RIP1 kinase (IC50 = 6.3 nM)
GSK3145095 has excellent activity in blocking RIP1 kinasedependent cellular responses and potently binds to RIP1 with exquisite kinase specificity. The inhibitor is also capable of encouraging a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures, highlighting its potential as a novel cancer therapy. [1]

GSK3145095 has excellent activity in blocking RIP1 kinasedependent cellular responses and potently binds to RIP1 with exquisite kinase specificity. The inhibitor is also capable of encouraging a tumor suppressive T cell phenotype in pancreatic adenocarcinoma organ cultures, highlighting its potential as a novel cancer therapy. [1]

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GSK3145095 potently inhibits the RIP1 kinase-dependent necroptosis pathway in human monocytic U937 cells (IC50 = 6.3 nM) [1].
In primary human neutrophils isolated from whole blood stimulated with TNF, caspase inhibitor (QVD-Oph), and SMAC mimetic (RMT 5265), GSK3145095 potently blocked cellular responses: inhibition of overall cell viability (measured by ATP levels, IC50 = 1.6 nM), inhibition of cell death (measured by LDH release, IC50 = 0.5 nM), and inhibition of RIP1-dependent inflammatory cytokine MIP-1β production (measured at protein level or mRNA expression, IC50 = 0.4 nM) [1].
In a human whole blood stimulation assay (TNF + QVD-Oph/zVAD.fmk + RMT 5265), GSK3145095 inhibited cytokine MIP-1β production with an IC50 of 5 nM. In a similar cynomolgus monkey whole blood assay, the IC50 was 16 nM [1].
GSK3145095 demonstrated a high degree of kinase selectivity. When tested at 10 µM against 359 kinases in a P33 radiolabeled assay and 456 kinases in a competition binding assay (KINOMEscan), it showed no inhibition of any kinase other than RIP1, representing a >1500-fold selectivity window based on its RIP1 potency [1].
GSK3145095 exhibited significantly reduced potency against non-primate RIP1. It was over 380-fold less potent in biochemical assays against non-primate RIP1 compared to primate RIP1. Correspondingly, in cellular assays, it showed a 340-fold reduction in potency for blocking necrotic death in mouse L929 cells (IC50 = 1.3 µM) compared to human U937 cells (IC50 = 6.3 nM) [1].
In ex vivo studies using patient-derived organotypic spheroids (PDOTS) from pancreatic adenocarcinoma, colorectal, breast, and gastric cancers, treatment with 0.5 nM GSK3145095 for 3 days led to a significant increase in effector-memory T cells (CD44+) and immunogenic CD4+ T cells (IFNγ+) compared to vehicle-treated samples, as analyzed by flow cytometry. A trend towards increased CD8+ cytolytic T cells and TNFα expression was also observed [1].

GSK3145095 also increased the survival rate (50%), but less than RI-962. Treatment with RI-962 or GSK3145095 remarkably reduced the TNFα-induced temperature loss (Fig. 8b) and the concentrations of proinflammatory cytokines (IL-1β and IL-6) in mice [2].
DSS treatment led to a rapid loss in mouse body weight from day 5 to day 11, and treatment with RI-962 or GSK3145095 strongly ameliorated this loss of body weight. Further, treatment with RI-962 or GSK3145095 markedly reduced the DSS-induced shortening of colon length (Fig. 9b, c). Histopathological analysis showed that RI-962 substantially decreased tissue damage in the colons of DSS-treated mice (Fig. 9d). In DSS-induced colitis, numerous S100a9-positive cells (a marker of inflammation) infiltrated into the mucosa and epithelial layer of the damaged colon (Fig. 9e), while no infiltration by S100a9-positive cells was observed in the colons of mice treated with RI-962 (Fig. 9e). More importantly, treatment with RI-962 or GSK3145095 dramatically increased the survival rate of DSS-treated mice (Fig. 9f; 40 mg/kg RI-962 or GSK3145095 survival rate, 100% vs vehicle: 16.7%) [2].

