PI3Kδ (IC50 = 4.6 nM); PI3Kα (IC50 = 16 nM); PI3Kβ (IC50 = 44 nM); PI3Kγ (IC50 = 49 nM); Autophagy

ZSTK474 inhibits all four PI3K isoforms in an ATP-competitive manner, according to analysis of Lineweaver-Burk plots. The four PI3K isoforms' Ki values revealed that ZSTK474 inhibited the PI3K isoform most potently with a Ki of 1.8 nM, while the other isoforms were inhibited with Ki values that were 4–10 times higher. ZSTK474 should be thought of as a pan-PI3K inhibitor as a result. Using ZSTK474 and LY294002, we also calculated the IC50 values for inhibiting the four PI3K isoforms. The IC50 values of ZSTK474 (16, 44, 4.6 and 49 nM for PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ, respectively) are shown to be consistent with the Ki values (6.7, 10.4, 1.8 and 11.7 nM for PI3Kα, PI3Kβ, PI3Kδ and PI3Kγ, respectively), which further supported the idea that ZSTK474 inhibits PI3Kδ most potently. ZSTK474 only moderately inhibits mTOR activity even at a concentration of 100 µM[1].

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The novel PI3K inhibitor, ZSTK474, showed potent antitumor activity in vivo against a human cancer xenograft without observable toxicity. However, the mode of its molecular action was not investigated in detail. Our previous study only suggested that ZSTK474 possibly competes with ATP for the ATP-binding pocket of PI3Kgamma. In the present study, we have used an in vitro homogenous time-resolved fluorescence kinase assay to examine whether ZSTK474 is indeed an ATP-competing inhibitor of PI3K, and also to determine whether the inhibitory activity of ZSTK474 was isoform-specific. Lineweaver-Burk plot analysis revealed that ZSTK474 inhibits all four PI3K isoforms in an ATP-competitive manner. Among all of the PI3K isoforms, PI3Kdelta was inhibited most potently by ZSTK474 with a K(i) of 1.8 nM, and the other isoforms were inhibited at higher doses. We have also used a kinase activity ELISA to determine whether ZSTK474 inhibits mammalian target of rapamycin, a key kinase acting downstream of PI3K to promote protein synthesis and cell proliferation. Even at a concentration of 100 microM, ZSTK474 inhibited mammalian target of rapamycin activity rather weakly. These results indicate that ZSTK474 is an ATP-competitive pan-class I PI3K inhibitor. [1]

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Competitive inhibition of class I PI3K isoforms by ZSTK474. [1]
ZSTK474 was previously reported to inhibit PI3K at nanomolar concentrations, and the molecular modeling analysis suggested that ZSTK474 might be an ATP‐competitive inhibitor that binds to the ATP‐binding pocket of PI3Kγ. To verify this notion, we used an in vitro assay to measure the PI3K activity at various ATP concentrations in the presence of increasing concentrations of ZSTK474. As shown in Fig. 1a, Lineweaver–Burk plot analysis revealed that ZSTK474 behaved as an ATP‐competitive inhibitor for all PI3K isoforms, as for each isoform the plots (straight lines) intersected on the 1/v axis. The Ki values of ZSTK474 for each PI3K isoform were shown by replotting the slope of each Lineweaver–Burk plot versus the respective ZSTK474 concentration (Fig. 1b). To determine the Ki values accurately, the data were best‐fitted to v = Vmax[ATP]/(Km(1 +[ZSTK474]/Ki) + [ATP]) using the GraphPad Prism 4 software program. As a result, the Ki values for PI3Kα, ‐β, ‐δ and ‐γ were determined as 6.7, 10.4, 1.8 and 11.7 nM, respectively.

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Comparison of inhibition of class I PI3K isoforms by ZSTK474 and LY294002. [1]
LY294002, a typical PI3K inhibitor, also competitively binds to the ATP‐binding pocket. Therefore, we compared the inhibition activities of ZSTK474 and LY294002 for each PI3K isoform. The dose–response inhibition profiles for both inhibitor are shown in Fig. 2. The IC50 values were calculated using GraphPad Prism 4 by fitting the data to a logistic curve. ZSTK474 was 30‐fold more potent than LY294002 in inhibiting the PI3K isoforms. Both inhibitors inhibited the PI3Kα and ‐δ isoforms more effectively than the PI3Kβ and ‐γ isoforms (Table 1).

