Diacylglycerol Kinase: R59022 inhibits diacylglycerol kinase in human platelets. At 10 μM, it inhibits thrombin-induced phosphatidic acid formation by approximately 40%. At 100 μM, approximately 80% inhibition is observed. [2]

59-022 (1 minute, 10 μM) strengthens the aggregation [2]. R 59-022 (30 uM, 0–60 minutes) causes basophils to release more normetins [3]. In HeLa and U87 cells, R 59-022 (40 uM, 30 minutes) activates PKC [4]. In Vero cells, R 59-022 (0-10 uM, 4 hours) bursts EBOV [5].

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Inhibition of Phosphatidic Acid Formation: R59022 (1-100 μM) concentration-dependently inhibited thrombin (1 unit/mL)-induced [³²P]phosphatidic acid formation in human platelets. At 10 μM, inhibition was approximately 40% (p < 0.05). At 100 μM, inhibition was approximately 80% (p < 0.01). R59022 alone (100 μM) had no significant effect on resting PA content. [2]
Potentiation of Platelet Aggregation: In 9 out of 10 experiments, R59022 (10 μM) potentiated aggregation induced by sub-maximal concentrations of thrombin (0.03-0.1 unit/mL). The compound did not affect shape change responses. [2]
Potentiation of ATP Secretion: In experiments with luciferin-luciferase reagent, R59022 (10 μM) potentiated the secretion of ATP from thrombin-stimulated platelets. [2]
Potentiation of 5-Hydroxytryptamine Secretion: R59022 (10 μM) significantly shifted the thrombin dose-response curve for [³H]5-hydroxytryptamine secretion to the left (p < 0.05), indicating potentiation of dense granule secretion. [2]
Increased 40kDa Protein Phosphorylation: R59022 (10 μM) significantly increased [³²P]phosphorylation of the 40kDa protein (a protein kinase C substrate) by 338% (p < 0.01) in platelets stimulated with threshold thrombin concentrations. [2]
No Effect on 20kDa Protein Phosphorylation: R59022 (10 μM) had no significant effect on [³²P]phosphorylation of the 20kDa protein (myosin light chain) in thrombin-stimulated platelets. [2]
Increased Diacylglycerol Formation: R59022 (10 μM) significantly increased [³H]diacylglycerol formation in platelets stimulated with thrombin (0.1 unit/mL), from 1035 ± 53 to 1356 ± 116 (p < 0.05). [2]
No Effect on Inositol Phosphate Formation: R59022 (10 μM) had no significant effect on thrombin (1 unit/mL)-induced [³H]inositol trisphosphate formation (380 ± 81 vs. 355 ± 67). [2]
No Effect on Intracellular Calcium Mobilization: R59022 (10 μM) had no significant effect on the dose-response curve for thrombin-induced intracellular Ca²⁺ mobilization, measured by fura2 fluorescence, in either the presence or absence of extracellular Ca²⁺. [2]
Minimal Effect on A23187-Induced Activation: In 3 out of 4 experiments, R59022 (10 μM) had no effect on secretion or aggregation induced by threshold concentrations of the calcium ionophore A23187 in the presence of indomethacin. In one experiment, slight potentiation was observed, but much less than with thrombin. [2]
Morphological Changes at High Concentrations: At concentrations above 10 μM, R59022 caused a decrease in light transmission reminiscent of thrombin-stimulated shape change, raising concerns about specificity at higher concentrations. [2]

The median survival of SCID mice transplanted with U87 GBM cells was significantly increased by R 59-022 (2 mg/kg, intraperitoneal injection, 12 days) [6].

