4-Deoxyphorbol

4-Deoxyphorbol inhibits HIV-1 infection in synergism with antiretroviral drugs and reactivates viral reservoirs through PKC/MEK activation synergizing with vorinostat.[Pubmed:32224142]

Biochem Pharmacol. 2020 Jul;177:113937.

Latent HIV reservoirs are the main obstacle to eradicate HIV infection. One strategy proposes to eliminate these viral reservoirs by pharmacologically reactivating the latently infected T cells. We show here that a 4-Deoxyphorbol ester derivative isolated from Euphorbia amygdaloides ssp. semiperfoliata, 4beta-dPE A, reactivates HIV-1 from latency and could potentially contribute to decrease the viral reservoir. 4beta-dPE A shows two effects in the HIV replication cycle, infection inhibition and HIV transactivation, similarly to other phorboids PKC agonists such PMA and prostratin and to other diterpene esters such SJ23B. Our data suggest 4beta-dPE A is non-tumorigenic, unlike the related compound PMA. As the compounds are highly similar, the lack of tumorigenicity by 4beta-dPE A could be due to the lack of a long side lipophilic chain that is present in PMA. 4beta-dPE activates HIV transcription at nanomolar concentrations, lower than the concentration needed by other latency reversing agents (LRAs) such as prostratin and similar to bryostatin. PKCtheta/MEK activation is required for the transcriptional activity, and thus, anti-latency activity of 4beta-dPE A. However, CD4, CXCR4 and CCR5 receptors down-regulation effect seems to be independent of PCK/MEK, suggesting the existence of at least two different targets for 4beta-dPE A. Furthermore, NF-kappab transcription factor is involved in 4beta-dPE HIV reactivation, as previously shown for other PKCs agonists. We also studied the effects of 4beta-dPE A in combination with other LRAs. When 4beta-dPE A was combined with another PKC agonists such as prostratin an antagonic effect was achieved, while, when combined with an HDAC inhibitor such as vorinostat, a strong synergistic effect was obtained. Interestingly, the latency reversing effect of the combination was synergistically diminishing the EC(50) value but also increasing the efficacy showed by the drugs alone. In addition, combinations of 4beta-dPE A with antiretroviral drugs as CCR5 antagonist, NRTIs, NNRTIs and PIs, showed a consistent synergistic effect, suggesting that the combination would not interefer with antiretroviral therapy (ART). Finally, 4beta-dPE A induced latent HIV reactivation in CD4 + T cells of infected patients under ART at similar levels than the tumorigenic phorbol derivative PMA, showing a clear reactivation effect. In summary, we describe here the mechanism of action of a new potent deoxyphorbol derivative as a latency reversing agent candidate to decrease the size of HIV reservoirs.

Analytical methods to access the chemical composition of an Euphorbia tirucalli anticancer latex from traditional Brazilian medicine.[Pubmed:30928500]

J Ethnopharmacol. 2019 Jun 12;237:255-265.