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Inhibition of mTOR by ZSTK474. [1]
Inhibition of mTOR by ZSTK474 was investigated using the K‐LISA assay. Wortmannin, which was previously reported to inhibit mTOR, was used as a positive control. As shown in Fig. 3, ZSTK474 did not inhibit mTOR at 0.1 µM, a concentration that is higher than the IC50 for PI3K inhibition; even at a concentration of 100 µM, ZSTK474 inhibited mTOR activity less than 40%, suggesting that ZSTK474 is a much weaker inhibitor for mTOR than for PI3K.

Treatment with ZSTK474 is examined in mice that have undergone MCAO at doses of 50, 100, 200, and 300 mg/kg. Since the 200 mg/kg dose produces significant improvement and no obvious toxic effects (P<0.01), mice are treated with ZSTK474 at a dose of 200 mg/kg/day daily for three post-MCAO days during the remaining experiments of this study. Neurological function is examined in mice suffered from MCAO followed by 24, 48, and 72 h of reperfusion. With the exception of the corner test, ZSTK474 group scores on neurological function are significantly higher than those of the control group[2].

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ZSTK474 alleviated neurological deficits and reduced infarct volume in the cerebral ischemia/reperfusion injury model. Presumably, ZSTK474 shifted the phenotype of microglia/macrophages to a restorative state, since this treatment decreased the secretion of pro-inflammatory factors and advanced the secretion of anti-inflammatory factors. These neuroprotective properties of ZSTK474 may be mediated by the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin complex 1 (mTORC1) pathway.
Conclusions: ZSTK474 can mediate a shift in microglia/macrophage phenotype and inhibit the inflammatory response in cerebral ischemia reperfusion injury of mice. These effects appeared to ensue via the PI3K/AKT/mTORC1 pathway. Therefore, ZSTK474 may represent a therapeutic intervention with potential for circumventing the catastrophic aftermath of ischemic stroke[3].

The linear phase of each kinetic reaction is defined at the respective enzyme amount (0.05, 0.1, 0.12 and 1 µg/mL for PI3Kα, PI3β, PI3δ and PI3γ, respectively) and reaction time (20 min). PI3K activity is assayed at various concentrations of ATP (5, 10, 25, 50, 100 µM) in the presence of increasing concentrations of ZSTK474. A Lineweaver-Burk plot is developed by plotting 1/v (the inverse of v, where v is obtained by subtracting the HTRF signal of the kinase test sample from the HTRF signal of the minus-enzyme control) versus 1/[ATP] (the inverse of the ATP concentration). PIP2 is incubated with ATP without kinase for the minus-enzyme control. To determine the Ki value (inhibition constant) of ZSTK474 for each PI3K isoform, the slope of the respective Lineweaver-Burk plot is replotted against the ZSTK474 concentration. GraphPad Prism 4 is used to analyze and calculate the Ki values[1].

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Phosphatidylinositol 3‐kinase HTRF assay. [1]
The principle of the PI3K HTRF assay has been described previously. Briefly, PI3K catalyzes the phosphorylation of PIP2 to PIP3 in the presence of ATP. The PIP3 product is detected by displacement of biotinylated PIP3 (biotin‐PIP3) from an energy transfer complex consisting of Europium‐labeled anti‐glutathione S‐transferase (GST) antibody, a GST‐tagged receptor for phosphoinositide‐1 (GRP1) pleckstrin homology (PH) domain, biotin‐PIP3 and streptavidin–allophycocyanin (APC). Excitation of Europium in the complex results in an energy transfer to the APC. Displacement of biotin‐PIP3 from the complex leads to a loss of energy transfer and a corresponding decrease in HTRF signal. The decreased signal is proportional to the amount of PIP3 produced in the reaction and therefore can be used to monitor the PI3K activity.

