PI3Kγ (IC50 = 16 nM); PI3Kα (IC50 = 3.2 μM); PI3Kβ (IC50 = 3.5 μM)

Eganelisib (IPI549) inhibits PI3Kγ with IC50 of 16 nM, with >100-fold selectivity over other lipid and protein kinases (PI3Kα IC50=3.2 μM, PI3Kβ IC50=3.5 μM, PI3Kδ IC50>8.4 μM). Eganelisib is evaluated for activity across all Class I PI3K isoforms. The activity of eganelisib is assessed across all Class I PI3K isoforms. The individual rates constants and for PI3K-α, β and δ are measured using equilibrium fluorescent titration to ascertain the binding affinity of Eganelisib for PI3K-γ. Eganelisib is a remarkably tight binder to PI3Kγ with a Kd of 290 pM and >58-fold weaker affinity for other Class I PI3K isoforms (PI3Kα Kd=17 nM, PI3Kβ Kd=82 nM, PI3Kδ Kd=23 M). Eganelisib exhibits excellent PI3K-γ potency (IC50=1.2 nM) and selectivity against other Class I PI3K isoforms (>146-fold) in PI3K-α, -β, -γ, and -δ dependent cellular phospho-AKT assays. Cellular IC50s for Class I PI3Kα (250 nM), PI3Kβ (240 nM), PI3Kγ (1.2 nM), PI3Kδ (180 nM) are determined in SKOV-3, 786-O, RAW 264.7, and RAJI cells, respectively, by monitoring inhibition of pAKT S473 by ELISA. Furthermore, Eganelisib dose dependently inhibits PI3Kγ dependent bone marrow-derived macrophage (BMDM) migration. Eganelisib is selective against a panel of 80 GPCRs, ion channels, and transporters at 10 μM[1]

Eganelisib (IPI549) exhibits advantageous pharmacokinetic characteristics and potent PI3K-mediated neutrophil migration inhibition. Eganelisib has excellent oral bioavailability, low clearance, and is distributed into tissues with a mean volume of distribution of 1.2 L/kg in vivo (in mice, rats, dogs, and monkeys). Overall, Eganelisib has a good pharmacokinetic profile that enables potent and precise PI3K- in vivo inhibition. The t1/2 of IPI-549 is 3.2, 4.4, 6.7, and 4.3 hours for mice, rats, dogs, and monkeys, respectively. In this model, oral administration of eganelisib at all of the tested doses significantly reduces neutrophil migration in a dose-dependent manner[1].

Biochemical Assay Materials:[1]
The Class I PI3K isoforms α, β, and δ were supplied with the p85α regulatory subunit and the γ isoform was supplied alone. The catalogue number (cat #) of the isoforms used were as follows PI3K-α: p110α/p85α cat #14-602-K, PI3K-β: p110β/p85α cat #14-603-K, PI3K-γ: p120γ cat #14-558-K, PI3K-δ: p110δ/p85α cat #14-604. The Class II PI3K isoforms were supplied as N-terminal 6xHis-tagged proteins; PI3K-C2α amino acids 299-end: cat # 14-906-K, PI3K-C2β full length: cat # 14-907-K and PI3K-C2γ: cat # 14-910-K. ATP disodium salt was prepared to a stock concentration of 30 mM in water, and neutralized to ~ pH 7.0 with sodium hydroxide (NaOH). Concentrations were determined via ultraviolet (UV) absorbance at 260 nm using an extinction coefficient of 15.4 x 103 M -1 cm -1 . Dioctanoyl-phosphatidylinositol-4,5-bis-phosphate (diC8PIP2) was made up as a stock solution of 5 mM in water, and stored at -20°C. Dioctanoyl-phosphatidylinositol (diC8PIP) was made up as a stock solution of 5 mM in water, and stored at – 20°C.

