Journal: iScience

Article Title: PIP4K2A/2B inhibitor suppresses tumor growth in a xenograft model of NSCLC

doi: 10.1016/j.isci.2026.115952

Figure Lengend Snippet: Improving mouse liver microsomal stability for the 2-amino-dihydropteridinone PIP4K2A/2B inhibitors (A) Three previously obtained and potent inhibitors share a dichlorophenol R2 side chain. The chiral center (C7) and the large hydrophobic R 1 side chain confer lipid kinase specificity. (B) The dichlorophenol in CC260 was replaced by various chemical groups with at least one aromatic ring and a (partial) negative charge. Some compounds have a 2-pyridinylmethyl R 1 side chain instead of CC260’s cyclopentylmethyl side chain. (C) The compounds’ solubility and mouse liver microsomal stability were compared. N.S., not soluble. The K i (s) for PIP4K2A and PIP4K2B were estimated from a single inhibitor (100 nM) and ATP (20 μM) concentration using the 32 P-based TLC lipid kinase assay.

Article Snippet: Crystal structure of PIP4K2A in complex with 066ATZ , Protein DataBank , PDB: 9OLE.

Techniques: Solubility, Concentration Assay, Kinase Assay

Journal: iScience

Article Title: PIP4K2A/2B inhibitor suppresses tumor growth in a xenograft model of NSCLC

doi: 10.1016/j.isci.2026.115952

Figure Lengend Snippet: 066ATZ maintains kinase selectivity and has improved in vivo half-life (A) The carboxylic acid moiety in 066A was replaced by various bioisosteres to reduce phase II metabolism. The K i (s) for PIP4K2A and PIP4K2B are shown in the parentheses (nM). (B) The concentration-inhibition curves for PIP4K2A and PIP4K2B ( n = 3, mean ± SEM). One representative set of autoradiographs of the TLC plates over a range of 066ATZ concentrations (0, 0.03, 0.16, 0.8, 4, and 20 μM) are shown. [ATP] is fixed at 20 μM in these experiments. (C) 066ATZ (0.5 μM) was profiled against 396 human protein kinases in the presence of 10 μM ATP. Only two protein kinases were significantly inhibited (CK2a, 57%; CK2a2, 90%). (D) 066ATZ (0.5 μM) was profiled against 17 lipid kinases in the presence of 10 μM ATP. (E) The time course of plasma 066ATZ concentration after IP injection into male C57BL/6 mice. Data are presented as mean ± SEM ( n = 3). Dose, 10 mg/kg; C max , 2,484 ng/mL (∼5.1 μM); t 1/2 , 0.5 h. (F) The effects of 066ATZ on the body weight and blood glucose levels (15 min and 30 min after IP injection) were tested in female nude mice. Each dose group (10, 30, and 100 mg/kg) contained 5 animals. Data are presented as mean ± SEM ( n = 5). A significant drop in blood sugar was observed in the two higher dose groups 30 min post injection. p values were calculated using Student’s t test in GraphPad Prism 10.0 (GraphPad Software, San Diego, CA).

Article Snippet: Crystal structure of PIP4K2A in complex with 066ATZ , Protein DataBank , PDB: 9OLE.

Techniques: In Vivo, Concentration Assay, Inhibition, Clinical Proteomics, Injection, Software

Journal: iScience

Article Title: PIP4K2A/2B inhibitor suppresses tumor growth in a xenograft model of NSCLC

