PKA (Ki = 10 μM); PKC (Ki = 4 μM)
cGMP-dependent protein kinase (cGPK) – Ki = 0.234 μM [1]
Myosin light-chain kinase (MLCK) – Ki > 10 μM [1]
cAMP-dependent protein kinase (cAPK) – Ki > 10 μM [1]
Protein kinase C (PKC) – Ki = 4.0 μM [1]
KT5823 targets cGMP-dependent protein kinase (PKG), a serine/threonine kinase that mediates many of the effects of cGMP. It has a Ki value of 0.23 μM for PKG. It also inhibits PKA and PKC with Ki values of 10 μM and 4 μM, respectively. It is cell-permeable.

KT5823 inhibits cGMP-dependent protein kinase.[1]
The effectiveness of the cGMP-dependent protein kinase inhibitor, KT5823, was investigated in human neutrophils. KT5823 did not inhibit the cGMP-dependent protein kinase mediated in vitro, or in situ phosphorylation of vimentin, a known substrate for this enzyme in activated neutrophils. In addition, KT5823 was shown to induce dramatic shape changes in neutrophils, suggesting it has an activating effect upon the cells.[2]

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Inhibiting basal PKG activity with KT5823 increased apoptotic DNA fragmentation by 9.8-fold. [3]

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These Bay 60-7550's effects were prevented by pretreatment with the PKG inhibitor KT5823. Moreover, the Bay 60-7550-induced downstream phosphorylation of cyclic AMP response element binding (pCREB) and brain-derived neurotrophic factor (BDNF) expression was also prevented (or partially prevented) byKT5823or the PKA inhibitor H89. [4]

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Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP- and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To distinguish among the different cGMP effectors, inhibitors of cGK and PKA have been developed including the K-252 compound KT5823 and the isoquinolinesulfonamide H89. KT5823, an in vitro inhibitor of cGK, has also been used in numerous studies with intact cells to implicate or rule out the involvement of this protein kinase in a given cellular response. However, the efficacy and specificity of KT5823 as cGK inhibitor in intact cells or tissues have never been demonstrated. Here, we analyzed the effects of both KT5823 and H89 on cyclic-nucleotide-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) in intact human platelets and rat mesangial cells. These two cell types both express high levels of cGK. KT5823 inhibited purified cGK. However, with both intact human platelets and rat mesangial cells, KT5823 failed to inhibit cGK-mediated serine 157 and serine 239 phosphorylation of VASP induced by nitric oxide, atrial natriuretic peptide, or the membrane-permeant cGMP analog, 8-pCPT-cGMP. KT5823 enhanced 8-pCPT-cGMP-stimulated VASP phosphorylation in platelets and did not inhibit forskolin-stimulated VASP phosphorylation in either platelets or mesangial cells. In contrast H89, an inhibitor of both PKA and cGK, clearly inhibited 8-pCPT-cGMP and forskolin-stimulated VASP phosphorylation in the two cell types. The data indicate that KT5823 inhibits purified cGK but does not affect a cGK-mediated response in the two different cell types expressing cGK I. These observations indicate that data that interpret the effects of KT5823 in intact cells as the major or only criteria supporting the involvement of cGK clearly need to be reconsidered[5].

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KT5823 (15 μM, 18 h) caused normal human skin fibroblasts (HSF55) to arrest in the G0/G1 phase, with an average of 84.5 ± 7.0% of cells accumulating with 2C DNA content. In contrast, transformed human skin fibroblasts (CT10-2C-T1) showed no cell cycle arrest under the same conditions. [1]
Increasing the concentration of KT5823 up to 30 μM did not induce a G2/M phase arrest in HSF55 cells. [1]
Release experiments: HSF55 cells arrested with KT5823 (15 μM for 18 h) entered S phase 7.2 ± 0.2 h after removal of the inhibitor, indicating an arrest point approximately 5.8 h after the G0/G1 transition. [1]
In vitro, KT5823 is a selective PKG inhibitor with a Ki of 0.23 μM. It also inhibits PKA and PKC with Ki values of 10 μM and 4 μM, respectively. It abolishes the cGMP-induced relaxation in smooth muscle cells with an IC₅₀ of 60 nM. It arrests human skin fibroblast cell cycle after the G0/G1 boundary.