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General Procedure for Class I and Class II PI3K Biochemical Assays at 3 mM ATP: [1]
Promega ADP-Glo Max assay kit was used to determine IC50 values for Class I α, β, δ, and γ and Class II α, β, and γ isoforms of human PI3 kinases. Samples of kinase (20 nM PI3K-α, PI3K-δ, PI3K-C2α, PI3K-C2β, and PI3K-C2γ or 40 nM PI3K-β and PI3K-γ) were incubated with compound for 15 minutes at room temperature in reaction buffer (15 mM HEPES pH 7.4, 20 mM NaCl, 1 mM EGTA, 0.02% Tween 20, 10 mM MgCl2, 0.2 mg/mL bovine-γ-globulins) followed by addition of ATP/diC8-PP2 mixture to give final concentrations of 3 mM ATP and 500 µM diC8PIP2 substrate (for Class I PI3Ks) or 500 µM diC8PIP substrate (for Class II PI3Ks). Reactions were incubated at room temperature for 2 hours followed by addition of 25 µL of Promega kit stop solution. After a 40-minute incubation at room temperature, 50 µL of Promega detection mix was added followed by incubation for 1 hour at room temperature. Plates were then read on Envision plate reader in luminescence mode. Data is converted to % inhibition and then plotted as % inhibition vs compound concentration and fit to a 4 parameter logistic equation to determine IC50 values.

At a density of 200,000 cells/200 L/well of RPMI-1640 with 10% FBS, SKOV-3 cells are seeded into 96-well cell culture-grade plates. Cells are incubated for the entire night at 5% CO2 and 37 °C. The cells are treated with substances, resulting in a final concentration of 0.5% DMSO, and then incubated for 30 minutes at 37 °C and 5% CO2. Following the aspiration of the media, 50 L of ice-cold lysis buffer is added to each well. After five minutes of incubation on ice, plates are centrifuged at 3000 rpm for five minutes at 4 °C.

The mice used in this study are C57BL/6J and Balb/c mice, aged 6 to 8 weeks. In the first day of the experiment, tumor cells are injected intradermally (i.d.) in the right flank. Once per day, 15 mg/kg of eganelisib is given orally through gavage. Beginning on day seven after tumor implantation and lasting until day twenty-one. Transportation (5% NMP, 95% PEG) is given to the control groups. Every second or third day, tumors are calibrated with a ruler to measure their volume (length, width, and height). when the total tumor volume reaches 2500 mm3, or when there are signs of distress in the animal. Tumors are then isolated and preserved in ice until needed[2].

[1]. Discovery of a Selective Phosphoinositide-3-Kinase (PI3K)-γ Inhibitor (IPI-549) as an Immuno-Oncology Clinical Candidate. ACS Med Chem Lett. 2016 Jul 22;7(9):862-7.

[2]. Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells. Nature. 2016 Nov 17;539(7629):443-447.

IPI-549 is under investigation in clinical trial NCT03795610 (Window of Opportunity Study of IPI-549 in Patients With Locally Advanced HPV+ and HPV- Head and Neck Squamous Cell Carcinoma).
Eganelisib is an orally bioavailable, highly selective small molecule inhibitor of the gamma isoform of phosphoinositide-3 kinase (PI3K-gamma) with potential immunomodulating and antineoplastic activities. Upon administration, eganelisib prevents the activation of the PI3K-gamma-mediated signaling pathways, which may lead to a reduction in cellular proliferation in PI3K-gamma-expressing tumor cells. In addition, this agent is able to modulate anti-tumor immune responses and inhibit tumor-mediated immunosuppression. Unlike other isoforms of PI3K, the gamma isoform is overexpressed in certain tumor cell types and immune cells; its expression increases tumor cell proliferation and survival. By selectively targeting the gamma isoform, PI3K signaling in normal, non-neoplastic cells is minimally or not affected, which results in a reduced side effect profile.

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Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.[1]

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Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.[2]

C30H24N8O2

528.58

528.2022

C, 68.17; H, 4.58; N, 21.20; O, 6.05

1693758-51-8

91933883

Yellow solid powder

3.2

123Ų

O=C1N(C2=CC=CC=C2)C([C@@H](NC(C3=C(N=CC=C4)N4N=C3N)=O)C)=CC5=CC=CC(C#CC6=CN(C)N=C6)=C51

XUMALORDVCFWKV-IBGZPJMESA-N

InChI=1S/C30H24N8O2/c1-19(34-29(39)26-27(31)35-37-15-7-14-32-28(26)37)24-16-22-9-6-8-21(13-12-20-17-33-36(2)18-20)25(22)30(40)38(24)23-10-4-3-5-11-23/h3-11,14-19H,1-2H3,(H2,31,35)(H,34,39)/t19-/m0/s1

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.73 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 (4.73 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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 (Please use freshly prepared in vivo formulations for optimal results.)