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The reduced potency of GSK3145095 against mouse RIP1 precluded its evaluation in rodent oncology models. Therefore, no in vivo efficacy data in animal models are reported in this literature [1].
Human in vivo predictions were made based on pharmacokinetic/pharmacodynamic (PK/PD) modeling. Doses of 273 mg once daily or 40 mg twice daily were predicted to maintain blood concentrations above the human whole blood IC90 for 24 hours, achieving 90% RIP1 inhibition [1].

ADP-Glo Activity Assay. [1]
The catalytic activity of RIP1 was quantified utilizing the Promega ADP-Glo kinase kit as previously described (Harris et al, 2016) using either a four-parameter curve fit or a tight binding curve fit for compounds whose potency was less than the detection limit of the assay (~ half the enzyme concentration). Data are presented as the mean IC50 from at least n=2 determinations[1].

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Compound 6 (GSK3145095) kinase selectivity and species selectivity profiles [1]
Percent Enzyme Inhibition against Reaction Biology Corporation (RBC) kinase Panel [1]
Compound 6 was tested at 10 M in duplicate against 359 kinases in the Reaction Biology Corporation (RBC) kinase panel.Control compound was tested in 10-dose IC50 mode with 3-fold serial dilution starting at 20 μM . Reactions were carried out at 10 μM ATP. Full protocol details are available at http://www.reactionbiology.com. Data is reported as % enzyme activity (relative to DMSO controls) in Table S1. Activity <50% (average of n =2) was observed for ABL1, ABL2/ARG, BLK, c-Src, DDR2, EPHA5, EPHB2, FGR, FRK/PTK5, FYN, LYN, LYN B, PEAK1 and YES/YES1. However full curve analysis for these 14 kinases against compound 6 at a top concentration of 30 M (see Table S2) found no inhibition indicating the initial single concentration findings were false positives. The kinase panel did not include RIP1.

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A biochemical ADP-Glo assay was used to measure RIP1 kinase inhibition potency, yielding an IC50 of 6.3 nM for GSK3145095 [1].
Kinase selectivity profiling was conducted using two independent screening platforms: a P33 radiolabeled assay screen against 359 kinases and a competition binding assay (KINOMEscan) against 456 kinases. GSK3145095 was tested at a concentration of 10 µM in both assays [1].
Binding kinetics were measured. The on-rate constant (k_on) was determined by stopped-flow kinetics to be 2.5 x 10^4 M^-1 s^-1. The off-rate constant (k_off) was measured by fluorescence polarization competitive binding to be 1.2 x 10^-4 s^-1, corresponding to a dissociation half-life (t_1/2) of 99 minutes [1].
The compound exhibited ATP-competitive inhibition, as it occupies the region where the α-phosphate of ATP binds, making binding mutually exclusive [1].

GSK3145095 is prepared in assay buffer, serially diluted 1:1.5 in a 22 point titration (high final concentration 3 μM), and added to a 384 white low volume Greiner plate. In assay buffer, 3.5 μL of each inhibitor concentration and 3.5 L of final 25 nM enzyme concentration are added to the plate. Following these additions, 3.5 μL of ATP (15.6 μM to 875 μM final) in assay buffer is added to the plate to start the reaction. At room temperature, the reaction proceeds for five hours.

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Cellular RIP1 inhibition was assessed in human monocytic U937 cells. The IC50 for blocking RIP1 kinase-dependent responses was 6.3 nM [1].
Primary human neutrophil assay: Neutrophils were isolated from human whole blood and treated with TNF, the caspase inhibitor QVD-Oph, and the SMAC mimetic RMT 5265 to drive the necrosis pathway in the presence or absence of GSK3145095. After incubation, multiple readouts were measured: overall cell viability using a cellular ATP assay, cell death by lactate dehydrogenase (LDH) release, and production of the inflammatory cytokine MIP-1β (both protein level and mRNA expression) [1].
Human and cynomolgus monkey whole blood stimulation assays: Whole blood was stimulated with TNF, caspase inhibitor (QVD-Oph or zVAD.fmk), and SMAC mimetic (RMT 5265). After incubation with GSK3145095, the production of cytokine MIP-1β was measured to determine inhibitory potency [1].
Mouse fibrosarcoma L929 cell assay: This assay was used to assess potency against mouse RIP1. Cells were treated to induce necrotic death, and the inhibitory effect of GSK3145095 was measured, yielding an IC50 of 1.3 µM [1].
Permeability and P-glycoprotein (P-gp) substrate evaluation was performed using relevant cell-based assays. GSK3145095 showed moderate cell permeability (6.7 x 10^-6 cm/s) and was identified as a substrate for the efflux transporter P-gp, which likely contributes to its low brain penetration [1].