However, these results of Bay 60-7550 on memory acquisition were suppressed by PKG inhibitor KT5823 and PKA inhibitor H89, both of which were managed 30 min prior to Bay 60-7550 (Figure 2B). Furthermore, PKG inhibitor KT5823 or PKA inhibitor H89 that used alone had no effect on acquisition ability in block 6 in Aβ1–42-treated mice (Figure 2C).[4]
The ameliorating results of high-dose Bay 60-7550 (3.0 mg/kg) both in long-term and short memories were blocked by pretreatment with KT5823 (Figure 3). H89 prevented high-dose Bay 60-7550 (3.0 mg/kg)-induced long-term memory retention (24 h after the training session) (Figures 3C,D), but not short-term memory retrieval (1 h after the training session) in the water maze test (p > 0.05, Figures 3A,B), while neither H89 nor KT5823 used alone impacted the learning and memory abilities in Aβ1–42-treated mice (Figure 3).[4]
PKG inhibitor KT5823 significantly prevented the effects of Bay 60-7550 on step-down latency both 1 and 3 h after the training session (Figures 4A,B). PKA inhibitor H89 significantly prevented the effects of Bay 60-7550 on step-down latency 3 h after the training session (Figure 4B), but not step-down latency 1 h after the training session (Figure 4A). Neither H89 nor KT5823 used alone impacted the memory ability in Aβ1–42-treated mice (Figures 4A,B).[4]
PKG inhibitor KT5823 blocked Bay 60-7550’s effects on BDNF expression in these two brain regions[4].
In vivo, KT5823 has the potential to modulate PKG-mediated signaling pathways. By inhibiting PKG, it could affect smooth muscle relaxation, platelet function, and neuronal signaling. However, specific in vivo efficacy data in animal models are not extensively detailed in the available literature.

The inhibition constants (Ki) for KT5823 against various kinases were previously reported (Kase, H., 1988; referenced in the paper). Values are as follows: cGMP-dependent protein kinase (cGPK) Ki = 0.234 μM; myosin light-chain kinase (MLCK) Ki > 10 μM; cAMP-dependent protein kinase (cAPK) Ki > 10 μM; protein kinase C (PKC) Ki = 4.0 μM. [1]
In vitro PKG kinase assays for KT5823 are performed using purified recombinant PKG enzyme. The kinase is incubated with a peptide substrate and ATP in the presence of varying concentrations of KT5823. Phosphorylation is detected using a luminescence-based or fluorescence-based assay. Ki or IC₅₀ values are calculated from dose-response curves.

Apoptotic DNA fragmentation induced by the PKG inhibitorKT5823 in unstressed HRE‐H9 cells[3]
We suspected that the anti‐apoptotic effects of cGMP (at basal levels) were mediated by partial activation of PKG (i.e. basal PKG activity) in HRE‐H9 cells. To test this, we used a potent and highly selective PKG inhibitor, KT5823. Exposure of HRE‐H9 cells to KT5823 in a wide range of concentrations for 24 h caused dose‐dependent increases in apoptotic DNA fragmentation, measured by CE‐LIF (Figure 10). Because normal medium (with serum) was used, the HRE‐H9 cells were under unstressed conditions (except for the exposure to KT5823). While KT5823 at 1 nmol/l and 10 nmol/l did not affect the levels of apoptotic DNA fragmentation, KT5823 at 100 nmol/l and 1 µmol/l (concentrations expected to selectively inhibit PKG; Ki = 234 nmol/l, Calbiochem catalogue 2003/2004) substantially increased DNA fragmentation, compared with controls (Figure 10). KT5823 at 100 nmol/l and 1 µmol/l caused 5.9‐ and 9.8‐fold increases in the DNA fragmentation levels respectively (Figure 11). The statistical significance of these effects was high (P < 0.001), due to the large pro‐apoptotic effects of this PKG inhibitor. The data suggest that the low‐level activation of PKG that occurs under normal culturing conditions, presumably because of the basal cGMP levels, may play an essential role in preventing spontaneous development of apoptosis in unstressed HRE‐H9 cells.