ETHNOPHARMACOLOGICAL RELEVANCE: Euphorbia tirucalli L. is widely used by Brazilian folk medicine, mainly for its anticancer activity. However, its commercialization was banned by The Brazilian National Sanitary Surveillance Agency (ANVISA) due to the presence of some compounds considered toxic, such as the diterpene esters. AIM OF THE STUDY: Chemical and biological analyses were performed with the Brazilian Euphorbia tirucalli latex to support its wide traditional use in Brazil. MATERIAL AND METHODS: Latex was collected by using two procedures, in a solution of dichloromethane: methanol (3:1, 100 mL) and in 100 mL of distilled water. The first procedure was concentrated as a crude extract and the second one was partitioned with hexane and dichloromethane. The partitions and crude extract were subjected to phytochemical analyses using three different methods: Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) with electrospray ionization sources in negative mode (ESI(-)) as well as in tandem mass spectrometry ESI(-) MS/MS and Atmospheric Pressure Chemical Ionization in positive mode (APCI(+)), Gas Chromatography coupled Mass Spectrometry (GC-MS) and Nuclear Magnetic Resonance (NMR) ((1)H-NMR and (13)C-NMR). The cytotoxic potential was evaluated using the crude extract in macrophages RAW 264.7 and Gastric Adenocarcinoma (AGS) cancer cells. The evaluation of immunomodulatory activity was made through the detection of Nitric Oxide (NO) and cytokines as Tumor necrosis factor alpha (TNF-alpha) and Interleukin-6 (IL-6). RESULTS: GC-MS showed the presence of some esters of fatty acids, for instance myristic, palmitic, stearic, oleic and linoleic acid and, mainly, triterpenes such as euphol and tirucallol. With NMR, most of the signals were related to triterpenoids euphol and tirucallol. However, when the latex was analyzed with ESI(-) FT-ICR MS, a wide variety of molecules from different classes of natural products (fatty acids, diterpenes, triterpenes, steroids) were found. On the other hand, when APCI(+)FT-ICR MS was used, the ion M(+.) At ratio mass-charge (m/z) 426.38567, related to triterpenes euphol and tirucallol masses, presented the most intense peak, with a mass error of -0.11, indicating high accuracy. Diterpene esters from 4-Deoxyphorbol and ingenol were identified only by ESI(-)FT-ICR MS and ESI(-)FT-ICR MS/MS. When evaluated biologically, the crude latex showed immunomodulatory activity, as it reduced the production of the pro-inflammatory cytokines TNF-alpha, IL-6 and NO, and the effect on NO reduction was more significant, obtaining in a similar result to the N(omega)-Nitro-L-Arginine Methyl Ester (L-NAME) standards, as well as significant cytotoxic activity with half inhibitory concentration (IC(50)) values of 69.43 +/- 1.29 mug/mL against AGS without damaging healthy ones. CONCLUSION: It was verified that the Brazilian Euphorbia tirucalli latex consists mainly of the triterpenes euphol and tirucallol, which may be the main cause of the anticancer activity attributed to the plant, but many other minor compounds could have been determined by the FT-ICR MS method, such as the diterpene esters. It has antitumor potential because it acts selectively against cancer cells and it also prevents the progression of tumors, because it carries an important immunomodulatory effect.

Environmentally Friendly Procedure Based on Supercritical Fluid Chromatography and Tandem Mass Spectrometry Molecular Networking for the Discovery of Potent Antiviral Compounds from Euphorbia semiperfoliata.[Pubmed:28925702]

J Nat Prod. 2017 Oct 27;80(10):2620-2629.

A supercritical fluid chromatography-based targeted purification procedure using tandem mass spectrometry and molecular networking was developed to analyze, annotate, and isolate secondary metabolites from complex plant extract mixture. This approach was applied for the targeted isolation of new antiviral diterpene esters from Euphorbia semiperfoliata whole plant extract. The analysis of bioactive fractions revealed that unknown diterpene esters, including jatrophane esters and phorbol esters, were present in the samples. The purification procedure using semipreparative supercritical fluid chromatography led to the isolation and identification of two new jatrophane esters (13 and 14) and one known (15) and three new 4-Deoxyphorbol esters (16-18). The structure and absolute configuration of compound 16 were confirmed by X-ray crystallography. This compound was found to display antiviral activity against Chikungunya virus (EC(50) = 0.45 muM), while compound 15 proved to be a potent and selective inhibitor of HIV-1 replication in a recombinant virus assay (EC(50) = 13 nM). This study showed that a supercritical fluid chromatography-based protocol and molecular networking can facilitate and accelerate the discovery of bioactive small molecules by targeting molecules of interest, while minimizing the use of toxic solvents.

Synthesis and biological evaluation of 12-aminoacylphorboids.[Pubmed:20121237]

J Nat Prod. 2010 Mar 26;73(3):447-51.

Spurred by the paradoxical anti-inflammatory activity of some aminoacylphorbol derivatives, the naturally occurring and epimeric N,N-dimethylvalinoyl-4alpha-4-Deoxyphorbol derivatives 3b and 3d have been prepared from 4alpha-4-Deoxyphorbol (3e), a byproduct of the isolation of phorbol from Croton oil and a phorboid polyol so far largely overlooked in terms of biological activity. The configuration of the side chain stereocenter was confirmed for both natural products, and to investigate the side chain structure-activity relationships within this class of compounds, their corresponding N,N-dimethylglycinate (3g) and nor (3h) and di-nor derivatives (3i, 3j) were also prepared. By using a PKC-sensitive model of HIV-1 latency (activation of HIV- gene expression in Jurkat-LAT-GFP cells), it was found that both 3b and 3d can activate PKC-dependent responses, while a series of experiments with isoform-specific PKC inhibitors showed that these compounds target PKCalpha and -delta. Both N,N-dimethylation and the presence of side chain alpha-substitution were critical for activity. Selective PKC binding, rather than COX inhibition, might explain the paradoxical anti-inflammatory activity of extracts containing aminoacylphorboids in the mouse ear edema assay.

