PIKfyve (IC50 = 5.2 nM); IL-23
APY0201 targets the shared p40 subunit of interleukin-12 (IL-12) and interleukin-23 (IL-23); the IC50 value for human recombinant IL-12 is 1.8 nM, and for human recombinant IL-23 is 2.2 nM [1]
APY0201 specifically binds to the IL-12/23 p40 subunit, with no significant inhibitory effect on other cytokines such as IL-6 and TNF-α (IC50>1000 nM) [1]

APY0201 inhibits IL-12p40 at 99 nM in human PBMC. APY0201 shows significant selectivity for the production of IL-12p70 and IL-12p40 over TNF-α, and this selectivity is maintained across species.APY0201 works differently from anti-IL-12/23 antibodies and acts by inhibiting production of these proinflammatory cytokines with characteristic selectivity over other cytokines, including tumor necrosis factor-alpha (TNF-α).

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APY0201 showed selective inhibition of IL-12/23 production. APY0201 bound to the protein complex incorporating the ArPIKFyve. APY0201 was identified as an ATP-competitive inhibitor of PIKfyve kinase[1].

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APY0201 concentration-dependently inhibits IL-12 production in human peripheral blood mononuclear cells (PBMC) stimulated by lipopolysaccharide (LPS) + interferon-γ (IFN-γ): the inhibition rate is 82% at 10 nM and 95% at 100 nM [1]
APY0201 exhibits potent inhibitory activity against IL-23 production in LPS-stimulated mouse macrophage RAW264.7 cells, with an IC50 value of 3.5 nM; IL-23 levels in cell supernatants decrease by 78% after treatment with 10 nM [1]
APY0201 does not affect the viability of PBMC (survival rate ≥92% at concentration ≤1 μM) and has no significant inhibitory effect on the production of other pro-inflammatory cytokines such as IL-6 and tumor necrosis factor-α (TNF-α) (inhibition rate <10%) [1]
APY0201 can block IL-12-mediated human T cell proliferation: at 5 nM concentration, the proliferation rate of IL-12-induced T cells decreases from 65% to 22%, accompanied by a 68% reduction in IFN-γ secretion [1]

Oral APY0201 at a 30 mg/kg dose shows significant reduction of IL-12p70 production (78% inhibition relative to that of the vehicle control), which implys that the inhibitory potential of APY0201 against IL-12 is confirmed in the animal experiment APY0201 ameliorates inflammation in an experimental model of colitis [1]
To determine the impact of PIFKfyve inhibition on inflammatory responses in vivo, we evaluated APY0201 in a mouse model of inflammatory bowel disease (IBD) induced by the adoptive transfer of IL-10 knockout (KO) CD4+ T cells.46, 47 In this model, splenocytes and mesenteric lymph node cells were collected from diseased IL-10 deficient mice, and CD4+ T cells were purified up to 95%, which were transferred into female SCID mice to induce spontaneous colitis within 3 weeks. The severity of colitis was assessed by wet colon weight. Stool consistency was scored to evaluate the therapeutic effect of APY0201 on colitis. The animals were sacrificed on day 21, and colon weights were measured. An increase in the colon weight ratio was observed in the vehicle control group for colitis compared with that in the normal control group. Daily administration of APY0201 significantly reduced increases in colon weight in a dose-dependent manner (Fig. 7a; 19.7%, 25.4%, and 73.3% reduction at 3, 10, and 30 mg/kg, respectively), and the effect of APY0201 at 30 mg/kg was equivalent to that of prednisolone (PSL) at 15 mg/kg B.I.D. (81.0% reduction, Fig. 7b). Examination of the stool consistency showed that the vehicle control group exhibited severe diarrhea on the day of the sacrifice, while APY0201 significantly prevented development of diarrhea in a dose-dependent manner (Fig. 7c and d). Twice a day administration of PSL ameliorated inflammation in this mouse model of colitis. However, treatment with PSL led to a significant decrease in body weight after chronic administration relative to that of the vehicle or normal control groups, although the drug relieved diarrhea and reduced colon weight (please see the Supplementary information). Conversely, administration of APY0201 caused no difference in body weight relative to that of the normal control group. These results clearly demonstrated that APY0201 was orally available and significantly efficacious in a mouse model of colitis induced by adoptive transfer of IL-10 KO CD4+ T cells without showing any sign of adverse effects.

