Synthesis and biological evaluation of solubilized sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474.

Replacing one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 with a variety of sulfonamide-linked solubilizing substituents produced a new class of active and potent PI3Kα inhibitors, with several derivatives demonstrating high PI3Kα enzyme potency and good cellular potency in two human derived cell lines. The overall results suggest a preference for linear and somewhat flexible solubilizing functions. From this series, compound 16, also known as SN32976, was selected for advanced preclinical evaluation.

Synthesis and Evaluation of Imidazo[1,2-a]pyridine Analogues of the ZSTK474 Class of Phosphatidylinositol 3-Kinase Inhibitors.

Using a scaffold-hopping approach, imidazo[1,2-a]pyridine analogues of the ZSTK474 (benzimidazole) class of phosphatidylinositol 3-kinase (PI3K) inhibitors have been synthesized for biological evaluation. Compounds were prepared using a heteroaryl Heck reaction procedure, involving the palladium-catalysed coupling of 2-(difluoromethyl)imidazo[1,2-a]pyridines with chloro, iodo or trifluoromethanesulfonyloxy (trifloxy) substituted 1,3,5-triazines or pyrimidines, with the iodo intermediates being preferred in terms of higher yields and milder reaction conditions. The new compounds maintain the PI3K isoform selectivity of their benzimidazole analogues, but in general show less potency.

Differential MicroRNA Expression between EGFR T790M and L858R Mutated Lung Cancer.

BACKGROUND: MicroRNAs (miRNAs) are short, non-coding RNAs that mediate post-transcriptional gene regulation. They are commonly deregulated in human malignancies, including non-small cell lung cancer (NSCLC). The aim of this study is to investigate miRNA expression in T790M-mutated NSCLC resistant to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors. METHODS: Six cases of resected NSCLC harboring the T790M mutation were examined. We performed miRNA time polymerase chain reaction (PCR) array profiling using EGFR T790M-mutated NSCLC and L858R-mutated NSCLC. Once identified, miRNAs that were differentially expressed between the two groups were validated by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: miRNA PCR array profiling revealed three up-regulated miRNAs whose expression levels were altered 4.0-fold or more in the EGFR T790M mutation group than in the L858R group: miR-1 (fold change, 4.384), miR-196a (fold change, 4.138), and miR-124 (fold change, 4.132). The three differentially expressed miRNAs were validated by qRT-PCR, and they were found to be overexpressed in the T790M group relative to L858R group. In particular, expression levels of miR-1 and miR-124 were significantly higher in the T790M group (p-value of miR-1 = .004, miR-124 = .007, miR-196a = .096). CONCLUSIONS: MiR-1, miR-124, and miR-196a are overexpressed in EGFR T790M mutated NSCLC.

Identification, structure modification, and characterization of potential small-molecule SGK3 inhibitors with novel scaffolds.

The serum and glucocorticoid-regulated kinase (SGK) family has been implicated in the regulation of many cellular processes downstream of the PI3K pathway. It plays a crucial role in PI3K-mediated tumorigenesis, making it a potential therapeutic target for cancer. SGK family consists of three isoforms (SGK1, SGK2, and SGK3), which have high sequence homology in the kinase domain and similar substrate specificity with the AKT family. In order to identify novel compounds capable of inhibiting SGK3 activity, a high-throughput screening campaign against 50,400 small molecules was conducted using a fluorescence-based kinase assay that has a Z' factor above 0.5. It identified 15 hits (including nitrogen-containing aromatic, flavone, hydrazone, and naphthalene derivatives) with IC50 values in the low micromolar to sub-micromolar range. Four compounds with a similar scaffold (i.e., a hydrazone core) were selected for structural modification and 18 derivatives were synthesized. Molecular modeling was then used to investigate the structure-activity relationship (SAR) and potential protein-ligand interactions. As a result, a series of SGK inhibitors that are active against both SGK1 and SGK3 were developed and important functional groups that control their inhibitory activity identified.

