RIP2 kinase
Receptor Interacting Protein 2 (RIP2) kinase.
GSK2983559 free acid RIP2 binding fluorescence polarization (FP) assay (human): IC50 = 5 nM.
RIP2 FP assay (rat): IC50 = 2 nM.
RIP2 ADP-Glo kinase activity assay (human): IC50 = 2 nM. [1]

GSK2983559 was found to be extremely selective in a screen against nonkinase targets (Eurofins, 104 receptor and ion channel binding assays, 35 enzyme and cell-based assays), inhibiting only the melatonin receptor MT3 at a concentration under 10 μM.[1].

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In a biochemical assay, GSK2983559 free acid was potent in both rat and human RIP2 binding FP assays and a human kinase activity assay (ADP-Glo) with IC50 values of 5 nM, 2 nM, and 2 nM, respectively. [1]
In HEK293 cells overexpressing NOD2, GSK2983559 free acid potently and dose-dependently inhibited MDP-stimulated IL-8 production with an IC50 of 4 nM. [1]
In primary human monocytes isolated from healthy volunteers, it inhibited MDP-stimulated TNFα production with an IC50 of 13 nM. [1]
In human whole blood (hWB), it inhibited MDP-stimulated TNFα production with an IC50 of 26 nM. This slight reduction in potency is attributed to plasma protein binding. [1]
The pathway specificity of GSK2983559 free acid was profiled in human monocytes. At 1 μM, it showed no inhibition of proinflammatory signaling upon activation of Toll-like receptors (TLR2, TLR4, TLR7) or cytokine receptors (IL-1R, TNFR). In contrast, activation of the NOD2 receptor by MDP was potently inhibited. [1]
In cultured inflamed intestinal mucosal biopsy samples from Crohn's disease (CD) or ulcerative colitis (UC) patients, GSK2983559 free acid reduced spontaneous production of proinflammatory cytokines IL-1β and IL-6 in a concentration-dependent manner. The magnitude of inhibition was comparable to that obtained with prednisolone dosed at 1 μM. The apparent potency in these explant cultures (IC50 ~ 10 nM) was comparable to that observed in MDP-stimulated human whole blood (IC50 = 26 nM). [1]
In human peripheral blood mononuclear cells (PBMCs), a selective RIP2 kinase inhibitor (compound 9, GSK214) was used to assess the effect on synergistic cytokine release following suboptimal stimulation of NOD2 in combination with TLR2. Pretreatment with the RIP2 kinase inhibitor followed by co-stimulation reduced cytokine levels to approximately those observed with TLR2 stimulation alone. Similar observations were made with TLR4, TLR5, and IL-1R. This data supports the hypothesis that RIP2 kinase inhibition blocks NOD2/TLR synergy. [1]

GSK2983559 (oral gavage; 3 and 10 mg/kg; once) substantially suppresses MDP-induced IL-6 in mice [2].

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The in vivo efficacy of the phosphate ester prodrug 3 (which delivers GSK2983559 free acid) was evaluated in a murine TNBS-induced colitis model. Mice were dosed orally with prodrug 3 at 0.25, 7.5, and 145 mg/kg b.i.d., corresponding to predicted 10%, 50%, and 90% inhibition at 24h, respectively. Administration at the 7.5 and 145 mg/kg b.i.d. doses exhibited efficacy similar to that of prednisolone as measured by summed colon scores (inflammation, edema, goblet cell depletion). These results support the hypothesis that inhibition of RIP2 kinase reduces GI inflammation associated with intestinal epithelial disruption. [1]

In a murine 2,4,6-trinitrobenzenesulfonic acid (TNBS) induced colitis model, administration of GSK2983559 at the 7.5 and 145 mg/kg b.i.d doses displays efficacy similar to that of prednisolone as measured by the sum of colon scores.[1].