The kinase reaction was carried out in a reaction mixture of 20 µL. Each class I PI3K isoform protein was incubated in the assay buffer containing 10 µM PIP2 and ATP (concentration as required) in a 384‐well plate at room temperature. The reaction was initiated by the addition of ATP, and stopped by adding 5 µL stop solution containing ethylenediaminetetraacetic acid and biotin‐PIP3 after 20 min. Then, 5 µL detection buffer was added, which contained the Europium‐labeled anti‐GST antibody, GST‐tagged GRP1 PH domain and streptavidin–APC. After incubation at room temperature for 14 h, the plate was read using the EnVision 2103 Multilabel Reader in time‐resolved fluorescence mode and the HTRF signal was determined according to the formula:

HTRF signal = 10 000 × (emission at 665 nm/emission at 620 nm).
Enzyme kinetic studies.  The linear phase of each kinetic reaction was defined at the respective enzyme amount (0.05, 0.1, 0.12 and 1 µg/mL for PI3Kα, ‐β, ‐δ and ‐γ, respectively) and reaction time (20 min). PI3K activity was assayed at various concentrations of ATP (5, 10, 25, 50, 100 µM) in the presence of increasing concentrations of ZSTK474. A Lineweaver–Burk plot was developed by plotting 1/v (the inverse of v, where v was obtained by subtracting the HTRF signal of the kinase test sample from the HTRF signal of the minus‐enzyme control) versus 1/[ATP] (the inverse of the ATP concentration). For the minus‐enzyme control, PIP2 was incubated with ATP in the absence of kinase. To determine the Ki value (inhibition constant) of ZSTK474 for each PI3K isoform, the slope of the respective Lineweaver–Burk plot was replotted against the ZSTK474 concentration. The Ki values were calculated by analysis using GraphPad Prism 4 and by fitting the curve to:
v = Vmax[ATP]/((Km(1 + [ZSTK474]/Ki) + [ATP]),
where v is the reaction velocity, Vmax is the maximal velocity, Km is the Michaelis constant, and [ZSTK474] and [ATP] are the concentrations of ZSTK474 and ATP, respectively.

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Determination of the IC50 of ZSTK474 and LY294002 for each class I PI3K isoform. [1]
Each PI3K isoform protein was incubated with a series of concentrations of ZSTK474 and LY294002 in the presence of 10 µM ATP. Other reaction conditions were as described above in the section ‘Enzyme kinetic studies’. The PI3K activity (% control) of a certain sample was calculated using the following formula:

PI3K activity (% control) = (sample – minus‐enzyme control)/(plus‐enzyme control – minus‐enzyme control) × 100.
For the plus‐enzyme control, the kinase was incubated with PIP2 and ATP in the absence of inhibitor. In the case of the minus‐enzyme control, PIP2 was incubated with ATP without kinase and inhibitor. For each PI3K isoform, the kinase activity was plotted as a function of the inhibitor concentration (ZSTK474 and LY294002). The IC50 values were calculated by fitting these data to a logistic curve using GraphPad Prism 4 software.

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K‐LISA mTOR assay.[1]
The K‐LISA mTOR assay is an enzyme‐linked immunosorbent assay (ELISA)‐based method that uses the p70S6K–GST fusion protein as the mTOR substrate. This substrate is first bound to a glutathione‐coated 96‐well plate, and then mTOR‐containing samples are incubated with ATP in the wells where active mTOR phosphorylates p70S6K at Thr389 (T389). To detect the phosphorylated substrate, the wells are first treated with anti‐p70S6K‐T389 antibody, followed by horseradish peroxidase (HRP)‐conjugated antibody and 3,3′,5,5′‐tetramethyl benzidine (TMB) substrate. Because the product of the HRP‐catalyzed reaction shows maximum absorbance at 450 nm, the activity of mTOR can be evaluated from the absorbance difference at 450 nm and 595 nm (background absorbance).

The assay was carried out according to the manufacturer's protocol. Briefly, 100 µL of recombinant p70S6K–GST fusion protein was preincubated at room temperature in the glutathione‐coated 96‐well plate and then removed 1 h later. Fifty microliters of ice‐chilled rat brain‐derived mTOR kinase in the presence of dimethylsulfoxide (DMSO; control), 5 µM wortmannin (positive control) or various concentrations of ZSTK474 was added to each well. The reaction was initiated by the addition of 50 µL kinase assay buffer containing 100 µM ATP, and incubated for 30 min at 30°C. After being washed, the plate was treated first with 100 µL of anti‐p70S6K‐T389 for 1 h and then with 100 µL of HRP‐conjugated antibody for 1 h to detect the T389‐phosphorylated p70S6K. Finally, 100 µL of TMB was added as HRP substrate and incubated for 20 min. The reaction was then stopped by the addition of 100 µL ELISA stop solution containing 2.5 N H2SO4. Absorbance was measured at 450 nm and 595 nm using a Benchmark Plus microplate spectrophotometer.