High-throughput screening campaigns against a PI3Kα isoform bearing the H1047R mutation identified potential inhibitors with novel scaffolds.

The phosphatidylinositol 3-kinase (PI3K) pathway is involved in many cellular functions including cell growth, metabolism, and transformation. Hyperactivation of this pathway contributes to tumorigenesis, therefore, PI3K is a major target for anticancer drug discovery. Since the PI3Kα isoform is implicated mostly in cancer, we conducted a high-throughput screening (HTS) campaign using a 3-step PI3K homogenous time-resolved fluorescence assay against this isoform bearing the H1047R mutation. A total of 288,000 synthetic and natural product-derived compounds were screened and of which, we identified 124 initial hits that were further selected by considering the predicted binding mode, relationship to known pan-assay interference compounds and previous descriptions as a lipid kinase inhibitor. A total of 24 compounds were then tested for concentration-dependent responses. These hit compounds provide novel scaffolds that can potentially be optimized to create novel PI3K inhibitors.

Synthesis and biological evaluation of sulfonamide analogues of the phosphatidylinositol 3-kinase inhibitor ZSTK474.

Replacement of one of the morpholine groups of the phosphatidylinositol 3-kinase (PI3K) inhibitor ZSTK474 (1) with sulfonamide containing substituents produced a new class of active and potent PI3Kα inhibitors. Solubility issues prevented all but the 6-amino derivative 17 from being evaluated in vivo, but the clear activity of this compound demonstrated that this class of PI3K inhibitor shows great promise.

Biological characterization of SN32976, a selective inhibitor of PI3K and mTOR with preferential activity to PI3Kα, in comparison to established pan PI3K inhibitors.

Multiple therapeutic agents have been developed to target the phosphatidylinositol 3-kinase (PI3K) signaling pathway, which is frequently dysregulated in cancer promoting tumor growth and survival. These include pan PI3K inhibitors, which target class Ia PI3K isoforms and have largely shown limited single agent activity with narrow therapeutic windows in clinical trials. Here, we characterize SN32976, a novel pan PI3K inhibitor, for its biochemical potency against PI3K isoforms and mTOR, kinase selectivity, cellular activity, pharmacokinetics, pharmacodynamics and antitumor efficacy relative to five clinically-evaluated pan PI3K inhibitors: buparlisib, dactolisib, pictilisib, omipalisib and ZSTK474. SN32976 potently inhibited PI3K isoforms and mTOR, displaying preferential activity for PI3Kα and sparing of PI3Kδ relative to the other inhibitors, while showing less off-target activity than the clinical inhibitors in a panel of 442 kinases. The major metabolites of SN32976 were also potent PI3K inhibitors with similar selectivity for PI3Kα as the parent compound. SN32976 compared favorably with the clinically-evaluated PI3K inhibitors in cellular assays, inhibiting pAKT expression and cell proliferation at nM concentrations, and in animal models, inducing a greater extent and duration of pAKT inhibition in tumors than pictilisib, dactolisib and omipalisib at similarly tolerated dose levels and inhibiting tumor growth to a greater extent than dactolisib and ZSTK474 and with similar efficacy to pictilisib and omipalisib. These results suggest that SN32976 is a promising clinical candidate for cancer therapy with enhanced kinase selectivity and preferential inhibition of PI3Kα compared to first generation pan PI3K inhibitors, while retaining comparable anticancer activity.

Novel pyrazolo[1,5-a]pyridines with improved aqueous solubility as p110α-selective PI3 kinase inhibitors.

As part of our investigation into pyrazolo[1,5-a]pyridines as novel p110α selective PI3 kinase inhibitors, we report a range of analogues with improved aqueous solubility by the addition of a basic amine. The compounds demonstrated comparable p110α potency and selectivity to earlier compounds but with up to 1000× greater aqueous solubility, as the hydrochloride salts. The compounds also displayed good activity in a cellular assay of PI3 kinase activity.

Unique microRNAs in lung adenocarcinoma groups according to major TKI sensitive EGFR mutation status.