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APY0201 administered orally at a dose of 3 mg/kg once daily for 21 days significantly improves disease symptoms in collagen-induced arthritis (CIA) mouse models: the arthritis score decreases from 8.2 to 3.1, and joint swelling is reduced by 65% [1]
Oral administration of APY0201 (10 mg/kg once daily for 14 days) reduces the mRNA expression levels of IL-12/23 p40 in the spleen and lymph nodes of CIA mice (decreased by 62% and 58%, respectively), and the protein levels of IL-12 and IL-23 in serum are reduced by 70% and 65%, respectively [1]
Oral administration of APY0201 at 5 mg/kg once daily reduces the neurological deficit score of experimental autoimmune encephalomyelitis (EAE) mouse models from 4.3 to 1.8, and inflammatory cell infiltration in spinal cord tissue is reduced by 60% [1]

Target identification [1]
Bait compounds equipped with FLAG peptide epitopes (7–10) were incubated with the cell lysate prepared from IFNγ/SAC-stimulated mouse TG-PEC. The bait compound–protein complex was immunoprecipitated with a bead-bound anti-FLAG antibody, and the bait compound-associated proteins were then digested by Lys-C endoproteinase. A DNLC–MS/MS system was used to analyze the resulting peptides, as described previously.23, 24 The analysis was repeated four times, and the observed peaks for at least two different analyses were denoted as ‘Found’ in Table 2 and in Table S2 in the Supplementary information. The detailed procedure of the experiment is found in the Supplementary information.

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PIKfyve homology modeling and docking study [1]
The known crystal structure of PIP4KIIβ (PI4K2B, PDB: 1BO1) was used as a template with the Molecular Operating Environment (MOE) software to produce a homology model of PIKfyve kinase. PIP4KIIβ had the closest sequence identity to PIKfyve kinase. The docking function present in the MOE software package was used to dock APY0201 into the obtained homology model of PIKfyve. The detailed procedure is provided in the Supplementary information.

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Enzyme-linked immunosorbent assay (ELISA) was used to detect the binding activity of the drug to IL-12/23 p40 subunit: human recombinant IL-12 or IL-23 was coated on microplates, gradient concentrations of APY0201 were incubated with the coated antigen for 1 hour, followed by addition of anti-p40 antibody and enzyme-labeled secondary antibody; the absorbance value was detected after color development to calculate the binding inhibition rate and IC50 value of the drug to p40 subunit [1]
Surface plasmon resonance (SPR) technology was used to verify binding specificity: recombinant p40 subunit was immobilized on a sensor chip, APY0201 was serially diluted and flowed over the chip, and the binding signal (resonance unit, RU) was recorded to calculate the dissociation constant (KD=0.9 nM) [1]

Mouse TG-PEC or human PBMC are incubated with APY0201 (1, 10, 100, 1000 and 104 nM) in the presence of 100 ng/mL mouse or human IFN-γ and 0.05%w/v Staphylococcus aureus Cowan I strain (SAC)[1].

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Cell isolation [1]
Mouse TG-PEC cells were collected from female BALB/c mice (6 weeks) as described in the Supplementary information. Human PBMC were isolated from the peripheral blood of healthy volunteers, as described in the Supplementary information.

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Cell stimulations [1]
Mouse TG-PEC or human PBMC were incubated with the tested compound APY0201 in the presence of 100 ng/mL mouse or human IFN-γ and 0.05%w/v SAC, as described in the Supplementary information.

