p110α (IC50 = 32 nM); p110α E545K (IC50 = 30 nM); p110α H1047R (IC50 = 43 nM); p110γ (IC50 = 3480 nM); PI3K-C2β (IC50 = 462 nM); PI4Kβ (IC50 = 236 nM)

The oncogenic forms of p110α, such as p110α E545K and p110α H1047R, are also potently inhibited by A66, with IC50 values of 30 nM and 43 nM, respectively. When compared to other class-I PI3K isoforms, A66 exhibits >100 fold selectivity for p110α, in contrast to PIK-75. Among the class-II PI3Ks, class-III PI3K and PI4Ks, A66 only exhibits limited cross-reactivity with the class-II PI3K PI3KC2β and the PI4Kβ isoform of PI4K with IC50 of 462 nM and 236 nM, respectively. The related kinases DNA-PK and mTOR as well as other lipid kinases are not inhibited by A66. When compared to PIK-75, A66 has a higher level of specificity when tested at 10 μM against two sizable panels of 110 protein kinases and 318 kinases. In some cell lines with H1047R mutations in PIK3CA and high levels of p110 and class-Ia PI3K activity, inhibition of p110α by A66 treatment is sufficient to block insulin signaling to Akt/PKB.[1] The highly transforming p85 iSH2 mutants KS459delN, DKRMN-S560del, and K379E experience a 75–80% reduction in focus formation after receiving 0.7 μM of A66, and all p85α iSH2 mutants have lessened Akt phosphorylation on T308.[2]

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Inhibitor specificity [1]
We first characterized A66 and confirmed it was a potent inhibitor of the wild-type and oncogenic forms of p110α but not other class-I PI3K isoforms (Table 1). We found A66 has a much greater degree of selectivity for p110α than PIK-75. Given the important roles of class-II PI3Ks, class-III PI3K [38] and PI4Ks (phosphoinositide 4-kinases) in growth factor signalling, we also assessed the activity of A66 towards these and found some limited cross-reactivity with the class-II PI3K PI3K-C2β and the PI4Kβ isoform of PI4K (Table 2). There was no inhibition of other lipid kinases or the related kinases DNA-PK and mTOR (Table 2). We also tested the inhibitory effects of 10 μM A66 effects on two large panels of 110 protein kinases (Supplementary Figure S1 at http://www.BiochemJ.org/bj/438/bj4380053add.htm) and 318 kinases (Supplementary Figure S2 at http://www.BiochemJ.org/bj/438/bj4380053add.htm). These show A66 is a very specific inhibitor of p110α, whereas PIK-75, the compound described previously as a p110α-selective inhibitor, inhibited a large number of protein kinases at this concentration (Supplementary Figure S1). Our data for TGX-221 and IC87114 generated using the HTRF assay agreed with previous studies using other assay methods and confirmed these are highly selective inhibitors of p110β and p110δ respectively (Table 1), although TGX-221 will cross-react with p110δ at higher concentrations. We report further that these inhibitors do not have any major effects on a panel of 110 protein kinases (Supplementary Fig 1).

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Effects of specifically inhibiting p110α on cell function [1]
To investigate the role of p110α in regulating proximal elements of PI3K-dependent signalling pathways, we determined the ability of various concentrations of the A66 S form to acutely block the activation of Akt/PKB in a range of cell lines as assessed by both phosphorylation of Ser473 and Thr308 (Figure 3). Loading was controlled for by reprobing for total PKB (see Supplementary Figure S3 at http://www.BiochemJ.org/bj/438/bj4380053add.htm). We found that phosphorylation of both Ser473 and Thr308 is sensitive to LY294002 in all cell lines tested, implying that class-I PI3K activity is required for activation of Akt/PKB. However, we found the amount of the A66 S form required to inhibit phosphorylation of Ser473 and Thr308 followed two distinct patterns, being either sensitive to inhibition by the A66 S form at concentrations consistent with it acting through p110α or being resistant. The most obvious feature of the sensitive cell lines was that they harboured H1047R mutations in PIK3CA, whereas all other cell lines were resistant. As a control we tested the effect of the A66 R form and found it was not able to inhibit the phosphorylation of Akt/PKB (see Supplementary Figure S4 at http://www.BiochemJ.org/bj/438/bj4380053add.htm).

