DNA-PK (IC50 = 2 nM); p110α (IC50 = 5.8 nM); p110γ (IC50 = 76 nM); p110δ (IC50 = 510 nM); p110β (IC50 = 1.3 μM); hsVPS34 (IC50 = 2.6 μM); PI3KC2β (IC50 = 1 μM); PI3KC2α (IC50 = 10 μM); mTORC1 (IC50 = 1 μM); mTORC2 (IC50 = 10 μM); ATM (IC50 = 2.3 μM); ATR (IC50 = 21 μM); PI4KIIIβ (IC50 = 50 μM)

PIK-75 shows the impressive potency and isoform selectivity at p110α while the corresponding IC50 values are 1300 nM, 76 nM and 510 nM for other PI3K isoforms, p110β, -γ, and -δ, respectively. Furthermore, when binding to purified p110α, Additionally, PIK-75 is a competitive inhibitor of the substrate PI with a Ki of 2.3 nM and a noncompetitive inhibitor of ATP when binding to purified p110. [1] DNA-PK is effectively inhibited by PIK-75 as well. PIK-75 (1 μM) reduces cell survival by significantly decreasing mitochondrial activity in unstimulated nonasthmatic airway smooth muscle (ASM) cells, asthmatic ASM cells, and lung fibroblasts. While in TGFβ stimulated ASM cells, PIK75 has no effects on non-asthmatic cells and only reduces mitochondrial activity in asthmatic cells. [2] According to a recent study, PIK-75 (10 nM) significantly reduces TNF-α-induced ADP-ribosyl cyclase activity and TNF-α-induced CD38 mRNA expression in human airway smooth muscle cells.[3]

In the ErbB3WT tumor model, PIK-75 lowers pAkt levels by 40% and lessens the in vitro chemotactic response to HRGβ1. Additionally, PIK-75 significantly lowers in vivo invasion and tumor cell motility in ErbB3WT primary tumors. [4] PIK-75 significantly impairs the insulin tolerance test (ITT), glucose tolerance test (GTT), and increases glucose production during the pyruvate tolerance test (PTT) in CD1 male mice.[5]

The PI3K inhibitor PIK-75 is dissolved at 10 mM in dimethyl sulfoxide and stored at −20°C until use. The PI3K enzyme's activity is assessed in 50 μL of 20 mM HEPES, pH 7.5, 5 mM MgCl2, 180 μM phosphatidyl inositol, and 2.5 μCi of [γ-32P]ATP. The reaction is sparked by the addition of 100 μM ATP. The enzyme reaction is stopped by adding 50 μL of 1 M HCl after it has been incubated at room temperature for 30 minutes. Then, phospholipids are extracted using 250 μL of 2 M KCl and 100 ml of a 1:1 chloroform/methanol mixture to extract the phospholipids for liquid scintillation counting. Using Prism version 5.00 for Windows, the concentration versus inhibition of enzyme activity curve is created by diluting inhibitors in 20% (v/v) dimethyl sulfoxide. The IC50 is then calculated using this analysis. An assay that detects ATP consumption is used for kinetic analysis. PI and ATP are used at various concentrations to measure the activity of the PI3K enzyme in 50 L of 20 mM HEPES, pH 7.5, and 5 mM MgCl2. After a 60-minute incubation at room temperature, the reaction is stopped by adding 50 μL of Kinase-Glo, followed by an additional 15 minutes of incubation. The Fluostar plate reader is then used to read the luminescence. Prism is used to analyze the outcomes.