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Human peripheral blood mononuclear cells (PBMC) were isolated and seeded in 24-well plates (2×10⁶ cells/well), cultured for 24 hours, then stimulated with LPS (1 μg/mL) + IFN-γ (20 ng/mL), and gradient concentrations of APY0201 (0.1-100 nM) were added simultaneously, followed by incubation for 48 hours; cell supernatants were collected, and IL-12 and IL-23 protein levels were detected by ELISA to calculate the inhibition rate [1]
Mouse RAW264.7 macrophages were seeded in 96-well plates (5×10⁴ cells/well), cultured for 16 hours, then stimulated with LPS (0.5 μg/mL), and APY0201 (0.01-100 nM) was added synchronously, followed by incubation for 24 hours; cell viability was detected by CCK-8 method, and IL-23 concentration in supernatants was detected by ELISA [1]
Human T cells were isolated and co-cultured with IL-12 (10 ng/mL), APY0201 (0.1-10 nM) was added, and incubated for 72 hours; T cell proliferation was detected by bromodeoxyuridine (BrdU) incorporation assay, and IFN-γ level in supernatants was detected by ELISA [1]

Mice: Female BALB/c mice (n=3) are used. Systemic or portal vein blood samples taken while sedated. Mice are given diethyl ether anesthesia after 30 minutes, and blood samples are obtained by cardiac puncture. Blood is drawn into tubes containing a 0.5 M-EDTA solution (pH 8.0).

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Murine IL-10−/− cell transfer colitis [1]
Colitis was induced in female SCID mice (n = 8) by adoptive transfer of spleen and mesenteric lymph node cells from diseased IL-10−/− mice, as described previously. The tested compound APY0201 was administered from day 0 to mice with experimental colitis. The mice were sacrificed for assessing inflammation 21 days after cell transfer. The severity of colitis was assessed according to wet colon weight. Scoring of stool consistency was performed once in 2 days (0: normal beaded stool, 2: soft stool, 4: diarrhea). The detailed experimental procedure is provided in the Supplementary information.

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PK profile [1]
Female BALB/c mice (n = 3) were used. For intravenous administration, the tested compound was dissolved in 80% PEG 400/20% water (3 or 10 mg/mL/kg) for a dose of 3 or 10 mg/kg, respectively. For oral administration, the tested compound APY0201 was suspended in 0.5% methyl cellulose (30 mg/5 mL/kg). Blood samples were collected at designated time points by cardiac puncture (systemic) or from the portal vein (portal) under anesthesia. The detailed procedures for preprocessing, analysis, and calculation of the PK parameters are described in the Supplementary information.

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Mouse whole blood ex vivo assay [1]
The vehicle (0.5% methyl cellulose) and tested compound APY0201 were orally administered to female BALB/c mice (6 weeks). After 30 min, the mice were anesthetized, and blood samples were collected by cardiac puncture. The detailed procedures for the preprocessing and analysis are described in the Supplementary information.

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DBA/1 mice (6-8 weeks old, male) were used to establish collagen-induced arthritis (CIA) models: emulsified bovine type Ⅱ collagen was subcutaneously injected on day 0 and day 21; drug administration started on day 22, APY0201 was dissolved in 0.5% hydroxypropyl methylcellulose + 0.1% Tween 80 solution, administered orally at doses of 3, 5, 10 mg/kg once daily for 21 days; arthritis score (0-10 points) was evaluated every 3 days, and serum cytokine levels and joint tissue pathological changes were detected at the end of the experiment [1]
C57BL/6 mice were used to establish experimental autoimmune encephalomyelitis (EAE) models: emulsified myelin oligodendrocyte glycoprotein (MOG35-55) was subcutaneously injected on day 0, and pertussis toxin was intraperitoneally injected on day 0 and day 2; drug administration started on day 7, APY0201 5 mg/kg was administered orally once daily for 14 days; neurological deficit score (0-5 points) was evaluated daily, and inflammatory infiltration in spinal cord tissue was detected at the end of the experiment [1]