BACKGROUND: Lung cancer is the leading cause of cancer mortality, despite development of therapeutic strategies. Altered expression of microRNAs(miRNAs) in human malignancies have been well recognized as diagnostic and prognostic indicators, including lung cancer. This study aims to delineate the clinicopathologic significance of three unique miRNAs in adenocarcinoma according to major sensitive EGFR mutation status. METHODS: One-hundred and three formalin-fixed paraffin-embedded (FFPE) tissues were collected from lung adenocarcinoma patients who underwent surgery and epidermal growth factor receptor (EGFR) mutation study. The samples were divided into three groups which include EGFR mutation in exons 19 and 21 and wild type. Some representative cases from each group were profiled using commercial miRNA microarray plates. Three significant miRNAs were selected and they were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), using collective cases of FFPE samples. RESULTS: We identified three microRNAs (miR-34c, miR-183, and miR-210) which showed significantly altered expression in all groups of lung adenocarcinoma by microarray study. Compared to normal control lung tissue, down-regulation of miR-34c and up-regulation of miR-183 and miR-210 were identified in caner groups (p < 0.05 for each). We validated the expression of three miRNAs by qRT-PCR. Expression levels of miR-34c, miR-183, and miR-210 were significantly different between normal control group and cancer groups (p = 0.034, <0.000, and 0.036, respectively). Moreover, expression level of miR-183 was significantly higher in EGFR mutation groups than wild type group (p = 0.028). Higher expression levels of three miRNAs were positively related to poor tumor differentiation. Increased expression of miR-183 was positively associated with lymphovascular invasion (p = 0.037). Aberrant expression of miR-210 was independently associated with T stage (p = 0.019), and TNM stage (p = 0.007). However, there was noted a limited statistical significance. In EGFR exon 19 mutation group, miR-34c high expression group showed poor overall survival than low expression one by univariate Kaplan-Meier method. (p = 0.035). CONCLUSIONS: Here, we show that miR-34c may act as a potential tumor suppressor gene and miR-183 and miR-210 have a potential oncogenic role in pulmonary adenocarcinoma. This study also suggests different miRNA expression between EGFR mutation group and wild type group. Consequently, further studies of the biology of miRNAs may lead to diagnostic and prognostic biomarkers in pulmonary adenocarcinoma.

Exploring the isoform selectivity of TGX-221 related pyrido[1,2-a]pyrimidinone-based Class IA PI 3-kinase inhibitors: synthesis, biological evaluation and molecular modelling.

A novel series of TGX-221 analogues was prepared and tested for their potency against the p110α, p110ß, and p110δ isoforms of the PI3K enzyme, and in two cellular assays. The biological results were interpreted in terms of a p110ß comparative model, in order to account for their selectivity towards this isoform. A CH2NH type linker is proposed to allow binding into the specificity pocket proposed to accommodate the high p110ß-selectivity of TGX-221, although there was limited steric tolerance for substituents on the pendant ring with the 2-position most favourable for substitution.

Oncogenic mutations of p110α isoform of PI 3-kinase upregulate its protein kinase activity.

In addition to lipid kinase activity, the class-I PI 3-kinases also function as protein kinases targeting regulatory autophosphorylation sites and exogenous substrates. The latter include a recently identified regulatory phosphorylation of the GM-CSF/IL-3 ßc receptor contributing to survival of acute myeloid leukaemia cells. Previous studies suggested differences in the protein kinase activity of the 4 isoforms of class-I PI 3-kinase so we compared the ability of all class-I PI 3-kinases and 2 common oncogenic mutants to autophosphorylate, and to phosphorylate an intracellular fragment of the GM-CSF/IL-3 ßc receptor (ßic). We find p110α, p110ß and p110γ all phosphorylate ßic but p110δ is much less effective. The two most common oncogenic mutants of p110α, H1047R and E545K have stronger protein kinase activity than wildtype p110α, both in terms of autophosphorylation and towards ßic. Importantly, the lipid kinase activity of the oncogenic mutants is still inhibited by autophosphorylation to a similar extent as wildtype p110α. Previous evidence indicates the protein kinase activity of p110α is Mn(2+) dependent, casting doubt over its role in vivo. However, we show that the oncogenic mutants of p110α plus p110ß and p110γ all display significant activity in the presence of Mg(2+). Furthermore we demonstrate that some small molecule inhibitors of p110α lipid kinase activity (PIK-75 and A66) are equally effective against the protein kinase activity, but other inhibitors (e.g. wortmannin and TGX221) show different patterns of inhibition against the lipid and protein kinases activities. These findings have implications for the function of PI 3-kinase, especially in tumours carrying p110α mutations.