ZSTK474 is applied to cells for 48 hours at progressively higher concentrations. By measuring changes in total cellular protein using a sulforhodamine B assay, the inhibition of cell proliferation is evaluated. Apoptosis is assessed by chromatin condensation or by flow cytometry. For chromatin condensation assay, cells are stained with Hoechst 33342 and examined by fluorescence microscopy. Morphologic changes induced by ZSTK474, such as the condensation of chromatin, are indicative of apoptosis. For flow cytometry analysis, cells are harvested, washed with ice-cold PBS, and fixed in 70% ethanol. Cells are then washed twice with ice-cold PBS again, treated with RNase A (500 μg/mL) at 37 °C for 1 hour, and stained with propidium iodide (25 μg/mL). The DNA content of the cells is analyzed with a flow cytometer.

Mice: Mice are randomly assigned to receive different doses of ZSTK474 (50, 100, 200, and 300 mg/kg) to determine the optimum dose; in our experiment, the optimum dose is 200 mg/kg. Then, mice are randomized into one of three groups: the ZSTK474-treated group (MCAO+ZSTK474), the control group (MCAO+PBS), or the group that received phosphate-buffered saline (PBS) as a sham operation. The mice in the ZSTK474-treated group receive the drug at the recommended dosage of 200 mg/kg. Mice are given an identical amount of PBS in the control and sham groups. For a total of three days, beginning six hours after the onset of focal ischemia and continuing daily for two additional days, all mice receive the same dose of medication via oral gavage.

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Treatment with ZSTK474 [3]
ZSTK474 was suspended in 5 % hydroxypropylcellulose in water as a solid dispersion. First, mice were randomly assigned to receive different doses of ZSTK474 (50, 100, 200, and 300 mg/kg) to determine the optimum dose; in our experiment, the optimum dose was 200 mg/kg (Additional file 1: Figure S1). Then mice were randomly assigned to one of three groups: a sham-operated group (phosphate-buffered saline, PBS); a control group (MCAO + PBS); a ZSTK474-treated group (MCAO + ZSTK474). In the ZSTK474-treated group, the mice were given the optimum dose of 200 mg/kg ZSTK474. In the sham-operated group and control group, mice were given an equivalent volume of PBS. All mice received that same dose daily via oral gavage beginning at 6 h after the onset of focal ischemia and continuing for two more days, i.e., for a total of 3 days.

[1]. ZSTK474 is an ATP-competitive inhibitor of class I phosphatidylinositol 3 kinase isoforms. Cancer Sci, 2007, 98(10), 1638-1642.

[2]. Prolonged inhibition of class I PI3K promotes liver cancer stem cell expansion by augmenting SGK3/GSK-3β/β-catenin signalling. J Exp Clin Cancer Res. 2018 Jun 25;37(1):122.

[3]. Class I PI3K inhibitor ZSTK474 mediates a shift in microglial/macrophage phenotype and inhibits inflammatory response in mice with cerebral ischemia/reperfusion injury. J Neuroinflammation. 2016 Aug 22;13(1):192.

ZSTK-474 is a triamino-1,3,5-triazine that is 1,3,5-triazine in which two of the hydrogens have been replaced by morpholin-4-yl groups while the third hydrogen has been replaced by a 2-(difluoromethyl)benzimidazol-1-yl group. It is an inhibitor of phosphatidylinositol 3-kinase. It has a role as an EC 2.7.1.137 (phosphatidylinositol 3-kinase) inhibitor and an antineoplastic agent. It is a member of morpholines, a member of benzimidazoles, a triamino-1,3,5-triazine and an organofluorine compound.
ZSTK474 has been used in trials studying the treatment of Neoplasms.
PI3K Inhibitor ZSTK474 is an orally available, s-triazine derivative, ATP-competitive phosphatidylinositol 3-kinase (PI3K) inhibitor with potential antineoplastic activity. PI3K inhibitor ZSTK474 inhibits all four PI3K isoforms. Inhibiting the activation of the PI3K/AKT kinase (or protein kinase B) signaling pathway results in inhibition of tumor cell growth and survival in susceptible tumor cell populations. Dysregulated PI3K signaling may contribute to tumor resistance to a variety of antineoplastic agents. This agent does not induce apoptosis but rather induces strong G(0)/G(1) arrest, which might contribute to its favorable efficacy in tumor cells.