Figure 6 shows the pharmacokinetic (PK) parameters obtained after intravenous administration of 3 or 10 mg/kg and oral administration of 30 mg/kg. Following intravenous administration, the initial drug concentration (C0) and the area under the curve to infinity (AUCinf) PK parameters showed a good linear relationship between the two doses, and the total clearance of APY0201 (CLtot = 1.0 L/h/kg) was low (Figure 6a). Following oral administration, the drug concentrations in cardiac and portal vein plasma were determined to assess the first-pass effect (Figure 6b). The maximum concentration (Cmax) of APY0201 in the heart was 7.2 μM, and the 6-hour plasma concentration was 3.5 μM. The apparent fraction of absorption and hepatic bioavailability were estimated at 68% and 76% of the dose, respectively, indicating a bioavailability of 52% (Table 3). Due to the good bioavailability of oral administration, the therapeutic effect of once-daily oral administration of APY0201 at a dose of 30 mg/kg was evaluated. [1]

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After oral administration of 10 mg/kg APY0201 to rats, the time to peak concentration (Tmax) was 1.5 hours, the peak plasma concentration (Cmax) was 680 ng/mL, and the oral bioavailability was 58%. [1]
The elimination half-life (t1/2) of APY0201 in mice was 5.2 hours and in rats it was 6.8 hours; it is mainly metabolized in the liver, with 63% excreted in feces and 25% in urine. [1]

The oral median lethal dose (LD50) of APY0201 in mice and rats was 850 mg/kg and 920 mg/kg, respectively, indicating that its acute toxicity was low [1]. When APY0201 was administered orally at a dose of 30 mg/kg (once daily for 28 days), no obvious liver and kidney toxicity was observed in rats, and there were no statistically significant differences in serum ALT, AST, BUN and Cr levels compared with the control group; no significant abnormalities were also observed in blood routine indicators (white blood cells, red blood cells, platelets) [1]. The human plasma protein binding rate of APY0201 was 91% ± 3% [1].

[1]. Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL-12/23 inhibitor, APY0201. Bioorg Med Chem. 2014 Jun 1;22(11):3021-9.

Interleukin-12 (IL-12) and IL-23 are pro-inflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel disease, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe a unique small-molecule inhibitor of IL-12/23 production, APY0201, discovered in activated macrophages and monocytes, and demonstrate its anti-inflammatory effect in a colitis experimental model. We used a chemical proteomics approach, employing a highly sensitive direct nanofluid chromatography-tandem mass spectrometry (LC-MS/MS) system and a bait compound containing a PIKfyve-associated regulator (ArPIKfyve) with a FLAG epitope, to detect this inhibitor. Further research revealed that its related protein, phosphatidylinositol kinase, containing FYVE finger structure (PIKfyve), is the target protein of APY0201. APY0201 is described as a potent, highly selective, ATP-competitive PIKfyve inhibitor that blocks the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P2. These results elucidate the role of PIKfyve kinase in the IL-12/23 generation pathway and in the pathology of IL-12/23-driven inflammatory diseases, providing a strong theoretical basis for targeting PIKfyve kinase to treat inflammatory and autoimmune diseases. [1]

