PIKfyve (IC50 = 5.2 nM); IL-23

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].

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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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

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.

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.

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.

The PK parameters obtained after intravenous administration at 3 or 10 mg/kg and oral dosing at 30 mg/kg are shown in Figure 6. For intravenous administration, the PK parameters of initial drug concentration (C0) and area under the curve to time infinity (AUCinf) showed good linearity between the two doses, and APY0201 showed low total body clearance (CLtot = 1.0 L/h/kg) (Fig. 6a). After oral administration, heart and portal plasma concentrations were determined to assess the first-pass effect (Fig. 6b). The maximum APY0201 concentration (Cmax) value in the heart was 7.2 μM, and the plasma concentration was 3.5 μM at 6 h. The apparent fraction absorbed and availability at the liver were estimated to be 68% and 76% of the dose, respectively, which indicated a good bioavailability of 52% (Table 3). Because good bioavailability with oral administration was obtained, APY0201 was examined for its oral therapeutic efficacy for once-daily dosing at 30 mg/kg.[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 proinflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct nanoflow LC-MS/MS system and bait compounds equipped with the FLAG epitope associated regulator of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P2. These results elucidate the function of PIKfyve kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.[1]

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APY0201 is an orally-available PIKfyve kinase inhibitor that is selective for all tested kinases, GPCRs, ion channels, and enzymes, and it is a powerful tool for understanding the role of PIKfyve and PtdIns(3,5)P2 in immunological and inflammatory responses. The administration of APY0201 can be used to assess PIKfyve function in immune cells and animals with normal architecture of the PIKfyve–ArPIKfyve–Sac3 complex. Exposure to APY0201 in vitro blocked production of IL-12/23, which highlighted the effect of selective inhibition of this kinase. The data presented here indicate a unique role of PIKfyve kinase in cytokine production. Because PIKfyve inhibition blocks production of IL-12 and IL-23 from activated macrophages, APY0201 may control the cytokine modulation function of these cell types and reduce the pathological proinflammatory cytokines IL-12/23 with negligible influence on other cytokines, including TNF-α. Moreover, the therapeutic impact of PIKfyve inhibition in a mouse model of IBD included reduction in inflammation without affecting body weight. In the in vitro assay using mouse whole blood for IL-12p70-inhibitory activity, APY0201 demonstrated potent inhibitory activity against IL-12p70 production with the IC50 value of 880 nM. At the therapeutic dose (30 mg/kg daily), the mean Cmax was 7.2 μM, which is sufficient to inhibit IL-12p70 production in whole blood almost completely, and the estimated plasma concentration at 12 h is still close to 880 nM, which indicated that an appropriate amount of APY0201 was maintained to inhibit IL-12p70 production for nearly a half day (Fig. 6b). Thus, the concentration of APY0201 at this experimental dose could be consistent with the observed therapeutic effect in the IL-10−/− cell transfer colitis model.
In summary, a chemical proteomics approach using a highly sensitive DNLC–MS/MS system and bait compounds equipped with the FLAG epitope was used in this investigation, and further study demonstrated that the associated protein PIKfyve kinase was the possible target protein of APY0201, which is a selective and potent IL-12/23 production inhibitor. The data by researchers at Novartis supported these discussions. Characterization showed that APY0201 was a potent, highly selective, ATP-competitive PIKfyve kinase inhibitor that strongly inhibited IL-12/23 production in vitro and ameliorated inflammation in an experimental model of colitis. The structure–activity relationship study on APY0201, its pharmacological profile in vitro and in vivo, the strategy for target identification, and the biological characterization of PIKfyve kinase as an anti-inflammatory drug target were presented. The results from this investigation should be useful in drug discovery targeted at novel agents for treating autoimmune and inflammatory diseases. Our findings provide a new understanding of the function of PIKfyve kinase in the IL-12/23 production pathway and IL-12/23–driven inflammatory disease pathologies. In addition, these findings support the development of a selective PIKfyve kinase inhibitor as a therapeutic modality for autoimmune disorders, such as IBD.[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.)