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Mouse study (Aβ1-42 model): Male ICR mice (8 weeks, 22-25 g) were used. KT5823 (source: Cayman Chemical) was microinjected bilaterally into the intracerebroventricular (i.c.v.) at a volume of 2 μl per mouse (dose not specified in the paper) 30 min prior to daily treatment with Bay 60-7550 (i.p.) for 14 days. The injection coordinates: anterior-posterior −1.7 mm from bregma, mediolateral ±0.8 mm, dorsoventral −2.0 mm from dura. Administration was performed daily during the 14-day treatment period. Behavioral tests (Morris water maze and step-down passive avoidance) were conducted 24 h after the last drug treatment, and then mice were sacrificed for biochemical assays (Western blot for CRF, GR, pCREB, CREB, BDNF). [4]
In the HRE-H9 cell study (no animal protocol for KT5823 alone), cells were cultured in DMEM with 4% FBS, penicillin/streptomycin at 37 °C, 5% CO2. For experiments, cells were seeded at 1×10⁶ cells per well in 6-well plates and treated with KT5823 for 24 h. [3]
In vitro cell-based assays for KT5823 are performed using smooth muscle cells or other cell types expressing PKG. Cells are cultured in appropriate media and treated with KT5823 at various concentrations. PKG activity is assessed by measuring the phosphorylation of PKG substrates by Western blot. Smooth muscle relaxation is measured using contraction assays. Cell cycle analysis is performed by flow cytometry.

The mice were treated with different doses of Bay 60-7550 (0.5, 1.0, 3.0 mg/kg/day, i.p.) or vehicle for 14 days after microinjection of Aβ1–42. PKG inhibitor KT5823 and PKA inhibitor H89 were microinjected bilaterally into the cerebroventricular, 30 min prior to treatment with Bay 60-7550. Antibodies against CRF, GR, p-CREB, CREB, and BDNF were obtained from Abcam[4].

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In vivo animal studies for KT5823 would typically involve rodent models of smooth muscle function or platelet function. The compound is administered intraperitoneally or intravenously. Smooth muscle relaxation is assessed by measuring blood pressure or vascular tone. Platelet function is assessed by aggregometry.

Specific pharmacokinetic properties of KT5823, such as half-life and oral bioavailability, are not extensively detailed in the available literature. It is cell-permeable. It is soluble in DMSO and is typically formulated for in vivo administration.

KT5823 is a selective inhibitor of protein kinase G (PKG) and is frequently used as a pharmacological tool to investigate cGMP/PKG signaling pathways. In the uterine epithelial cell study (HRE-H9), basal PKG activity was shown to be anti-apoptotic; inhibition by KT5823 triggered spontaneous apoptosis, suggesting that normal basal PKG activity is necessary for cell survival. In the Alzheimer’s disease mouse model, the memory-enhancing effects of the PDE2 inhibitor Bay 60-7550 were dependent on PKG activation, as KT5823 completely blocked these effects. This indicates that the cGMP/PKG pathway plays a crucial role in cognitive function and HPA axis regulation. [3][4]
Comprehensive toxicological data for KT5823 are not widely available in public literature. As a research compound, it is intended for laboratory use only and is not for human therapeutic use. Standard safety precautions should be followed when handling this compound. The compound is supplied with a purity of ≥98%.

[1]. Multiple kinase arrest points in the G1 phase of nontransformed mammalian cells are absent in transformed cells. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8626-30.
[2]. KT5823 activates human neutrophils and fails to inhibit cGMP-dependent protein kinase phosphorylation of vimentin. Res Commun Chem Pathol Pharmacol. 1991 Oct;74(1):3-14.
[3]. Guanylyl cyclase inhibitors NS2028 and ODQ and protein kinase G (PKG) inhibitor KT5823 trigger apoptotic DNA fragmentation in immortalized uterine epithelial cells: anti-apoptotic effects of basal cGMP/PKG. Mol Hum Reprod. 2003 Dec;9(12):775-83.
[4]. Ruan L, et al. Phosphodiesterase-2 Inhibitor Bay 60-7550 Ameliorates Aβ-Induced Cognitive and Memory Impairment via Regulation of the HPA Axis. Front Cell Neurosci. 2019 Oct 2;13:432.
[5]. KT5823 inhibits cGMP-dependent protein kinase activity in vitro but not in intact human platelets and rat mesangial cells. J Biol Chem. 2000 Oct 27;275(43):33536-41.