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We have also shown that ZSTK474 inhibited mTOR much less effectively than the PI3K isoforms. As mTOR is a serine–threonine kinase containing a conserved PI3K domain, PI3K inhibitors are expected to be cross reactive to mTOR. In fact, other PI3K inhibitor such as LY294002 and PI‐103 are known to inhibit mTOR significantly. However, ZSTK474 did not inhibit mTOR. Our previous study showed that ZSTK474 inhibits PI3K more effectively than 139 other protein kinases. Taken together, these results further demonstrate the superior specificity of ZSTK474 to PI3K. The contribution of this selectivity to its reduced toxicity and its higher potency in vivo in animal models remains to be clarified. In conclusion, we have demonstrated ZSTK474 is an ATP‐competitive inhibitor of all class I PI3K isoforms. ZSTK474 was indicated to be a pan‐class I PI3K inhibitor.[1]

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Background: Microglia/macrophages play a critical role in the inflammatory and immune processes of cerebral ischemia/reperfusion injury. Since microglia/macrophages can reversibly shift their phenotype toward either a "detrimental" or a "restorative" state in the injured central nervous system (CNS), compounds mediate that shift which could inhibit inflammation and restore the ability to alleviate cerebral ischemia/reperfusion injury would have therapeutic potential.

Methods: Transient middle cerebral artery occlusion was induced in male C57BL/6 mice. Mice were randomly separated into a sham-operated group, a control group, and a ZSTK474-treated group. We investigated the effect of ZSTK474 by assessing neurological deficits, infarct volume, and histopathological changes. We then determined the potential mechanism by immunofluorescent staining, quantitative real-time polymerase chain reaction (PCR), and Western blot analysis. The Tukey's test or Mann-Whitney U test was used to compare differences among the groups.

Results: ZSTK474 alleviated neurological deficits and reduced infarct volume in the cerebral ischemia/reperfusion injury model. Presumably, ZSTK474 shifted the phenotype of microglia/macrophages to a restorative state, since this treatment decreased the secretion of pro-inflammatory factors and advanced the secretion of anti-inflammatory factors. These neuroprotective properties of ZSTK474 may be mediated by the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin complex 1 (mTORC1) pathway.

Conclusions: ZSTK474 can mediate a shift in microglia/macrophage phenotype and inhibit the inflammatory response in cerebral ischemia reperfusion injury of mice. These effects appeared to ensue via the PI3K/AKT/mTORC1 pathway. Therefore, ZSTK474 may represent a therapeutic intervention with potential for circumventing the catastrophic aftermath of ischemic stroke.[3]

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In conclusion, our results demonstrated that ZSTK474 induced a shift in microglial/macrophage phenotype, inhibited inflammatory responses, reduced infarct volume, and improved neurological function in an experimental model of cerebral ischemia/reperfusion injury. These effects appeared be mediated by the PI3K/AKT/mTORC1 pathway. Our findings established a sound foundation for developing a therapeutic strategy that may benefit the patients who endure ischemic stroke.[3]

C19H21F2N7O2

417.4125

417.172

C, 54.67; H, 5.07; F, 9.10; N, 23.49; O, 7.67

475110-96-4

19545-26-7

11647372

White to off-white solid powder

1.6±0.1 g/cm3

640.3±65.0 °C at 760 mmHg

341.0±34.3 °C

0.0±1.9 mmHg at 25°C

1.711

-0.21

0

10

4

30

539

0

FC([H])(C1=NC2=C([H])C([H])=C([H])C([H])=C2N1C1=NC(=NC(=N1)N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])N1C([H])([H])C([H])([H])OC([H])([H])C1([H])[H])F

HGVNLRPZOWWDKD-UHFFFAOYSA-N

InChI=1S/C19H21F2N7O2/c20-15(21)16-22-13-3-1-2-4-14(13)28(16)19-24-17(26-5-9-29-10-6-26)23-18(25-19)27-7-11-30-12-8-27/h1-4,15H,5-12H2

4-[4-[2-(difluoromethyl)benzimidazol-1-yl]-6-morpholin-4-yl-1,3,5-triazin-2-yl]morpholine

ZSTK-474; ZSTK474; ZSTK 474

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: ~21 mg/mL (~50.3 mM)
Water: <1 mg/mL (slightly soluble or insoluble)
Ethanol: <1 mg/mL

Formulation 1: 0.5% hydroxyethyl cellulose: 30mg/mL
Formulation 2: suspended in 5% hydroxypropyl cellulose (Please use freshly prepared in vivo formulations for optimal results.)