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Rottlerin (NSC-56346; NSC-94525), a naturally occuring compound isolated from Mallotus Philippinensis with the potential to be used as a chemotherapeutic agent for adrenocortical carcinoma, is a novel and specific protein kinase C (PKC) inhibitor with IC50 values for PKCδ of 3-6 μM, PKCα,β,γ of 30-42 μM, PKCε,η,ζ of 80-100 μM. Rottlerin acts as a direct mitochondrial uncoupler, and stimulates autophagy by targeting a signaling cascade upstream of mTORC1. Rottlerin induces apoptosis via caspase 3 activation. Rottlerin is an angiogenesis inhibitor and an inhibitor of protein kinase Cdelta (PKCdelta) and calmodulin kinase III.
Physicochemical Properties
| Molecular Formula | C30H28O8 |
| Molecular Weight | 516.53852 |
| Exact Mass | 516.178 |
| Elemental Analysis | C, 69.76; H, 5.46; O, 24.78 |
| CAS # | 82-08-6 |
| PubChem CID | 5281847 |
| Appearance | Brown to reddish brown solid powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 800.4±65.0 °C at 760 mmHg |
| Melting Point | 200 °C |
| Flash Point | 266.0±27.8 °C |
| Vapour Pressure | 0.0±2.9 mmHg at 25°C |
| Index of Refraction | 1.682 |
| LogP | 8.66 |
| Hydrogen Bond Donor Count | 5 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 6 |
| Heavy Atom Count | 38 |
| Complexity | 921 |
| Defined Atom Stereocenter Count | 0 |
| SMILES | CC1=C(C(=C(C(=C1O)C(=O)C)O)CC2=C(C(=C3C(=C2O)C=CC(O3)(C)C)C(=O)/C=C/C4=CC=CC=C4)O)O |
| InChi Key | DEZFNHCVIZBHBI-ZHACJKMWSA-N |
| InChi Code | InChI=1S/C30H28O8/c1-15-24(33)19(27(36)22(16(2)31)25(15)34)14-20-26(35)18-12-13-30(3,4)38-29(18)23(28(20)37)21(32)11-10-17-8-6-5-7-9-17/h5-13,33-37H,14H2,1-4H3/b11-10+ |
| Chemical Name | (E)-1-(6-((3-Acetyl-2,4,6-trihydroxy-5-methylphenyl)methyl)-5,7-dihydroxy-2,2-dimethyl-2H-1-benzopyran-8-yl)-3-phenyl-2-propen-1-one |
| Synonyms | Mallotoxin; NSC 56346; rottlerin; Mallotoxin; 82-08-6; Kamalin; UNII-E29LP3ZMUH; E29LP3ZMUH; EINECS 201-395-4; NSC 94525; NSC56346; NSC94525; Kamalin; NSC-56346; NSC-94525. |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
PKC; PKCδ(IC50 = 3-6 μM); PKCα,β,γ (IC50 = 30-42 μM); PKCε,η,ζ(IC50 = 80-100 μM) Rottlerin is a dose-dependent inhibitor of PKCθ (IC50 ≈ 1.25 μM) and PKCδ (IC50 ≥ 6.0 μM). At concentrations < 6.0 μM, it shows selective inhibition for PKCθ over PKCδ and PKCε/λ. |
| ln Vitro |
In primary HMVECs, rottlerin (20 μM, 2/6/24 hours) significantly and time-dependently lowers the levels of cyclin D-1 mRNA [2]. In HMVEC, rottlerin (20 μM) causes cell growth [2]. Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF-7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D-1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα-stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D-1 and endothelin-1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme-1 expression, thus contributing to the drop in endothelin-1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis[2]. Rottlerin inhibited HIV-1 replication in T cells. In MT-2 cells, IC50 was 5.2 μM, resulting in more than 20-fold reduction. In Jurkat cells, IC50 was 2.2 μM, with more than 20-fold reduction. In anti-CD3/CD28-activated peripheral blood lymphocytes (PBLs), IC50 was 4.4 μM, leading to more than 4-fold reduction. Rottlerin (3.0 μM) significantly inhibited HIV-1 proviral integration in MT-2 cells. Rottlerin induced a dose-dependent inhibition of HIV-1 LTR transactivation in basal conditions and upon PMA activation in Jurkat cells. It also inhibited NF-κB-dependent luciferase expression and Tat-mediated HIV-1 transactivation. Stable RNA interference of PKCθ mRNA in Jurkat and MT-2 cells (achieving 60-70% knockdown) led to more than 3-fold and 4-fold reduction in HIV-1 replication, respectively, compared to control cells. Transient co-transfection of