and mTOR (mTORC1 and mTORC2). GDC-0980 has been tested in phase I studies where the phase I extension cohort showed two objective responses among 26 patients with mesothelioma.¹⁷ Despite these encouraging results, this drug will not be explored further because of side effects observed in another clinical trial.¹⁸ This, however, should not deter us for trying to find means to improve the antitumor effect of this class of agents. We have previously shown that PI3K/mTOR signaling inhibition sensitizes mesothelioma cells to drugs that are effluxed via ABCG2 transporter by inhibiting the function of ABCG2.¹⁹ In this study, we aimed at identifying the underlying mechanisms responsible for sensitivity versus resistance towards PI3K/mTOR inhibition in a large panel of mesothelioma cell lines. We observed that PI3K/mTOR inhibition increases autophagic rate, which constitutes an efficient mechanism of resistance by inducing growth arrest and survival. However, blocking autophagy, which per se affects cell growth, is synthetically lethal when combined with PI3K/mTOR inhibitors by a mechanism involving receptor-interacting protein kinase 1 (RIP1)-dependent cell death.     

Optimization of a series of multi-isoform PI3 kinase inhibitors

Optimization of the cellular and pharmacological activity of a novel series of PI3 kinase inhibitors targeting multiple isoforms is described.     

The PI3K/AKT/mTOR signaling pathway inhibitors enhance radiosensitivity in cancer cell lines

Molecular Biology Reports - Malignant tumors have become the most dangerous disease in recent years. Chemotherapy is the most effective treatment for this disease; however, the problem of drug...     

4-Methylpteridinones as orally active and selective PI3K/mTOR dual inhibitors

Pteridinones were designed based on a non-selective kinase template. Because of the uniqueness of the PI3K and mTOR binding pockets, a methyl group was introduced to C-4 position of the peteridinone core to give compounds with excellent selectivity for PI3K and mTOR. This series of compounds were further optimized to improve their potency against PI3Kα and mTOR. Finally, orally active compounds with improved solubility and robust in vivo efficacy in tumor growth inhibition were identified as well.     

Structure-based optimization of morpholino-triazines as PI3K and mTOR inhibitors

A virtual screen of our in-house database using various fingerprint techniques returned several triazine hits which were found to be mTOR inhibitors with a slight selectivity over PI3Kα. Using structure-guided lead optimization the inhibitory activity towards mTOR and PI3Kα was increased to the low nanomolar range. Exploiting shape differences in the binding-site allowed for the design of mTOR selective inhibitors. Focus on ligand efficiency ensured the inhibitors retained a low molecular weight and desirable drug-like properties.     

Virtual screening of naphthoquinone analogs for potent inhibitors against the cancer-signaling PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR pathway is one of the most commonly disrupted signaling pathways that plays a role in the development and pathogenicity of multiple cancers. Therefore, the critical proteins of this pathway have been targeted for anticancer therapy. The scientific community has increasingly been realizing the anti-cancer therapeutic potential of naphthoquinone analogs. These compounds constitute a major class of diverse sets of plant metabolites, which include various natural products and synthetic compounds with proven anticancer activity. The current study involved structural computational biology approaches to explore compounds from a diverse pool of naphthoquinone analogs that can inhibit key cancer-signaling proteins phosphoinositide 3-kinase (PI3K), protein kinase B, PKB (AKT), and mammalian target of rapamycin (mTOR). The novel compound identified commonly among the top 10 dock score lists of PI3K, AKT, and mTOR was selected for further study and proposed as a potential inhibitor of the 3 cancer-signaling proteins and an anticancer agent. Further, to check the docking accuracy and potential of the compound, post docking analyses, namely, binding comparison with the native ligand, the role of the interacting residue role in binding, predicted binding energy and dissociation constant calculations, etc., were performed. All these measures showed good-quality binding, and thus provide weight to our prediction of the novel compound as a pan PI3K/AKT/mTOR inhibitor and an anticancer agent. Finally, to compare the binding and similarity in the active sites of the 3 protein kinases, a ligand-based active site alignment was performed and analyzed. Thus, the study proposed a novel naphthoquinone analog as a potential anticancer drug, and provided comparative structural insight into its binding to the 3 protein kinases.     