In vivo, at 1 hour and 6 hours after dosing, a single dose of A66 at 100 mg/kg results in a significant decrease in the phosphorylation of Akt/PKB and p70 S6 kinase, but not of ERK. A66 causes SK-OV-3 xenografted tumors to grow significantly more slowly than the well-known pan-PI3K inhibitor BEZ-235, with average TGI of 45.9% and 29.9%, respectively, when administered at doses of 100 mg/kg once daily (QD) for 21 days or 75 mg/kg twice daily (BID) for 16 days. While causing a non-significant reduction in tumor volume in the U87MG xenograft model, QD dosing of A66 also causes a significant reduction in tumor volume in the HCT-116 xenograft model with a TGI of 77.2%.[1] In male CD1 mice, administration of A66 at 10 mg/kg results in significant impairments in the ITT (insulin tolerance test) and GTT (glucose tolerance test), as well as an increase in glucose production during a PTT (pyruvate tolerance test) that is almost as severe as that caused by pan-PI3K inhibitors. [3]

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On the basis of the pharmacokinetic and pharmacodynamic findings, A66 S was dosed QD at 100 mg/kg of body weight for up to 21 days or BID at 75 mg/kg of body weight for 16 days in tumour efficacy studies. Both dosing strategies induced a significant delay in growth of SK-OV-3 xenografted tumours, which was even greater than that induced by the well-established pan-PI3K inhibitor BEZ-235 (Figure 7A). At the final day of dosing, the average TGI for A66 S form was 45.9% of control (QD; P<0.05) and 29.9% of control (BID; P<0.01) (Table 4). QD A66 S was well tolerated in this xenograft model with minimum body weight loss; however BID treatment was associated with moderate body weight loss and two deaths, although it is not clear whether the deaths were due to drug toxicity or other causes since these mice did not show significant body weight loss (Figure 7B). In comparison, BEZ-235 induced a non-significant reduction in tumour growth and was even less tolerated, with moderate body weight loss and four deaths. QD dosing of A66 S in an HCT-116 xenograft model also induced a significant reduction in tumour volume with a TGI of 77.2% of control (P<0.01) at the end of dosing, but caused a non-significant reduction in tumour volume in the U87MG xenograft model (Table 4 and Supplementary Figure S5 at http://www.BiochemJ.org/bj/438/bj4380053add.htm). In contrast, BEZ-235 significantly reduced U87MG tumour growth (TGI=61.1% of control; P<0.05), but had no effect on HCT-116 tumours. The drugs were well tolerated in both the U87MG model, despite the toxicity with the same dose level of BEZ-235 in the SK-OV-3 study, and in the HCT-116 model, where a lower dose (10 mg/kg of body weight) of BEZ-235 was used due to the moderate body weight loss of control-treated mice. [1]

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The present study is the first to examine the effect of a selective p110α inhibitor (A66) on glucose metabolism in vivo. We find that A66 impairs all measures of in vivo insulin action, almost to the same level as the pan-PI3K inhibitors. This provides strong pharmacological evidence that p110α is the most important isoform in the pathways acutely regulating glucose metabolism, and that functional redundancy between PI3K isoforms is unlikely to be a major feature of major pathways regulating glucose metabolism in vivo. The effects of A66 on glucose metabolism are a phenocopy of mice heterozygous for global expression of a kinase-dead form of p110α. However, even though A66 is inhibiting p110α globally, the results of the present study are also remarkably similar to those seen in mice in which the Pik3ca gene had been deleted either acutely or chronically only in liver. Taken together with our PTT results this suggests that a major site of action of the p110α in regulating the effects of insulin on glucose metabolism is in liver [3].

The PI3K (human) HTRF Assay is used to calculate IC50 values. Invitrogen provides the p85α/p110δ . All other isoforms are made on-site by combining full-length human p85α with the appropriate full-length human catalytic subunit, which is marked with a histidine tag at the N-terminus to facilitate purification. The concentration at which the PI3Ks are used is titrated between their EC65 and EC80 values. Using an antibody to p85α N-SH2 (N-Src homology 2) domain, PI3K activity in immunoprecipitates is measured. The National Centre for Protein Kinase Profiling and Invitrogen Drug Discovery Services conduct assays for additional lipid kinases and protein kinases[1].