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Purified DGKα Kinase Assay: FLAG-DGKα was affinity-purified from HeLa cells overexpressing the enzyme. Liposomes were prepared with PC, DAG (dioleoyl), and PS (55:5:40 mol%) by drying lipids in chloroform, hydrating in buffer (50 mM MOPS pH 7.5, 100 mM NaCl, 5 mM MgCl2), freeze-thaw cycling, and extrusion through 100 nm polycarbonate filters. R59022 was incorporated into liposomes by adding to lipids in chloroform at 0.5 or 2.0 mol% before drying. Kinase reactions contained buffer B, 1 mM CaCl2, 1 mM DTT, purified enzyme, 4.75 mM lipids, and were initiated with 10 μL of 10 mM [γ-32P]ATP (final volume 100 μL). Reactions proceeded for 15 min at 25°C. Activity was measured as incorporation of 32P into PA. [4]
DGK Activity in Cell Homogenates: HEK 293T cells were transiently transfected with FLAG-tagged DGK isoenzymes. After 48 h, cells were harvested and homogenized. Kinase assays contained buffer B, 1 mM DTT, 2 mM lipids (PC:DAG:PS liposomes with indicated DAG species), and 5 μg protein from homogenate. For DGKα and DGKβ, 1 mM CaCl2 was added. Reactions were initiated with [γ-32P]ATP. [4]
5-HTR Binding Assays: Radioligand competition binding assays were performed by the NIMH Psychoactive Drug Screening Program. Primary screen used 10 μM compound to determine % inhibition of radioligand binding. For receptors with significant inhibition, Ki values were determined in secondary assays using the Cheng-Prusoff equation. Functional assays (agonist/antagonist) were performed to determine mode of action. [4]

Western Blot Analysis [4]
Cell Types: HeLa Cell
Tested Concentrations: 40 uM
Incubation Duration: 30 minutes
Experimental Results: Phosphorylation of PKC downstream targets increased approximately 2.5-fold.

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Platelet Preparation: Blood from drug-free volunteers was collected with sterile citrate anticoagulant. Platelet-rich plasma was obtained by centrifugation at 200g for 20 min. Platelets were separated by centrifugation at 800g for 10 min in the presence of prostacyclin. Platelets were resuspended in modified Tyrode buffer (135 mM NaCl, 12 mM NaHCO₃, 2.8 mM KCl, 0.35 mM NaH₂PO₄, 1 mM MgCl₂, 5 mM Hepes, 5 mM glucose, pH 7.3). For labeling experiments, platelets were incubated with [³²P]Pi (1 mCi/3 mL) for 1 h, [³H]inositol (50 μCi/mL) for 3 h, [³H]5-hydroxytryptamine (10 μCi) for 45-60 min, [³H]arachidonic acid (50 μCi) for 45-60 min, or fura2 AM (6 μM) for 45-60 min in platelet-rich plasma before centrifugation. All experiments were performed in the presence of indomethacin (10 μM). [2]
Aggregation and Secretion Studies: Experiments were carried out in a Chronolog Lumi-aggregometer (0.5 mL final volume, stirring, 37°C). R59022 or solvent was added 60 seconds before thrombin or A23187, and platelets were left for a further 60 seconds. For ATP secretion measurements, luciferin-luciferase reagent (0.2 mg) was included. Light transmission was recorded throughout. [2]
Phosphatidic Acid and Protein Phosphorylation: For [³²P]-labeled platelets, reactions were terminated by taking a 50 μL sample for protein phosphorylation analysis (SDS-PAGE, 11% gel), and the remaining portion was transferred to 1.8 mL chloroform/methanol (1:2 v/v) for PA measurement by TLC. [2]
Inositol Phosphates and Diacylglycerol: For [³H]inositol- or [³H]arachidonic acid-labeled platelets, reactions were terminated by transfer to 1.8 mL chloroform/methanol/HCl (50:100:1 by vol.). Inositol phosphates were separated by Dowex anion-exchange chromatography. [³H]DG was measured by TLC, identified by co-chromatography with DG prepared from phosphatidylinositol incubated with platelet phospholipase C. [2]
5-Hydroxytryptamine Secretion: [³H]5-hydroxytryptamine secretion was measured as previously described. [2]
Calcium Measurements: Fura2 studies were carried out in a Perkin-Elmer single-beam spectrofluorimeter (model LS-3) at 37°C with constant stirring. Excitation at 350 or 380 nm, emission at 510 nm. Intracellular Ca²⁺ concentrations were estimated by the ratio method or by single wavelength readings as described by Grynkiewicz et al. [2]

Animal/Disease Models: SCID (severe combined immunodeficient) mouse implanted with U87 GBM cells [6]
Doses: 10 mg/kg
Route of Administration: intraperitoneal (ip) injection
Experimental Results: Median survival increased and tumor volume diminished.