Mitochondrial activity is measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay after stimulation with TGF with or without inhibitors for 48 hours. Before serial dilution (1:2) in duplicates, harvested washed cells are resuspended in DMEM-lO% FCS and aliquoted (500 μL) into 24-well cluster plates. Immediately after dilution, 100 μL of an appropriate MTT concentration (dissolved in PBS and filtered through a 0.2 μm filter before use to remove any blue formazan product) is added to each well. The cells are then incubated for 3.5 hours at 37 °C. 500 μL of 10% sodium dodecyl sulfate (SDS) in 0.01 M HCl are added to each well to dissolve the resulting blue formazan product over the course of 16 hours at 37°C. In a 96-well microplate, a sample (150 μL) from each duplicate well is transferred, and the optical density is calculated using automated spectrophotometry in comparison to a reagent blank (no cells).A reference wavelength of 690 nm and a test wavelength of 570 nm are used to measure absorbance. Results from three to six wells from each treatment are averaged for each primary cell culture, and data are expressed as absorbance 570 to 690 nm.

MTLn3 cells are injected into the right fourth mammary fat pad from the head of female severe-combined immunodeficient/NCr mice.
≤1 μM
Administered via i.p.

[1]. Mol Pharmacol. 2011 Oct;80(4):657-64.

[2]. J Pharmacol Exp Ther. 2011 May;337(2):557-66.

[3]. Am J Respir Cell Mol Biol. 2012 Oct;47(4):427-35.

[4]. Oncogene. 2012 Feb 9;31(6):706-15.

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

The combination of molecular modeling and X-ray crystallography has failed to yield a consensus model of the mechanism for selective binding of inhibitors to the phosphoinositide 3-kinase (PI3K) p110 α-isoform. Here we have used kinetic analysis to determine that the p110α-selective inhibitor 2-methyl-5-nitro-2-[(6-bromoimidazo[1,2-α]pyridin-3-yl)methylene]-1-methylhydrazide-benzenesulfonic acid (PIK-75) is a competitive inhibitor with respect to a substrate, phosphatidylinositol (PI) in contrast to most other PI3K inhibitors, which bind at or near the ATP site. Using sequence analysis and the existing crystal structures of inhibitor complexes with the p110γ and -δ isoforms, we have identified a new region of nonconserved amino acids (region 2) that was postulated to be involved in PIK-75 p110α selectivity. Analysis of region 2, using in vitro mutation of identified nonconserved amino acids to alanine, showed that Ser773 was a critical amino acid involved in PIK-75 binding, with an 8-fold-increase in the IC(50) compared with wild-type. Kinetic analysis showed that, with respect to PI, the PIK-75 K(i) for the isoform mutant S773D increased 64-fold compared with wild-type enzyme. In addition, a nonconserved amino acid, His855, from the previously identified region 1 of nonconserved amino acids, was found to be involved in PIK-75 binding. These results show that these two regions of nonconserved amino acids that are close to the substrate binding site could be targeted to produce p110α isoform-selective inhibitors.[1]

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The phosphatidylinositol 3-kinase (PI3K) signal transduction pathway is implicated in the airway remodeling associated with asthma. The class IA PI3K isoforms are known to be activated by growth factors and cytokines. Because this pathway is a possible site of pharmacological intervention for treating the disease, it is important to know which isoforms contribute to this process. Therefore, we used a pharmacological approach to investigate the roles of the three class IA PI3K isoforms (p110α, p110β, and p110δ) in airway remodeling using airway smooth muscle (ASM) cells derived from asthmatic subjects and ASM cells and lung fibroblasts from nonasthmatic subjects. These studies used the inhibitors N'-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylidene]-N,2-dimethyl-5-nitrobenzenesulfonohydrazide (PIK75) (which selectively inhibits p110α), 7-methyl-2-(4-morpholinyl)-9-[1-(phenylamino)ethyl]-4H-pyrido[1,2-a]pyrimidin-4-one (TGX221) (which selectively inhibits p110β), and 2-[(6-amino-9H-purin-9-yl)methyl]-5-methyl-3-(2-methylphenyl)-4(3H)-quinazolinone (IC87114) (which selectively inhibits p110δ). Cells were stimulated with transforming growth factor-β (TGFβ) and/or 10% fetal bovine serum in the presence or absence of inhibitor or vehicle control (dimethyl sulfoxide). PIK75, but not TGX221 or IC87114, attenuated TGFβ-induced fibronectin deposition in all cell types tested. PIK75 and TGX221 each decreased secretion of vascular endothelial growth factor and interleukin-6 in nonasthmatic ASM cells and lung fibroblasts, whereas TGX221 was not as effective in asthmatic ASM cells. In addition, PIK75 decreased cell survival in TGFβ-stimulated asthmatic, but not nonasthmatic, ASM cells. In conclusion, specific PI3K isoforms may play a role in pathophysiological events relevant to airway wall remodeling.[2]

