NAT10 (N-acetyltransferase 10); E3 ligase

NAT10 Degradation: In SiHa cells, NP1192 induced NAT10 degradation rates of 28%, 32%, 40%, and 43% at concentrations of 1.25, 2.5, 5, and 10 μM, respectively, with a maximum of 69% degradation at 20 μM after 36 hours. In murine U14 cervical cells, it achieved nearly 70% reduction after 36 hours. NP1192 did not exhibit the classic "hook effect" even at concentrations as high as 20 μM [1].
Comparison with Remodelin: NP1192 exhibited significantly greater efficacy in reducing CCA cell viability, with a 26.8% lower IC50 value in SiHa cells (IC50 = 7.814 μM for NP1192 vs 10.67 μM for Remodelin). NP1192 also demonstrated superior antiproliferative and anti-invasive effects compared to Remodelin [1].
Mechanism of Degradation: Co-treatment with the proteasome inhibitor MG132 significantly impeded NP1192-mediated degradation of NAT10, whereas the lysosomal inhibitor bafilomycin A1 had no discernible effect. This confirms that NP1192 facilitates NAT10 degradation through the ubiquitin-proteasome system (UPS) rather than via lysosomal pathways [1].
Effect on HIF-1α and PD-L1: NP1192 treatment decreased HIF-1α protein levels and resulted in pronounced downregulation of PD-L1. NAT10 knockdown or NP1192 treatment led to decreased HIF1A mRNA stability and a reduction in global nascent protein translation. NP1192 also decreased ac4C modification of HIF1A mRNA, as confirmed by acRIP-qPCR [1].
Effect on Glycolysis and Metabolism: Treatment with NP1192 significantly decreased lactate levels and reduced glucose uptake in SiHa and U14 cells, while ATP production increased markedly, suggesting a shift toward oxidative phosphorylation. JC-1 fluorescence staining revealed reduced red:green ratios, supporting enhanced mitochondrial activity. Seahorse extracellular flux analysis showed that the glycolytic capacity of NP1192-treated CCa cells remained markedly impaired compared to controls [1].
Effect on Cell Cycle, Proliferation, and Invasion: NP1192 treatment led to cell cycle arrest (enrichment of cell cycle-related pathways, particularly in cell division and G1/S transition) and reduced colony formation and invasive capacity (Transwell assay) of SiHa cells [1].
Activity in Patient-Derived Organoids (PDOs): NP1192 treatment had a negligible impact on the viability of normal human epithelial foreskin fibroblast (HFF-1) organoids but exhibited a clear dose-dependent inhibitory effect on cancer organoids, with IC50 values of 9.947 μM for ovarian cancer (OV), 3.048 μM for cervical cancer (CESC), and 13.76 μM for glioblastoma (GBM) [1].
Resistance Profile: Remodelin-resistant SiHa cells were generated, but the relative resistance index (RI) for NP1192 was not explicitly provided in the text. The RI was defined as the ratio of the IC50 in resistant cells to that in parental cells [1].

---

NP1192 (0-20 μM, 24-48 h) induced the degradation of NAT10 in SiHa (human) and U14 (mouse) cervical cancer cell lines in a dose- and time-dependent manner via the ubiquitin-proteasome system (UPS) rather than the lysosomal pathway [1]. NP1192 (36 h) inhibited the growth of cervical cancer (SiHa) cells with an IC50 of 7.814 μM (76.2 μM in resistant cells) and also showed dose-dependent efficacy in organoids, with IC50 values of 9.947, 3.048 and 13.76 μM for ovarian cancer (OV), cervical cancer (CESC) and glioblastoma (GBM) organoids, respectively [1]. NP1192 (20 μM, 0-72 h and 2 weeks) significantly reduced the viability of cervical cells, induced cell cycle arrest, and inhibited their invasive and clonogenic abilities [1]. NP1192 (0.195-50 μM, 1-8 days) significantly reduced the size and number of organoids in tumor models (ovarian cancer, cervical cancer and glioblastoma), while the corresponding normal organoids were unaffected, indicating that it has very low cytotoxicity to non-malignant tissues [1]. NP1192 (20 μM, 36 hours) degraded NAT10 and thus impaired the expression of HIF-1α, reprogramming the hypoxic tumor microenvironment and metabolism of cervical cancer cells, which was manifested as reduced glucose uptake and lactate production and increased ATP levels, thereby disrupting the glycolysis process [1].