doi: 10.1016/j.isci.2026.115952

Figure Lengend Snippet: The crystal structure of 066ATZ in complex with PIP4K2A (A) Fo-Fc map, contoured at 3.0 sigma, confirms the presence of the bound inhibitor. The apo PIP4K2A structure (PDB: 7N6Z ), with solvents and ions removed, was used in rigid body refinement and phase calculation. (B) The binding mode of 066ATZ is identical to those of other 2-amino-dihydropteridinone inhibitors. The newly installed 2-methylpyridine forms a water-mediated hydrogen bond with the backbone carbonyl of Ile-194. The nitrogen in the thiazole ring forms water-mediated hydrogen bonds with the side chain of Asn-198. Although disordered in the crystal, the side chain of Lys-209 is predicted to form a salt bridge with N1 of the tetrazole group. (C) The PIP4K2A/066ATZ (blue and green) and PIP4K2B/CC260 (gray and orange; PDB: 7N81 ) structures were superimposed based on the proteins’ N-lobe backbone atoms only. PIP4K2B has a slightly wider gap between the N- and C-lobes, as illustrated by the shift of the peptide segment between Ile-358 and Leu-361 (PIP4K2A numbering). Protein residues in contact with the bound inhibitors were labeled (PIP4K2B in black; those omitted for clarity were listed in the boxes on either side of the diagram). The protein backbone in all experimentally determined PIP4K2B structures has a sharp bend immediately following Leu-361, which is absent in PIP4K2A. (D) PIP4K2B demonstrates some interdomain conformational flexibility. In the diagram, the N-lobes of apo (gray; PDB: 7N80 ) and AMPPNP-bound (purple; PDB: 6K4H ) PIP4K2Bs were superimposed, revealing a slight movement in the C-lobe. , None of the solved PIP4K2A structures has this flexibility.

Article Snippet: Crystal structure of PIP4K2A in complex with 066ATZ , Protein DataBank , PDB: 9OLE.

Techniques: Binding Assay, Labeling

Journal: iScience

Article Title: PIP4K2A/2B inhibitor suppresses tumor growth in a xenograft model of NSCLC

doi: 10.1016/j.isci.2026.115952

Figure Lengend Snippet: 066ATZ reduces tumor growth rate in a xenograft model of NSCLC (A) The growth curves of H1975∗ xenograft tumor in R2G2 mice. Data are presented as mean ± SEM ( n = 5). 066ATZ (100 mg/kg) or vehicle was IP injected 5 times a week after the tumors reached ∼150 mm 3 . One animal died in the treatment group on day 39 for an unknown cause when the tumor reached a size of ∼550 mm 3 . All other mice were sacrificed when the tumors reached ∼1,000 mm 3 . On day 24, the average tumor volume was significantly smaller in the treatment group ( p < 0.01). p values were calculated using Student’s t test (two-tailed unpaired t test) with Welch’s correction in GraphPad Prism 10.0 (GraphPad Software, San Diego, CA). (B) The growth curves of H1975∗ xenograft tumor in R2G2 mice after DOX-induced PIP4K2A/2B knockdown. Data are presented as mean ± SEM ( n = 5). When the tumors reached ∼150 mm 3 , DOX was administered to the mice continuously through drinking water (1 mg/mL). Tumor tissues were harvested at the end of the experiment, homogenized, and analyzed by WB (shown below the growth curves). (C) In vitro , neither 066ATZ (20 μM) nor DOX (0.2 μg/mL) affected the growth rate of cultured H1975∗ cells. Data are presented as mean ± SEM ( n = 6). The cell numbers were measured by CellTiter-Glo.

Article Snippet: Crystal structure of PIP4K2A in complex with 066ATZ , Protein DataBank , PDB: 9OLE.

Techniques: Injection, Two Tailed Test, Software, Knockdown, In Vitro, Cell Culture

Journal: iScience

Article Title: PIP4K2A/2B inhibitor suppresses tumor growth in a xenograft model of NSCLC

doi: 10.1016/j.isci.2026.115952

Figure Lengend Snippet: Representative IHC and IF images of H1975∗ xenograft tumor (A) IHC staining of tumor tissue (animal #2 in the vehicle group) with F4/80 antibody and hematoxylin. In all animals, the tumor cells (blue) are surrounded by a dense layer of murine F4/80+ macrophages (brown). In some animals (regardless of treatment), macrophages are also found in dense clusters within the tumor. Nevertheless, there is no correlation between macrophage infiltration and tumor growth rate. Scale bars, 500 (4× panel) or 100 μm (20× panel). (B) IF staining reveals that most F4/80-stained cells (green) are also positive for CD206 (red), a marker highly expressed by M2a and M2c macrophages. Scale bars, 100 μm. (C) IHC staining with CD31 antibody reveals that 066ATZ treatment is associated with enhanced angiogenesis (animal #1 in the vehicle group and animal #10 in the treatment group are shown). Scale bars, 100 μm.