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A fluorescence polarization (FP) assay was used to measure binding of GSK2983559 free acid to human and rat RIP2 kinase. The assay determined the IC50 values for binding. [1]
An ADP-Glo kinase activity assay was used to measure the inhibitory activity of GSK2983559 free acid against human RIP2 kinase. The assay determined the IC50 for inhibition of kinase activity. [1]
A kinase selectivity panel screening 300 kinases at 1 μM was performed for compound 5. It inhibited 4 kinases between 70-90% (STK10, VEGFR2, EPHA6, ABL2) and 1 kinase (VEGFR3) at >90% inhibition. [1]
A kinase selectivity panel screening 344 kinases at 10 μM was performed for the prodrug, compound 3, to understand off-target liabilities in the GI tract. VEGFR3 was inhibited by more than 90%, and an additional 11 kinases were inhibited in the range of 70-89%. [1]
Cocrystallization of GSK2983559 free acid with RIP2 kinase confirmed a type 1 binding mode in the ATP pocket, with the hinge hydrogen bond interaction of the quinazoline N1 with Met98 and the sulfone hydrogen bond interaction with Ser25 in the glycine rich loop and the interaction of the benzothiazole nitrogen with Asp164 of the DFG loop each important for binding activity. [1]

For the HEK293 cell assay, cells overexpressing NOD2 were stimulated with MDP (1 μg/mL) in the presence of varying concentrations of GSK2983559 free acid. After incubation, IL-8 production was measured. Test compound inhibition was expressed as percent inhibition of internal assay high and low controls. [1]
For the primary human monocyte assay, cells isolated from normal healthy volunteers were stimulated with MDP (1 μg/mL) in the presence of varying concentrations of GSK2983559 free acid. TNFα production was then assessed. [1]
For the human whole blood assay, blood was stimulated with MDP in the presence of varying concentrations of GSK2983559 free acid, and TNFα production was measured. [1]
For the human PBMC synergy assay, cells were isolated using a Ficoll-Paque method and resuspended in supplemented RPMI-1640. Test compounds (including a selective RIP2 inhibitor) were preincubated for 30 min before ligands (MDP for NOD2 and Pam2CSK4 for TLR2, at EC10 to EC30 ranges) were added. Plates were incubated for 4 h. Supernatants were assessed for human proinflammatory cytokines. A similar approach was used for MDP with LPS (TLR4), Flagellin (TLR5), and IL-1β. [1]
For the inflamed intestinal mucosal biopsy explant assay, biopsies from Crohn's disease or ulcerative colitis patients were cultured overnight in the presence of medium alone, prednisolone (1 μM), or indicated concentrations of GSK2983559 free acid. Cytokine concentration (IL-1β, IL-6) was assessed by immunoassay. [1]

Animal/Disease Models: C57BL/6 mice (female) were injected with MDP (100 μg) [2]
Doses: 3 and 10 mg/kg
Route of Administration: po (oral gavage); 3 and 10 mg/kg;
Experimental Results: in a dose-dependent manner Suppresses serum IL-6 levels.

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For the TNBS-induced colitis model, colitis was induced in male mice by intrarectal administration of 100 μL of TNBS (4 mg) in 50% ethanol on day 0. Animals were dosed with test article (prodrug 3) or vehicle from day -2 to day 5 via oral gavage (po). The dose levels were 0.25, 7.5, and 145 mg/kg, administered twice daily (b.i.d.). On day 5, animals were sacrificed, and the colon was excised. The colon was trimmed for histological evaluation. Tissues were scored for inflammation, edema, goblet cell depletion, and epithelial damage (0=none to 4=severe), and the scores were summed. [1]
For the tissue distribution study, prodrug 3 was infused for 4 hours (total dose 2.1 mg/kg) to rats before a range of terminal tissue samples were taken, and compound 5 concentrations were determined and compared to blood levels. [1]
For an in vivo study to define the site of cleavage of prodrug 3, GI tissue samples were assessed from rats at 15, 30, 60, and 120 minutes post-dose. Prior to collection, contents of the stomach and intestines were washed to assess levels of parent 5 and prodrug 3. Homogenized tissue samples from the stomach and intestines were also analyzed. [1]