Cell Culture and Transfection. [2]
Fertilized chicken eggs (white Leghorn) were obtained from Charles River Breeding Laboratories. Primary CEF were prepared and cultured as described previously. For transfection, cells were plated at 80% confluence in F-10 containing 5.8% iron-supplemented FCS and 1% L-glutamine–penicillin–streptomycin solution. On the following day, CEF were transfected with the RCAS vectors using the dimethyl sulfoxide/Polybrene method. After two passages in the presence of serum, the cells were harvested for further analysis.

HEK 293-T cells were cultured in DMEM supplemented with 10% FCS and 1% L-glutamine–penicillin–streptomycin solution. Transfections were carried using lipofectamine-PLUS according to the manufacturer's protocol. HEK293T cells in MP6 plates at 70% confluency were washed once with Opti-MEM medium and incubated in 0.8 mL of Opti-MEM. A total of 1 μg of plasmid DNA was mixed with 0.1 mL of Opti-MEM and 2 μL of Lipofectamine PLUS for 15 min at room temperature. Opti-MEM (0.1 mL) with 6 μL of Lipofectamine was added to the DNA-PLUS mixture and incubated for 15 min at room temperature. The mixture of DNA, PLUS, and Lipofectamine was added to the cells and incubated overnight. The second day, the medium was changed to DMEM containing 10% FCS and 1% L-glutamine–penicillin–streptomycin solution. Forty hours after transfection, the cells were collected, lysed, and analyzed for specific proteins.

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Focus Assay. [2]
Focus assays with infectious retroviral vectors were performed as previously described. CEF were transfected with the appropriate RCAS constructs using the dimethyl sulfoxide/Polybrene method and overlayed with nutrient agar every other day for 2 to 3 wk until focus formation was observed. The plates were stained with crystal violet, and foci of transformed cells were counted. To examine inhibition of focus formation by different compounds, 10 μM LY294002, 100 nM NVP-BEZ-235, 2 nM rapamycin, 5 μM ZK-93, 250 nM TGX221, 5 μM IC87114, or 5 μM AS604850 were added to the nutrient agar in every overlay.

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Cell Proliferation. [2]
After transfection CEF were split into proliferation assay media containing F-10 supplemented with 2% FCS and 1% chicken serum. At the second split cells were seeded into a 96-well plate at 4,000 cells per well. On days 1 to 5 after seeding, CEF were incubated with 10 μg/mL Resazurin-Na in proliferation assay media for 4 h at 37 °C. Fluorescence was determined at an excitation wavelength of 560 nm and an emission of 590 nm.

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Western Blot and Immunoprecipitation. [2]
Western blotting was performed as previously described, with minor modifications. Cells were lysed in modified Nonidet P-40 lysis buffer (20 mM Tris-Cl, 150 mM NaCl, 1 mM MgCl2, 1% Nonidet P-40, and 10% glycerol with 1 mM PMSF, 1 mM DTT, 50 mM NaF, 1 mM Na3VO4, 50 mM β-glycerophosphate, and a protease inhibitor mixture). After centrifugation for 10 min at 18,000 × g at 4 °C, the protein concentration of the supernatant was determined. For the examination of inhibitor signaling, the cells were treated with 250 nM TGX-221 or 5 μM IC87114 for 2 h in the serum-containing condition ahead of collecting. For immunoprecipitation, cell lysates containing 40 μg of protein were incubated with anti-FLAG M2 Agarose overnight at 4 °C. Agarose beads were washed four times with lysis buffer and heated to 95 °C before separation on an SDS/PAGE gel. After transfer to Immobilon P membranes these membranes were blocked with 5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 2 h at room temperature and then incubated with a dilution of 1:2,000 of anti-FLAG or 1:1,000 of anti-p110α or anti-p110β primary antibody overnight. Membranes were washed three times in TBS-T and incubated with peroxidase-coupled goat anti-mouse or goat anti-rabbit antibody for 1 h in 5% BSA/TBS-T at room temperature. The reactive bands were visualized by SuperSignal West Pico Chemiluminescent substrate. For Western blotting, cell lysates containing 10 μg of total protein were separated on SDS/PAGE gels and transferred to Immobilon P membranes. Membranes were incubated with 1:1,000 dilutions of primary antibodies directed against p85, pAkt (T308), Akt, p4E-BP, 4E-BP, and β-actin. Western blots were developed as described above. Anti-FLAG antibody (Flag-M2 F3165) was purchased from xxx. Anti-p110α antibody (#4255), anti-p110 β antibody (#3011S), anti-p85 antibody (#4292), anti-Akt (#2967), anti-phospho-Akt (Thr-308) (#9275S), anti-4E-BP1 (#9452), and anti-phospho-4E-BP1 (Ser-65) (#9451S) antibodies were obtained from xyz.