PBLs with plasmids for PKCθ shRNA and HIV-1 proviral clone resulted in more than 6-fold reduction in HIV-1 replication. Rottlerin did not inhibit HIV-1 replication in PKCθ non-expressing U87.CD4.CXCR4 cells at concentrations within the low cytotoxicity range. Rottlerin did not interfere with viral entry, as it inhibited VSV-pseudotyped HIV-1 infection (independent of CD4/CXCR4) and did not significantly modify cell surface expression of CD4, CXCR4, or CCR5. Rottlerin (3.0 μM) did not modify the amount of early (R/U5) and late (LTR/gag) HIV-1 reverse transcription products in MT-2 cells. Rottlerin (3.0 μM) reduced phosphorylation of PKCθ at Thr538, prevented its translocation to lipid rafts, and repressed the PKCθ pathway effector NF-κB by inhibiting IκBα phosphorylation/degradation and NF-κB nuclear binding activity. |
| ln Vivo |
In Balb C nude mice, Rottlerin (20 mg/kg, once daily, five days a week, for six weeks) inhibits the growth of AsPC-1 pancreatic tumors without causing toxicity [3]. Rottlerin activates caspase-3 and cleaves poly(ADP-ribose) polymerase (PARP) to induce apoptosis. Rottlerin-treated mice showed a significant inhibition in tumor growth which was associated with suppression of cell proliferation, activation of capase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibited the markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epithelial-mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug and Snail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated from Kras(G12D) mice showed a significant inhibition in Akt, Shh and Notch pathways compared to control groups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiple signaling pathways which are constitutively active in pancreatic cancer. Taken together, our data show that the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch and Shh signaling pathways, and provide a new therapeutic approach with translational potential for humans.[3] |
| Enzyme Assay |
Rottlerin, a compound from Mallotus philippinensis, is shown to inhibit protein kinases with some specificity for PKC. To some extent, the novel inhibitor is able to differentiate between PKC isoenzymes, with IC50 values for PKC delta of 3-6 microM, PKC alpha,beta,gamma of 30-42 microM and PKC epsilon,eta,zeta of 80-100 microM. Inhibition of PKC appears, at least in part, to be due to a competition between rottlerin and ATP. Among the protein kinases tested, only CaM-kinase III is suppressed by rottlerin as effectively as PKC delta. The chemical structure of rottlerin might serve as a basis for the development of novel inhibitors with improved selectivity for a distinct PKC isoenzyme, such as PKC delta, or for CaM-kinase III [1]. PKCθ and PKCδ enzymatic activity was assayed using an immunoprecipitation kinase assay. Jurkat cells were incubated with inhibitors for 18 hours, lysed, and cytosolic extracts were immunoprecipitated with anti-PKCθ or anti-PKCδ antibodies. The immunoprecipitates were incubated with a PKCε peptide substrate, phosphatidylserine, diacylglycerol, and [γ-32P]ATP for 10 minutes at 30°C. The reaction was stopped by spotting onto phosphocellulose P81 paper, washed with phosphoric acid, and radioactivity was counted. Inhibition of enzymatic activity was analyzed, and IC50 was calculated. |
| Cell Assay |