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The ADP-ribosyl cyclase activity of CD38 generates cyclic ADP-ribose, a Ca(2+)-mobilizing agent. In human airway smooth muscle (HASM) cells, TNF-α mediates CD38 expression through mitogen-activated protein kinases and NF-κB and AP-1. The phosphatidylinositol-3 kinase/Akt (PI3K/Akt) pathway is involved in TNF-α signaling and contributes to airway hyperresponsiveness and airway remodeling. We hypothesized that PI3Ks mediate CD38 expression and are involved in the differential induction of CD38 by TNF-α in asthmatic HASM cells. HASM cells were treated with pan-PI3K inhibitors (LY294002 or wortmannin) or class I-selective (GDC0941) or isoform-selective PI3K inhibitors (p110α-PIK-75 and p110β-TGX-221) with or without TNF-α. HASM cells were transfected with a catalytically active form of PI3K or phosphatase and tensin homolog (PTEN) or nontargeting or p110 isoform-targeting siRNAs before TNF-α exposure. CD38 expression and activation of Akt, NF-κB, and AP-1 were determined. LY294002 and wortmannin inhibited TNF-α-induced Akt activation, whereas only LY294002 inhibited CD38 expression. P110 expression caused Akt activation and basal and TNF-α-induced CD38 expression, whereas PTEN expression attenuated Akt activation and CD38 expression. Expression levels of p110 isoforms α, β, and δ were comparable in nonasthmatic and asthmatic HASM cells. Silencing of p110α or -δ, but not p110β, resulted in comparable attenuation of TNF-α-induced CD38 expression in asthmatic and nonasthmatic cells. NF-κB and AP-1 activation were unaltered by the PI3K inhibitors. In HASM cells, regulation of CD38 expression occurs by specific class I PI3K isoforms, independent of NF-κB or AP-1 activation, and PI3K signaling may not be involved in the differential elevation of CD38 in asthmatic HASM cells.[3]

C16H14BRN5O4S.HCL

488.74

486.971

C, 39.32; H, 3.09; Br, 16.35; Cl, 7.25; N, 14.33; O, 13.09; S, 6.56

372196-77-5

PIK-75;372196-67-3

45265864

White to off-white solid powder

1.7±0.1 g/cm3

1.701

3.84

1

7

4

28

679

0

BrC1C([H])=C([H])C2=NC([H])=C(/C(/[H])=N/N(C([H])([H])[H])S(C3C([H])=C(C([H])=C([H])C=3C([H])([H])[H])[N+](=O)[O-])(=O)=O)N2C=1[H].Cl[H]

VOUDEIAYNKZQKM-MYHMWQFYSA-N

InChI=1S/C16H14BrN5O4S.ClH/c1-11-3-5-13(22(23)24)7-15(11)27(25,26)20(2)19-9-14-8-18-16-6-4-12(17)10-21(14)16;/h3-10H,1-2H3;1H/b19-9+;

N-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylideneamino]-N,2-dimethyl-5-nitrobenzenesulfonamide;hydrochloride

PIK-75 hydrochloride; PIK-75 HCl; PIK75 HCl; PIK 75 HCl

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

Solubility in Formulation 1: ≥ 1.1 mg/mL (2.25 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 11.0 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: ≥ 1.1 mg/mL (2.25 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 11.0 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: ≥ 1.1 mg/mL (2.25 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 11.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: PIK-75 HCl; PIK75 HCl; PIK 75 HCl.

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