Xenograft Model Efficacy: In a murine cervical cancer (U14) xenograft model, the combination of NP1192 (25 mg/kg, peritumoral injection every 2 days) with anti-PD-L1 antibody (2.5 mg/kg, every 3 days) significantly inhibited tumor growth compared to NP1192 or anti-PD-L1 alone. This combination also achieved the most pronounced reduction (>80%) in tumor lactate levels, with more than a 40% decline in the NP1192 monotherapy group. In vivo bioluminescence imaging corroborated these findings [1].
Metabolic Imaging: 18F-FDG-PET-CT scans revealed minimal metabolic uptake in the combination-treated group, indicating that NP1192 effectively suppresses tumor metabolic activity [1].
Immune Modulation: Immunoflow cytometry of tumor tissues showed that NP1192 combined with anti-PD-L1 blockade resulted in the highest frequency of CD8+ tumor-infiltrating lymphocytes (TILs), increased IFN-γ production (ELISA), significantly reduced cancer-promoting regulatory T cells (Tregs, CD4+CD25+FOXP3+), and decreased the proportion of M2 macrophages (CD11b+F4/80+CD206+), indicating a shift toward M1 macrophage polarization. NP1192 monotherapy also significantly reduced myeloid-derived suppressor cells (MDSCs, CD11b+LY-6G+) [1].
Single-Cell RNA-seq (scRNA-seq) Analysis: scRNA-seq on tumor samples from NP1192-treated mice revealed that NP1192 treatment significantly reduced the proportion of immunosuppressive M2 macrophages and MDSCs, while increasing M1 macrophages. In CD8+ TILs, NP1192-treated tumors exhibited fewer exhausted CD8+ TILs (Tex) and more cytotoxic effector-like CD8+ TILs (Teff, including IFN-γ-producing cells). Pseudotime analysis suggested that NP1192 sustains CD8+ TILs in an initial activation state [1].

---

NP1192 (25 mg/kg, intraperitoneal injection, once every 2 days for one week) can synergize with anti-PD-L1 antibody to inhibit tumor growth, inhibit glycolysis and enhance CD8+ T effector cell immune function in U14-luc xenograft model [1].

No direct enzyme assays (e.g., using purified NAT10 enzyme) were described for NP1192 in the provided document. The study focuses on cellular degradation and downstream effects.

Western Blot Analysis for NAT10 Degradation: SiHa, U14, and HFF-1 cells were treated with NP1192 or control. RIPA lysis buffer with protease inhibitor was used to extract total protein. Proteins were separated on polyacrylamide gels, transferred to PVDF membranes, blocked with milk, and incubated with primary antibodies (anti-NAT10, anti-PD-L1, anti-HIF-1α, anti-β-tubulin) overnight, followed by secondary antibody incubation. Target proteins were detected via chemiluminescence [1].
Cell Viability (CCK-8) Assay: NAT10-knockdown SiHa cells and SiHa cells treated with DMSO, Remodelin, or NP1192 for 36 hours were seeded into 96-well plates (2000-3000 cells/well). After adherence, a 1:10 mixture of CCK8 reagent and medium was added, incubated for 2 hours, and absorbance at 450 nm was measured to calculate IC50 values [1].
Colony Formation Assay: NAT10-knockdown SiHa cells and SiHa cells treated with DMSO, Remodelin, or NP1192 for 36 hours were seeded into 6-well plates (2000-3000 cells/well) and incubated for at least 2 weeks. Colonies were fixed with formalin, stained with crystal violet, and counted [1].
Transwell Invasion Assay: Matrigel-coated Transwell chambers were used. NAT10-knockdown SiHa cells and SiHa cells treated with DMSO, Remodelin, or NP1192 for 36 hours were resuspended in serum-free medium and added to the top compartment. After 12 hours, cells on the bottom membrane surface were fixed, stained, and counted [1].
Flow Cytometry for Cell Cycle: Cells were fixed with 70% ethanol, treated with a solution containing PI and RNase A, and analyzed via flow cytometry to determine cell cycle distribution [1].
mRNA Stability Assay: SiHa and NAT10-KD SiHa cells were treated with actinomycin D (5 μM) for 0, 4, or 8 hours. Changes in HIF1A mRNA half-life were evaluated via qPCR [1].
Nascent Protein Synthesis Assay: Cells were fixed with methanol, dyed with a Click-iT Plus OPP Protein Synthesis Assay Kit, and stained with DAPI. Cells were observed under a confocal laser scanning microscope [1].
Lactic Acid, ATP, and 2-NBDG Uptake Assays: SiHa and U14 cells were treated with 20 μM NP1192 or DMSO for 36 hours. Assays were performed according to kit instructions, and data were analyzed via GraphPad Prism 9 [1].
Seahorse Assay: SiHa and U14 cells were treated with 20 μM NP1192 or DMSO for 36 hours. The assay was performed according to the Seahorse XF Glycolysis Test Kit manual [1].
JC-1 Staining Assay: SiHa and U14 cells were treated with 20 μM NP1192 or DMSO for 36 hours. The assay was performed according to the mitochondrial membrane potential detection kit manual [1].
ROS Staining Assay: SiHa and U14 cells were treated with 20 μM NP1192 or DMSO for 36 hours. The assay was performed according to the ROS Assay Kit manual [1].
acRIP-qPCR: acRIP-qPCR results showed a decrease in the IP/input percentage of SiHa groups treated with 20 μM NP1192 for 36 hours and NAT10-knockdown compared to DMSO treatment, indicating decreased ac4C modification of target RNAs [1].
CD8+ T Cell Coculture Assay: Fresh CD8+ T cells were extracted from mouse spleens, activated with anti-CD3/CD28 and IL-2, and cocultured with U14 cells at a 10:1 effector-to-target ratio with different drugs for 24 hours. CD8+ T-cell subtypes were detected by flow cytometry. Tumor cell killing was assessed via CFSE-based cytotoxicity, Calcein AM/PI costaining, and CCK8 assays. IFN-γ and TNF-α levels in supernatant were measured by ELISA [1].