Western blot analysis[2] Cell Types: primary HMVEC (human microvascular endothelial cells). Tested Concentrations: 20μM. Incubation Duration: 2, 6, 24 hrs (hours). Experimental Results: Cyclin D-1 mRNA levels were Dramatically diminished in a time-dependent manner. After 2 hrs (hours) of treatment, mRNA levels diminished to 50% of control, after 6 hrs (hours) to approximately 40%, and after 24 hrs (hours) to 20%. A similar trend was observed at the protein level, with a decrease of approximately 50% after 2 hrs (hours), an 80% decrease after 6 hrs (hours), and a decrease to almost undetectable levels after 24 hrs (hours). Cell proliferation assay [2] Cell Types: primary HMVEC (human microvascular endothelial cells). Tested Concentrations: 20μM. Incubation Duration: 24/48 hrs (hours). Experimental Results: demonstrated strong growth inhibition, with thymidine incorporation diminished by approximately 75% and 80%, respectively, relative to control cells (DMSO 0.1%). For HIV-1 infection assays, PBLs activated with anti-CD3/CD28 or PHA/IL-2 for 3 days, or MT-2 cells, were pretreated with Rottlerin for 30 minutes and then infected with HIV-1 NL4.3 strains for 2 hours. After washing, the compound was added again and left in culture for 2-7 days. Supernatants were collected for p24 antigen measurement, and cells were lysed for Renilla or luciferase activity quantification. IC50 was determined using a 96-well plate format with recombinant HIV-1 NL4.3-Renilla and increasing concentrations of Rottlerin, measuring Renilla activity 48 hours post-infection. Cell viability was determined using a luminescent cell viability assay quantifying ATP levels in metabolically active cells after incubation with Rottlerin for 72 hours. Cell viability was also assessed by bright-field microscopy and flow cytometry with propidium iodide staining. Cell proliferation was measured using a non-radioactive cell proliferation assay. PBLs were cultured with or without PHA and Rottlerin for 72 hours, followed by addition of a tetrazolium compound mixture. Absorbance was measured at 490 nm after 3 hours. For analysis of HIV-1 reverse transcription, MT-2 cells infected with HIV-1 NL4.3-wt for 18 hours were used to extract total DNA. Semiquantitative PCR was performed to amplify short (R/U5) and long (LTR/gag) reverse transcriptase products. For analysis of HIV-1 proviral integration, a semiquantitative nested Alu-PCR assay was performed on genomic DNA extracted from MT-2 cells infected with HIV-1 NL4.3-wt for 18 hours. For transcriptional activity assays, Jurkat cells were transfected with LTR-LUC or 3κB-LUC reporter vectors, treated with Rottlerin and/or PMA, and luciferase activity was measured after 18 hours. For DNA affinity immunoblotting, nuclear extracts from Jurkat cells were incubated with a biotin-labeled probe containing κB consensus motifs. Protein-DNA complexes were captured with streptavidin-agarose, separated by SDS-PAGE, and analyzed by immunoblotting for p65/RelA. For generation of stable PKCθ knockdown cells, Jurkat and MT-2 cells were co-transfected with shRNA plasmids (pGeneClip-iPKCθ-1 and pGeneClip-iPKCθ-3) by electroporation and selected with puromycin. |
| Animal Protocol |
Animal/Disease Models: Balb C nude mice (4-6 weeks old) were injected with AsPC-1 cells (2×106 cells mixed with Matrigel, 50:50 ratio) [3]. Doses: 0 or 20 mg/kg. Route of Administration: Administer one time/day, 5 days a week, for 6 weeks. Experimental Results: Inhibited the growth of AsPC-1 pancreatic tumors in Balb C nude mice and had no effect on the body weight of AsPC-1 tumor-bearing mice. |
| Toxicity/Toxicokinetics |
rat LDLo oral 750 mg/kg Indian Journal of Physiology and Pharmacology., 3(168), 1959 [PMID:13841348] Cytotoxicity of Rottlerin was evaluated in resting PBLs, Jurkat, and MT-2 cells incubated with increasing doses for 72 hours. It induced cytotoxicity at ≥15 μM in PBLs and ≥10 μM in Jurkat cells. No significant cytotoxicity was observed in MT-2 cells even at 100 μM. Half-maximal cytotoxic concentration (CC50) was calculated using a sigmoidal dose-response formula. Rottlerin (3.0 μM) did not significantly reduce cell viability during long-term treatment (7 days) in HIV-1 infected MT-2 cells and even increased cell viability more than 2-fold by protecting cells from HIV-induced cytopathic effect/syncytia formation. Rottlerin diminished PHA-induced T cell proliferation in a dose-dependent manner, keeping proliferation at the level of resting cells at 3.0 μM. |