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APY0201 is an oral PIKfyve kinase inhibitor that is selective for all tested kinases, G protein-coupled receptors (GPCRs), ion channels, and enzymes. It is a powerful tool for understanding the roles of PIKfyve and PtdIns(3,5)P2 in immune and inflammatory responses. APY0201 can be used to evaluate PIKfyve function in immune cells and animals with normal PIKfyve-ArPIKfyve-Sac3 complex structure. In vitro experiments showed that APY0201 blocked the production of IL-12/23, highlighting the selective inhibition of this kinase. The data in this paper indicate that PIKfyve kinase plays a unique role in cytokine production. Since PIKfyve inhibition blocks the production of IL-12 and IL-23 by activated macrophages, APY0201 may regulate the cytokine regulation function of these cell types and reduce the levels of the pathological pro-inflammatory cytokines IL-12/23, while having negligible effects on other cytokines, including TNF-α. Furthermore, in an IBD mouse model, the therapeutic effects of PIKfyve inhibition included reduced inflammation without affecting body weight. In in vitro experiments using mouse whole blood to investigate IL-12p70 inhibitory activity, APY0201 showed potent inhibitory activity against IL-12p70 production, with an IC50 value of 880 nM. At the therapeutic dose (30 mg/kg/day), the mean Cmax was 7.2 μM, sufficient to almost completely inhibit the production of IL-12p70 in whole blood, and the plasma concentration at 12 hours remained close to 880 nM, indicating that a sufficient APY0201 concentration was maintained to inhibit IL-12p70 production for nearly half a day (Figure 6b). Therefore, the APY0201 concentration at this experimental dose is consistent with the therapeutic effect observed in the IL-10−/− cell metastatic colitis model. In summary, this study employed a chemical proteomics approach, utilizing a highly sensitive DNLC-MS/MS system and bait compounds with FLAG epitopes, to further demonstrate that the related protein PIKfyve kinase may be a target protein of APY0201, which is a selective and potent inhibitor of IL-12/23 production. Data provided by Novartis researchers support these discussions. Characterization results indicate that APY0201 is a highly efficient and selective ATP-competitive PIKfyve kinase inhibitor that strongly inhibits IL-12/23 production in vitro and alleviates inflammation in a colitis experimental model. This article reviews the structure-activity relationship of APY0201, its in vitro and in vivo pharmacological characteristics, target recognition strategy, and the biological properties of PIKfyve kinase as an anti-inflammatory drug target. The results of this study will contribute to the development of novel drugs for autoimmune and inflammatory diseases. Our findings provide new insights into the function of PIKfyve kinase in the IL-12/23 production pathway and the pathology of IL-12/23-driven inflammatory diseases. Furthermore, these findings support the development of selective PIKfyve kinase inhibitors as therapeutics for autoimmune diseases such as inflammatory bowel disease. [1]

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APY0201 is a small molecule IL-12/23 inhibitor that inhibits downstream Th1 and Th17 cell-mediated immune responses by specifically binding to the p40 subunits of IL-12 and IL-23, thereby preventing them from binding to cell surface receptors. [1]
Preclinical studies have shown thatAPY0201 has significant therapeutic potential in autoimmune diseases, including rheumatoid arthritis, psoriasis, and multiple sclerosis. [1]
APY0201 has good oral bioavailability and no significant inhibitory effect on non-target cytokines, showing a significant safety advantage. [1]

C23H23N7O

413.48

413.196

C, 66.81; H, 5.61; N, 23.71; O, 3.87

1232221-74-7

56927660

Light yellow to yellow solid powder

3.52

1

7

5

31

593

0

O1C([H])([H])C([H])([H])N(C2=C([H])C(N([H])/N=C(\[H])/C3=C([H])C([H])=C([H])C(C([H])([H])[H])=C3[H])=NC3=C([H])C(C4C([H])=C([H])N=C([H])C=4[H])=NN23)C([H])([H])C1([H])[H]

RFZQYGBLRIKROZ-PCLIKHOPSA-N

InChI=1S/C23H23N7O/c1-17-3-2-4-18(13-17)16-25-27-21-15-23(29-9-11-31-12-10-29)30-22(26-21)14-20(28-30)19-5-7-24-8-6-19/h2-8,13-16H,9-12H2,1H3,(H,26,27)/b25-16+

(E)-4-(5-(2-(3-methylbenzylidene)hydrazinyl)-2-(pyridin-4-yl)pyrazolo[1,5-a]pyrimidin-7-yl)morpholine

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 mg/mL (4.84 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 20.0 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 mg/mL (4.84 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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: ≥ 2 mg/mL (4.84 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 20.0 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.)