Enzyme activity effects of N-terminal His-tag attached to catalytic sub-unit of phosphoinositide-3-kinase.

NTT (N-terminal tags) on the catalytic (p110) sub-unit of PI 3-K (phosphoinositol 3-kinase) have previously been shown to increase cell signalling and oncogenic transformation. Here we test the impact of an NT (N-terminal) His-tag on in vitro lipid and protein kinase activity of all class-1 PI 3-K isoforms and two representative oncogenic mutant forms (E545K and H1047R), in order to elucidate the mechanisms behind this elevated signalling and transformation observed in vivo. Our results show that an NT His-tag has no impact on lipid kinase activity as measured by enzyme titration, kinetics and inhibitor susceptibility. Conversely, the NT His-tag did result in a differential effect on protein kinase activity, further potentiating the elevated protein kinase activity of both the helical domain and catalytic domain oncogenic mutants with relation to p110 phosphorylation. All other isoforms also showed elevated p110 phosphorylation (although not statistically significant). We conclude that the previously reported increase in cell signalling and oncogenic-like transformation in response to p110 NTT is not mediated via an increase in the lipid kinase activity of PI 3-K, but may be mediated by increased p110 autophosphorylation and/or other, as yet unidentified, intracellular protein/protein interactions. We further observe that tagged recombinant protein is suitable for use in in vitro lipid kinase screens to identify PI 3-K inhibitors; however, we recommend that in vivo (including intracellular) experiments and investigations into the protein kinase activity of PI 3-K should be conducted with untagged constructs.

Synthesis and biological evaluation of novel phosphatidylinositol 3-kinase inhibitors: Solubilized 4-substituted benzimidazole analogs of 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474).

A range of 4-substituted derivatives of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di-(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) were prepared in a search for more soluble analogs. 4-Aminoalkoxy substituents provided the most potent derivatives, with the 4-O(CH2)3NMe2 analog (compound 14) being identified as displaying the best overall activity in combination with good aqueous solubility (25 mg/mL for the hydrochloride salt). This compound was tested in a U87MG xenograft model, but displayed less potency than ZSTK474 as a result of an unfavorable pharmacokinetic profile.

Discovery of pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors.

We have made a novel series of pyrazolo[1,5-a]pyridines as PI3 kinase inhibitors, and demonstrated their selectivity for the p110α isoform over the other Class Ia PI3 kinases. We investigated the SAR around the pyrazolo[1,5-a]pyridine ring system, and found compound 5x to be a particularly potent example (p110α IC(50) 0.9nM). This compound inhibits cell proliferation and phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity, and showed in vivo activity in an HCT-116 human xenograft model.

Novel pyrazolo[1,5-a]pyridines as p110α-selective PI3 kinase inhibitors: Exploring the benzenesulfonohydrazide SAR.

Structure-activity relationship studies of the pyrazolo[1,5-a]pyridine class of PI3 kinase inhibitors show that substitution off the hydrazone nitrogen and replacement of the sulfonyl both gave a loss of p110α selectivity, with the exception of an N-hydroxyethyl analogue. Limited substitutions were tolerated around the phenyl ring; in particular the 2,5-substitution pattern was important for PI3 kinase activity. The N-hydroxyethyl compound also showed good inhibition of cell proliferation and inhibition of phosphorylation of Akt/PKB, a downstream marker of PI3 kinase activity. It had suitable pharmacokinetics for evaluation in vivo, and showed tumour growth inhibition in two human tumour cell lines in xenograft studies. This work has provided suggestions for the design of more soluble analogues.