| References |
[1]. Rottlerin, a novel protein kinase inhibitor. Biochem Biophys Res Commun. 1994 Feb 28;199(1):93-8. [2]. Rottlerin exhibits antiangiogenic effects in vitro. Chem Biol Drug Des. 2011 Jun;77(6):460-70. [3]. Rottlerin suppresses growth of human pancreatic tumors in nude mice, and pancreatic cancer cells isolated from KrasG12D mice. Cancer Letters 353 (2014) 32-40. [4]. Protein kinase Ctheta is a specific target for inhibition of the HIV type 1 replication in CD4+ T lymphocytes. J Biol Chem. 2011 Aug 5;286(31):27363-77. [5]. Kinase inhibitors tyrphostin 9 and rottlerin block early steps of rabies virus cycle. Antiviral Res. 2019 Aug;168:51-60. |
| Additional Infomation |
Rottlerin is a chromenol that is 2,2-dimethyl-2H-chromene substituted by hydroxy groups at positions 5 and 7, a 3-acetyl-2,4,6-trihydroxy-5-methylbenzyl group at position 6 and a (1E)-3-oxo-1-phenylprop-1-en-3-yl group at position 8. A potassium channel opener, it is isolated from Mallotus philippensis. It has a role as an antineoplastic agent, an apoptosis inducer, a metabolite, a K-ATP channel agonist, an antihypertensive agent and an anti-allergic agent. It is an enone, a chromenol, a benzenetriol, a methyl ketone and an aromatic ketone. Rottlerin has been reported in Mallotus philippensis with data available. \n\nThe purpose of the study was to examine the molecular mechanisms by which rottlerin inhibited growth of human pancreatic tumors in Balb C nude mice, and pancreatic cancer cells isolated from Kras(G12D) mice. AsPC-1 cells were injected subcutaneously into Balb c nude mice, and tumor-bearing mice were treated with rottlerin. Cell proliferation and apoptosis were measured by Ki67 and TUNEL staining, respectively. The expression of components of Akt, Notch, and Sonic Hedgehog (Shh) pathways were measured by the immunohistochemistry, Western blot analysis, and/or q-RT-PCR. The effects of rottlerin on pancreatic cancer cells isolated from Kras(G12D) mice were also examined. Rottlerin-treated mice showed a significant inhibition in tumor growth which was associated with suppression of cell proliferation, activation of capase-3 and cleavage of PARP. Rottlerin inhibited the expression of Bcl-2, cyclin D1, CDK2 and CDK6, and induced the expression of Bax in tumor tissues compared to untreated control. Rottlerin inhibited the markers of angiogenesis (Cox-2, VEGF, VEGFR, and IL-8), and metastasis (MMP-2 and MMP-9), thus blocking production of tumorigenic mediators in tumor microenvironment. Rottlerin also inhibited epithelial-mesenchymal transition by up-regulating E-cadherin and inhibiting the expression of Slug and Snail. Furthermore, rottlerin treatment of xenografted tumors or pancreatic cancer cells isolated from Kras(G12D) mice showed a significant inhibition in Akt, Shh and Notch pathways compared to control groups. These data suggest that rottlerin can inhibit pancreatic cancer growth by suppressing multiple signaling pathways which are constitutively active in pancreatic cancer. Taken together, our data show that the rottlerin induces apoptosis and inhibits pancreatic cancer growth by targeting Akt, Notch and Shh signaling pathways, and provide a new therapeutic approach with translational potential for humans.