---

Western Blot Analysis[1]
Cell Types: SiHa, U14, and HFF-1 cells
Tested Concentrations: 0, 1.25, 2.5, 5, 10 and 20 μM
Incubation Duration: 24, 36 and 48 h
Experimental Results: Led to a substantial depletion of total NAT10 protein in SiHa (human) and U14 (murine) CCa cell lines in a a dose-and time-dependent manner. Induced degradation rates of 28%, 32%, 40%, and 43% in SiHa cells at concentrations of 1.25, 2.5, 5, and 10 μM, respectively, with a maximum of 69% degradation at 20 μM after 36 h and only 20% degradation after 24 h. Led to a nearly 70% reduction in the NAT10 protein content in murine U14 cervical cells after 36 h. Did not exhibit the classical hook effect even at concentrations as high as 20 μM. Failed to degrade NAT10 significantly when co-treated with MG132, whereas Bafilomycin A1 exerted no discernible effect on its degradation efficacy. Substantially induced minimal cytotoxicity in nonmalignant HFF-1 cells with low NAT10 expression. Decreased HIF-1α protein levels. Reversed the induction of hypoxia-related genes under hypoxic conditions, such as HIF1A and PD-L1, at both mRNA and protein levels.

---

Cell Proliferation Assay[1]
Cell Types: SiHa cells and NAT10-KD SiHa cells
Tested Concentrations: 20 μM
Incubation Duration: 2 weeks
Experimental Results: Led to a substantial decrease in colony formation capacity, with the magnitude of inhibition being only second to that in NAT10-knockdown cells.

---

Cell Invasion Assay[1]
Cell Types: SiHa cells and NAT10-KD SiHa cells
Tested Concentrations: 20 μM
Incubation Duration: 36 h
Experimental Results: Substantially hindered cell invasion with a significant decrease in the number of cells crossing the basement membrane.

