PI3K-IN-1 (Voxtalisib Analogue) is a dual inhibitor of mTOR/PI3K, mostly for p110γ , also inhibits DNA-PK and mTOR.

PI3Kγ:9 nM, PI3Kα:39 nM, DNA-PK:150 nM, PI3Kβ:113 nM, PI3Kδ:43 nM

In the GBM 39-luc xenografted nude mice model, oral administration of XL765 was able to inhibit the growth of tumor cells while also enhancing survival rates. In the BxPC-3 xenograft mouse model, combining XL765 (30 mg/kg) with chloroquine (50 mg/kg) demonstrated the capability to suppress tumor cell growth.

XL765 inhibits Class I PI3K, specifically targeting p110α (IC50=39 nM), p110β (IC50=113 nM), p110γ (IC50=9 nM), and p110δ (IC50=43 nM). It also suppresses mTOR (IC50 = 157 nM) by significantly reducing the phosphorylation of mTOR targets, including S6, S6K, and 4EBP1. In MIAPaCa-2 cells, treatment with XL765 leads to the accumulation of autophagic vacuoles.

Radioligand Binding Assays: Each tube in the A3 AR competitive binding assay contains 100 μL of membrane suspension (20 μg of protein), 50 μL of [125I]4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide (0.5 nM), and 50 μL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.4) containing 10 mM MgCl2 and 1 mM EDTA. Nonspecific binding is determined using 10 mM 5′-N-ethylcarboxamidoadenosine in the buffer. The mixtures are incubated at 25°C for 60 min. Binding reactions are terminated by filtration through Whatman GF/B filters under reduced pressure using a MT-24 cell harvester. Filters are washed three times with 9 mL of ice-cold buffer. Radioactivity is determined using a Beckman γ-counter, and the percent inhibition is calculated.

Cells are treated with XL765 24 hours after plating and harvested for apoptosis or autophagy assays at 24, 48, or 72 hours after XL765 treatment. Apoptosis is determined by total percentage of annexin V-positive cells by fluorescence-activated cell sorting (FACS). Acidic vesicular organelles (AVOs) are detected in XL765-treated cells by vital staining with acridine orange. The degree of AVO formation is expressed as fold increase of acridine orange fluorescence intensity (FL3) in XL765-treated cells versus control cells. (Only for Reference)