Mice: Subcutaneous inoculation of 5×106 U87MG, SK-OV-3, or HCT-116 cells in PBS is performed on the right flank of age-matched, pathogen-free Rag1-/- or NIH-III mice. Based on the formula (L×w2)×π/6 (where L is the longest tumour diameter and w is the perpendicular diameter), tumour volume (mm3) is calculated using the tumor diameter as measured by electronic calipers. While BEZ-235 is administered in 10% ethanol, A66 is given in 20% 2-hydroxypropyl-β-cyclodextrin in water. The A66 dosing vehicle is given to control mice only. The drugs are administered intraperitoneally at a dose volume of 10 mL/kg of body weight as the free base equivalent. When tumors have grown to a diameter of about 8 to 9 mm, mice are given a single dose of A66 or the control substance for tumor pharmacodynamic studies. The tumors are removed, biopulverized, and the protein concentration is measured before the animals are killed 1 or 6 hours after the last dose.

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Xenograft methods [1]
Age-matched specific pathogen-free Rag1−/− or NIH-III mice were subcutaneously inoculated on the right flank with 5×106 U87MG, SK-OV-3 or HCT-116 cells in PBS. Tumour diameter as measured by electronic calipers was used to calculate tumour volume (mm3) based on the formula (L×w2)×π/6 (where L=longest tumour diameter and w=perpendicular diameter). A66 was administered in 20% 2-hydroxypropyl-β-cyclodextrin in water, whereas BEZ-235 was administered in 10% ethanol. Control mice were administered the A66 dosing vehicle alone. The drugs were dosed by intraperitoneal injection as the free base equivalent at a dosing volume of 10 ml/kg of body weight. For tumour pharmacodynamic studies, mice were administered a single dose of A66 or the control vehicle when tumours reached approximately 8–9 mm in diameter. Animals were killed 1 or 6 h after dosing and the tumours were removed, biopulverized and assayed for protein concentration. For antitumour efficacy studies, dosing began when tumours were well established, averaging approximately 7 mm in diameter. Doses were administered once daily (QD) or twice daily (BID) with injections separated by a minimum of approximately 8 h. Different dosing schedules were used for the three xenograft models depending on the rate of tumour growth and the body weight tolerance of control mice. Animals were dosed daily for 21 days or twice daily for 16 days (SK-OV-3), daily for 14 days (U87MG) and daily for 7 days (HCT-116). Animals were monitored daily for any signs of emerging toxicity and body weight was recorded. Mice were killed if they developed moderate signs of toxicity or if body weight loss exceeded 20% of starting weight. TGI (tumour growth inhibition) was calculated on the final day of dosing by determining the relative tumour size of drug-treated mice as a percentage of the average relative tumour size of control mice. The statistical significance of TGI values was determined by one-way ANOVA with Bonferroni multiple comparison analysis using GraphPad Prism 5.02.

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GTT, ITT and PTT [3]
GTTs, ITTs and PTTs, as well as determinations of insulin levels, were performed as described previously, except that male CD1 mice were used instead of rats. For GTTs and PTTs the mice were starved overnight and for the ITT food was withdrawn 2 h prior to the start of the experiments. Drugs were dosed intraperitoneally 1 h after the end of the dark cycle and 1 h prior to the intraperitoneal dosing with glucose or pyruvate (2 g/kg of body mass) or insulin (0.75 unit/kg of body mass).

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Metabolic cage studies [3]
Oxymax/CLAMS (Columbus Instruments) was used to quantify oxygen consumption (V̇O2), CO2 production (V̇CO2), BMR (basal metabolic rate), food intake, water intake and animal movement as described previously. BMR was expressed as a function of lean body mass as recommended in a previous study. All data were normalized to total lean mass using the EchoMRI-100 quantitative magnetic resonance system as described previously. Animals were acclimatized for 24 h in cages and the data were collected over the following 24 h.