B7-H4-mediated immunoresistance is supressed by PI3K/Akt/mTOR pathway inhibitors

B7-H4 plays an important role in tumor immune evasion. In previous studies we have found that B7-H4 can translocate to the nucleus, and the exposure to PI3K inhibitor Ly294002 affects B7-H4 subcellular distribution. In this study we report the role of PI3K/Akt pathway in the B7-H4 subcellular distribution and the effect of PI3K/Akt inhibitors on B7-H4-mediated immunoresistance. The involvement of PI3K/Akt pathway in B7-H4 subcellular distribution was evident in experiments with wortmannin, while MDM2 inhibitor nutlin-3 and the mTOR inhibitor rapamycin were used to dissect the signaling downstream of Akt. Wortmannin and rapamycin demonstrated similar effects on B7-H4 subcellular distribution. Exposure to any of these inhibitors decreased levels of membrane B7-H4 while at the same time inducing its nuclear accumulation, while exposure to nutlin-3 had no effect on B7-H4 subcellular distribution. In the T cell proliferation assay, both wortmannin and rapamycin effectively inhibited B7-H4 WT/293 cells-mediated T cell proliferation while exerting no effect on Mock/293 cells. PI3K/Akt/mTOR plays a role in B7-H4 subcellular distribution, while MDM2 does not take part in it. Moreover, we show that wortmannin and rapamycin inhibit B7-H4-mediated tumor immunoresistance through regulating B7-H4 subcellular distribution. Taken together, these results suggest that PI3K/Akt/mTOR inhibitors might be used for adjuvant therapy aimed at inhibition of immune evasion.     

The immune receptor Tim-3 mediates activation of PI3 kinase/mTOR and HIF-1 pathways in human myeloid leukaemia cells

The T-cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated protein that is highly expressed in human acute myeloid leukaemia cells. As an acute myeloid leukaemia antigen, it could therefore be considered as a potential target for immune therapy and highly-specific drug delivery. However, a conceptual understanding of its biological role is required before consideration of this protein for therapeutic settings. Here, we reveal the detailed mechanism of action underlying the biological responses mediated by the Tim-3 receptor in myeloid cells. Our studies demonstrate that Tim-3 triggers growth factor type responses in acute myeloid leukaemia cells by activating a phosphatidylinositol-3 kinase (PI-3K)/mammalian target of rapamycin (mTOR) pathway. In addition, the receptor activates hypoxic signalling pathways upregulating glycolysis and pro-angiogenic responses. These findings suggest that Tim-3 could be used as a potential therapeutic target for immune therapy and drug delivery in human acute myeloid leukaemia cells.     

Combined pharmacophore modeling, 3D-QSAR and docking studies to identify novel HDAC inhibitors using drug repurposing

Abstract

Histone deacetylases (HDACs), a critical family of epigenetic enzymes, has emerged as a promising target for antitumor drugs. Here, we describe our protocol of virtual screening in identification of novel potential HDAC inhibitors through pharmacophore modeling, 3D-QSAR, molecular docking and molecular dynamics (MD) simulation. Considering the limitation of current virtual screening works, drug repurposing strategy was applied to discover druggable HDAC inhibitor. The ligand-based pharmacophore and 3D-QSAR models were established, and their reliability was validated by different methods. Then, the DrugBank database was screened, followed by molecular docking. MD simulation (100?ns) was performed to further study the stability of ligand binding modes. Finally, results indicated the hit DB03889 with high in silico inhibitory potency was suitable for further experimental analysis.

Communicated by Ramaswamy H. Sarma     

Quinazolines with intra-molecular hydrogen bonding scaffold (iMHBS) as PI3K/mTOR dual inhibitors

Intra-molecular hydrogen bonding was introduced to the quinazoline motif to form a pseudo ring (intra-molecular H-bond scaffold, iMHBS) to mimic our previous published core structures, pyrido[2.3-D]pyrimidin-7-one and pteridinone, as PI3K/mTOR dual inhibitors. This design results in potent PI3K/mTOR dual inhibitors and the purposed intra-molecular hydrogen bonding structure is well supported by co-crystal structure in PI3Kγ enzyme. In addition, a novel synthetic route was developed for these analogs.     