Nitrogen-containing phorbol esters from Croton ciliatoglandulifer and their effects on cyclooxygenases-1 and -2.[Pubmed:16792405]

J Nat Prod. 2006 Jun;69(6):887-90.

Four new phorbol derivatives, 12-O-[(2R)-N,N-dimethyl-3-methylbutanoyl]-4-Deoxyphorbol 13-acetate (1), 12-O-[(2S)-N,N-dimethyl-3-methylbutanoyl]-4-Deoxyphorbol 13-acetate (2), 12-O-[3-methyl-2-butenoyl]-4-Deoxyphorbol 13-acetate (3), and 12-O-[(2R)-N,N-dimethyl-3-methylbutanoyl]phorbol 13-acetate (4), along with six known compounds, were isolated from the aerial parts of Croton ciliatoglandulifer. An anti-inflammatory activity of a hexane extract of this plant was demonstrated against ear edema in mice produced by 12-O-tetradecanoylphorbol 13-acetate, and compounds 1, 4, and 3beta-O-acetyloleanolic acid (5) were active when evaluated against cyclooxygenases-1 and -2.

Tigliane diterpenes from the latex of Euphorbia obtusifolia with inhibitory activity on the mammalian mitochondrial respiratory chain.[Pubmed:12639752]

J Ethnopharmacol. 2003 Apr;85(2-3):279-82.

Six diterpenes isolated from the latex of Euphorbia obtusifolia Poir. (Euphorbiaceae) were evaluated for their inhibition of the NADH oxidase activity in submitochondrial particles from beef heart. 4,20-Dideoxyphorbol-12,13-bis(isobutyrate) was the most potent inhibitor and showed an inhibitory concentration with IC(50) value of 2.6+/-0.3mM. In the present study, some structure-activity trends are suggested for the inhibitory activity of the mammalian mitochondrial respiratory chain of these natural product derivatives of 4-Deoxyphorbol esters.

Dietary cancer risk from conditional cancerogens (tumor promoters) in produce of livestock fed on species of spurge (Euphorbiaceae). V. Skin irriitant and tumor-promoting diterpene ester toxins of the tigliane and ingenane type in the herbs Euphorbia nubica and Euphorbia helioscopia contaminating fodder of livestock.[Pubmed:11206270]

J Cancer Res Clin Oncol. 2001 Jan;127(1):40-7.

Irritant diterpene ester toxins were isolated from Euphorbia nubica and E. helioscopia, which are contaminants of the green fodder of livestock in Egypt. Fractionations of methanol extracts of aerial parts of both plants were monitored by the irritation unit on the mouse ear. Plant extracts were subjected to multiplicative distribution methods, yielding irritant hydrophilic fractions that were further purified by column chromatography. Final purification of the materials was achieved by TLC (silica gel) followed by HPLC, or by TLC alone. In this way, from E. nubica, five Euphorbia factors (Nu1-Nu5) were isolated and characterized as short-chain polyfunctional diterpene esters of tigliane-type parent alcohols. The two weak irritants Nul and Nu3 were triesters of 4-deoxy(4alpha)phorbol. Nu2 was shown to be a triester of the stereoisomeric tigliane-type parent alcohol 4-Deoxyphorbol. Weak irritant Nu4 probably is a positional isomer of Nu2. Nu5 was characterized as a short-chain triester of 4,20-dideoxy-5xi-hydroxyphorbol. From E. helioscopia, six short- to medium-chain polyfunctional diterpene esters of the ingenane type, generally containing unsaturated acids were obtained, i.e., four irritant esters of ingenol (Euphorbia factors H1, H2, H5, and H6) and two esters of 20-deoxyingenol (non-irritant Euphorbia substance HS4, and irritant Euphorbia factor H8). All irritant Euphorbia factors of the tigliane and ingenane diterpene ester type described in this investigation are considered to be more or less active tumor promoters, i.e., conditional (non-genotoxic) cancerogens. The Euphorbia factors assayed exhibited moderate (H1) to low (H8) relative tumor-promoting potency in comparison to the ingenane prototype DTE tumor promoter 3-TI.