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Analysis of drug levels [3]
Pharmacological kinetics studies were undertaken in fed CD1 male mice (30 g body mass). Animals were administered with the stated PI3K inhibitors via oral gavage or intraperitoneal injection, and terminal blood samples were collected in EDTA blood collection tubes at 15 min, and 1, 2, 4, 6 and 24 h post-drug exposure. All drugs were dissolved in DMSO. Blood was centrifuged (2000 g for 10 min and 4°C) and plasma isolated for drug quantification. Drug quantification was undertaken using LC-MS/MS (liquid chromatography tandem MS). Briefly, 300 μl of 100% methanol was added to 100 μl of plasma. The samples were gently mixed and centrifuged (2000 g for 10 min and 4°C). The supernatant was removed and 50 μl was added into vials for LC-MS/MS. The ion-source type was ESI (electrospray ionization) with the following conditions: spray voltage (5500 V), sheath gas pressure (50 units), ion sweep gas pressure (0.0 unit), auxillary gas pressure (2 units), capillary temperature [370°C and the capillary offset at 35 V. HPLC kinetex columns were used (100 mm × 3 mm, 2.6u C18(2)-HST; Phenomenex]. The run method was isocratic 10% (0.1% formic acid in water) and 90% methanol. The flow rate was 0.2 ml/min. Retention times were 2.64 min (PI-103), 2.76 min (TGX221) and 2.35 min (IC87114). Unknown concentrations were determined from the standard curve and internal standard.

First, we determined the optimal dosing strategy for xenograft studies by investigating the drug pharmacokinetics after a dose of 10 mg/kg of body weight by intraperitoneal injection in CD-1 mice. Despite a short half-life of only 0.42 h, the large Cmax (8247 nM) of A66 S that was reached 30 min after dosing ensured that the AUC0-inf (area under the curve from zero time to infinity) (6809 nM·h) was similar to that of BEZ-235 (7333 nM·h), which has a longer half-life of 2.73 h (Table 3). Furthermore, we tested the effect of the A66 S form on SK-OV-3 tumour tissue in vivo using a single dose of 100 mg/kg of body weight to determine whether a long-lasting effect of the drug could be achieved on target tissues (Figure 6). These studies show that A66 S causes a profound reduction in the phosphorylation of Akt/PKB and p70 S6 kinase, but not of ERK (extracellular-signal-regulated kinase), at both 1 and 6 h after dosing (Figure 6). This is consistent with A66 S having a full inhibitory effect on PI3K signalling in the tumours during this time. In the present study, levels of A66 S in plasma were determined to be 21.1±1.2 μM and 9.1±1.1 μM at 1 and 6 h after drug injection, whereas levels of A66 S in the tumour were 22.7±2.1 μM and 16.0±1.3 μM at the same time points. Thus, the retention of drug in the tumour is likely to explain the persistence of the inhibitory effect. [1]

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Pharmacokinetic methods [1]
Age-matched specific pathogen-free male CD-1 mice were administered a single dose of A66 (10 mg/kg of body weight) in 20% 2-hydroxypropyl-β-cyclodextrin in water or BEZ-235 in 15% (v/v) DMSO, 20% (v/v) 0.1 M HCl, 0.7% Tween 20 and 64.3% (v/v) saline. Mice were killed at five or six time points after dosing (n=3/time point) and blood was removed by cardiac puncture into EDTA collection tubes. Blood samples were centrifuged for 10 min at 6000 rev./min at 20 °C and the plasma supernatant was retained. Methanol was added to the plasma for protein extraction. Quantitative analysis was performed on an Agilent 6460 triple quadrupole LC-MS/MS (tandem MS) using multiple reaction monitoring and electrospray ionization. For chromatographic separation, an Agilent Zorbax SB-C18 column (2.1 mm×50 mm; 5 μm) was used with a mobile phase gradient of 20–100% methanol in 0.1% formic acid and 5 mM ammonium formate at a flow rate of 0.4 ml/min. Plasma drug concentrations were quantified against a calibration curve of known drug concentrations ranging from 10 to 10000 nM, with quality controls included at 65, 650 and 6500 nM. To prevent contamination from previous samples, a methanol slug was run between each plasma sample. Pharmacokinetic parameters were determined by noncompartmental analysis using WinNonlin 5.3 software.

[1]. Biochem J. 2011 Aug 15;438(1):53-62.

[2]. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15547-52.

[3]. Biochem J. 2012 Feb 15;442(1):161-9.

(2S)-N1-[5-(2-tert-butyl-4-thiazolyl)-4-methyl-2-thiazolyl]pyrrolidine-1,2-dicarboxamide is a proline derivative.