Allosteric and ATP-competitive kinase inhibitors of mTOR for cancer treatment

Over the past few years a number of components of the PI3K/mTOR pathway have been the subject of intense drug discovery activities both in pharmaceutical companies and in academia. This review article summarizes progress made in the identification and development of allosteric and ATP-competitive kinase inhibitors of mTOR and their potential therapeutic use in oncology.     

Ligand-based 3-D pharmacophore generation and molecular docking of mTOR kinase inhibitors

The 3-D structure of the human mTOR kinase domain was modeled based on the crystal structure of PI3Kγ using comparative modeling methods, and the ATP-binding site of mTOR was characterized. The mTOR kinase 3-D model structure is similar to the structure of the PI3Kγ kinase domain, and exhibits great similarity to PI3Kγ at the active site of the kinase. Pharmacophore generation, the docking of mTOR inhibitors, and molecular dynamics (MD) simulations of mTOR–inhibitor docked complexes were carried out in this work. The best pharmacophore model generated from 27 ATP-competitive mTOR inhibitors comprised two hydrogen-bond acceptors, one aromatic ring, and one hydrophobic feature. These 27 inhibitors were docked into the ATP-binding site comprising the DFG motif, and the interactions in each protein–inhibitor complex were characterized. Mapping the pharmacophore model onto the docked inhibitors explained the specificity of the features of the pharmacophore and how they were arranged inside the active site of mTOR kinase. MD studies revealed important structural features, such as the large hydrophobic pocket “HP” and hydrophilic pocket “A1,” and the solvent-exposed hydrophilic pocket “A2” at the active site of mTOR. Our results provide structural models of mTOR–inhibitor complexes and clues that should aid in the design of better mTOR kinase inhibitors.     

The PI3K/AKT/mTOR Signaling Pathway Is Overactivated in Primary Aldosteronism

Background

To date, the available non-invasive remedies for primary aldosteronism are not satisfactory in clinical practice. The phosphoinositide 3-kinase (PI3Ks)/protein kinase B (PKB or AKT)/mammalian target of rapamycin (mTOR) signaling pathway is essential for tumorigenesis and metastasis in many types of human tumors, including renal cancer, adrenal carcinoma and pheochromocytoma. The possibility that this pathway is also necessary for the pathogenesis of primary aldosteronism has not yet been explored. To answer this question, we investigated the activity of the PI3K/AKT/mTOR signaling pathway in normal adrenal glands (NAGs), primary aldosteronism (PA) patients and NCI-H295R cells.

Methodology/Principal Findings

Between January 2005 and December 2011, we retrospectively reviewed the records of 45 patients with PA. We compared clinical characteristics (age, gender and biochemical data) and the expression of phospho-AKT (p-AKT), phospho-mTOR (p-mTOR), phospho-S6 (p-S6) and vascular endothelial growth factor (VEGF) by immunohistochemical staining and western blotting, analyzing 30 aldosterone-producing adenomas (APAs), 15 idiopathic hyperaldosteronism (IHA) tissues and 12 NAGs following nephrectomy for renal tumors (control group). Compared with the control group, most of the PA patients presented with polydipsia, polyuria, resistant hypertension, profound hypokalemia, hyperaldosteronemia and decreased plasma renin activity. Compared with normal zona glomerulosa, the levels of p-AKT, p-mTOR, p-S6 and VEGF were significantly upregulated in APA and IHA. No significant differences were found between APA and IHA in the expression of these proteins. Additionally, positive correlations existed between the plasma aldosterone levels and the expression of p-AKT and p-mTOR. In vitro studies showed that mTOR inhibitor rapamycin could inhibit cell proliferation in NCI-H295R cells in a dose- and time-dependent manner. Furthermore, this inhibitor also decreased aldosterone secretion.

Conclusions

Our data suggest that the PI3K/AKT/mTOR signaling pathway, which was overactivated in APA and IHA compared with normal zona glomerulosa, may mediate aldosterone hypersecretion and participate in the development of PA.