KT 5823 is an organic heterooctane compound with the structure 1H,1'H-2,2'-biindole, wherein the nitrogen atom is formally oxidatively coupled to the 2 and 5 positions of methyl(3R)-3-methoxy-2-methyltetrahydrofuran-3-carboxylic acid ester (2S,3R,5R product), and the 3 and 3' positions of the biindole moiety are also formally oxidatively coupled to the 3 and 4 positions of 1-methyl-1,5-dihydro-2H-pyrrole-2-one. It is an EC 2.7.11.12 (cGMP-dependent protein kinase) inhibitor. It is a γ-lactam, organic heterooctane compound, methyl ester, hemiacetal, and indolecarbazole. Many signal transduction pathways are mediated by the second messengers cGMP and cAMP, cGMP and cAMP-dependent protein kinases (cGK and PKA), phosphodiesterases, and ion channels. To differentiate between different cGMP effectors, cGK and PKA inhibitors have been developed, including the K-252 compound KT5823 and isoquinoline sulfonamide H89. KT5823, an in vitro cGK inhibitor, has also been used in many intact cell studies to identify or rule out the role of this protein kinase in specific cellular responses. However, the efficacy and specificity of KT5823 as a cGK inhibitor in intact cells or tissues have not been confirmed. This study analyzed the effects of KT5823 and H89 on cyclic nucleotide-mediated vasodilatory stimulating phosphoprotein (VASP) phosphorylation in intact human platelets and mouse mesangial cells. Both cell types highly express cGK. KT5823 inhibited purified cGK. However, in intact human platelets and mouse mesangial cells, KT5823 failed to inhibit cGK-mediated VASP serine 157 and serine 239 phosphorylation induced by nitric oxide, atrial natriuretic peptide, or the membrane permeability cGMP analog 8-pCPT-cGMP. KT5823 enhanced phosphorylation of VASP in 8-pCPT-cGMP-stimulated platelets, but did not inhibit phosphorylation of VASP in forosclin-stimulated platelets or mesangial cells. In contrast, H89, a dual inhibitor of PKA and cGK, significantly inhibited both 8-pCPT-cGMP and forosclin-stimulated VASP phosphorylation in both cell types. The data suggest that KT5823 inhibits purified cGK, but does not affect cGK-mediated responses in two different cell types expressing cGK I. These observations suggest that the role of KT5823 in intact cells should be reconsidered as the primary or sole criterion for supporting cGK involvement. [5]

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KT5823 is a specific inhibitor of cGMP-dependent protein kinase. It was used as a biochemical probe to demonstrate that multiple kinase-mediated events are required for G1 phase progression in normal human fibroblasts, and that these mechanisms are absent or bypassed in simian virus 40-transformed cells. The G1 arrest induced by KT5823 occurs later (≈6 h after G0/G1) than that induced by staurosporine (≈2.5 h) or by KT5926/K252b (≈3.8 h). This suggests that cGPK activity is required at a specific point in mid-G1 phase for normal cell cycle progression. [1]
KT5823 is a selective PKG inhibitor. It is a staurosporine-related protein kinase inhibitor. It has a Ki of 0.23 μM for PKG. This product is for research use only.

495.179

C, 70.29; H, 5.09; N, 8.48; O, 16.14

126643-37-6

108152

White to off-white solid powder

1.52g/cm3

629.2ºC at 760mmHg

334.3ºC

9.67E-16mmHg at 25°C

1.763

4.589

0

5

3

37

1020

3

QTYMDECKVKSGSM-YMUMJAELSA-N

InChI=1S/C29H25N3O5/c1-28-29(36-4,27(34)35-3)13-20(37-28)31-18-11-7-5-9-15(18)22-23-17(14-30(2)26(23)33)21-16-10-6-8-12-19(16)32(28)25(21)24(22)31/h5-12,20H,13-14H2,1-4H3/t20-,28+,29+/m1/s1

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)

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.)