Following oral administration of the phosphate prodrug 3, GSK2983559 free acid (parent 5) was efficiently delivered with no prodrug observed in systemic circulation in rats. [1]
Low dose PK parameters of GSK2983559 free acid following administration of prodrug 3 in rat (Sprague-Dawley, oral 2.0 mg/kg): oral Cmax 560 ng/mL, oral AUC 170 ng·h/mL, Cl 30 mL min-1 kg-1, Vdss 2.1 L/kg, T1/2 2.4 h, F 77%. [1]
Low dose PK parameters in dog (Beagle, oral 2.0 mg/kg): oral Cmax 840 ng/mL, oral AUC 4400 ng·h/mL, Cl 11 mL min-1 kg-1, Vdss 2.0 L/kg, T1/2 2.9 h, F 100%. [1]
Low dose PK parameters in minipig (Göttingen, oral 2.0 mg/kg): oral Cmax 340 ng/mL, oral AUC 1100 ng·h/mL, Cl 11 mL min-1 kg-1, Vdss 2.1 L/kg, T1/2 4.2 h, F 58%. [1]
The prodrug 3 demonstrated dose linearity in oral exposure across multiple preclinical PK species (rat and minipig) as shown in a dose linearity plot. [1]
The in vitro stability of the phosphate ester prodrug 3 was evaluated in intestinal S9 fractions and whole blood, showing rapid cleavage (T1/2 in rat intestinal S9 = 3.3 min, dog = 8.0 min, human = 18.3 min; T1/2 in rat blood = 35 min, dog = 21 min, human = 25 min). [1]
The in vitro hepatic clearance of GSK2983559 free acid in hepatocytes was low to moderate across species: rat <0.48 (mL/min)/g liver, dog 0.65 (mL/min)/g liver, minipig 0.55 (mL/min)/g liver, human <0.48 (mL/min)/g liver. [1]
GSK2983559 free acid was shown to be stable in the presence and absence of NADPH cofactor in human liver S9 fractions, suggesting it is not a substrate for aldehyde oxidase (AO). [1]
A metabolite identification study in rat, minipig, and human hepatocytes confirmed that prodrug 3 is efficiently converted to GSK2983559 free acid (5) across species, and that 5 is further metabolized via both phase I and phase II metabolic pathways (oxidation to a carboxylic acid, hydroxylation, hydrogenation or ring opening, and glucuronidation). No human-specific metabolites were detected. [1]
In a rat tissue distribution study, parent compound 5 distributed into a range of tissues including the colon and skin. Brain and ocular levels were low, likely due to active extrusion via P-glycoprotein (P-gp). The brain to blood ratio was 0.065:1. [1]
Human pharmacokinetics for GSK2983559 free acid was predicted via allometric scaling, with a predicted terminal half-life of 6 h, clearance of 7.1 mL min-1 kg-1, volume of distribution of 2.8 L/kg, and bioavailability of 70%. [1]

In a 7-day rat dose range finding study, compound 5 exhibited non-dose linear toxicokinetics, precluding adequate toxicology evaluation at the highest dose. This lack of dose linearity is believed to be a consequence of poor solubility in fasted state simulated intestinal fluid (FaSSIF: 4 μg/mL for compound 5), leading to compound precipitation in the gastrointestinal tract as dose increased. Dose-normalized AUC decreased from 0.44 to 0.21 kg·h/L as dose increased from 20 to 200 mg/kg. [1]
The blood free fraction for GSK2983559 free acid was similar across species: rat 11%, dog 8.9%, minipig 6.8%, human 6.9%. [1]
The valine ester prodrug (8) suffered from activity against the hERG ion channel (7.3 μM) and inhibition of P450 enzyme CYP3A4 (126 nM), with potential for time-dependent CYP3A4 inhibition (2.9-fold shift). The phosphate prodrug 3 did not possess either hERG or CYP3A4 activity (IC50 > 30 μM). [1]
In a screen against non-kinase targets (104 receptor and ion channel binding assays and 35 enzyme and cell-based assays), both parent 5 and prodrug 3 were highly selective, inhibiting only melatonin receptor MT3 at a concentration under 10 μM. As MT3 is primarily expressed in the brain and parent 5 has low brain penetration, this activity is unlikely to result in any pharmacology. [1]
Six-week safety assessment studies in rat and minipig with prodrug 3 provided sufficient therapeutic windows to facilitate progression into clinical trials. [1]