Macrocyclic diterpenoids from Euphorbia semiperfoliata.[Pubmed:9644058]

J Nat Prod. 1998 Jun 26;61(6):749-56.

In addition to known compounds, the aerial parts of E. semiperfoliata afforded an abietanolide (3), 13 jatrophane polyesters (4-9, 12, 14-19), two 4-Deoxyphorbol diesters (23, 24), and a pair of epimeric diterpenes (21, 22) with a novel carbon skeleton, which was named euphoperfoliane. Structures were determined by spectroscopic analysis, and the main conformational features of jatropha-6(17),11-dienes are discussed in detail. The obtained isolation yield of several jatrophanes was unprecedented within the spurges (Euphorbia spp.), making E. semiperfoliata a unique source of macrocyclic diterpenoids.

Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection.[Pubmed:8608235]

Blood. 1996 Feb 15;87(4):1446-57.

Four novel Epstein-Barr virus (EBV)-carrying T-cell lines, designated SIS, AIK-T8, AIK-T4, and SKN, were established from peripheral blood lymphocytes (PBL) of patients with severe chronic active EBV infection, in the presence of interleukin-2 and 4-Deoxyphorbol ester. AIK-T8 and -T4 were derived from a single patient. Cell marker and genotype analyses showed that SIS, AIK-T8, and AIK-T4 had mature T-cell phenotypes with clonally rearranged T-cell receptor (TCR) genes, whereas SKN had an immature T-cell phenotype without TCR gene rearrangement. None of the cell lines expressed B, natural killer, or myeloid antigens or had Ig gene rearrangement. All lines carried EBV genomes in a single episomal form. SIS, AIK-T8, and SKN showed the same phenotype, TCR gene configuration, and/or EBV clonotype as their source or biopsied materials; therefore, they represented EBV-infected T cells proliferating in the patients. TCR gene and EBV episomal structures similar to those of AIK-T4 were not found in its source PBL, probably due to the few parental clones in vivo. All lines expressed EBV-encoded small RNA (EBER) 1, nuclear antigen (EBNA) 1, and latent membrane protein (LMP) 1, -2A, and -2B, but not other EBNAs that could be recognized by EBV-specific immune T cells. EBV replicative antigens were rarely expressed or induced. Such EBV latency reflects the in vivo situation, in which the T cells may evade immune surveillance and be insensitive to antiherpesvirus drugs. Collectively, the data suggest that EBV can target and latently infect T cells at any stage of differentiation in vivo, thus potentially causing uncontrolled T-cell proliferation. These cell lines will facilitate further analyses of possible EBV-induced oncogenicity in T cells.

[Epstein-Barr virus-specific immunity in asymptomatic carriers of human T-cell leukemia virus type 1].[Pubmed:7774883]

Hokkaido Igaku Zasshi. 1995 Mar;70(2):315-28.

Adult T-cell leukemia (ATL) patients are immunosuppressed as evidenced by anergy to recall antigens and the occurrence of opportunistic infections. The immunosuppression appears to be a critical factor or a predictive sign for the development of ATL in carriers of human T-cell leukemia virus type 1 (HTLV-1). This study was aimed at assessing the immune status of asymptomatic HTLV-1 carriers with the immunity specific to Epstein-Barr virus (EBV), a ubiquitous human herpesvirus with oncogenic potential. Forty-three asymptomatic HTLV-I carriers were examined for their EBV serology and EBV-specific cytotoxic T-cell (EBV-CTL) activity, in comparison with 10 HTLV-I-non-infected normal controls. Both carriers and controls were all positive for EBV capsid antigen (VCA) IgG. Significantly elevated titer of VCAIgG and lower titer of EBV-determined nuclear antigen (EBNA) antibodies were observed in asymptomatic HTLV-I carriers, suggesting reactivation of EBV. Among the HTLV-I carriers, 9 (20.9%) had reduced activity of EBV-CTL as revealed by lower incidence of regression of in vitro EBV-induced B-cell transformation. Accordingly, asymptomatic HTLV-I carriers were divided into three groups: the carriers with reduced EBV-specific cellular immunity (group I), the carriers showing normal cellular immunity but aberrant EBV-specific antibody titers (group II), and the carriers with normal EBV-specific cellular immunity and serology (group III). Higher positive rate of anti-HTLV-I Tax antibody was found in the former two groups (44.4% and 56.5%, respectively) compared with group III (18.2%). An immunosuppressive agent, 4-Deoxyphorbol ester induced a remarkable decrease of EBV-CTL activity in the carriers of group II and III at the concentration that affected none of the normal controls. These findings indicate that asymptomatic HTLV-I carriers suffer stepwise impairment of EBV-specific immunities, which may be caused by HTLV-I infection.