The DSS-induced IBD experiment[2]
DSS (3% w/v) was administered in drinking water ad libitum for 7 d (from day 0 to day 7). DSS solution was replaced three times on day 2, day 4, and day 6. C57BL/6 female mice were injected intraperitoneally with vehicle, RI-962 (40 mg/kg), or GSK3145095 (40 mg/kg) for 10 d (from day 0 to day 9). Three mice in each group were killed at random on day 7, and distal colon tissues were collected for analysis. The mice weight and survival rate were recorded daily.

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Pharmacokinetic studies were conducted in rats, dogs, and cynomolgus monkeys. For intravenous (iv) administration, GSK3145095 was formulated in a vehicle containing 20% Cavitron and 5% DMSO in PBS. Doses were 1.0 mg/kg (rat), 1.1 mg/kg (dog), and 0.93 mg/kg (monkey). Blood samples were collected at various time points to determine pharmacokinetic parameters [1].
For oral (po) administration, GSK3145095 was formulated in a vehicle containing 6% Cavitron and 5% DMSO in PBS. Doses were 2.1 mg/kg (rat), 2.0 mg/kg (dog), and 1.9 mg/kg (monkey). Blood samples were collected to determine T_max, C_max, AUC, and bioavailability [1].
A rat tissue distribution study was conducted following a 4-hour intravenous infusion. Tissues (brain, liver, kidney, colon, heart, skin) were collected and drug concentrations were measured and compared to blood concentrations [1].

GSK3145095 showed low to moderate clearance in preclinical animal models: 27 ± 5 mL/min/kg in rats (35% of hepatic blood flow), 9.8 ± 1.8 mL/min/kg in dogs, and 6.4 ± 0.5 mL/min/kg in monkeys [1]. The steady-state volume of distribution (Vd_ss) was moderate: 1.8 ± 0.3 L/kg in rats, 1.1 ± 0.2 L/kg in dogs, and 1.8 ± 0.1 L/kg in monkeys [1]. The terminal half-life (t1/2) was 2.2 ± 0.8 h in rats (intravenous injection), 1.7 ± 0.2 h in dogs (intravenous injection), and 4.2 ± 0.6 h in monkeys (intravenous injection) [1].
Oral bioavailability was high in different animal models: 84 ± 8% in rats, 78 ± 3% in dogs, and 88 ± 13% in monkeys [1].
Free fractions of plasma concentrations were 13% in rats, 12% in dogs, 12% in monkeys, and 8.1% in humans [1].
In vitro metabolic stability in rat hepatocytes was moderate, while metabolic stability in monkey and human hepatocytes was low [1].
Metabolite identification studies in rat, monkey, and human hepatocytes showed that GSK3145095 can be metabolized via phase I (hydroxylation) and phase II (glucuronidation) pathways. Trace amounts of glutathione (GSH) conjugates were detected in rat and monkey hepatocyte incubation media, but not in human hepatocyte incubation media. No human-specific metabolites were identified [1].
Tissue distribution in rats showed that, compared with blood, GSK3145095 had higher concentrations in the liver and kidneys (7-8 times), comparable concentrations in the colon, heart, and skin (0.7-3 times), and lower brain permeability (only 6% of blood concentration) [1].
Based on allometric growth ratios and in vitro-in vivo extrapolation, the predicted human pharmacokinetic parameters for GSK3145095 include: high bioavailability, low clearance, moderate volume of distribution, and a terminal half-life of approximately 3.3 hours [1].

[1]. ACS Med Chem Lett. 2019 May 9;10(6):857-862.

[2]. Generative deep learning enables the discovery of a potent and selective RIPK1 inhibitor. Nat Commun . 2022 Nov 12;13(1):6891.