Article Snippet: Crystal structure of PIP4K2A in complex with 066ATZ , Protein DataBank , PDB: 9OLE.

Techniques: Immunohistochemistry, Staining, Marker

Journal: iScience

Article Title: PIP4K2A/2B inhibitor suppresses tumor growth in a xenograft model of NSCLC

doi: 10.1016/j.isci.2026.115952

Figure Lengend Snippet: PIP4K2A/2B inactivation reduces macrophage’s activity in promoting tumor cell growth (A) A schematic diagram illustrating the time course of macrophage (MP) differentiation and polarization in vitro . , THP-1∗ cells were treated with 200 nM PMA for 24 h to induce differentiation. The adherent cells were collected and replated (∼2,000,000 cells per well) on day 2. In the knockdown experiment, DOX (200 ng/mL) was added on day 3 and maintained throughout the experiment. In the compound treatment experiment, 066ATZ (20 μM) was added to the macrophage culture on day 5. Neither DOX nor 066ATZ affected macrophage viability. The following reagents were added on day 6 to induce macrophage polarization (only M2a polarization was shown in the diagram): M1, IFNγ (100 units/ml) and LPS (100 ng/mL); M2a, IL-4 (10 units/ml) and LPS (100 ng/mL); M2b, IgG-OVA complex and LPS (100 ng/mL); M2c, IL-10 (10 ng/mL); M2d, NECA (5 μM) and LPS (100 ng/mL). After removing polarization solutions on day 7, serum-free DMEM/F12 medium was added, and culture supernatant was collected after 24 h. The WB shown next to the time course diagram confirms DOX-inducible knockdown of PIP4K2A and PIP4K2B in THP-1∗ macrophages. M2a polarization caused an increase of CCL18 secretion, which was detected by ELISA. Data are presented as mean ± SEM ( n = 3). (B) The conditioned media from unpolarized macrophages (M0) and differently polarized macrophages (M1, M2a-d) promoted H1975 cell growth. Data are presented as mean ± SEM ( n = 6). The cell numbers were determined by CellTiter-Glo. The control medium was collected from a well without macrophages. During the 3 days growth period, H1975 cell number doubled in the control medium. To account for this residual growth, the difference in final cell numbers was used to quantify the growth promoting effect of macrophage conditioned medium. (C) Knockdown of PIP4K2A/2B in M2a polarized macrophage eliminated its growth promoting activity (∗∗∗, p < 0.0001). Data are presented as mean ± SEM ( n = 6). Neither DOX-induced knockdown nor 066ATZ affected macrophage viability. p values were calculated using Student’s t test in GraphPad Prism 10.0 (GraphPad Software, San Diego, CA). (D) The effect of PIP4K2A/2B knockdown in other macrophages did not reach statistical significance, although in M1 polarized macrophages the knockdown appeared to produce a small reduction. Data are presented as mean ± SEM ( n = 6). (E) Treatment with 066ATZ (20 μM), or with a more potent PIP4K inhibitor, BAY-091 (2 μM), reduced M2a macrophage’s growth promoting activity (∗∗, p < 0.001; ∗∗∗, p < 0.0001). Data are presented as mean ± SEM ( n = 6). CK2α/α′ inhibitor CX-4945 (0.5 μM) had no effect on the conditioned medium of M2a macrophages. p values were calculated using Student’s t test in GraphPad Prism 10.0 (GraphPad Software, San Diego, CA). (F) M2a conditioned medium promoted the growth of other cell lines: Calu-3 (lung cancer), MIA PaCa-2 (pancreatic cancer), HCT-116 (colon cancer), 22Rv1 (prostate cancer), and U-87MG (glioblastoma). Knockdown of PIP4K2A/2B in the M2a macrophage reduced these growth promoting activities (∗, p < 0.01; ∗∗, p < 0.001; ∗∗∗, p < 0.0001). Data are presented as mean ± SEM ( n = 6). p values were calculated using Student’s t test in GraphPad Prism 10.0 (GraphPad Software, San Diego, CA).

Article Snippet: Crystal structure of PIP4K2A in complex with 066ATZ , Protein DataBank , PDB: 9OLE.

Techniques: Activity Assay, In Vitro, Knockdown, Enzyme-linked Immunosorbent Assay, Control, Software