Murine Xenograft Model (U14 cells): Female C57BL/6J mice (6-8 weeks old) were implanted subcutaneously with U14 cells (1x10^7 cells/mouse). After 7 days, mice received one of five treatments: DMSO; Remodelin (25 mg/kg); anti-PD-L1 antibody (2.5 mg/kg); NP1192 (25 mg/kg); or anti-PD-L1 antibody + NP1192. Remodelin and NP1192 were injected peritumorally every 2 days; anti-PD-L1 antibody was injected every 3 days. Tumors were measured every 2 days. After 1 week, mice were euthanized, and tumors were removed for analysis [1].
In Vivo Imaging Study: U14 cells stably expressing firefly luciferase were implanted into C57BL/6J mice. After tumor stabilization, the luciferase substrate was injected every 2 days, and fluorescence values were obtained via an in vivo imaging system. Treatment groups were the same as above [1].
Immunoflow Cytometry: After euthanasia, tumor and peritumoral tissues from the xenograft model were analyzed with an immunoflow kit using specific antibodies for various immune cell markers (CD11b, F4/80, CD206, CD86, CD4, CD25, FOXP3, CD45, LY-6G/LY-6C, etc.) [1].
Pharmacokinetic Study in Rats: Male SD rats (180-220 g) were administered NP1192 orally (10 mg/kg) or intravenously (2 mg/kg). Blood samples were collected from the tail vein at various time points (0.0833, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours). Drug concentrations were determined by LC-MS/MS [1].
Acute Toxicity Study: Six-week-old male C57BL/6J mice were administered NP1192 intraperitoneally at doses of 100, 500, and 1000 mg/kg. Mouse survival was monitored for 14 days consecutively [1].

---

Animal/Disease Models: Female C57BL/6J mice (6-8 weeks old) subcutaneously injected with U14-luc cells[1]
Doses: 25 mg/kg
Route of Administration: i.p., every 2 days for a week
Experimental Results: Significantly reduced tumor volume and tumor burden compared to DMSO at 25 mg/kg. Significantly inhibited tumor growth when combined with anti-PD-L1 antibody (2.5 mg/kg). Showed the most pronounced reduction (>80%) in tumor lactate levels when combined with anti-PD-L1 antibody (2.5 mg/kg). ¹⁸F-FDG PET–CT imaging revealed minimal metabolic uptake when combined with anti-PD-L1 antibody, suggesting effective suppresses tumor metabolic activity. Enhanced the CD8+ Teff cell population, reprograms the TME, and potentiates antitumor immunity when combined with anti-PD-L1 blockade. Did not cause significant weight loss or systemic toxicity both alone and in combination.

Rat Pharmacokinetics: In male SD rats, following oral administration (10 mg/kg) and intravenous administration (2 mg/kg) of NP1192, the drug plasma concentration-time curve was determined. Specific PK parameters (e.g., T1/2, Cmax, AUC, F%) were provided in Supplementary Table S1 of the original paper but not detailed in the main text [1].

Acute Toxicity: In a 14-day acute toxicity study, male C57BL/6J mice were administered intraperitoneal injections of NP1192 at doses of 100, 500, and 1000 mg/kg. No significant body weight changes were observed in the treated groups (Figure S2) [1].
Cytotoxicity in Normal Cells/Organoids: NP1192 exposure had a negligible impact on the viability of normal human epithelial foreskin fibroblast (HFF-1) organoids, indicating minimal cytotoxicity to nonmalignant tissues [1].
Off-Target Toxicity: The PROTAC design strategy aimed to reduce off-target toxicity compared to small-molecule inhibitors, and the favorable safety profile was corroborated by findings from in vitro and in vivo evaluations [1].

[1]. https://pubs.acs.org/action/showCitFormats?doi=10.1021/acscentsci.5c00812&ref=pdf

Mechanism of Action (MOA): NP1192 is a PROTAC that simultaneously binds to NAT10 and an E3 ubiquitin ligase (using pomalidomide as the E3 ligand), leading to ubiquitination and subsequent proteasomal degradation of NAT10. This degrader action is superior to the inhibition by Remodelin. NAT10 degradation abrogates its ac4C RNA modification activity, particularly on HIF1A mRNA, reducing its stability and translation. This leads to decreased HIF-1α protein levels, which in turn suppresses hypoxia-driven glycolysis (reduced lactate production and glucose uptake) and downregulates PD-L1 expression. The overall effect is a reversal of the immunosuppressive tumor microenvironment (TME) and reinvigoration of CD8+ effector T-cell function [1].
Synergy with Immunotherapy: NP1192 synergizes with anti-PD-L1 blockade to enhance antitumor immunity. The combination therapy reshapes the immune cell repertoire by increasing CD8+ Teff cells and M1 macrophages, while decreasing Tex cells, Tregs, and MDSCs [1].
Clinical Potential: NP1192 is positioned as a promising cancer immunotherapeutic agent with a dual metabolic-immune strategy to overcome resistance to immune checkpoint blockade (ICB) therapy in hypoxic tumors [1].

C39H42N8O5S

734.87

2966791-41-1

Typically exists as solids at room temperature

NP-1192; NP 1192

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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