GSK3145095, an oral small molecule receptor-interacting serine/threonine protein kinase 1 (RIPK1; receptor-interacting protein 1; RIP1) inhibitor, possesses potential antitumor and immunomodulatory activities. Upon administration, GSK3145095 blocks RIPK1-mediated signaling pathways, thereby reducing the recruitment and migration of CXC motif chemokine ligand 1 (CXCL1)-driven immunosuppressive myeloid-derived suppressor cells (MDSCs) in the tumor microenvironment (TME). This enables effector cells, such as natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), to kill and eliminate cancer cells. RIPK1, a serine/threonine kinase, typically plays a crucial role in inflammation and cell death induced by tissue damage and pathogen recognition. It is overexpressed in certain cancer types and may be associated with the promotion of tumorigenesis and the immunosuppressive properties of the tumor microenvironment (TME).

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GSK3145095 (Compound 6) is a clinical candidate RIP1 kinase inhibitor with the chemical name (S)-5-benzyl-N-(7,9-difluoro-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azapyrrol-3-yl)-1H-1,2,4-triazol-3-carboxamide[1].
It is a type III kinase inhibitor, and its cocrystal structure (PDB: 6RLN) confirms that it binds to the allosteric lipophilic pocket behind the RIP1 ATP binding site. Key interactions include hydrogen bonds between the amide carbonyl group and the Asp156 backbone NH, water-mediated hydrogen bonds between the triazole nitrogen and the Met67 carbonyl oxygen, and potentially long hydrogen bonds between the lactam nitrogen and the Leu90 backbone carbonyl group[1].
This compound was discovered and optimized from a DNA-encoded compound library screening. Due to differences in safety, tolerability, and dosage requirements, GSK3145095 was specifically developed for oncology, unlike the RIP1 inhibitor GSK2982772 (compound 3) for inflammatory diseases [1].
GSK3145095 was selected for clinical development due to its potent RIP1 inhibitory activity, superior kinase selectivity, and favorable preclinical pharmacokinetics and development potential [1].
At the time of this publication, GSK3145095 is undergoing a Phase I clinical trial for the treatment of pancreatic adenocarcinoma and other specific solid tumors [1].
The rationale for its use in treating pancreatic cancer is that studies have shown that RIP1 kinase inhibition can reverse macrophage-mediated adaptive immune tolerance in the tumor microenvironment, promote the formation of tumor suppressor T cell phenotypes, and make tumors more sensitive to immune checkpoint blockade therapy [1].

C20H17F2N5O2

397.378090620041

397.135

C, 60.45; H, 4.31; F, 9.56; N, 17.62; O, 8.05

1622849-43-7

1622849-43-7

118557502

White to off-white solid powder

3.1

3

6

4

29

603

1

C1CC2=C(C(=CC(=C2)F)F)NC(=O)[C@H]1NC(=O)C3=NNC(=N3)CC4=CC=CC=C4

ATQAGKAMBISZQM-HNNXBMFYSA-N

InChI=1S/C20H17F2N5O2/c21-13-9-12-6-7-15(19(28)25-17(12)14(22)10-13)23-20(29)18-24-16(26-27-18)8-11-4-2-1-3-5-11/h1-5,9-10,15H,6-8H2,(H,23,29)(H,25,28)(H,24,26,27)/t15-/m0/s1

5-benzyl-N-[(3S)-7,9-difluoro-2-oxo-1,3,4,5-tetrahydro-1-benzazepin-3-yl]-1H-1,2,4-triazole-3-carboxamide

GSK3145095; GSK-3145095; GSK 3145095; 1622849-43-7; UNII-B4D3WPS7JY; B4D3WPS7JY; GSK-3145095; 5-benzyl-N-[(3S)-7,9-difluoro-2-oxo-1,3,4,5-tetrahydro-1-benzazepin-3-yl]-1H-1,2,4-triazole-3-carboxamide; CHEMBL4452233; (S)-5-benzyl-N-(7,9-difluoro-2-oxo-2,3,4,5-tetrahydro-1H-benzo[b]azepin-3-yl)-4H-1,2,4-triazole-3-carboxamide;

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: 250~79 mg/mL (~198.8 mM)
Ethanol: ~6 mg/mL (~15.1 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.23 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 20.8 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: ≥ 2.08 mg/mL (5.23 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 20.8 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: ≥ 2.08 mg/mL (5.23 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 20.8 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.)