Cryptic dysfunction of cellular immunity in asymptomatic human immunodeficiency virus (HIV) carriers and its actualization by an environmental immunosuppressive factor.[Pubmed:7772730]

In Vivo. 1994 Nov-Dec;8(6):1019-22.

Peripheral blood lymphocytes from 4 asymptomatic HIV-1 carriers with normally retained EBV-specific cytotoxic T-cell activity were exposed to EBV and incubated with 0.2 ng/ml 4-Deoxyphorbol ester, an immunosuppressive substance derived from an African plant Euphorbia tirucalli. The regression of EBV-induced B-cell transformation by EBV-specific cytotoxic T-cells was significantly impaired in the presence of a small amount of 4-Deoxyphorbol ester, but not so in 5 HIV-1-seronegative healthy counterparts. When the EBV-specific cytotoxic T-cells from the asymptomatic carriers were exposed to 0.2ng/ml 4-Deoxyphorbol ester and incubated with 51Cr-labeled autologous EBV-transformed B lymphocytes, the released radioactivity was significantly smaller than that of the healthy counterparts. The results suggest that the cellular immunity of the asymptomatic HIV-1 carriers is cryptically impaired, and the cryptic immunological dysfunction is actualized by exposure to a small amount of the immunosuppressive substance, a dose which does not affect the immunity of uninfected healthy individuals.

African Burkitt's lymphoma: a plant, Euphorbia tirucalli, reduces Epstein-Barr virus-specific cellular immunity.[Pubmed:8074496]

Anticancer Res. 1994 May-Jun;14(3A):933-6.

Endemicity of Burkitt's lymphoma (BL) coincides with profusion of a plant Euphorbia tirucalli in tropical Africa. E. tirucalli contains 4-Deoxyphorbol ester that enhances Epstein-Barr virus (EBV) infection of B lymphocytes. In this study, we found that 4-Deoxyphorbol ester reduced EBV-specific cytotoxic T-cell function. Furthermore, the B lymphocytes dually exposed to EBV and 4-Deoxyphorbol ester were resistant to EBV-specific T cell cytotoxicity, through down-regulation of latent membrane protein 1 (LMP1), the major target to EBV-specific cytotoxic T-cells. These immunologic findings strengthen the notion that E. tirucalli may be an important environmental risk factor for the genesis of African BL.

Chromosome translocation and c-MYC activation by Epstein-Barr virus and Euphorbia tirucalli in B lymphocytes.[Pubmed:1673740]

Lancet. 1991 May 18;337(8751):1190.

Dual exposure to Epstein-Barr virus and purified 4-Deoxyphorbol ester derived from the plant Euphorbia tirucalli induced a high frequency of chromosomal rearrangements in human B lymphocytes in vitro. Rearrangements most commonly affected chromosome 8, the chromosome most often showing structural changes in Burkitt's lymphoma (BL) cells. E tirucalli is indigenous in parts of Africa where BL is endemic and may be an important risk factor for the disease.

Sapintoxin A. A fluorescent phorbol ester that is a potent activator of protein kinase C but is not a tumour promoter.[Pubmed:3690506]

Cancer Lett. 1987 Dec;38(1-2):165-70.

In this communication we report on the activity of the naturally occurring, highly fluorescent phorbol ester Sapintoxin A (12-o-[2-methylaminobenzoate]-4-Deoxyphorbol 13-acetate). This compound potently activates the enzyme protein kinase C (PKC) (Ka 76 nM) but is neither a complete nor second-stage tumour promoter in traditional Berenblum tests. Sapintoxin A has properties in common with promoters such as 12-o-tetradecanoylphorbol 13-acetate (TPA) in that it will induce erythema in vivo, induce lymphocyte mitogenesis in vitro and cause aggregation of human and rabbit platelets. Accordingly, Sapintoxin A is a suitable negative control compound for biochemical studies concerning the involvement of PKC in tumour promotion and cell proliferation.