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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/p110β/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.[1]

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Cancer-specific mutations in the iSH2 (inter-SH2) and nSH2 (N-terminal SH2) domains of p85alpha, the regulatory subunit of phosphatidylinositide 3-kinase (PI3K), show gain of function. They induce oncogenic cellular transformation, stimulate cellular proliferation, and enhance PI3K signaling. Quantitative determinations of oncogenic activity reveal large differences between individual mutants of p85alpha. The mutant proteins are still able to bind to the catalytic subunits p110alpha and p110beta. Studies with isoform-specific inhibitors of p110 suggest that expression of p85 mutants in fibroblasts leads exclusively to an activation of p110alpha, and p110alpha is the sole mediator of p85 mutant-induced oncogenic transformation. The characteristics of the p85 mutants are in agreement with the hypothesis that the mutations weaken an inhibitory interaction between p85alpha and p110alpha while preserving the stabilizing interaction between p85alpha iSH2 and the adapter-binding domain of p110alpha. [2]

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In in vitro studies class-I PI3Ks (phosphoinositide 3-kinases), class-II PI3Ks and mTOR (mammalian target of rapamycin) have all been described as having roles in the regulation of glucose metabolism. The relative role each plays in the normal signalling processes regulating glucose metabolism in vivo is less clear. Knockout and knockin mouse models have provided some evidence that the class-I PI3K isoforms p110α, p110β, and to a lesser extent p110γ, are necessary for processes regulating glucose metabolism and appetite. However, in these models the PI3K activity is chronically reduced. Therefore we analysed the effects of acutely inhibiting PI3K isoforms alone, or PI3K and mTOR, on glucose metabolism and food intake. In the present study impairments in glucose tolerance, insulin tolerance and increased hepatic glucose output were observed in mice treated with the pan-PI3K/mTOR inhibitors PI-103 and NVP-BEZ235. The finding that ZSTK474 has similar effects indicates that these effects are due to inhibition of PI3K rather than mTOR. The p110α-selective inhibitors PIK75 and A66 also induced these phenotypes, but inhibitors of p110β, p110δ or p110γ induced only minor effects. These drugs caused no significant effects on BMR (basal metabolic rate), O2 consumption or water intake, but BEZ235, PI-103 and PIK75 did cause a small reduction in food consumption. Surprisingly, pan-PI3K inhibitors or p110α inhibitors caused reductions in animal movement, although the cause of this is not clear. Taken together these studies provide pharmacological evidence to support a pre-eminent role for the p110α isoform of PI3K in pathways acutely regulating glucose metabolism.[3]

C17H23N5O2S2

393.5268

393.129

C, 51.89; H, 5.89; N, 17.80; O, 8.13; S, 16.29

1166227-08-2

1166227-08-2

42636535

Off-white to light yellow solid powder

1.4±0.1 g/cm3

1.640

0.63

2

6

4

26

556

1

S1C([H])=C(C2=C(C([H])([H])[H])N=C(N([H])C(N3C([H])([H])C([H])([H])C([H])([H])[C@@]3([H])C(N([H])[H])=O)=O)S2)N=C1C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

HBPXWEPKNBHKAX-NSHDSACASA-N

InChI=1S/C17H23N5O2S2/c1-9-12(10-8-25-14(20-10)17(2,3)4)26-15(19-9)21-16(24)22-7-5-6-11(22)13(18)23/h8,11H,5-7H2,1-4H3,(H2,18,23)(H,19,21,24)/t11-/m0/s1

(2S)-1-N-[5-(2-tert-butyl-1,3-thiazol-4-yl)-4-methyl-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide

A-66; A66; 1166227-08-2; A66; (S)-N1-(2-(tert-butyl)-4'-methyl-[4,5'-bithiazol]-2'-yl)pyrrolidine-1,2-dicarboxamide; A 66; CHEMBL3218581; (2S)-1-N-[5-(2-tert-butyl-1,3-thiazol-4-yl)-4-methyl-1,3-thiazol-2-yl]pyrrolidine-1,2-dicarboxamide; 1,2-Pyrrolidinedicarboxamide, N1-[2-(1,1-dimethylethyl)-4'-methyl[4,5'-bithiazol]-2'-yl]-, (2S)-; A 66

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: ~79 mg/mL (~200.7 mM)
Water: ~1 mg/mL (~2.5 mM)
Ethanol: ~4 mg/mL (~26.8 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.35 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 15% Captisol: 8mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)