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Bone Marrow-Derived Macrophage Isolation:** Mice were euthanized, tibia and femur bones collected, centrifuged to obtain bone marrow cells. Cells were filtered, differentiated into macrophages in 20% L929-conditioned media for 7 days. All animal procedures approved by University of Ottawa Animal Care Committee. No in vivo drug dosing studies were performed. [5]

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Bone Marrow-Derived Macrophage Isolation: Mice were euthanized, tibia and femur bones collected, centrifuged to obtain bone marrow cells. Cells were filtered, differentiated into macrophages in 20% L929-conditioned media for 7 days. All animal procedures approved by University of Ottawa Animal Care Committee. No in vivo drug dosing studies were performed. [5]

Specificity Concerns at High Concentrations: At concentrations above 10 μM, R59022 caused a decrease in light transmission reminiscent of thrombin-stimulated shape change, raising concerns about non-specific effects or loss of selectivity at higher concentrations. For this reason, 10 μM was used as the maximum concentration for functional studies. [2]

[1]. R 59 022, a diacylglycerol kinase inhibitor. Its effect on diacylglycerol and thrombin-induced C kinase activation in the intact platelet. J Biol Chem. 1985 Dec 15;260(29):15762-70.

[2]. A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets. Biochem J. 1987 May 1;243(3):809-13.

[3]. Influence of phorbol esters, and diacylglycerol kinase and lipase inhibitors on noradrenalinerelease and phosphoinositide hydrolysis in chromaffin cells. Br J Pharmacol. 1990 Nov;101(3):521-6.

[4]. Dual activities of ritanserin and R59022 as DGKα inhibitors and serotonin receptor antagonists. Biochem Pharmacol. 2017 Jan 1;123:29-39.

[5]. A Diacylglycerol Kinase Inhibitor, R-59-022, Blocks Filovirus Internalization in Host Cells. Viruses. 2019 Mar 1;11(3). pii: E206.

[6]. Diacylglycerol kinase α is a critical signaling node and novel therapeutic target in glioblastoma and other cancers. Cancer Discov. 2013 Jul;3(7):782-97.

6-[2-[4-[(4-fluorophenyl)-phenylmethylene]-1-piperidinyl]ethyl]-7-methyl-5-thiazo[3,2-a]pyrimidinone is a diarylmethane.

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Background: R59022 is a diacylglycerol kinase inhibitor. It was used in this study to investigate the role of protein kinase C and diacylglycerol in platelet activation by thrombin. By inhibiting DG kinase, it prevents the metabolism of DG to phosphatidic acid, leading to accumulation of DG and enhanced activation of protein kinase C. [2]
Mechanism of Action (Rationale): The study hypothesized that if DG is a second messenger in platelet activation, inhibiting its metabolism should potentiate platelet responses to sub-maximal thrombin stimulation, analogous to methylxanthine potentiation of cAMP-mediated responses. The results support this hypothesis. [2]
Evidence Against PA as Second Messenger: The study provides evidence against phosphatidic acid having a second messenger role in platelets, as R59022 inhibited PA formation without affecting Ca²⁺ mobilization, while potentiating functional responses. [2]
Selectivity at 10 μM: The study established that 10 μM R59022 is below the threshold for inducing shape change-like effects and can be used as a relatively selective inhibitor of DG kinase in platelet studies. [2]

C27H26FN3OS

459.58

459.178

93076-89-2

R 59-022 hydrochloride;93076-98-3

3012

White to off-white solid powder

1.26g/cm3

619.8ºC at 760mmHg

328.6ºC

1.654

5.281

0

5

5

33

868

0

O=C1N2C(SC=C2)=NC(C)=C1CCN1CC/C(=C(\C2C=CC(F)=CC=2)/C2C=CC=CC=2)/CC1

MFVJXLPANKSLLD-UHFFFAOYSA-N

InChI=1S/C27H26FN3OS/c1-19-24(26(32)31-17-18-33-27(31)29-19)13-16-30-14-11-22(12-15-30)25(20-5-3-2-4-6-20)21-7-9-23(28)10-8-21/h2-10,17-18H,11-16H2,1H3

6-[2-[4-[(4-fluorophenyl)-phenylmethylidene]piperidin-1-yl]ethyl]-7-methyl-[1,3]thiazolo[3,2-a]pyrimidin-5-one

R59022 R 59022 R-59022

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 : ~62.5 mg/mL (~135.99 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.44 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 25.0 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: ≥ 2.5 mg/mL (5.44 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 25.0 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: ≥ 2.5 mg/mL (5.44 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 25.0 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.)