[3] \nIntegration of HIV-1 genome in CD4(+) T cells produces latent reservoirs with long half-life that impedes the eradication of the infection. Control of viral replication is essential to reduce the size of latent reservoirs, mainly during primary infection when HIV-1 infects CD4(+) T cells massively. The addition of immunosuppressive agents to highly active antiretroviral therapy during primary infection would suppress HIV-1 replication by limiting T cell activation, but these agents show potential risk for causing lymphoproliferative disorders. Selective inhibition of PKC, crucial for T cell function, would limit T cell activation and HIV-1 replication without causing general immunosuppression due to PKC being mostly expressed in T cells. Accordingly, the effect of rottlerin, a dose-dependent PKC inhibitor, on HIV-1 replication was analyzed in T cells. Rottlerin was able to reduce HIV-1 replication more than 20-fold in MT-2 (IC(50) = 5.2 μM) and Jurkat (IC(50) = 2.2 μM) cells and more than 4-fold in peripheral blood lymphocytes (IC(50) = 4.4 μM). Selective inhibition of PKC, but not PKCδ or -ζ, was observed at <6.0 μM, decreasing the phosphorylation at residue Thr(538) on the kinase catalytic domain activation loop and avoiding PKC translocation to the lipid rafts. Consequently, the main effector at the end of PKC pathway, NF-κB, was repressed. Rottlerin also caused a significant inhibition of HIV-1 integration. Recently, several specific PKC inhibitors have been designed for the treatment of autoimmune diseases. Using these inhibitors in combination with highly active antiretroviral therapy during primary infection could be helpful to avoid massive viral infection and replication from infected CD4(+) T cells, reducing the reservoir size at early stages of the infection.[4]\n \nRabies virus (RABV) is a neurotropic virus that causes fatal encephalitis in humans and animals and still kills up to 59,000 people worldwide every year. To date, only preventive or post-exposure vaccination protects against the disease but therapeutics are missing. After screening a library of 80 kinases inhibitors, we identified two compounds as potent inhibitors of RABV infection: tyrphostin 9 and rottlerin. Mechanism of action studies show that both inhibitors interfere with an early step of viral cycle and can prevent viral replication. In presence of tyrphostin 9, the viral entry through endocytosis is disturbed leading to improper delivery of viral particles in cytoplasm, whereas rottlerin is inhibiting the transcription, most likely by decreasing intracellular ATP concentration, and therefore the replication of the viral genome.[5] Rottlerin (also known as Mallotoxin) is a cell-permeable PKC inhibitor used as a research tool. Its inhibitory effect on HIV-1 replication is sustained from the beginning of infection in long-term culture. The study uses Rottlerin to demonstrate the importance of PKCθ in HIV-1 replication in T cells, not to promote it as a clinical candidate. The compound has a narrow therapeutic range and is intended for research only. PKCθ is selectively expressed in T cells and is crucial for T cell receptor-mediated activation and NF-κB signaling. Specific inhibition of PKCθ could limit T cell activation and HIV-1 replication without causing general immunosuppression. The authors suggest that specific PKCθ inhibitors, designed for autoimmune diseases, could be evaluated in combination with HAART during primary HIV-1 infection to reduce massive viral replication and latent reservoir establishment. |
Solubility Data
| Solubility (In Vitro) | DMSO : ~12.5 mg/mL (~24.20 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 1.25 mg/mL (2.42 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 12.5 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. Solubility in Formulation 2: ≥ 1.25 mg/mL (2.42 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 3: 22 mg/mL (42.59 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.9360 mL | 9.6798 mL | 19.3596 mL | |
| 5 mM | 0.3872 mL | 1.9360 mL | 3.8719 mL | |
| 10 mM | 0.1936 mL | 0.9680 mL | 1.9360 mL |