[1]. Discovery of a First-in-Class Receptor Interacting Protein 2 (RIP2) Kinase Specific Clinical Candidate, 2-((4-(Benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethyl Dihydrogen Phosphate, for the Treatment of Inflammat.

[2]. Design, synthesis, and structure-activity relationship of novel RIPK2 inhibitors. Bioorg Med Chem Lett. 2022 Sep 2;75:128968.

GSK2983559 free acid (compound 5) is derived from a 4-aminoquinazoline chemotype. Optimization of this series led to inhibitors 4 and 5, with compound 5 showing an IC50 of 26 nM in human whole blood MDP-stimulated TNFα production while maintaining selectivity against the hERG ion channel. [1]
A prodrug approach was envisioned to address the solubility-driven pharmacokinetic issues (DCS 2b status) of compound 5. Over 50 prodrugs were designed, and the phosphate ester (compound 3) was selected for further profiling. The prodrug possesses a weakly basic N1 nitrogen in addition to acidic hydroxyl groups in the phosphate moiety, allowing for preparation of multiple salt forms (HCl, bisodium, hemi-calcium) all with excellent FaSSIF and FeSSIF solubilities. The combination of high prodrug solubility and high parent permeability (MDCK cells, 166 nm/s for parent 5) drives rapid absorption of parent 5 across the intestinal wall. [1]
The proposed mechanism for the efficacy of a RIP2 kinase inhibitor in blocking spontaneous proinflammatory release in inflamed IBD biopsy samples may be due to effects on cross-talk and synergistic signaling between NOD2 and other PRRs like TLR2, TLR4, TLR5, and IL-1R. [1]
Based on human PK/PD predictions, prodrug 3 dosed twice daily at 103 mg or with a single dose of 745 mg is predicted to deliver parent 5 (GSK2983559 free acid) to achieve 90% inhibition over 24 h (Ctrough > 90% inhibition). [1]
GSK2983559 free acid (as the prodrug 3) was advanced into phase I clinical trials in early 2018. [1]

C21H23N4O7PS2

538.5298

538.074

C, 46.84; H, 4.31; N, 10.40; O, 20.80; P, 5.75; S, 11.91

1579965-12-0

GSK2983559;GSK2983559 active metabolite;1423186-80-4

73386708

Light yellow to yellow solid powder

1.5±0.1 g/cm3

823.6±75.0 °C at 760 mmHg

451.9±37.1 °C

0.0±3.1 mmHg at 25°C

1.675

0.55

3

12

9

35

878

0

S(C1=CC2=C(N=CN=C2C=C1OCCOP(=O)(O)O)NC1C=CC2=C(C=1)N=CS2)(C(C)(C)C)(=O)=O

MJLYDVMFNHZMLV-UHFFFAOYSA-N

InChI=1S/C21H23N4O7PS2/c1-21(2,3)35(29,30)19-9-14-15(10-17(19)31-6-7-32-33(26,27)28)22-11-23-20(14)25-13-4-5-18-16(8-13)24-12-34-18/h4-5,8-12H,6-7H2,1-3H3,(H,22,23,25)(H2,26,27,28)

2-[4-(1,3-benzothiazol-5-ylamino)-6-tert-butylsulfonylquinazolin-7-yl]oxyethyl dihydrogen phosphate

GSK2983559; GSK-2983559; GSK 2983559; GSK2983559 free acid

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)

DMSO: ~5 mg/mL (~9.3 mM)

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.

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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).

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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)

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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).

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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

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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.

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