Synthesis and biological evaluation of novel analogues of the pan class I phosphatidylinositol 3-kinase (PI3K) inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474).

A structure-activity relationship (SAR) study of the pan class I PI 3-kinase inhibitor 2-(difluoromethyl)-1-[4,6-di(4-morpholinyl)-1,3,5-triazin-2-yl]-1H-benzimidazole (ZSTK474) identified substitution at the 4 and 6 positions of the benzimidazole ring as having significant effects on the potency of substituted derivatives. The 6-amino-4-methoxy analogue displayed a greater than 1000-fold potency enhancement over the corresponding 6-aza-4-methoxy analogue against all three class Ia PI 3-kinase enzymes (p110α, p110ß, and p110δ) and also displayed significant potency against two mutant forms of the p110α isoform (H1047R and E545K). This compound was also evaluated in vivo against a U87MG human glioblastoma tumor xenograft model in Rag1(-/-) mice, and at a dose of 50 mg/kg given by ip injection at a qd × 10 dosing schedule it dramatically reduced cancer growth by 81% compared to untreated controls.

A drug targeting only p110α can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types.

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110α in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110α have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110α/p110ß/p110δ in all cell lines tested. However, inhibition of p110α alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110α inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110α alone has the potential to block growth factor signalling and reduce growth in a subset of tumours.

Protective effect of rebamipide against Helicobacter pylori-CagA-induced effects on gastric epithelial cells.

BACKGROUND: Helicobacter pylori CagA dysregulates cell signaling pathways and leads to targeted transcriptional up-regulation of genes implicated in gastric cell injury. The aim of this study was to determine the effects of rebamipide on CagA-induced effects on gastric epithelial cells. We investigated the effects of rebamipide treatment (pre- or post-treatment before or after CagA transfection) on CagA-induced gastric cell injury. METHOD: We evaluated the morphologic changes (hummingbird phenotype) associated with ZO-1 mislocalization by confocal microscopy, IL-8 production by ELISA, and NF-κB activation by luciferase assay in AGS gastric epithelial cells and MDCK cells. RESULTS: Transfection of CagA into gastric epithelial cells induced morphologic changes (hummingbird phenotype), ZO-1 mislocalization, and IL-8 production in gastric epithelial cells. Pre-treatment with rebamipide inhibits CagA-induced effects on gastric epithelial cells, including morphologic changes (hummingbird phenotype) associated with ZO-1 mislocalization, IL-8 production, and NF-κB activity. CONCLUSIONS: These results suggest that rebamipide might have a potential role in the protection of H. pylori CagA-induced effects on gastric epithelial cells.

Organ culture at the air-liquid interface maintains structural and functional integrities of inflammatory and fibrovascular cells of nasal polyps.

BACKGROUND: Both inflammatory and fibrovascular cells play an important role in development of nasal polyps (NPs). In this study, we have developed a culture system to maintain structural and functional integrities of submucosal cells in vitro. METHODS: NP tissue was cultured on a gelatin sponge at air-liquid (AL) interface or was cultured in submerging. Tissues were analyzed for survival, structural integrity, and vascular endothelial growth factor (VEGF) expression. RESULTS: Most cells of NPs cultured in submerging died within 3 days. In culture at the AL interface, epithelium as well as submucosa kept structural integrity during the culture period. RT-PCR and immunohistochemistry showed that submucosa cells displayed VEGF expression, which is a major inducer of angiogenesis and edema of NPs. CONCLUSION: Our study is the first demonstration that organ culture of NPs at the AL interface retains integrity of both epithelium and submucosa and this culture method possibly will be used to study the pathogenesis of NPs.